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P1 SHOX and limb development

  1. Top of page
  2. P1 SHOX and limb development
  3. P2 Developmental mechanisms underlying polydactyly in the mouse mutant, Doublefoot
  4. P3 Migration behaviour of myogenic precursor cells in the limb bud with respect to dorsoventral patterning
  5. P4 Axial myogenic cells differentiate and express fast and slow myosin heavy chains early, but splitting into individual muscles is then delayed in the chick embryo (Gallus)
  6. P5 Difference of chondrogenic pattern of cultured limb mesenchyme taken from various part of the limb bud
  7. P6 An approach to the role of hedgehogs in vascular development via the chicken mutant talpid3
  8. P7 Fin development in a cartilaginous fish and the origin of vertebrate limbs
  9. P8 Contributions of neural crest and paraxial mesoderm to craniofacial morphogenesis in the chick embryo
  10. P9 Functions of ALX4 and MSX2 in ossification of the skull vault
  11. P10 Tooth formation in reaggreated dental mesenchyme of mice
  12. P11 Morphological evidence for the role of epithelial tissue during mouse tongue development
  13. P12 From ectodermal bud to epidermal appendage: does keratin expression play a role in early mammary gland development?
  14. P13 An embryological study of the anorectal system in mice
  15. P14 Contrast agents as an aid to the magnetic resonance microimaging examination of chick embryos
  16. P15 Functional characterisation of mouse pre-Sertoli cells cultured on reconstituted basement membrane
  17. P16 On the estimation and comparison of intracellular gold labelling densities: a simple and efficient method for quantitative immunoelectron microscopy
  18. P17 Quantitative analysis of placental microstructure in pregnancies complicated by pre-eclampsia with and without intrauterine growth restriction
  19. P18 Human endometrial stroma following exposure to the levonorgestrel intrauterine system (LNG-IUS)
  20. P19 Sex dimorphism in anterior pituitary glucocorticoid feedback revealed by annexin 1 knockout in mice
  21. P20 Effects of knockout of neuronal nitric oxide synthase (nNOS) on pituitary gonadotrophs
  22. P21 The vasculature of the spinal cord of the wobbler mutant mouse: a stereological study
  23. P22 Radial glial cell interactions in the developing spinal cord: an immunohistochemical and confocal analysis
  24. P23 An ultrastructural investigation of the early rat spinal ventral commissure

E. Tiecke 1, R. Blaschke 2, C. Tickle 1 and G. Rappold 2

1 Division of Cell and Developmental Biology, Wellcome Trust Biocentre, University of Dundee, UK; and 2 Institute of Human Genetics, University of Heidelberg, Germany

SHOX is a member of the homeobox-containing gene family and is highly conserved amongst species as diverse as fish, chicken and humans. SHOX was first cloned in 1997 and shown to be causative for idiopathic short stature. It is located in the pseudoautosomal region (PAR1) and haploinsufficiency of SHOX has been implicated in short stature and additional skeletal malformations frequently observed in Turner syndrome. SHOX mutations have also been shown to underlie other phenotypes such as Leri-Weill and Langer syndrome.

We have performed an in situ hybridisation analysis of SHOX expression during limb development in chicken embryos. We find that SHOX is expressed in a central region of the early limb bud, leaving a rim of non-expressing cells around it. When these buds are sectioned expression is found to be in a thin layer of mesenchyme just under the ectoderm. In later stages, expression is restricted to the proximal two thirds of the limb bud and eventually becomes expressed in the digital rays with stronger expression dorsally.

We have started to explore how SHOX expression is controlled. When the apical ridge of a chick limb bud was removed, SHOX expression extended to the distal edge of the bud. We are currently testing whether Fgfs play a role in inhibiting SHOX expression in distal mesenchyme.

P2 Developmental mechanisms underlying polydactyly in the mouse mutant, Doublefoot

  1. Top of page
  2. P1 SHOX and limb development
  3. P2 Developmental mechanisms underlying polydactyly in the mouse mutant, Doublefoot
  4. P3 Migration behaviour of myogenic precursor cells in the limb bud with respect to dorsoventral patterning
  5. P4 Axial myogenic cells differentiate and express fast and slow myosin heavy chains early, but splitting into individual muscles is then delayed in the chick embryo (Gallus)
  6. P5 Difference of chondrogenic pattern of cultured limb mesenchyme taken from various part of the limb bud
  7. P6 An approach to the role of hedgehogs in vascular development via the chicken mutant talpid3
  8. P7 Fin development in a cartilaginous fish and the origin of vertebrate limbs
  9. P8 Contributions of neural crest and paraxial mesoderm to craniofacial morphogenesis in the chick embryo
  10. P9 Functions of ALX4 and MSX2 in ossification of the skull vault
  11. P10 Tooth formation in reaggreated dental mesenchyme of mice
  12. P11 Morphological evidence for the role of epithelial tissue during mouse tongue development
  13. P12 From ectodermal bud to epidermal appendage: does keratin expression play a role in early mammary gland development?
  14. P13 An embryological study of the anorectal system in mice
  15. P14 Contrast agents as an aid to the magnetic resonance microimaging examination of chick embryos
  16. P15 Functional characterisation of mouse pre-Sertoli cells cultured on reconstituted basement membrane
  17. P16 On the estimation and comparison of intracellular gold labelling densities: a simple and efficient method for quantitative immunoelectron microscopy
  18. P17 Quantitative analysis of placental microstructure in pregnancies complicated by pre-eclampsia with and without intrauterine growth restriction
  19. P18 Human endometrial stroma following exposure to the levonorgestrel intrauterine system (LNG-IUS)
  20. P19 Sex dimorphism in anterior pituitary glucocorticoid feedback revealed by annexin 1 knockout in mice
  21. P20 Effects of knockout of neuronal nitric oxide synthase (nNOS) on pituitary gonadotrophs
  22. P21 The vasculature of the spinal cord of the wobbler mutant mouse: a stereological study
  23. P22 Radial glial cell interactions in the developing spinal cord: an immunohistochemical and confocal analysis
  24. P23 An ultrastructural investigation of the early rat spinal ventral commissure

A. Crick 1,2, J.M. Brown 1 and G.M. Morriss-Kay 1

1 Department of Human Anatomy and Genetics, University of Oxford; and 2 Department of Plastic Surgery, Radcliffe Infirmary, Oxford, UK

The preaxial polydactylous mouse mutant Doublefoot (Dbf) is exceptional in showing normal Shh expression although it has 6–8 triphalangeal digits per limb. However genes down-stream of Shh, including its own receptor and direct transcriptional target Patched (Ptc1) which in the wild-type all have posterior domains of expression, are all ectopically expressed throughout the distal limb bud mesenchyme in Dbf. Grafts of posterior, mid-distal and anterior Dbf limb bud mesenchyme into anterior chick limb buds in ovo all induce digit duplications. Thus there is ectopic polarising activity and activation of the Shh pathway in the absence of ectopic expression of Shh itself. Indian hedgehog (Ihh) is another of the 3 vertebrate homologues of the Drosophila gene hedgehog (hh), and is normally associated with patterning of the cartilage anlagen later in development of the limb. Ihh has recently been mapped to the Dbf interval, although Ihh is not itself the locus of the Dbf mutation which has not yet been identified. Using polarising grafts and whole mount in situ hybridisation we have shown that Ihh is precociously and ectopically expressed throughout the distal limb bud mesenchyme in Dbf and that this pattern of expression correlates spatially and temporally with the ectopic polarising activity and activation of the hedgehog pathway. Cell proliferation and programmed cell death are important cellular mechanisms involved in the morphogenesis of the mouse limb bud. Polydactylous mouse mutants, including Dbf, have limb buds that are abnormally enlarged along the anteroposterior axis. Using bromodeoxyuridine incorporation followed by immunohistochemical detection on paraffin embedded sections, and whole mount acridine orange staining, we have shown that the normal posterior bias for cell proliferation is gradually reversed and that the region of programmed cell death known as the anterior necrotic zone is absent in the Dbf limb bud. In summary, the ectopic Ihh expression correlates spatially and temporally with the appearance of the abnormally enlarged limb bud, the ectopic polarising activity, the ectopic expression of genes downstream in the hedgehog pathway, the increase in cell proliferation and the reduction in programmed cell death seen in the Dbf limb bud.

P3 Migration behaviour of myogenic precursor cells in the limb bud with respect to dorsoventral patterning

  1. Top of page
  2. P1 SHOX and limb development
  3. P2 Developmental mechanisms underlying polydactyly in the mouse mutant, Doublefoot
  4. P3 Migration behaviour of myogenic precursor cells in the limb bud with respect to dorsoventral patterning
  5. P4 Axial myogenic cells differentiate and express fast and slow myosin heavy chains early, but splitting into individual muscles is then delayed in the chick embryo (Gallus)
  6. P5 Difference of chondrogenic pattern of cultured limb mesenchyme taken from various part of the limb bud
  7. P6 An approach to the role of hedgehogs in vascular development via the chicken mutant talpid3
  8. P7 Fin development in a cartilaginous fish and the origin of vertebrate limbs
  9. P8 Contributions of neural crest and paraxial mesoderm to craniofacial morphogenesis in the chick embryo
  10. P9 Functions of ALX4 and MSX2 in ossification of the skull vault
  11. P10 Tooth formation in reaggreated dental mesenchyme of mice
  12. P11 Morphological evidence for the role of epithelial tissue during mouse tongue development
  13. P12 From ectodermal bud to epidermal appendage: does keratin expression play a role in early mammary gland development?
  14. P13 An embryological study of the anorectal system in mice
  15. P14 Contrast agents as an aid to the magnetic resonance microimaging examination of chick embryos
  16. P15 Functional characterisation of mouse pre-Sertoli cells cultured on reconstituted basement membrane
  17. P16 On the estimation and comparison of intracellular gold labelling densities: a simple and efficient method for quantitative immunoelectron microscopy
  18. P17 Quantitative analysis of placental microstructure in pregnancies complicated by pre-eclampsia with and without intrauterine growth restriction
  19. P18 Human endometrial stroma following exposure to the levonorgestrel intrauterine system (LNG-IUS)
  20. P19 Sex dimorphism in anterior pituitary glucocorticoid feedback revealed by annexin 1 knockout in mice
  21. P20 Effects of knockout of neuronal nitric oxide synthase (nNOS) on pituitary gonadotrophs
  22. P21 The vasculature of the spinal cord of the wobbler mutant mouse: a stereological study
  23. P22 Radial glial cell interactions in the developing spinal cord: an immunohistochemical and confocal analysis
  24. P23 An ultrastructural investigation of the early rat spinal ventral commissure

H. Schweizer 1,2, R.L. Johnson 2 and B. Brand-Saberi 1

1 Institute of Anatomy, University of Freiburg, Germany; and 2 Department of Biochemistry and Molecular Biology, M.D. Anderson Cancer Center, Texas, USA

Limb buds develop from somatopleure derived stationary mesenchyme as well as from cells invading the limb bud proximally. The largest population of these cells are myogenic precursor cells which are derived from the lateral dermomyotomes.

After detachment under the influence of SF/HGF, myogenic precursor cells migrate in a proximodistal direction and populate a dorsal and ventral zone. The patterning mechanism leading to the segregation of dorsal and ventral myogenic cells is at present not understood.

We have addressed the problem of dorsoventral patterning of muscle precursor cells in the limb buds using quail-chick chimeras and transgenic mice.

Although cells seem to be capable of migrating either ventrally or dorsally, they are restricted to the positions they attain during early migration and keep to these during later development.

Furthermore we addressed the question of differences between dorsal and ventral migration behaviour. This may be important because in knockout mice flexor and extensor muscles are differentially affected by targeting of specific genes such as Lbx1 or Mox2. We also used information from transgenic mice carrying a LacZ reporter gene under the control of the endogenous Lmx-1b locus for histological analysis of dorsal and ventral tissues.

P4 Axial myogenic cells differentiate and express fast and slow myosin heavy chains early, but splitting into individual muscles is then delayed in the chick embryo (Gallus)

  1. Top of page
  2. P1 SHOX and limb development
  3. P2 Developmental mechanisms underlying polydactyly in the mouse mutant, Doublefoot
  4. P3 Migration behaviour of myogenic precursor cells in the limb bud with respect to dorsoventral patterning
  5. P4 Axial myogenic cells differentiate and express fast and slow myosin heavy chains early, but splitting into individual muscles is then delayed in the chick embryo (Gallus)
  6. P5 Difference of chondrogenic pattern of cultured limb mesenchyme taken from various part of the limb bud
  7. P6 An approach to the role of hedgehogs in vascular development via the chicken mutant talpid3
  8. P7 Fin development in a cartilaginous fish and the origin of vertebrate limbs
  9. P8 Contributions of neural crest and paraxial mesoderm to craniofacial morphogenesis in the chick embryo
  10. P9 Functions of ALX4 and MSX2 in ossification of the skull vault
  11. P10 Tooth formation in reaggreated dental mesenchyme of mice
  12. P11 Morphological evidence for the role of epithelial tissue during mouse tongue development
  13. P12 From ectodermal bud to epidermal appendage: does keratin expression play a role in early mammary gland development?
  14. P13 An embryological study of the anorectal system in mice
  15. P14 Contrast agents as an aid to the magnetic resonance microimaging examination of chick embryos
  16. P15 Functional characterisation of mouse pre-Sertoli cells cultured on reconstituted basement membrane
  17. P16 On the estimation and comparison of intracellular gold labelling densities: a simple and efficient method for quantitative immunoelectron microscopy
  18. P17 Quantitative analysis of placental microstructure in pregnancies complicated by pre-eclampsia with and without intrauterine growth restriction
  19. P18 Human endometrial stroma following exposure to the levonorgestrel intrauterine system (LNG-IUS)
  20. P19 Sex dimorphism in anterior pituitary glucocorticoid feedback revealed by annexin 1 knockout in mice
  21. P20 Effects of knockout of neuronal nitric oxide synthase (nNOS) on pituitary gonadotrophs
  22. P21 The vasculature of the spinal cord of the wobbler mutant mouse: a stereological study
  23. P22 Radial glial cell interactions in the developing spinal cord: an immunohistochemical and confocal analysis
  24. P23 An ultrastructural investigation of the early rat spinal ventral commissure

P.J. Adds and L.G. Robson

Department of Neuroscience, Barts and the London, Queen Mary's School of Medicine and Dentistry, London, UK

The epaxial muscles form the postvertebral muscle groups, and develop from the myotome of the somites. The myotome develops from the myogenic precursors that come from the dorsomedial lip of the dermomyotome. These myogenic cells differentiate in a cranial to caudal sequence, with the first myotomal muscle fibres differentiating in the cranial somites at Hamburger and Hamilton stage 10. Differentiation then proceeds caudally until stage 23–24. Although we know the differentiation sequence of the myotome, little is known about the subsequent development into the individual muscles, and this is the aim of this study. Here we use triple labelling to simultaneously follow the development of the tendons and the muscle fibre types in the epaxial muscles. This labelling reveals that the myotome is divided into the epimere and hypomere regions by tenascin labelling the intermuscular septum by stage 26–27. Tenascin labelling surrounds the epimere muscle mass, but this muscle mass does not split into the individual epaxial muscles until stage 28–29 when tenascin labelling is found within the epimere. Before any splitting of the myotome has occurred, there are specific regions of fast and slow myosin heavy chain (MyHC) expression. There are 2 regions that only contain fast MyHC fibres, one is located at the dorso-medial lip of the myotome, and the second is located more ventrally and lies next to the sclerotome. This study reveals that the formation of the individual epaxial muscles in the chick is delayed when compared to the limb and face, where by stage 28–29 the formation of the individual muscles is well under way, even though the myogenic cells of the myotome have differentiated considerably earlier. Studies are ongoing to elucidate further the process of epaxial muscle formation.

P5 Difference of chondrogenic pattern of cultured limb mesenchyme taken from various part of the limb bud

  1. Top of page
  2. P1 SHOX and limb development
  3. P2 Developmental mechanisms underlying polydactyly in the mouse mutant, Doublefoot
  4. P3 Migration behaviour of myogenic precursor cells in the limb bud with respect to dorsoventral patterning
  5. P4 Axial myogenic cells differentiate and express fast and slow myosin heavy chains early, but splitting into individual muscles is then delayed in the chick embryo (Gallus)
  6. P5 Difference of chondrogenic pattern of cultured limb mesenchyme taken from various part of the limb bud
  7. P6 An approach to the role of hedgehogs in vascular development via the chicken mutant talpid3
  8. P7 Fin development in a cartilaginous fish and the origin of vertebrate limbs
  9. P8 Contributions of neural crest and paraxial mesoderm to craniofacial morphogenesis in the chick embryo
  10. P9 Functions of ALX4 and MSX2 in ossification of the skull vault
  11. P10 Tooth formation in reaggreated dental mesenchyme of mice
  12. P11 Morphological evidence for the role of epithelial tissue during mouse tongue development
  13. P12 From ectodermal bud to epidermal appendage: does keratin expression play a role in early mammary gland development?
  14. P13 An embryological study of the anorectal system in mice
  15. P14 Contrast agents as an aid to the magnetic resonance microimaging examination of chick embryos
  16. P15 Functional characterisation of mouse pre-Sertoli cells cultured on reconstituted basement membrane
  17. P16 On the estimation and comparison of intracellular gold labelling densities: a simple and efficient method for quantitative immunoelectron microscopy
  18. P17 Quantitative analysis of placental microstructure in pregnancies complicated by pre-eclampsia with and without intrauterine growth restriction
  19. P18 Human endometrial stroma following exposure to the levonorgestrel intrauterine system (LNG-IUS)
  20. P19 Sex dimorphism in anterior pituitary glucocorticoid feedback revealed by annexin 1 knockout in mice
  21. P20 Effects of knockout of neuronal nitric oxide synthase (nNOS) on pituitary gonadotrophs
  22. P21 The vasculature of the spinal cord of the wobbler mutant mouse: a stereological study
  23. P22 Radial glial cell interactions in the developing spinal cord: an immunohistochemical and confocal analysis
  24. P23 An ultrastructural investigation of the early rat spinal ventral commissure

T. Miura

Department of Anatomy and Developmental Biology, Kyoto University Graduate School of Medicine, Japan

It has been speculated that a reaction-diffusion mechanism is working to make the periodic pattern of chondrogenesis during digit formation in limb development. One drawback in this hypothesis is that digits appear sequentially from posterior to anterior direction (except the fifth digit) in vivo, whereas the entire structure should appear simultaneously in the reaction-diffusion model. To explain the sequential appearance of digits we hypothesised that the parameter of the reaction term in the reaction-diffusion model should be different at the ZPA region, and undertook micromass culture of various parts of the mouse embryonic d 10.5 limb bud. The chondrogenic area appeared like spots when we cultured cells from the posterior part of the limb bud, whereas the chondrogenic area appeared like stripes when we cultured cells from other parts of the limb bud. To confirm this tendency, we developed an automated method to detect quantitatively the continuity of the periodic pattern using an image-processing technique, and confirmed that a statistically significant difference can be detected between the chondrogenic patterns generated by cells from different part of the limb bud. This kind of change in pattern can be reproduced in a computer simulation by changing the reaction term of the reaction-diffusion model, and we can similarly reproduce the sequential appearance of structures by changing this parameter of one end of the limb field. A similar kind of pattern change could be obtained using all-transretinoic acid, but currently we could not detect a change due to Sonic Hedgehog treatment. These results suggest that the ZPA region can influence the reaction-diffusion mechanism in the limb bud in vivo.

P6 An approach to the role of hedgehogs in vascular development via the chicken mutant talpid3

  1. Top of page
  2. P1 SHOX and limb development
  3. P2 Developmental mechanisms underlying polydactyly in the mouse mutant, Doublefoot
  4. P3 Migration behaviour of myogenic precursor cells in the limb bud with respect to dorsoventral patterning
  5. P4 Axial myogenic cells differentiate and express fast and slow myosin heavy chains early, but splitting into individual muscles is then delayed in the chick embryo (Gallus)
  6. P5 Difference of chondrogenic pattern of cultured limb mesenchyme taken from various part of the limb bud
  7. P6 An approach to the role of hedgehogs in vascular development via the chicken mutant talpid3
  8. P7 Fin development in a cartilaginous fish and the origin of vertebrate limbs
  9. P8 Contributions of neural crest and paraxial mesoderm to craniofacial morphogenesis in the chick embryo
  10. P9 Functions of ALX4 and MSX2 in ossification of the skull vault
  11. P10 Tooth formation in reaggreated dental mesenchyme of mice
  12. P11 Morphological evidence for the role of epithelial tissue during mouse tongue development
  13. P12 From ectodermal bud to epidermal appendage: does keratin expression play a role in early mammary gland development?
  14. P13 An embryological study of the anorectal system in mice
  15. P14 Contrast agents as an aid to the magnetic resonance microimaging examination of chick embryos
  16. P15 Functional characterisation of mouse pre-Sertoli cells cultured on reconstituted basement membrane
  17. P16 On the estimation and comparison of intracellular gold labelling densities: a simple and efficient method for quantitative immunoelectron microscopy
  18. P17 Quantitative analysis of placental microstructure in pregnancies complicated by pre-eclampsia with and without intrauterine growth restriction
  19. P18 Human endometrial stroma following exposure to the levonorgestrel intrauterine system (LNG-IUS)
  20. P19 Sex dimorphism in anterior pituitary glucocorticoid feedback revealed by annexin 1 knockout in mice
  21. P20 Effects of knockout of neuronal nitric oxide synthase (nNOS) on pituitary gonadotrophs
  22. P21 The vasculature of the spinal cord of the wobbler mutant mouse: a stereological study
  23. P22 Radial glial cell interactions in the developing spinal cord: an immunohistochemical and confocal analysis
  24. P23 An ultrastructural investigation of the early rat spinal ventral commissure

M.G. Davey 1, I.R. Paton 2, D.R. Morrice 2, D.W. Burt 2 and C. Tickle 1

1 Division of Cell and Developmental Biology, School of Life Sciences, University of Dundee; and 2 Roslin Institute, Midlothian, UK

The polydactylous chicken mutant talpid3 has an abnormality in Hedgehog signalling. We have shown that this leads to the ectopic localisation of Shh protein in the limb and this has important consequences on limb patterning. Futhermore there is an abnormal vasculature pattern in the talpid3 embryos including that of the limb (Mohammed 1986). Through analysis of talpid3 we have examined the effects that abnormal hedgehog signalling has on the vasculature. We have further characterised the role of Shh in patterning the vasculature of the limb through manipulations of the normal wing bud. Recent evidence has shown that hedgehogs play an important role in the patterning of the vasculature of the zebrafish (Lawson et al. in press) and this is consistent with the abnormal patterning of the vasculature in the talpid3 limb.

P7 Fin development in a cartilaginous fish and the origin of vertebrate limbs

  1. Top of page
  2. P1 SHOX and limb development
  3. P2 Developmental mechanisms underlying polydactyly in the mouse mutant, Doublefoot
  4. P3 Migration behaviour of myogenic precursor cells in the limb bud with respect to dorsoventral patterning
  5. P4 Axial myogenic cells differentiate and express fast and slow myosin heavy chains early, but splitting into individual muscles is then delayed in the chick embryo (Gallus)
  6. P5 Difference of chondrogenic pattern of cultured limb mesenchyme taken from various part of the limb bud
  7. P6 An approach to the role of hedgehogs in vascular development via the chicken mutant talpid3
  8. P7 Fin development in a cartilaginous fish and the origin of vertebrate limbs
  9. P8 Contributions of neural crest and paraxial mesoderm to craniofacial morphogenesis in the chick embryo
  10. P9 Functions of ALX4 and MSX2 in ossification of the skull vault
  11. P10 Tooth formation in reaggreated dental mesenchyme of mice
  12. P11 Morphological evidence for the role of epithelial tissue during mouse tongue development
  13. P12 From ectodermal bud to epidermal appendage: does keratin expression play a role in early mammary gland development?
  14. P13 An embryological study of the anorectal system in mice
  15. P14 Contrast agents as an aid to the magnetic resonance microimaging examination of chick embryos
  16. P15 Functional characterisation of mouse pre-Sertoli cells cultured on reconstituted basement membrane
  17. P16 On the estimation and comparison of intracellular gold labelling densities: a simple and efficient method for quantitative immunoelectron microscopy
  18. P17 Quantitative analysis of placental microstructure in pregnancies complicated by pre-eclampsia with and without intrauterine growth restriction
  19. P18 Human endometrial stroma following exposure to the levonorgestrel intrauterine system (LNG-IUS)
  20. P19 Sex dimorphism in anterior pituitary glucocorticoid feedback revealed by annexin 1 knockout in mice
  21. P20 Effects of knockout of neuronal nitric oxide synthase (nNOS) on pituitary gonadotrophs
  22. P21 The vasculature of the spinal cord of the wobbler mutant mouse: a stereological study
  23. P22 Radial glial cell interactions in the developing spinal cord: an immunohistochemical and confocal analysis
  24. P23 An ultrastructural investigation of the early rat spinal ventral commissure

M. Tanaka 1, A. Munsterberg 1,4, W.G. Anderson 3, A.R. Prescot 2, N. Hazon 3 and C. Tickle 1

1 Division of Cell and Developmental Biology, and 2 Division of Cell Biology and Immunology, Wellcome Trust Biocentre, University of Dundee; 3School of Biology, Division of Environmental and Evolutionary Biology, Gatty Marine Lab, University of St. Andrews; and 4School of Biological Sciences, University of East Anglia, UK

Recent fossil finds and experimental analysis of chick and mouse embryos prompted us to re-explore the lateral fin fold theory which suggests that two pairs of limbs in tetrapods evolved by subdivision of an elongated single fin. We examined fin development in embryos of the primitive cartilaginous fish, Scyliorhinus canicula (dogfish) by scanning electron microscopy and investigated expression of genes known to be involved in limb positioning, identity and patterning in higher vertebrates. Although we did not detect lateral fin folds in dogfish embryos, Engrailed-1 expression suggests the body is compartmentalised dorsoventrally. Furthermore, specification of limb identity occurs via Tbx4 and Tbx5 genes as in higher vertebrates. In contrast, unlike higher vertebrates, we could not detect Shh transcripts in dogfish fin buds, although dHand (a gene involved in establishing Shh) is expressed. In S. canicula, the main fin axis seems to lie parallel to the body axis. Freeing fins from the body axis and establishing a separate limb axis has been proposed to be a crucial step in evolution of tetrapod limbs. We suggest that Shh plays a critical role in this process.

P8 Contributions of neural crest and paraxial mesoderm to craniofacial morphogenesis in the chick embryo

  1. Top of page
  2. P1 SHOX and limb development
  3. P2 Developmental mechanisms underlying polydactyly in the mouse mutant, Doublefoot
  4. P3 Migration behaviour of myogenic precursor cells in the limb bud with respect to dorsoventral patterning
  5. P4 Axial myogenic cells differentiate and express fast and slow myosin heavy chains early, but splitting into individual muscles is then delayed in the chick embryo (Gallus)
  6. P5 Difference of chondrogenic pattern of cultured limb mesenchyme taken from various part of the limb bud
  7. P6 An approach to the role of hedgehogs in vascular development via the chicken mutant talpid3
  8. P7 Fin development in a cartilaginous fish and the origin of vertebrate limbs
  9. P8 Contributions of neural crest and paraxial mesoderm to craniofacial morphogenesis in the chick embryo
  10. P9 Functions of ALX4 and MSX2 in ossification of the skull vault
  11. P10 Tooth formation in reaggreated dental mesenchyme of mice
  12. P11 Morphological evidence for the role of epithelial tissue during mouse tongue development
  13. P12 From ectodermal bud to epidermal appendage: does keratin expression play a role in early mammary gland development?
  14. P13 An embryological study of the anorectal system in mice
  15. P14 Contrast agents as an aid to the magnetic resonance microimaging examination of chick embryos
  16. P15 Functional characterisation of mouse pre-Sertoli cells cultured on reconstituted basement membrane
  17. P16 On the estimation and comparison of intracellular gold labelling densities: a simple and efficient method for quantitative immunoelectron microscopy
  18. P17 Quantitative analysis of placental microstructure in pregnancies complicated by pre-eclampsia with and without intrauterine growth restriction
  19. P18 Human endometrial stroma following exposure to the levonorgestrel intrauterine system (LNG-IUS)
  20. P19 Sex dimorphism in anterior pituitary glucocorticoid feedback revealed by annexin 1 knockout in mice
  21. P20 Effects of knockout of neuronal nitric oxide synthase (nNOS) on pituitary gonadotrophs
  22. P21 The vasculature of the spinal cord of the wobbler mutant mouse: a stereological study
  23. P22 Radial glial cell interactions in the developing spinal cord: an immunohistochemical and confocal analysis
  24. P23 An ultrastructural investigation of the early rat spinal ventral commissure

D.J.R. Evans 1, I.G. Thomas 1, S. Thomas 1, A. Prabhu 1 and M. Zhang 2

1 School of Biosciences, Cardiff University, Wales, UK; and 2 Department of Anatomy and Structural Biology, University of Otago, Dunedin, New Zealand

Fate mapping of cranial neural crest and paraxial mesoderm cell populations have already been investigated extensively using quail-chick transplantation, however controversies still remain about some of the precise derivatives of each population and the relations between these precursor cells during morphogenesis. In the present study we utilised the LacZ reporter gene, encoding for bacterial β-galactosidase to label and follow individual neural crest and paraxial mesoderm populations. The gene was introduced into cells via a replication deficient retroviral vector. Injections of retrovirus were made into precisely recorded regions of mesoderm of stage 10 to 12 White Leghorn chick embryos. Following cervical dislocation, at stage 36, embryos were fixed and stained for β-galactosidase to identify infected cells and their descendants. Where injections were made adjacent to the level of the junction of the mesencephalon and metencephalon, crest cells gave rise to a precise pattern of derivatives including skeletal elements and connective tissues, whereas mesoderm gave rise to myogenic and endothelial cells. In contrast injections made into mesoderm at the level of rhombomere 3 (a neural crest-free region) showed that mesoderm could also give rise to precursors of connective tissues. Injection of lacZ retroviruses at the interface between migrating neural crest cells and underlying myogenic mesoderm revealed that the precursors of neural crest and mesoderm-derived tissues in the branchial arches arise and develop in close registration. However, our data also showed that muscle precursors and adjacent migrating neural crest cells, although retaining this arch registration, do not maintain nearest neighbour relations with labelled myotubes never found to be attached to labelled crest derived skeletal structures or associated features. Overall our results illustrate the precise contributions of neural crest and paraxial mesoderm to specific regions within the developing avian head and highlight some of the relationships between these precursor populations during craniofacial morphogenesis.

Supported by the Wellcome Trust.

P9 Functions of ALX4 and MSX2 in ossification of the skull vault

  1. Top of page
  2. P1 SHOX and limb development
  3. P2 Developmental mechanisms underlying polydactyly in the mouse mutant, Doublefoot
  4. P3 Migration behaviour of myogenic precursor cells in the limb bud with respect to dorsoventral patterning
  5. P4 Axial myogenic cells differentiate and express fast and slow myosin heavy chains early, but splitting into individual muscles is then delayed in the chick embryo (Gallus)
  6. P5 Difference of chondrogenic pattern of cultured limb mesenchyme taken from various part of the limb bud
  7. P6 An approach to the role of hedgehogs in vascular development via the chicken mutant talpid3
  8. P7 Fin development in a cartilaginous fish and the origin of vertebrate limbs
  9. P8 Contributions of neural crest and paraxial mesoderm to craniofacial morphogenesis in the chick embryo
  10. P9 Functions of ALX4 and MSX2 in ossification of the skull vault
  11. P10 Tooth formation in reaggreated dental mesenchyme of mice
  12. P11 Morphological evidence for the role of epithelial tissue during mouse tongue development
  13. P12 From ectodermal bud to epidermal appendage: does keratin expression play a role in early mammary gland development?
  14. P13 An embryological study of the anorectal system in mice
  15. P14 Contrast agents as an aid to the magnetic resonance microimaging examination of chick embryos
  16. P15 Functional characterisation of mouse pre-Sertoli cells cultured on reconstituted basement membrane
  17. P16 On the estimation and comparison of intracellular gold labelling densities: a simple and efficient method for quantitative immunoelectron microscopy
  18. P17 Quantitative analysis of placental microstructure in pregnancies complicated by pre-eclampsia with and without intrauterine growth restriction
  19. P18 Human endometrial stroma following exposure to the levonorgestrel intrauterine system (LNG-IUS)
  20. P19 Sex dimorphism in anterior pituitary glucocorticoid feedback revealed by annexin 1 knockout in mice
  21. P20 Effects of knockout of neuronal nitric oxide synthase (nNOS) on pituitary gonadotrophs
  22. P21 The vasculature of the spinal cord of the wobbler mutant mouse: a stereological study
  23. P22 Radial glial cell interactions in the developing spinal cord: an immunohistochemical and confocal analysis
  24. P23 An ultrastructural investigation of the early rat spinal ventral commissure

I. Antonopoulou 1, L. Mavrogiannis 2, A.O.M. Wilkie 2 and G.M. Morriss-Kay 1

1 Department of Human Anatomy and Genetics, University of Oxford; and 2 Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, UK

Parietal foramina (PFM) are defects of the human skull vault, mainly characterised by delayed ossification of the parietal bones. Loss-of-function mutations in 2 genes encoding homeobox-containing transcription factors, ALX4 and MSX2, have been detected in patients with PFM. In this study we have used mice with loss-of-function mutations in Alx4 and Msx2 to investigate whether these 2 genes operate in the same or parallel pathways. Combined Alx4/Msx2 mutants were generated by crossing the 2 carrier strains. Mutant genotypes were identified by tail-tip biopsy and PCR. Newborn mice were collected at P0 and P2, and stained for Alizarin red and Alcian blue for skeletal analysis. Embryos from E14 to E16 were used for in situ hybridisation (whole heads and sections), using digoxygenin-labelled riboprobes. Skeletal staining revealed that loss of one allele of Msx2 was without effect, but a progressively more severe retardation of skull vault ossification occurred in pups with the genotypes Alx4+/−/Msx2+/−, Alx4+/−/Msx2−/− and Alx4−/−/Msx2+/−. In pups with the genotype Alx4−/−/Msx2−/− the entire roof of the skull vault was missing. The phenotypes of these compound mutants were not merely composites of the individual Alx4 and Msx2 mutant phenotypes, but included novel abnormalities involving nonskeletal tissues such the heart. In situ hybridisation did not reveal altered levels of expression of Alx4 in Msx2−/− mutants, or of Msx2 in Alx4−/− mutants. Expression of genes implicated in osteogenesis (Fgfr1 and Spp1) was decreased in Alx4−/− but not Msx2−/− mutants. These observations suggest that Alx4 and Msx2 play similar but not indentical roles in transcriptional activation leading to intramembranous ossification in the skull vault. The presence of one copy of either Alx4 or Msx2 also appears to be required for normal heart development.

All procedures were carried out according to Home Office regulations. This study is supported by an Anatomical Society studentship to I.A., an MRC studentship and a grant from the Alexander S. Onassis Foundation to L.M., and grants from Action Research (G.M.M.-K.) and the Wellcome Trust (A.O.M.W.).

P10 Tooth formation in reaggreated dental mesenchyme of mice

  1. Top of page
  2. P1 SHOX and limb development
  3. P2 Developmental mechanisms underlying polydactyly in the mouse mutant, Doublefoot
  4. P3 Migration behaviour of myogenic precursor cells in the limb bud with respect to dorsoventral patterning
  5. P4 Axial myogenic cells differentiate and express fast and slow myosin heavy chains early, but splitting into individual muscles is then delayed in the chick embryo (Gallus)
  6. P5 Difference of chondrogenic pattern of cultured limb mesenchyme taken from various part of the limb bud
  7. P6 An approach to the role of hedgehogs in vascular development via the chicken mutant talpid3
  8. P7 Fin development in a cartilaginous fish and the origin of vertebrate limbs
  9. P8 Contributions of neural crest and paraxial mesoderm to craniofacial morphogenesis in the chick embryo
  10. P9 Functions of ALX4 and MSX2 in ossification of the skull vault
  11. P10 Tooth formation in reaggreated dental mesenchyme of mice
  12. P11 Morphological evidence for the role of epithelial tissue during mouse tongue development
  13. P12 From ectodermal bud to epidermal appendage: does keratin expression play a role in early mammary gland development?
  14. P13 An embryological study of the anorectal system in mice
  15. P14 Contrast agents as an aid to the magnetic resonance microimaging examination of chick embryos
  16. P15 Functional characterisation of mouse pre-Sertoli cells cultured on reconstituted basement membrane
  17. P16 On the estimation and comparison of intracellular gold labelling densities: a simple and efficient method for quantitative immunoelectron microscopy
  18. P17 Quantitative analysis of placental microstructure in pregnancies complicated by pre-eclampsia with and without intrauterine growth restriction
  19. P18 Human endometrial stroma following exposure to the levonorgestrel intrauterine system (LNG-IUS)
  20. P19 Sex dimorphism in anterior pituitary glucocorticoid feedback revealed by annexin 1 knockout in mice
  21. P20 Effects of knockout of neuronal nitric oxide synthase (nNOS) on pituitary gonadotrophs
  22. P21 The vasculature of the spinal cord of the wobbler mutant mouse: a stereological study
  23. P22 Radial glial cell interactions in the developing spinal cord: an immunohistochemical and confocal analysis
  24. P23 An ultrastructural investigation of the early rat spinal ventral commissure

E.-J. Kim , Y. Hitoshi , J.-Y. Kim and H.S. Jung

Division of Histology, Department of Oral Biology, College of Dentistry, Yonsei University, South Korea

Induction of tooth formation by dental epithelium and ectomesenchyme is of fundamental importance and occurs throughout tooth development. Before the bud stage dental epithelium has the potential to induce tooth formation, whereas dental mesenchyme has the potential to induce tooth formation after the bud stage.

In this study different numbers of dissociated mesenchymal cells were recombined with pieces of epithelium of the same size, and we then observed which kinds of teeth they produced.

Using the reaggregated system in kidney capsules, we obtained reaggregated teeth. After 3 wk the control teeth had 6–7 cusps. They were mesially inclined crests, and 6 of them were tall and 1 of them was short, whereas the reaggregated teeth had various types. We distinguished 3 types of cuspal pattern of reaggregated teeth. 8 of our samples belonged to the M1-like of type 1. 2 of them had the crater-like structure of type 2. Most of them were slope-like of type 3. We examined the reaggregated tooth by H-E staining of paraffin sections after decalcification. We detected tooth cusps, dentinal tubule running to dentin surface, and mantle dentin in dentin surface similar to the control tooth. We attempted in situ hybridisation of Bmp4, which was expressed in the epithelium of the reaggregated tooth. When incisor epithelium was recombined with the reaggregated mesenchyme of a molar, we obtained an M-1 like reaggregated tooth. When DiI was injected into the mesenchyme cells just below the enamel knot, the DiI-labeled cells spread through the mesenchyme of the reaggregated tooth, showing no predetermination of dental mesenchymal cells.

In summary, the cuspal pattern of the reaggregated tooth may be associated with mesenchymal cell numbers. Moreover, we predict that diversity of tooth pattern might be determined by cell numbers in the dental mesenchyme.

P11 Morphological evidence for the role of epithelial tissue during mouse tongue development

  1. Top of page
  2. P1 SHOX and limb development
  3. P2 Developmental mechanisms underlying polydactyly in the mouse mutant, Doublefoot
  4. P3 Migration behaviour of myogenic precursor cells in the limb bud with respect to dorsoventral patterning
  5. P4 Axial myogenic cells differentiate and express fast and slow myosin heavy chains early, but splitting into individual muscles is then delayed in the chick embryo (Gallus)
  6. P5 Difference of chondrogenic pattern of cultured limb mesenchyme taken from various part of the limb bud
  7. P6 An approach to the role of hedgehogs in vascular development via the chicken mutant talpid3
  8. P7 Fin development in a cartilaginous fish and the origin of vertebrate limbs
  9. P8 Contributions of neural crest and paraxial mesoderm to craniofacial morphogenesis in the chick embryo
  10. P9 Functions of ALX4 and MSX2 in ossification of the skull vault
  11. P10 Tooth formation in reaggreated dental mesenchyme of mice
  12. P11 Morphological evidence for the role of epithelial tissue during mouse tongue development
  13. P12 From ectodermal bud to epidermal appendage: does keratin expression play a role in early mammary gland development?
  14. P13 An embryological study of the anorectal system in mice
  15. P14 Contrast agents as an aid to the magnetic resonance microimaging examination of chick embryos
  16. P15 Functional characterisation of mouse pre-Sertoli cells cultured on reconstituted basement membrane
  17. P16 On the estimation and comparison of intracellular gold labelling densities: a simple and efficient method for quantitative immunoelectron microscopy
  18. P17 Quantitative analysis of placental microstructure in pregnancies complicated by pre-eclampsia with and without intrauterine growth restriction
  19. P18 Human endometrial stroma following exposure to the levonorgestrel intrauterine system (LNG-IUS)
  20. P19 Sex dimorphism in anterior pituitary glucocorticoid feedback revealed by annexin 1 knockout in mice
  21. P20 Effects of knockout of neuronal nitric oxide synthase (nNOS) on pituitary gonadotrophs
  22. P21 The vasculature of the spinal cord of the wobbler mutant mouse: a stereological study
  23. P22 Radial glial cell interactions in the developing spinal cord: an immunohistochemical and confocal analysis
  24. P23 An ultrastructural investigation of the early rat spinal ventral commissure

J.-Y. Kim 1, T. Mochizuko 2, A. Keiichi 2 and H.S. Jung 1

1 Division of Histology, Department of Oral Biology, College of Dentistry, Yonsei University, South Korea; and 2 Unit of Functional Anatomy, Graduate School, Tokyo Medical and Dental University, Japan

We investigated the basic processes of development of tongue papillae as part of a study aimed at understanding epithelial-mesenchymal interactions during mouse embryonic development.

Using scanning electron microscopy (SEM) and histological methods we examined the morphological changes associated with normal development of tongue papillae. From embryonic day 12 (E12) to E17, morphogenesis of fungiform papillae occurs in a stereotyped pattern on the dorsal surface of the tongue. Development of fungiform papillae began with epithelial thickening and then formed a concave structure of epithelium and condensed mesenchyme. Formation of tongue papillae shared a pattern of morphological change similar to other epithelial appendages.

We examined expression of the signalling molecules bmp2, bmp4 and shh which are known to play an important role in development of other epithelial appendages. At E12 bmp2, bmp4 and shh had a broad distribution on the dorsal surface of the tongue, restricted to the epithelial layer. At E13 signalling molecules were expressed in a restricted region of the dorsal epithelium.

E14 expression of bmp2 and bmp4 had decreased but shh was maintained and had a punctate pattern in the epithelium. By comparing the SEM and histological studies, we recognised that the regions expressing bmp2, bmp4 and shh coincided with the regions of fungiform papillae formation.

In summary, development of fungiform papillae should be a useful model for studying epithelial-mesenchymal interactions.

P12 From ectodermal bud to epidermal appendage: does keratin expression play a role in early mammary gland development?

  1. Top of page
  2. P1 SHOX and limb development
  3. P2 Developmental mechanisms underlying polydactyly in the mouse mutant, Doublefoot
  4. P3 Migration behaviour of myogenic precursor cells in the limb bud with respect to dorsoventral patterning
  5. P4 Axial myogenic cells differentiate and express fast and slow myosin heavy chains early, but splitting into individual muscles is then delayed in the chick embryo (Gallus)
  6. P5 Difference of chondrogenic pattern of cultured limb mesenchyme taken from various part of the limb bud
  7. P6 An approach to the role of hedgehogs in vascular development via the chicken mutant talpid3
  8. P7 Fin development in a cartilaginous fish and the origin of vertebrate limbs
  9. P8 Contributions of neural crest and paraxial mesoderm to craniofacial morphogenesis in the chick embryo
  10. P9 Functions of ALX4 and MSX2 in ossification of the skull vault
  11. P10 Tooth formation in reaggreated dental mesenchyme of mice
  12. P11 Morphological evidence for the role of epithelial tissue during mouse tongue development
  13. P12 From ectodermal bud to epidermal appendage: does keratin expression play a role in early mammary gland development?
  14. P13 An embryological study of the anorectal system in mice
  15. P14 Contrast agents as an aid to the magnetic resonance microimaging examination of chick embryos
  16. P15 Functional characterisation of mouse pre-Sertoli cells cultured on reconstituted basement membrane
  17. P16 On the estimation and comparison of intracellular gold labelling densities: a simple and efficient method for quantitative immunoelectron microscopy
  18. P17 Quantitative analysis of placental microstructure in pregnancies complicated by pre-eclampsia with and without intrauterine growth restriction
  19. P18 Human endometrial stroma following exposure to the levonorgestrel intrauterine system (LNG-IUS)
  20. P19 Sex dimorphism in anterior pituitary glucocorticoid feedback revealed by annexin 1 knockout in mice
  21. P20 Effects of knockout of neuronal nitric oxide synthase (nNOS) on pituitary gonadotrophs
  22. P21 The vasculature of the spinal cord of the wobbler mutant mouse: a stereological study
  23. P22 Radial glial cell interactions in the developing spinal cord: an immunohistochemical and confocal analysis
  24. P23 An ultrastructural investigation of the early rat spinal ventral commissure

A.M. Al-Dahmash and E.B. Lane

Cancer Research UK Cell Structure Research Group, School of Life Sciences, University of Dundee, UK

During early mammalian development ectodermal structures (e.g. mammary gland, sweat gland and hair follicles) develop as thickenings of the ectoderm. These then form solid knobs of epithelial cells protruding into the mesenchyme more deeply. Later these knobs change their shapes and form the epidermal appendages. Although there is a rapid advance in the understanding of the general mechanisms behind these developmental processes, the changes within the cells of these appendages at the early stages that enable them to reach their ultimate morphology is unknown.

To understand and study the molecular events that contribute to this developmental process we have chosen the mouse mammary gland as a model for our study. Our aim is to understand the changes in the cytoskeleton network, in particular the intermediate filaments, during these developmental events.

Keratin expression is linked to the state of epithelial differentiation and has been shown to be involved in cell, and subsequently tissue, physical stability and resilience. Despite this, little is known about the expression or the role of keratins in mammary gland development. In addition it is not known if the mesenchyme has any direct influence that leads to changes in the bud cells’ keratin expression.

In this study the expression of keratins, desmosome, hemidesmosome, and extracellular matrix proteins have been examined at different stages of embryonic mouse mammary gland development by immunohistochemistry. The results show that there are site specific expression patterns of some keratins and desmosomal proteins within the bud. (A recent publication by Nanba et al. Develop. Growth Differ. 43, 2001, has confirmed our desmosomal results). In addition a loss of expression of a number of extracellular matrix proteins and hemidesmosome proteins is observed around the mammary gland.

Loss of cell-cell or cell-substrate adhesion may be needed to reposition cells to form new structures. Alterations in the expression of structural proteins including those which are in the extracellular matrix junctions may change the physical properties of the epithelia making them more or less plastic as appropriate, which may help determine the way a tissue changes shape.

P13 An embryological study of the anorectal system in mice

  1. Top of page
  2. P1 SHOX and limb development
  3. P2 Developmental mechanisms underlying polydactyly in the mouse mutant, Doublefoot
  4. P3 Migration behaviour of myogenic precursor cells in the limb bud with respect to dorsoventral patterning
  5. P4 Axial myogenic cells differentiate and express fast and slow myosin heavy chains early, but splitting into individual muscles is then delayed in the chick embryo (Gallus)
  6. P5 Difference of chondrogenic pattern of cultured limb mesenchyme taken from various part of the limb bud
  7. P6 An approach to the role of hedgehogs in vascular development via the chicken mutant talpid3
  8. P7 Fin development in a cartilaginous fish and the origin of vertebrate limbs
  9. P8 Contributions of neural crest and paraxial mesoderm to craniofacial morphogenesis in the chick embryo
  10. P9 Functions of ALX4 and MSX2 in ossification of the skull vault
  11. P10 Tooth formation in reaggreated dental mesenchyme of mice
  12. P11 Morphological evidence for the role of epithelial tissue during mouse tongue development
  13. P12 From ectodermal bud to epidermal appendage: does keratin expression play a role in early mammary gland development?
  14. P13 An embryological study of the anorectal system in mice
  15. P14 Contrast agents as an aid to the magnetic resonance microimaging examination of chick embryos
  16. P15 Functional characterisation of mouse pre-Sertoli cells cultured on reconstituted basement membrane
  17. P16 On the estimation and comparison of intracellular gold labelling densities: a simple and efficient method for quantitative immunoelectron microscopy
  18. P17 Quantitative analysis of placental microstructure in pregnancies complicated by pre-eclampsia with and without intrauterine growth restriction
  19. P18 Human endometrial stroma following exposure to the levonorgestrel intrauterine system (LNG-IUS)
  20. P19 Sex dimorphism in anterior pituitary glucocorticoid feedback revealed by annexin 1 knockout in mice
  21. P20 Effects of knockout of neuronal nitric oxide synthase (nNOS) on pituitary gonadotrophs
  22. P21 The vasculature of the spinal cord of the wobbler mutant mouse: a stereological study
  23. P22 Radial glial cell interactions in the developing spinal cord: an immunohistochemical and confocal analysis
  24. P23 An ultrastructural investigation of the early rat spinal ventral commissure

K. Akita , K. Yamaguchi , C. Sasaki and T. Sato

Unit of Functional Anatomy, Graduate School, Tokyo Medical and Dental University, Japan

To understand the great diversity of congenital anorectal malformations in man, knowledge of the normal embryogenesis of this area should be essential. To elucidate the principles of normal cloacal (anal) development we studied the morphology of the region in normal mouse embryos. Programmed cell death is a common event during morphogenesis and occurs according to precise temporal sequences and spatial patterns during development. We examined serial sagittal sections of mouse embryos (E11.5-13.5) to understand the typical cloacal embryogenesis with special reference to the patterns of the programmed cell death (PCD). PCD was detected in the endodermal layer of the urorectal septum, resulting in separation between the urogenital and anorectal regions of the caudal end of the hindgut. In addition PCD was detected in the mesenchyme surrounding the endodermal layer of the caudal end of the hindgut and cranial end of the tailgut up to E12.5, subsequently the hindgut and tailgut became bent at a right angle following the ventral outgrowth of the genital tubercle. During these processes, the tail became relatively thinner, and the dorsal expansion of the cloaca membrane continuously occurred. Then the dorsal part of this membrane and the dorsal wall of the caudalmost region of the hindgut disintegrated in the formation of the anal opening by PCD at the cranial end of the tailgut.

The anal opening has been described as being made in the posterior half of the primitive cloaca, however the anal opening is made at the junction between the hindgut and the tailgut as a new hole. Therefore, the anal development might be a characteristic process in mammals except for Monotremata. In addition, PCD patterns during the normal development might suggest the various patterns of the abnormal development in anorectal system.

P14 Contrast agents as an aid to the magnetic resonance microimaging examination of chick embryos

  1. Top of page
  2. P1 SHOX and limb development
  3. P2 Developmental mechanisms underlying polydactyly in the mouse mutant, Doublefoot
  4. P3 Migration behaviour of myogenic precursor cells in the limb bud with respect to dorsoventral patterning
  5. P4 Axial myogenic cells differentiate and express fast and slow myosin heavy chains early, but splitting into individual muscles is then delayed in the chick embryo (Gallus)
  6. P5 Difference of chondrogenic pattern of cultured limb mesenchyme taken from various part of the limb bud
  7. P6 An approach to the role of hedgehogs in vascular development via the chicken mutant talpid3
  8. P7 Fin development in a cartilaginous fish and the origin of vertebrate limbs
  9. P8 Contributions of neural crest and paraxial mesoderm to craniofacial morphogenesis in the chick embryo
  10. P9 Functions of ALX4 and MSX2 in ossification of the skull vault
  11. P10 Tooth formation in reaggreated dental mesenchyme of mice
  12. P11 Morphological evidence for the role of epithelial tissue during mouse tongue development
  13. P12 From ectodermal bud to epidermal appendage: does keratin expression play a role in early mammary gland development?
  14. P13 An embryological study of the anorectal system in mice
  15. P14 Contrast agents as an aid to the magnetic resonance microimaging examination of chick embryos
  16. P15 Functional characterisation of mouse pre-Sertoli cells cultured on reconstituted basement membrane
  17. P16 On the estimation and comparison of intracellular gold labelling densities: a simple and efficient method for quantitative immunoelectron microscopy
  18. P17 Quantitative analysis of placental microstructure in pregnancies complicated by pre-eclampsia with and without intrauterine growth restriction
  19. P18 Human endometrial stroma following exposure to the levonorgestrel intrauterine system (LNG-IUS)
  20. P19 Sex dimorphism in anterior pituitary glucocorticoid feedback revealed by annexin 1 knockout in mice
  21. P20 Effects of knockout of neuronal nitric oxide synthase (nNOS) on pituitary gonadotrophs
  22. P21 The vasculature of the spinal cord of the wobbler mutant mouse: a stereological study
  23. P22 Radial glial cell interactions in the developing spinal cord: an immunohistochemical and confocal analysis
  24. P23 An ultrastructural investigation of the early rat spinal ventral commissure

X. Li , M. Tanaka , M. Davies , C. Tickle and S. Chudek

School of Life Sciences, University of Dundee, UK

As part of an evaluation of the use of magnetic resonance microimaging (MRM) in the study of the development of chick embryos, we are attempting to image live quail chick embryos in the egg. MRM images are maps of the concentration of hydrogen nuclei (i.e. water) throughout the sample. In general bright areas correspond to high concentrations of water and dark areas to lower concentrations. In many cases the intensities are also affected by tissue type so that even when the water concentration is equal there is image contrast allowing differing tissues to be observed. However this is not always the case.

Chick embryos within the egg are submerged in a medium that, to the MRM scanner, is very similar to that of the embryo. This provides very little contrast and it can be very difficult to observe the embryo in situ.

The addition of a paramagnetic contrast agent (a metal such as gadolinium in an organic ligand cage) causes electronic effects within the sample and therefore strongly influences the contrast of the area into which it has been introduced. Gadotetridol, a contrast agent used in human MRI, was injected into both the egg medium and to the embryo. Both techniques allowed the embryo to be observed, however injection into the embryo gave the best contrast. We also found that contrast varied with time after injection and with concentration. The observations of the live embryo in situ compared favourably with those seen in excised embryos of the same age.

P15 Functional characterisation of mouse pre-Sertoli cells cultured on reconstituted basement membrane

  1. Top of page
  2. P1 SHOX and limb development
  3. P2 Developmental mechanisms underlying polydactyly in the mouse mutant, Doublefoot
  4. P3 Migration behaviour of myogenic precursor cells in the limb bud with respect to dorsoventral patterning
  5. P4 Axial myogenic cells differentiate and express fast and slow myosin heavy chains early, but splitting into individual muscles is then delayed in the chick embryo (Gallus)
  6. P5 Difference of chondrogenic pattern of cultured limb mesenchyme taken from various part of the limb bud
  7. P6 An approach to the role of hedgehogs in vascular development via the chicken mutant talpid3
  8. P7 Fin development in a cartilaginous fish and the origin of vertebrate limbs
  9. P8 Contributions of neural crest and paraxial mesoderm to craniofacial morphogenesis in the chick embryo
  10. P9 Functions of ALX4 and MSX2 in ossification of the skull vault
  11. P10 Tooth formation in reaggreated dental mesenchyme of mice
  12. P11 Morphological evidence for the role of epithelial tissue during mouse tongue development
  13. P12 From ectodermal bud to epidermal appendage: does keratin expression play a role in early mammary gland development?
  14. P13 An embryological study of the anorectal system in mice
  15. P14 Contrast agents as an aid to the magnetic resonance microimaging examination of chick embryos
  16. P15 Functional characterisation of mouse pre-Sertoli cells cultured on reconstituted basement membrane
  17. P16 On the estimation and comparison of intracellular gold labelling densities: a simple and efficient method for quantitative immunoelectron microscopy
  18. P17 Quantitative analysis of placental microstructure in pregnancies complicated by pre-eclampsia with and without intrauterine growth restriction
  19. P18 Human endometrial stroma following exposure to the levonorgestrel intrauterine system (LNG-IUS)
  20. P19 Sex dimorphism in anterior pituitary glucocorticoid feedback revealed by annexin 1 knockout in mice
  21. P20 Effects of knockout of neuronal nitric oxide synthase (nNOS) on pituitary gonadotrophs
  22. P21 The vasculature of the spinal cord of the wobbler mutant mouse: a stereological study
  23. P22 Radial glial cell interactions in the developing spinal cord: an immunohistochemical and confocal analysis
  24. P23 An ultrastructural investigation of the early rat spinal ventral commissure

S. Mackay , C.L. Ballingall and R.A. Smith

Institute of Biomedical and Life Sciences, University of Glasgow, UK

Somatic cells of the mouse testis are believed to be the target of the testis-determining gene which acts cell-autonomously triggering differentiation along the Sertoli cell pathway. Testis differentiation is first apparent around 12 d post coitum (dpc) when pre-Sertoli cells aggregate to form testicular cords. Isolated mouse pre-Sertoli cells, cultured on reconstituted basement membrane (RBM), show a polarised epithelial phenotype and form cord-like aggregations (Mackay et al. J. Electron Microsc. 48, 1999). Such cultures provide a model system permitting an investigation of factors involved in gonadal differentiation, such as cytokines and cell-cell interactions. This study aimed to determine whether the structural differentiation achieved in vitro is matched by functional differentiation. As anti-Müllerian hormone is the earliest known secretory product of pre-Sertoli cells, its detection within cultures provides a good indication of functional state.

Mouse embryos were removed from pregnant CBA strain females killed by CO2 asphyxiation at either 15 (n = 6) or 19 dpc (n = 11). Pre-Sertoli cell isolation was achieved by disrupting testes both enzymically and mechanically. Cells were plated onto RBM (ECM gel; Sigma, Poole, Dorset), prepared as a gel either directly on the culture dish or on a Thermanox coverslip. Cultures were maintained in Dulbecco's medium, supplemented with 10% fetal calf serum and 0.1% gentamycin, changed every 48 h and monitored by daily phase contrast microscopy. After 7 d cultures were fixed in 4% paraformaldehyde for immunocytochemistry. An anti-AMH goat polyclonal IgG (Santa Cruz Biotechnology, Inc., Santa Cruz, California) was used, with optimum results obtained using a 1:500 dilution. The primary antibody was omitted as a control. Reconstituted basement membrane cultures without coverslips provided the best conditions for AMH immunocytochemistry. Though immunostaining was found in controls without gel, the result was much stronger in the presence of the RBM gel. Cultures established from 19dpc testes appeared to stain more positively than those from younger testes. This was surprising, since AMH levels in vivo are at their highest when Müllerian duct regression is occurring and decline before birth. A possible explanation is that the 19dpc cultures formed a greater number of cord-like aggregates leading to a corresponding increase in AMH production.

P16 On the estimation and comparison of intracellular gold labelling densities: a simple and efficient method for quantitative immunoelectron microscopy

  1. Top of page
  2. P1 SHOX and limb development
  3. P2 Developmental mechanisms underlying polydactyly in the mouse mutant, Doublefoot
  4. P3 Migration behaviour of myogenic precursor cells in the limb bud with respect to dorsoventral patterning
  5. P4 Axial myogenic cells differentiate and express fast and slow myosin heavy chains early, but splitting into individual muscles is then delayed in the chick embryo (Gallus)
  6. P5 Difference of chondrogenic pattern of cultured limb mesenchyme taken from various part of the limb bud
  7. P6 An approach to the role of hedgehogs in vascular development via the chicken mutant talpid3
  8. P7 Fin development in a cartilaginous fish and the origin of vertebrate limbs
  9. P8 Contributions of neural crest and paraxial mesoderm to craniofacial morphogenesis in the chick embryo
  10. P9 Functions of ALX4 and MSX2 in ossification of the skull vault
  11. P10 Tooth formation in reaggreated dental mesenchyme of mice
  12. P11 Morphological evidence for the role of epithelial tissue during mouse tongue development
  13. P12 From ectodermal bud to epidermal appendage: does keratin expression play a role in early mammary gland development?
  14. P13 An embryological study of the anorectal system in mice
  15. P14 Contrast agents as an aid to the magnetic resonance microimaging examination of chick embryos
  16. P15 Functional characterisation of mouse pre-Sertoli cells cultured on reconstituted basement membrane
  17. P16 On the estimation and comparison of intracellular gold labelling densities: a simple and efficient method for quantitative immunoelectron microscopy
  18. P17 Quantitative analysis of placental microstructure in pregnancies complicated by pre-eclampsia with and without intrauterine growth restriction
  19. P18 Human endometrial stroma following exposure to the levonorgestrel intrauterine system (LNG-IUS)
  20. P19 Sex dimorphism in anterior pituitary glucocorticoid feedback revealed by annexin 1 knockout in mice
  21. P20 Effects of knockout of neuronal nitric oxide synthase (nNOS) on pituitary gonadotrophs
  22. P21 The vasculature of the spinal cord of the wobbler mutant mouse: a stereological study
  23. P22 Radial glial cell interactions in the developing spinal cord: an immunohistochemical and confocal analysis
  24. P23 An ultrastructural investigation of the early rat spinal ventral commissure

T.M. Mayhew

School of Biomedical Sciences, University of Nottingham, UK

The use of transmission electron microscopy and particulate gold for immunochemical localisation of intracellular antigens is well established. A common practice when seeking to quantify gold labelling in different intracellular compartments is to calculate labelling densities (LD). These relate gold counts to the sizes of compartments and are proportional to antigen concentrations. The sizes of compartments may be expressed as volumes or profile areas (organelles) or as trace lengths (membranes, filaments). Consequently, LD may be expressed as golds per µm3, per µm2 or per ìm. Recently, we introduced a new method for quantifying gold labelling (Mayhew et al. J. Microsc. 205, 2002). This involved calculating a relative labelling index (RLI) for each compartment and then testing statistically for preferential (nonrandom) distributions. RLI = 1 for random labelling and RLI > 1 for preferential labelling. Here we provide a simplification of the LD approach in which compartment sizes are estimated stereologically by randomly superimposing on micrographs lattice arrays of test points or test lines. Given random sampling, test point counts are proportional to profile areas whilst counting chance intersections between test lines and traces of membranes or filaments provides data proportional to trace length. It follows that estimation of LD can be simplified by expressing values as golds per test point (organelles) or per intersection (membranes/filaments). Furthermore, the LD value obtained by dividing total golds (on all compartments) by total points/intersections (on all compartments) is the one which is to be expected if all compartments contain equal densities of antigen (LDexp). Also, test point/intersection totals on different compartments provide a convenient predicted distribution, namely, that which would be obtained if gold particles were distributed randomly. It follows that observed distributions of gold particles can be compared with predicted point/intersection distributions by a chi-squared analysis. If the value of total chi-squared suggests that compartments are not labelled randomly, the LD of a given compartment X (LDX) can be divided by the expected density (LDexp) to obtain a relative labelling index for that compartment (RLIX). A given compartment is preferentially labelled if it meets two criteria: (1) its partial chi-squared makes a substantial contribution to total chi-squared, and (2) its value of RLIX is greater than 1 (i.e. the observed is greater than the expected labelling density). This approach provides a simple, efficient, and robust method for comparing labelling densities in different organelle or membrane compartments.

P17 Quantitative analysis of placental microstructure in pregnancies complicated by pre-eclampsia with and without intrauterine growth restriction

  1. Top of page
  2. P1 SHOX and limb development
  3. P2 Developmental mechanisms underlying polydactyly in the mouse mutant, Doublefoot
  4. P3 Migration behaviour of myogenic precursor cells in the limb bud with respect to dorsoventral patterning
  5. P4 Axial myogenic cells differentiate and express fast and slow myosin heavy chains early, but splitting into individual muscles is then delayed in the chick embryo (Gallus)
  6. P5 Difference of chondrogenic pattern of cultured limb mesenchyme taken from various part of the limb bud
  7. P6 An approach to the role of hedgehogs in vascular development via the chicken mutant talpid3
  8. P7 Fin development in a cartilaginous fish and the origin of vertebrate limbs
  9. P8 Contributions of neural crest and paraxial mesoderm to craniofacial morphogenesis in the chick embryo
  10. P9 Functions of ALX4 and MSX2 in ossification of the skull vault
  11. P10 Tooth formation in reaggreated dental mesenchyme of mice
  12. P11 Morphological evidence for the role of epithelial tissue during mouse tongue development
  13. P12 From ectodermal bud to epidermal appendage: does keratin expression play a role in early mammary gland development?
  14. P13 An embryological study of the anorectal system in mice
  15. P14 Contrast agents as an aid to the magnetic resonance microimaging examination of chick embryos
  16. P15 Functional characterisation of mouse pre-Sertoli cells cultured on reconstituted basement membrane
  17. P16 On the estimation and comparison of intracellular gold labelling densities: a simple and efficient method for quantitative immunoelectron microscopy
  18. P17 Quantitative analysis of placental microstructure in pregnancies complicated by pre-eclampsia with and without intrauterine growth restriction
  19. P18 Human endometrial stroma following exposure to the levonorgestrel intrauterine system (LNG-IUS)
  20. P19 Sex dimorphism in anterior pituitary glucocorticoid feedback revealed by annexin 1 knockout in mice
  21. P20 Effects of knockout of neuronal nitric oxide synthase (nNOS) on pituitary gonadotrophs
  22. P21 The vasculature of the spinal cord of the wobbler mutant mouse: a stereological study
  23. P22 Radial glial cell interactions in the developing spinal cord: an immunohistochemical and confocal analysis
  24. P23 An ultrastructural investigation of the early rat spinal ventral commissure

T.M. Mayhew 1, C. Ohadike 1, P.N. Baker 2, I.P. Crocker 2, I.R. Johnson 3, C. Mitchell 3 and S.S. Ong 3

Schools of 1Biomedical Sciences and 2HumanDevelopment, University of Nottingham; and 3Maternal and Fetal Health Research Centre, University of Manchester, UK

The aim of this study was to quantify placental morphology in pregnancies complicated by pre-eclampsia with and without intrauterine growth restriction, paying particular attention to the dimensions and composition of peripheral (intermediate + terminal) villi. Placentas from control pregnancies (n = 9) and cases of pre-eclampsia (PE, n = 5), intrauterine growth restriction (IUGR, n = 5) and pre-eclampsia with intrauterine growth restriction (PE+IUGR, n = 5) were randomly sampled for microscopic evaluation. PE was defined by significant maternal proteinuria (greater than 300 mg/L in a 24 h collection in the absence of urinary tract infection) and a blood pressure in a previously normotensive woman of higher than 140/90 mmHg on 2 or more occasions. IUGR cases were identified by deficient fetal growth on ultrasound scans and the final decider was an individualised birth weight ratio (IBR) below 10. IBR relates to a predicted birth weight calculated using factors including fetal sex, gestational age, parity, ethnic origin, maternal age, maternal height and booking weight. It provides a better correlation with perinatal outcomes than birthweight for gestational age alone. Most IBR values in IUGR were below 5. Only one IUGR case was confirmed as having absent end-diastolic flow in the umbilical artery. Formalin-fixed, wax-embedded sections stained by the Masson trichrome method were analysed stereologically in order to estimate the volumes of placental components and the surfaces and diameters of villi and fetal capillaries. Group comparisons were drawn using 2-way analysis of variance. Fetal weights were reduced in all complicated pregnancies but only IUGR was accompanied by a significantly smaller placenta. PE had no main effect on placental morphology and (except for trophoblast thickness) there were no interaction effects involving PE. In contrast, IUGR was associated with a placenta which had reduced volumes of intervillous space and all types of villi (stem, intermediate, terminal). The impoverished growth of peripheral villi affected all tissues (trophoblast, stroma, capillaries) and was accompanied by smaller exchange surface areas and a thicker trophoblastic epithelium. Mean diameters of villi and capillaries were not affected. This study emphasises the importance of proper categorisation of subjects when studying cases of PE and IUGR. We conclude that IUGR, but not PE, produces substantial changes in placental morphology including impoverished growth of villi and fetal microvessels. These changes are likely to reduce placental oxygen diffusive conductances and contribute to fetal hypoxic stress.

P18 Human endometrial stroma following exposure to the levonorgestrel intrauterine system (LNG-IUS)

  1. Top of page
  2. P1 SHOX and limb development
  3. P2 Developmental mechanisms underlying polydactyly in the mouse mutant, Doublefoot
  4. P3 Migration behaviour of myogenic precursor cells in the limb bud with respect to dorsoventral patterning
  5. P4 Axial myogenic cells differentiate and express fast and slow myosin heavy chains early, but splitting into individual muscles is then delayed in the chick embryo (Gallus)
  6. P5 Difference of chondrogenic pattern of cultured limb mesenchyme taken from various part of the limb bud
  7. P6 An approach to the role of hedgehogs in vascular development via the chicken mutant talpid3
  8. P7 Fin development in a cartilaginous fish and the origin of vertebrate limbs
  9. P8 Contributions of neural crest and paraxial mesoderm to craniofacial morphogenesis in the chick embryo
  10. P9 Functions of ALX4 and MSX2 in ossification of the skull vault
  11. P10 Tooth formation in reaggreated dental mesenchyme of mice
  12. P11 Morphological evidence for the role of epithelial tissue during mouse tongue development
  13. P12 From ectodermal bud to epidermal appendage: does keratin expression play a role in early mammary gland development?
  14. P13 An embryological study of the anorectal system in mice
  15. P14 Contrast agents as an aid to the magnetic resonance microimaging examination of chick embryos
  16. P15 Functional characterisation of mouse pre-Sertoli cells cultured on reconstituted basement membrane
  17. P16 On the estimation and comparison of intracellular gold labelling densities: a simple and efficient method for quantitative immunoelectron microscopy
  18. P17 Quantitative analysis of placental microstructure in pregnancies complicated by pre-eclampsia with and without intrauterine growth restriction
  19. P18 Human endometrial stroma following exposure to the levonorgestrel intrauterine system (LNG-IUS)
  20. P19 Sex dimorphism in anterior pituitary glucocorticoid feedback revealed by annexin 1 knockout in mice
  21. P20 Effects of knockout of neuronal nitric oxide synthase (nNOS) on pituitary gonadotrophs
  22. P21 The vasculature of the spinal cord of the wobbler mutant mouse: a stereological study
  23. P22 Radial glial cell interactions in the developing spinal cord: an immunohistochemical and confocal analysis
  24. P23 An ultrastructural investigation of the early rat spinal ventral commissure

J. Treacy 1, C.J. McGavigan 2, I.T. Cameron 4, S. Campbell 2, M.A. Warren 3, T.C. Li 5 and P. Dockery 1

1 Department of Anatomy, University College Cork, Ireland; 2 Department of Obstetrics and Gynaecology, University of Glasgow, Glasgow Royal Infirmary, UK; 3 Department of Biomedical Science, University of Sheffield, UK; 4 Maternal, Fetal and Neonatal Physiology Group, Fetal Origins of Adult Disease (FOAD) Division, Princess Anne Hospital, Southampton, UK; and 5 Jessops Hospital for Women, Sheffield, UK

The levonorgestrel intrauterine system (LNG-IUS or Mirena®) is an efficient contraceptive device which also brings about a profound reduction in menstrual blood loss. It is therefore a recognised treatment for menorrhagia. A common cause of discontinuation of treatment is the associated breakthrough bleeding which occurs in the early phase of use. Stereological methods were employed to document stromal morphology in women treated with LNG-IUS prior to hysterectomy. The results were compared with data obtained from biopsies taken from untreated women at various stages of the menstrual cycle (Dockery et al. Human Reproduction4, 1998). All patients provided written informed consent.

Endometrial stroma was examined in 2 groups of women: hysterectomy samples from women exposed to LNG-IUS for 6 to 10 wk (n = 7), and well timed normal luteal phase biopsy specimens taken 2, 4, 6, 8,10 and 12 d after the luteinising hormone (LH) surge (n = 4 or 5 on each day). A number of morphological parameters were estimated including nuclear volume and the volume weighted star volume using the digital stereology programme, Kinetic Imaging Sterelogy (Kinetic Imaging Ltd.) Previously we have shown, that there was little overall difference in the ultrastructure of endothelial cells in the LNG-IUS group compared with the control group (Treacy et al. J. Anat, 200, 2002). The vascular basal lamina was thinner in the LNG-IUS group and there was also a borderline significant reduction in vessel wall thickness compared with the controls. In the present study the star volume of the stromal extracellular space in the LNG-IUS group was significantly smaller than that of the control group on all days of the menstrual cycle studied (P < 0.05). The volume weighted mean (± SE) star volume at LH+2 was 60067 ± 8004 µm3 rising to a maximum of 64574 ± 7978 µm3 at LH+10, whereas the value for the LNG-IUS group was 17548 ± 4272 µm3. This is consistent with the decidual transformation of the stroma in the LNG-IUS group. These changes in vessel wall thickness and alterations in the stroma into which the vessels are embedded may be responsible for some of the breakthrough bleeding seen after short- term exposure to intrauterine LNG.

We acknowledge support from Schering Healthcare UK.

P19 Sex dimorphism in anterior pituitary glucocorticoid feedback revealed by annexin 1 knockout in mice

  1. Top of page
  2. P1 SHOX and limb development
  3. P2 Developmental mechanisms underlying polydactyly in the mouse mutant, Doublefoot
  4. P3 Migration behaviour of myogenic precursor cells in the limb bud with respect to dorsoventral patterning
  5. P4 Axial myogenic cells differentiate and express fast and slow myosin heavy chains early, but splitting into individual muscles is then delayed in the chick embryo (Gallus)
  6. P5 Difference of chondrogenic pattern of cultured limb mesenchyme taken from various part of the limb bud
  7. P6 An approach to the role of hedgehogs in vascular development via the chicken mutant talpid3
  8. P7 Fin development in a cartilaginous fish and the origin of vertebrate limbs
  9. P8 Contributions of neural crest and paraxial mesoderm to craniofacial morphogenesis in the chick embryo
  10. P9 Functions of ALX4 and MSX2 in ossification of the skull vault
  11. P10 Tooth formation in reaggreated dental mesenchyme of mice
  12. P11 Morphological evidence for the role of epithelial tissue during mouse tongue development
  13. P12 From ectodermal bud to epidermal appendage: does keratin expression play a role in early mammary gland development?
  14. P13 An embryological study of the anorectal system in mice
  15. P14 Contrast agents as an aid to the magnetic resonance microimaging examination of chick embryos
  16. P15 Functional characterisation of mouse pre-Sertoli cells cultured on reconstituted basement membrane
  17. P16 On the estimation and comparison of intracellular gold labelling densities: a simple and efficient method for quantitative immunoelectron microscopy
  18. P17 Quantitative analysis of placental microstructure in pregnancies complicated by pre-eclampsia with and without intrauterine growth restriction
  19. P18 Human endometrial stroma following exposure to the levonorgestrel intrauterine system (LNG-IUS)
  20. P19 Sex dimorphism in anterior pituitary glucocorticoid feedback revealed by annexin 1 knockout in mice
  21. P20 Effects of knockout of neuronal nitric oxide synthase (nNOS) on pituitary gonadotrophs
  22. P21 The vasculature of the spinal cord of the wobbler mutant mouse: a stereological study
  23. P22 Radial glial cell interactions in the developing spinal cord: an immunohistochemical and confocal analysis
  24. P23 An ultrastructural investigation of the early rat spinal ventral commissure

E.C. Wang 1, J.F. Morris 1, H.C. Christian 1, J.C. Buckingham 2 and R.J. Flower 3

1Department of Human Anatomy and Genetics, University of Oxford;2 Imperial College of Science, Technology and Medicine, London; and 3The William Harvey Research Institute, St Bartholomew's and The Royal London School of Medicine and Dentistry, London, UK

nnexin 1 (Anx 1) is a member of the annexin family of phospholipid- and calcium-binding proteins which has a well-demonstrated role in mediating the early delayed (30 min–3 h) inhibitory feedback effects of glucocorticoids in the pituitary. Anx 1 is located in folliculostellate (FS) cells and glucocorticoids act on these cells to externalise and stimulate the synthesis of Anx 1 which then acts on high affinity Anx 1-binding sites on the endocrine cells. The stress response is notably sexually dimorphic, with females being more susceptible except during pregnancy and lactation. We have examined the corticotrophs of the anterior pituitary in wild-type and Anx 1−/− animals to determine the effects of removal of Anx 1 in male and female mice. Our prediction was that the lack of inhibition would cause hyperactivity of the corticotrophs.

Anterior pituitary tissue was removed from Anx 1 +/+, +/− and −/− adult mice (n= 4 per group), fixed by immersion, and examined by confocal immunocytochemistry to determine the number of corticotrophs and by electron microscopy to examine the size, secretory granule population and secretory machinery of immunoidentified cells. Plasma corticosterone was measured by radioimmunoassay. No differences in any of the histological parameters could be detected in the female mice. By contrast in the male Anx 1 −/− mice there were approximately 3 times the number of corticotrophs as in the wild-type animals. However the corticotrophs in Anx 1 −/− mice were smaller (cytoplasm and nucleus) and had reduced numbers of secretory vesicles (the reduced number of vesicles parallelling the reduction in cell size). The amount of rough endoplasmic reticulum and Golgi was also reduced in the Anx 1 −/− male animals. In general data for the male Anx 1 +/− animals was intermediate between that of the wild-type and homozygotes. In female but not male Anx 1 −/− mice plasma corticosterone levels were increased compared to wild-type.

Many of these data were unexpected. One possible interpretation is that the Anx 1 −/− females are more resistant to glucocorticoid feedback at the pituitary than the males. The striking increase in the number of corticotrophs in the male Anx 1 −/− animals could suggest that, in males, a lack of glucocorticoid feedback via annexin has caused increased proliferation of the corticotrophs. However, in both cases the mechanism(s) remain to be determined.

P20 Effects of knockout of neuronal nitric oxide synthase (nNOS) on pituitary gonadotrophs

  1. Top of page
  2. P1 SHOX and limb development
  3. P2 Developmental mechanisms underlying polydactyly in the mouse mutant, Doublefoot
  4. P3 Migration behaviour of myogenic precursor cells in the limb bud with respect to dorsoventral patterning
  5. P4 Axial myogenic cells differentiate and express fast and slow myosin heavy chains early, but splitting into individual muscles is then delayed in the chick embryo (Gallus)
  6. P5 Difference of chondrogenic pattern of cultured limb mesenchyme taken from various part of the limb bud
  7. P6 An approach to the role of hedgehogs in vascular development via the chicken mutant talpid3
  8. P7 Fin development in a cartilaginous fish and the origin of vertebrate limbs
  9. P8 Contributions of neural crest and paraxial mesoderm to craniofacial morphogenesis in the chick embryo
  10. P9 Functions of ALX4 and MSX2 in ossification of the skull vault
  11. P10 Tooth formation in reaggreated dental mesenchyme of mice
  12. P11 Morphological evidence for the role of epithelial tissue during mouse tongue development
  13. P12 From ectodermal bud to epidermal appendage: does keratin expression play a role in early mammary gland development?
  14. P13 An embryological study of the anorectal system in mice
  15. P14 Contrast agents as an aid to the magnetic resonance microimaging examination of chick embryos
  16. P15 Functional characterisation of mouse pre-Sertoli cells cultured on reconstituted basement membrane
  17. P16 On the estimation and comparison of intracellular gold labelling densities: a simple and efficient method for quantitative immunoelectron microscopy
  18. P17 Quantitative analysis of placental microstructure in pregnancies complicated by pre-eclampsia with and without intrauterine growth restriction
  19. P18 Human endometrial stroma following exposure to the levonorgestrel intrauterine system (LNG-IUS)
  20. P19 Sex dimorphism in anterior pituitary glucocorticoid feedback revealed by annexin 1 knockout in mice
  21. P20 Effects of knockout of neuronal nitric oxide synthase (nNOS) on pituitary gonadotrophs
  22. P21 The vasculature of the spinal cord of the wobbler mutant mouse: a stereological study
  23. P22 Radial glial cell interactions in the developing spinal cord: an immunohistochemical and confocal analysis
  24. P23 An ultrastructural investigation of the early rat spinal ventral commissure

E. Fryer and J.F. Morris

Department of Human Anatomy and Genetics, University of Oxford, UK

Pituitary gonadotrophs are regulated by a complex of influences which include hypothalamic GnRH, feedback of gonadal steroids and peptides, and local paracrine influences from lactotrophs. One local regulator would appear to be nitric oxide (NO). Gonadotrophs express neuronal nitric oxide synthase (nNOS); the folliculo-stellate cells which are intermingled with the endocrine cells express both nNOS and probably also inducible NOS (iNOS). NO has also been implicated in the stimulation of GnRH release However the effects of NO on the gonadotrophs are controversial and both stimulation (SNP increases release of LH and FSH; Pinilla et al. 1998) and inhibition (NO donors inhibit and nNOS inhibitors stimulate GnRH-induced LH release; Ceccatelli et al. 1993). One study on nNOS −/− mice has reported reduced fertility in the mice, but attributed this to an action at the ovary, because no difference could be detected in basal or GnRH-stimulated LH release (Klein et al 1998). We have therefore examined the gonadotroph cells of nNOS −/− and +/+ mice, comparing the number of cells, the size of the cells, and their secretory vesicle population.

Male and female wild-type and nNOS −/− mice (n= 4 per group) were examined. Both the wild-type and the −/− females had estrous cycles which were often irregular, but animals were selected when the vaginal smear clearly indicated proestrus in a 4 or 5 d cycle. The animals were terminally anaesthetised, cardiac blood taken for hormone assay, and the tissues prepared for confocal microscopy to detect the number of LH-immunoreactive gonadotrophs, and for electron microscopy to analyse the secretory status of the cells.

Knockout of nNOS had no apparent effect on the number of gonadotrophs. In female but not male animals the gonadotrophs were significantly smaller and contained less rough endoplasmic reticulum than those in wild-type mice. The secretory vesicles were smaller in female than in male mice of both genotypes, but in both sexes the vesicles were larger in the knockout than the wild-type animals. These data suggest that in both sexes there is a reduction in secretory vesicle turnover (indicated by the larger vesicles) and in the females significantly less active gonadotrophs (smaller, less rough ER). Such changes are consistent either with a reduction in NO-stimulated GnRH release from the hypothalamus or with a diminished intrapituitary stimulatory effect of NO. The respective roles of gonadotroph- and follicostellate-derived NO must await further experiments.

P21 The vasculature of the spinal cord of the wobbler mutant mouse: a stereological study

  1. Top of page
  2. P1 SHOX and limb development
  3. P2 Developmental mechanisms underlying polydactyly in the mouse mutant, Doublefoot
  4. P3 Migration behaviour of myogenic precursor cells in the limb bud with respect to dorsoventral patterning
  5. P4 Axial myogenic cells differentiate and express fast and slow myosin heavy chains early, but splitting into individual muscles is then delayed in the chick embryo (Gallus)
  6. P5 Difference of chondrogenic pattern of cultured limb mesenchyme taken from various part of the limb bud
  7. P6 An approach to the role of hedgehogs in vascular development via the chicken mutant talpid3
  8. P7 Fin development in a cartilaginous fish and the origin of vertebrate limbs
  9. P8 Contributions of neural crest and paraxial mesoderm to craniofacial morphogenesis in the chick embryo
  10. P9 Functions of ALX4 and MSX2 in ossification of the skull vault
  11. P10 Tooth formation in reaggreated dental mesenchyme of mice
  12. P11 Morphological evidence for the role of epithelial tissue during mouse tongue development
  13. P12 From ectodermal bud to epidermal appendage: does keratin expression play a role in early mammary gland development?
  14. P13 An embryological study of the anorectal system in mice
  15. P14 Contrast agents as an aid to the magnetic resonance microimaging examination of chick embryos
  16. P15 Functional characterisation of mouse pre-Sertoli cells cultured on reconstituted basement membrane
  17. P16 On the estimation and comparison of intracellular gold labelling densities: a simple and efficient method for quantitative immunoelectron microscopy
  18. P17 Quantitative analysis of placental microstructure in pregnancies complicated by pre-eclampsia with and without intrauterine growth restriction
  19. P18 Human endometrial stroma following exposure to the levonorgestrel intrauterine system (LNG-IUS)
  20. P19 Sex dimorphism in anterior pituitary glucocorticoid feedback revealed by annexin 1 knockout in mice
  21. P20 Effects of knockout of neuronal nitric oxide synthase (nNOS) on pituitary gonadotrophs
  22. P21 The vasculature of the spinal cord of the wobbler mutant mouse: a stereological study
  23. P22 Radial glial cell interactions in the developing spinal cord: an immunohistochemical and confocal analysis
  24. P23 An ultrastructural investigation of the early rat spinal ventral commissure

V. Bane 1, D.K.C. Tay 2, Y. Tang 2, M. Morais 2, L.L. Vacca-Galloway 2 and P. Dockery 1

1Department of Anatomy, University College Cork, Ireland; and 2Department of Anatomy, The University of Hong Kong, China

The homozygous Wobbler mouse represents a useful model for the investigation of human inherited motoneuron diseases, including amyotrophic lateral sclerosis (ALS). The clinico-pathological features of ALS differ from the Wobbler MND because of the lack of involvement of the human corticospinal tract. Nevertheless the pathogenesis of the mouse and human diseases may have some important common features.

Previous pathological reports have indicated that swollen and vacuolated motoneuron cell bodies are the most predominant feature characterising the wobbler mouse motoneuron disease. Here we extend our earlier studies (Dockery et al. J. Anat, 191, 1997; Treacy et al. 2002) which documented the light and electron microscopical changes in neuronal volumes in the wobbler mutant.

In this study we used stereological methods to document the vasculature of the spinal cord in wobbler mutants and control animals at 3 wk (young, stage 1) and 3 mo of age (old, stage 4) using stereological methods (n = 6 for each group). All animals were anaesthetised and sacrificed by cardiac perfusion, with appropriate ethics committee approval.

A 3-way analysis (3AOV) of variance of the diffusion distance of the grey and white matter of the spinal cord showed a significant effect of location (P < 0.05) but no significant effect of age condition or interaction. Ultrastructural studies on the grey matter revealed no significant effect of age or condition on endothelial thickness and the Vv of mitochondria:cell and nucleus:cell. These results highlight the highly conservative nature of the vasculature in the murine spinal cord.

P22 Radial glial cell interactions in the developing spinal cord: an immunohistochemical and confocal analysis

  1. Top of page
  2. P1 SHOX and limb development
  3. P2 Developmental mechanisms underlying polydactyly in the mouse mutant, Doublefoot
  4. P3 Migration behaviour of myogenic precursor cells in the limb bud with respect to dorsoventral patterning
  5. P4 Axial myogenic cells differentiate and express fast and slow myosin heavy chains early, but splitting into individual muscles is then delayed in the chick embryo (Gallus)
  6. P5 Difference of chondrogenic pattern of cultured limb mesenchyme taken from various part of the limb bud
  7. P6 An approach to the role of hedgehogs in vascular development via the chicken mutant talpid3
  8. P7 Fin development in a cartilaginous fish and the origin of vertebrate limbs
  9. P8 Contributions of neural crest and paraxial mesoderm to craniofacial morphogenesis in the chick embryo
  10. P9 Functions of ALX4 and MSX2 in ossification of the skull vault
  11. P10 Tooth formation in reaggreated dental mesenchyme of mice
  12. P11 Morphological evidence for the role of epithelial tissue during mouse tongue development
  13. P12 From ectodermal bud to epidermal appendage: does keratin expression play a role in early mammary gland development?
  14. P13 An embryological study of the anorectal system in mice
  15. P14 Contrast agents as an aid to the magnetic resonance microimaging examination of chick embryos
  16. P15 Functional characterisation of mouse pre-Sertoli cells cultured on reconstituted basement membrane
  17. P16 On the estimation and comparison of intracellular gold labelling densities: a simple and efficient method for quantitative immunoelectron microscopy
  18. P17 Quantitative analysis of placental microstructure in pregnancies complicated by pre-eclampsia with and without intrauterine growth restriction
  19. P18 Human endometrial stroma following exposure to the levonorgestrel intrauterine system (LNG-IUS)
  20. P19 Sex dimorphism in anterior pituitary glucocorticoid feedback revealed by annexin 1 knockout in mice
  21. P20 Effects of knockout of neuronal nitric oxide synthase (nNOS) on pituitary gonadotrophs
  22. P21 The vasculature of the spinal cord of the wobbler mutant mouse: a stereological study
  23. P22 Radial glial cell interactions in the developing spinal cord: an immunohistochemical and confocal analysis
  24. P23 An ultrastructural investigation of the early rat spinal ventral commissure

D. Barry , J.P. Fraher and K. McDermott

Department of Anatomy, Biosciences Research Institute, University College Cork, Ireland

Gliogenesis in the rat spinal cord has been studied extensively in the past few decades particularly with the advent of electronmicroscopy and immunocytochemistry. However the exact nature of glial cell development remains unresolved. The factors which affect gliogenesis, lineage relationships and differentiation, require further analysis both in vitro and in vivo. Foremost among these is the phenotypic fate of radial glial cells. Their primary purpose, at least in the cerebral cortex, is to act as guidance conduits for migrating neurones. However radial glia have other diverse and important functions in the developing central nervous system, which sees them not only as a transient population of neuronal guides but as prevalent precursors that can parent both neurons and glia, and as key elements in the region-specific differentiation and patterning of the CNS (Campbell & Gotz, Trends Neur. Sci. 25, 2002). Antibodies directed against vimentin, GFAP, nestin, 2F7 (a novel cell surface marker of immature and mature neurones) and GAP-43 detected the presence and distribution of glial and neuronal precursor cells from embryonic day 13 to postnatal day 6. Dual immunofluorescent labelling allowed the interactions between radial glial cells and axons to be examined at different developmental ages. Radial glial like structures appear to participate in the formation of complex boundaries in the dorsal and ventral midline at early and late embryonic ages. The transformation of radial glia into cells of astrocytic morphology was also studied. Dual labelling revealed an emerging population of cells expressing GFAP only in the white matter, as well as cells expressing both vimentin and GFAP. By examining the temporal distribution, and differentiation of radial glia, this study extends previous immunocytochemical studies in order to determine the precise phenotypic fate and function of radial glia in the developing rat spinal cord.

P23 An ultrastructural investigation of the early rat spinal ventral commissure

  1. Top of page
  2. P1 SHOX and limb development
  3. P2 Developmental mechanisms underlying polydactyly in the mouse mutant, Doublefoot
  4. P3 Migration behaviour of myogenic precursor cells in the limb bud with respect to dorsoventral patterning
  5. P4 Axial myogenic cells differentiate and express fast and slow myosin heavy chains early, but splitting into individual muscles is then delayed in the chick embryo (Gallus)
  6. P5 Difference of chondrogenic pattern of cultured limb mesenchyme taken from various part of the limb bud
  7. P6 An approach to the role of hedgehogs in vascular development via the chicken mutant talpid3
  8. P7 Fin development in a cartilaginous fish and the origin of vertebrate limbs
  9. P8 Contributions of neural crest and paraxial mesoderm to craniofacial morphogenesis in the chick embryo
  10. P9 Functions of ALX4 and MSX2 in ossification of the skull vault
  11. P10 Tooth formation in reaggreated dental mesenchyme of mice
  12. P11 Morphological evidence for the role of epithelial tissue during mouse tongue development
  13. P12 From ectodermal bud to epidermal appendage: does keratin expression play a role in early mammary gland development?
  14. P13 An embryological study of the anorectal system in mice
  15. P14 Contrast agents as an aid to the magnetic resonance microimaging examination of chick embryos
  16. P15 Functional characterisation of mouse pre-Sertoli cells cultured on reconstituted basement membrane
  17. P16 On the estimation and comparison of intracellular gold labelling densities: a simple and efficient method for quantitative immunoelectron microscopy
  18. P17 Quantitative analysis of placental microstructure in pregnancies complicated by pre-eclampsia with and without intrauterine growth restriction
  19. P18 Human endometrial stroma following exposure to the levonorgestrel intrauterine system (LNG-IUS)
  20. P19 Sex dimorphism in anterior pituitary glucocorticoid feedback revealed by annexin 1 knockout in mice
  21. P20 Effects of knockout of neuronal nitric oxide synthase (nNOS) on pituitary gonadotrophs
  22. P21 The vasculature of the spinal cord of the wobbler mutant mouse: a stereological study
  23. P22 Radial glial cell interactions in the developing spinal cord: an immunohistochemical and confocal analysis
  24. P23 An ultrastructural investigation of the early rat spinal ventral commissure

S. Lane , K. McDermott , P. Dockery and J. Fraher

Department of Anatomy and Biosciences Research Institute, University College, Cork, Ireland

The spinal ventral commissure (VC) is a uniquely precocious glial segregating element of the presumptive spinal cord. It forms immediately ventral to the floor plate at the midline of the neural tube. The floor plate is a crucial component of the developing neural tube, one of its functions being to organise midline symmetry. It does this through the complex interactions of diffusible, membrane bound and extra cellular matrix bound ligands and their receptors. Unlike the well studied floor plate the glial segregating system of the VC is not a well defined structure. It could provide a substrate from which substances might influence transmedian axon growth and might influence migrating axons directly through cell-cell contacts. The aim of this study is to characterise the cells of the VC and to establish their relationship to crossing commissural axons as a step toward understanding their role in midline development.

The morphology of the early rat VC at embryonic days 12, 13, 14, 16 and 18 was examined at light and electron microscopic levels. The nature of glial cell perikarya and their relationship to the cells of the floor plate and to the glia limitans was examined. Morphometric parameters such as the branching patterns of constituent VC cells were investigated. An analysis of the ultrastructural characteristics of VC glial cells undertaken to gain an insight into the changing nature of these cells over time revealed features typical of cells undergoing differentiation. The axons crossing in the VC were also examined, as were the relationships between these axons and the glial matrix of the ventral commissure.