SEARCH

SEARCH BY CITATION

Keywords:

  • adenohypophysis;
  • infundibulum;
  • monotreme;
  • neurohypophysis;
  • preoptic area

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References

The living monotremes (platypus and echidnas) are distinguished by the development of their young in a leathery-shelled egg, a low and variable body temperature and a primitive teat-less mammary gland. Their young are hatched in an immature state and must deal with the external environment, with all its challenges of hypothermia and stress, as well as sourcing nutrients from the maternal mammary gland. The Hill and Hubrecht embryological collections have been used to follow the structural development of the monotreme hypothalamus and its connections with the pituitary gland both in the period leading up to hatching and during the lactational phase of development, and to relate this structural maturation to behavioural development. In the incubation phase, development of the hypothalamus proceeds from closure of the anterior neuropore to formation of the lateral hypothalamic zone and putative medial forebrain bundle. Some medial zone hypothalamic nuclei are emerging at the time of hatching, but these are poorly differentiated and periventricular zone nuclei do not appear until the first week of post-hatching life. Differentiation of the pituitary is also incomplete at hatching, epithelial cords do not develop in the pars anterior until the first week, and the hypothalamo-neurohypophyseal tract does not appear until the second week of post-hatching life. In many respects, the structure of the hypothalamus and pituitary of the newly hatched monotreme is similar to that seen in newborn marsupials, suggesting that both groups rely solely on lateral hypothalamic zone nuclei for whatever homeostatic mechanisms they are capable of at birth/hatching.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References

The monotremes are a group of mammals consisting of the Ornithorhynchidae (represented by the platypus, Ornithorhynchus anatinus) and the Tachyglossidae (short- and long-beaked echidnas; Tachyglossus aculeatus and species of genus Zaglossus, respectively). The monotremes are distinguished from therians by unusual features that relate to homeostasis and reproduction. They have a body temperature that is lower than in placental mammals and may vary with ambient temperature (Griffiths, 1978), but the most striking feature of the group is their reproduction. Monotreme development encompasses three distinct phases: an intrauterine period of uncertain duration (but lasting between 15 and 27 days; Temple-Smith & Grant, 2001; Holland & Jackson, 2002; Rismiller & McKelvey, 2003; Hawkins & Battaglia, 2009); an incubation phase of 10–11 days within a leathery-shelled egg (Hawkins & Battaglia, 2009; Renfree et al. 2009) finishing with hatching at an immature state; and a lactational phase of 5–7 months (Holland & Jackson, 2002; Rismiller & McKelvey, 2003; Hawkins & Battaglia, 2009). The newly hatched monotreme develops in either a shallow maternal pouch (short-beaked echidnas) or a vegetation nest (platypus). The hatchling faces the challenges of breaking through the egg membranes, repeatedly locating the teat-less areola, stimulating milk let-down and sucking milk (Griffiths, 1968, 1978; Griffiths et al. 1969). The young monotreme also has poor thermoregulation for at least the first 2 months of post-hatching life (Griffiths, 1968, 1978).

The hypothalamus of the adult monotreme is structurally and chemoarchitecturally similar to that of therians, although the supraoptic nucleus is small (Ashwell et al. 2006). On the other hand, the adult pituitary of the echidna has some unusual structural features, in that the infundibular cavity extends into the pars nervosa, which is sacculated rather than knob-like (Griffiths, 1968). In this respect the monotreme pars nervosa is said to be more like that of sauropsids than therians (Griffiths, 1968). Furthermore, the infundibular cavity of the short-beaked echidna is lined by ependymal cells whose processes intermingle with the processes of pituicytes, as they do in the pars nervosa of the domestic chicken (Griffiths, 1940).

The aim of the present study was to follow the structural development of the hypothalamus and pituitary from embryonic age to adulthood to answer several questions: (i) how does development of the hypothalamus and pituitary during the incubation phase provide the hatchling with the necessary neural machinery for life outside the egg; (ii) how does the early structural development of the hypothalamus and pituitary in the monotremes compare with that in therians; and (iii) how does the structural development of the hypothalamus and pituitary during the lactational phase correlate with the progressive acquisition of behavioural independence?

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References

This study is based on 22 platypus and 10 short-beaked echidna specimens held at the Museum für Naturkunde (MfN), Berlin (Tables 1 and 2). All of the monotreme material is archival and was collected during the late 19th and early 20th centuries. Most of the material is part of the J. P. Hill embryological collection, but seven platypus and one echidna specimen were originally from collections in American museums [American Museum of Natural History (AMNH), National Museum of Natural History (USNM)] and had been sectioned by J. Zeller (Zeller, 1989).

Table 1.   Summary of platypuses used
No.Crown-rump length (mm)aDorsal contour length (mm)Head length (mm)Estimated agebStainc
  1. aFor embryonic ages this is listed as GL (greatest length) in museum records. na, not available.

  2. bEstimated on the basis of 10 days between egg-laying and hatching for pre-hatching ages (H-1, H-2, etc.) and using tables in Manger et al. (1998) for post-hatching specimens. H-x, pre-hatching days (x); PH, post-hatching.

  3. cH&E, haematoxylin and eosin; H, haematoxylin; AB&NR, Alcian blue and nuclear red; H&E/Azan, sections alternately stained.

M436.5nanaH-9H&E
M37Sag8.5nanaH-6.5H&E
M378.5nanaH-6.5H
M398.5nanaH-6.5H&E
M39x8.5nanaH-6.5H&E
M389nanaH-6H&E
M409nanaH-6H
M419nanaH-6H&E
M079nanaH-6AB&NR
M4210nanaH-5H&E
M4416.7528.06.0PH0 to PH2Haem
AMNH 20196929.374.014.2PH6Azan
M4533.056.0naPH7H&E
MO3844.012222.5PH11Azan
AMNH 20203065193naPH40Azan
MO397018031.0PH42Azan
AMNH 20200281.3215naPH45Azan
AMNH 2020038724041.7PH49Azan
AMNH 201311na26043.0PH52Azan
AMNH 201312na30055.5PH110Azan
USNM 22111216033360PH140Azan
Adultna400100AdultH&E/Azan
Table 2.   Summary of short-beaked echidnas used
No.Crown-rump length (mm)aEstimated agebStain
  1. aFor embryonic ages this is listed as GL (greatest length) in museum records.

  2. bEstimated on the basis of 10 days between egg-laying and hatching for prehatching ages (H-1, H-2, etc.). PH ages are estimated on the basis of the relationship between CRL and weight and with reference to Rismiller & McKelvey (2003).

  3. cEstimated from available sections.

M1555.5cH-10H&E
MOF142c6cH-9H&E
M1576.5cH-9H&E
MOF1616.6H-9H&E
M1537.3H-8H&E
M1547.5cH-7.5H&E
MO558H-7H&E
M15812.5H-2H&E
M16225PH6Carmine
Adult400AdultAzan

Details of collection and processing of the material are not available, but the embryonic monotreme material had probably been collected in the eastern states of Australia, fixed by immersion in aldehyde fixatives for several days and stored in alcohol. The echidna specimens are probably the subspecies Tachyglossus aculeatus aculeatus. The tissue had been embedded in paraffin and sectioned at 8- or 10-μm thickness, usually in the transverse (coronal) or horizontal plane (except for M37Sag, cut in the sagittal plane), before being stained with haematoxylin, haematoxylin and eosin, or alcian blue and nuclear red. Post-hatching platypus specimens had been embedded in paraffin (M44, M45, MO38, AMNH201969) or celloidin (all others) and sectioned at thicknesses of 10 μm (M44, AMNH 201969, M45, MO38), 35 μm (AMNH202030, MO39, AMNH201311, AMNH201312), 35–50 μm (AMNH202002, AMNH 202003) or 35–80 μm (USNM 221112, adult platypus) and stained with azan. Post-hatching echidnas were embedded in paraffin or celloidin (adult) and sectioned at 15 or 35 μm and stained with haematoxylin and eosin, carmine or azan.

Most of the sectioned material was photographed with the aid of either a Zeiss Axioplan2 fitted with an AxioCam MRc5 camera, or a Leica M420 macroscope fitted with an Apozoom 1 : 6 lens and Leica DFC490 camera. All images were calibrated by photographing a scale bar at the same magnification. Measurements of the thickness of developmental zones have been made with the aid of imagej version 1.37 software. All measurements have been made on tissues that have been subject to dehydration, so all distances must be subject to some degree of shrinkage. The magnitude of shrinkage is likely to change during maturation depending on the maturation of ultrastructural elements but differential shrinkage is unlikely to make a significant difference to the conclusions that are reached.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References

Summary of the phases of monotreme development

Monotreme development differs somewhat from that seen in the more familiar marsupials and placentals, so it is worth summarising its phases before covering the results. There are three phases of monotreme development: intra-uterine (pre-embyronic development before laying of the egg), incubation (development within the egg after laying), and lactational (development after hatching). The precise duration of the intra-uterine phase is not known, but incubation lasts about 10–11 days (Hawkins & Battaglia, 2009; Renfree et al. 2009) with progression from neural tube closure to the completion of organogenesis (i.e. all organs are present, even if still immature). Incubation can be split into three subphases of about 3–4 days’ duration each (Hughes & Hall, 1998; Renfree et al. 2009; Werneburg & Sánchez-Villagra, 2011). The first is an early pharyngeal arch subphase (standard event stages 12–15; Ina), the second is a mid-incubation late pharyngeal arch subphase (stages 16–20; Inb), and the third is a late pre-hatching stage (stages 21–24; Inc). The anterior neuropore closes at stage 13 [about 5.5 mm crown-rump length (CRL)]. Both platypus and echidna reach 14–15 mm CRL at hatching (Renfree et al. 2009). The lactational period is 114–145 days for captive platypus (Holland & Jackson, 2002; Hawkins & Battaglia, 2009). The lactational phase for echidna starts with 55 days’ growth in the pouch, followed by growth in the nursery burrow, with weaning at day 205 (Rismiller & McKelvey, 2003).

Development of the hypothalamus and pituitary during early to mid-incubation

During the first third of incubation, the neuroepithelium of the hypothalamus is only 80 μm thick and no post-mitotic cells have settled outside the neuroepithelium. At the beginning of this phase, the anterior neuropore is in the process of closing (Fig. 1A) and the optic stalk (os) has extended 300 μm from the secondary prosencephalon. On the basis of the position of the os, one can assign putative neuroepithelial zones (Fig. 1B,C) for the future parts of the hypothalamus (i.e. a preoptic area neuroepithelium rostral to the os; ootoeminential domain caudal to the os and tuberal neuropeithelium still further caudally; based on maps for placental embryos, Puelles & Rubenstein, 1993; Puelles et al. 2004). The developing pituitary is only rudimentary at this stage, in that the Rathke’s pouch is a relatively shallow (20–30 μm from orifice to fundus) extension of the roof of the oral cavity and the infundibular recess is a thin-walled shallow depression (Fig. 1D).

image

Figure 1.  Development of the monotreme hypothalamus and pituitary in the first third of incubation as shown in photomicrographs of horizontal sections. The optic stalk (os) projects from the forebrain before closure of the anterior neuropore (anp) (A), but the wall of the developing hypothalamus remains at a rudimentary neuroepithelial stage until after 6.5 mm CRL (B,C). Rathke’s pouch (Rathke) and the infundibular recess are only simple recesses by 6.6 mm CRL (D). 3V, third ventricle; InfR, infundibular recess; poa, preoptic area neuroepithelium; oem, ootoeminential domain; Oral, oral cavity; tu, tuberal region neuroepithelium.

Download figure to PowerPoint

From the end of the first third of incubation (i.e. 8.0–8.5 mm CRL; Figs 2 and 3), the two species begin to differ slightly in the pace of hypothalamic and pituitary development, even though the process appears to be the same. In the short-beaked echidna at this size, neuroepithelial domains that will give rise to the rostrocaudal regions of the adult hypothalamus can be distinguished in the walls of the ventral third ventricle (Fig. 2A–C). In addition, post-mitotic cells have begun to settle in large numbers in the mantle layer external to the neuroepithelium. The monotreme hypothalamus appears to share the lateral-to-medial neurogenetic gradient observed in therian hypothalamus (Altman & Bayer, 1986; Cheng et al. 2002) in that these neurons are destined for the nuclei of the lateral zone (e.g. LH, lateral hypothalamus). A diffuse zone of fibres that is probably the medial forebrain bundle can also be distinguished running along the rostrocaudal axis of the hypothalamus (Fig. 2B,C). Development of the pituitary is much more advanced for the anterior lobe compared to the posterior. The infundibular recess is narrow and the infundibulum and posterior pituitary are still thin-walled (Fig. 2D,E) but the pars anterior of the anterior pituitary has begun to expand rapidly and is being invaded by vasculature (Fig. 2E). Rathke’s pouch is a transverse slit approximately 200 μm wide whose anterior wall is developing into the pars intermedia, whereas the lateral wall will give rise to the pars tuberalis.

image

Figure 2.  The hypothalamus and pituitary of the echidna at the beginning of the middle third of incubation as seen in horizontal sections. Anterior is to the top and the midline is on the left side of images (A–D). Photomicrographs (A) and (B) show sections through the middle and superior part of the hypothalamic anlage at 8.0 mm CRL. Discrete regions of the hypothalamic neuroepithelium can be distinguished (ml, lateral mammillary neuroepithelium; mm, medial mammillary neuroepithelium; pa, paraventricular area neuroepithelium; poa, preoptic area neuroepithelium; tm, tuberomammillary area neuroepithelium; tu, tuberal area neuroepithelium) and neurons (of the presumptive lateral hypothalamic zone – LH) have begun to settle in the mantle region. Separation of cells in the lateral zone suggests that fibres of the medial forebrain bundle (mfb) have begun to penetrate the mantle, but there is no fornix visible. In the more inferior (tuberal and infundibular) part of the hypothalamus (C,D) the wall of the infundibular recess arises from the posterior ventricular wall. The lumen of Rathke’s pouch has a simple transverse slit surrounding the pars tuberalis of the adenohypophysis (E), but cell nests of the pars anterior have begun to form and have been invaded by vasculature (v). The neurohypophysis (PPit, posterior pituitary) is rudimentary at this stage (E). Anterior is to the top in all photomicrographs. The midline is to the left in (A–C). ictd, internal carotid artery; InfR, infundibular recess; lge, lateral ganglionic eminence; mge, medial ganglionic eminence; os, optic stalk; Rathke, Rathke’s pouch; V3V, ventral third ventricle.

Download figure to PowerPoint

image

Figure 3.  Photomicrographs of sections in the sagittal plane through the developing forebrain of the platypus at 8.5 mm CRL. The hypothalamus and pituitary of the platypus are slightly less advanced than those illustrated in Fig. 2. As can be seen in a section, a few hundred μm from the midline (A) neuroepithelial regions of the hypothalamus can be distinguished, but a more lateral section (B) shows that only a few neurons of the lateral zone have settled in the mantle layer. In a midline section (C), the pituitary structure is still rudimentary for both anterior (APit) and posterior (PPit) parts. bas, basilar artery; D3V, dorsal third ventricle; IVF, interventricular foramen; LTerm, lamina terminalis; ML, lateral mammillary nucleus; p1, p2, p3, prosomeres 1, 2, 3; rm, retromammillary neuroepithelium of hypothalamus; spt, septum. All other abbreviations are as Figs 1 and 2.

Download figure to PowerPoint

By contrast, the hypothalamus and pituitary of the platypus appear to be less advanced than in the echidna at a similar CRL. At 8.5 mm CRL in the platypus (Fig. 3), the hypothalamic neuroepithelial domains in the wall of the ventral third ventricle as seen in the echidna, can also be identified (Fig. 3A), but there are two significant differences. First, the nuclei in the mantle layer of the platypus hypothalamus (Fig. 3B) are much smaller than in the echidna at a similar body length, suggesting that generation of hypothalamic neurons is delayed by 1–2 mm CRL (probably about 1–2 developmental days) relative to the echidna. Secondly, the state of maturation of the platypus pituitary at this stage is much less advanced than in the echidna of a similar body length. Although Rathke’s pouch in the platypus has elongated to a length of 400 μm, the walls of both Rathke’s pouch and the infundibular recess are only 40–50 μm thick and there is no development of the pars anterior or tuberalis.

Towards the end of the mid-incubation phase (10.0 mm CRL), the differentiation of the platypus hypothalamus and pituitary reaches a level comparable to that already described for the 8.0 mm CRL echidna. Nuclei of the lateral zone (including presumptive LH) have formed in the mantle layer alongside the preoptic, tuberal and tuberomammillary neuroepithelium and a putative medial forebrain bundle (mfb) can be seen (Fig. 4A,B). Significantly, apart from the putative mfb and unidentified fibre bundles in the marginal zone, no other tracts associated with the mature hypothalamus (e.g. fornix or mammillothalamic tract) can be found. In the more caudal hypothalamus (Fig. 4C), three recesses of the caudal third ventricle can be identified in the coronal section: infundibular, mammillary and retromammillary; but very few post-mitotic cells have settled into the mantle layer of the more caudal parts of the hypothalamus. Differentiation of the pituitary in the 10.0-mm CRL platypus (Fig. 4D–G) is also comparable to that described above for the 8.0-mm CRL echidna. The degree of differentiation of the pars anterior and extent of its vascularisation are both similar to that in the younger echidna, and Rathke’s pouch has a similar transverse orientation (with a 300–400-μm-wide slit). The posterior pituitary is undifferentiated at this stage (Fig. 4G).

image

Figure 4.  Photomicrographs of coronal sections through a 10 mm CRL platypus show the hypothalamus (A–C) and pituitary (D–G). Both structures are shown in a rostral to caudal sequence. More neurons have settled in the mantle layer of the hypothalamus (A–C), but the process is not greatly advanced from that seen at 8.0 mm CRL in the echidna (compare with Fig. 2). The pars anterior has expanded (D,E), but Rathke’s pouch remains wide (F,G) and the posterior pituitary is still rudimentary (G). ah, anterior hypothalamic neuroepithelium; dm?, putative dorsomedial hypothalamic nucleus neuroepithelium; ictd, internal carotid artery; RMR, retromammillary recess; sch, suprachiasmatic nucleus neuroepithelium. All other abbreviations are the same as for previous figures. Scale bar in (D) also applies to (E–G).

Download figure to PowerPoint

There is no platypus specimen available for the immediately pre-hatching period, but the existing echidna specimen at this age (12.5 mm CRL; Fig. 5) gives an idea of the state of differentiation of the hypothalamus and pituitary leading up to the critical event of hatching. The neuroepithelium is still thick at this stage (more than 100 μm thick in some places), but settling of neurons in the mantle has progressed significantly. Not only are the components of the lateral zone [e.g. LH; lateral preoptic area (LPO)] identifiable, components of the medial zone (e.g. AH, anterior hypothalamic nucleus; DM, dorsomedial hypothalamic nucleus; VMH, ventromedial hypothalamic nucleus) have also begun to settle in the mantle zone. Cytoarchitectural characteristics of nuclei in the lateral hypothalamic zone, e.g. the large neurons of the magnocellular nucleus of the lateral hypothalamus (MCLH) are also emerging at this time (Fig. 5E). The anterior pituitary at this immediately pre-hatching age (Fig. 5F) is not greatly different in appearance from that seen in the 8.0-mm CRL echidna (compare with Fig. 2) but the posterior pituitary has developed a lobar structure (Fig. 5G) not seen at the earlier age. Fibre tract development is not apparently advanced from earlier ages.

image

Figure 5.  Photomicrographs of sections in the coronal plane show the structure of the hypothalamus (rostral to caudal sequence – A–F) and pituitary (rostral – G, caudal – H) in an echidna in the period immediately before hatching. The neuroepithelium is still thick, but nuclei of both the lateral and medial zones of the hypothalamus have settled in the mantle layer and cytoarchitectural differentiation of nuclei like the magnocellular nucleus of the lateral hypothalamus (MCLH) is underway. The posterior pituitary is developing as a solid mass at the rostral end of the infundibular stalk (InfS) but there is no hypothalamo-neurohypophyseal tract as yet. AH, anterior hypothalamus; cp, cerebral peduncle; DM, putative dorsomedial hypothalamic nucleus; HDB, nucleus of horizontal limb of diagonal band; LPO, lateral preoptic nucleus; MPO, medial preoptic nucleus; PLH, posterior nucleus of lateral hypothalamus; RCh, retrochiasmatic nucleus; VMH, putative ventromedial hypothalamic nucleus. All other abbreviations are the same as for previous figures. Scale bar in (A) also applies to (B–F). Scale bar in (G) also applies to (H).

Download figure to PowerPoint

Development of the hypothalamus and pituitary in the lactational phase

In the first week after hatching, neurons of the medial group of hypothalamic nuclei continue to settle in the mantle layer (Fig. 6A–C) and the putative fornix is visible for the first time at approximately 2 days after hatching in the platypus (Fig. 6B). The first neurons of the paraventricular nucleus, which ultimately lies at the boundary between periventricular and medial zones of the hypothalamus, is seen for the first time (Fig. 6A–C). The hypothalamic neuroepithelium is still up to 100 μm thick in several sites (in particular the anterior hypothalamus and arcuate regions), suggesting the continued active production of neurons, presumably for the periventricular zone of the hypothalamus. The structure of the anterior pituitary of the platypus is much the same as that seen before hatching (Fig. 6D), but cords of epithelial cells are visible in the echidna anterior pituitary at the end of the first post-hatching week (Fig. 6E).

image

Figure 6.  Photomicrographs of sections through the hypothalamus and pituitary of a platypus (A, B, D) and echidna (C, E) during the first week of post-hatching life. Although the neuroepithelium remains up to 100 μm thick in parts of the hypothalamus, all neurons of the lateral and medial zones, and some components of the periventricular zone (e.g. Arc, arcuate nuclei; Pa, paraventricular nucleus), have been produced. The fornix (f) is seen for the first time (B,C). Cords of epithelial cells are visible in the pars anterior of the echidna (e). hs, hypothalamic sulcus. All other abbreviations are the same as for previous figures.

Download figure to PowerPoint

By the second week of post-hatching life (Fig. 7A–C), neuropil has increased in the lateral and medial zones of the hypothalamus, thereby separating the constituent neurons. The putative hypothalamo-neurophypophyseal tract (Fig. 7B,E) is seen for the first time and the differentiation of the mammillary and retromammillary regions has advanced sufficiently that all the constituent nuclei can be identified (Fig. 7C). The anterior pituitary has attained a mature appearance (Fig. 7D), but Rathke’s pouch remains large (Fig. 7E). Invasion of the posterior pituitary by the putative hypothalamo-neurohyphyseal tract has expanded the region (Fig. 7E) and the extension of the infundibular recess into the posterior pituitary is visible at this stage.

image

Figure 7.  Photomicrographs of coronal sections through the hypothalamus (A–C) and pituitary (D,E) of a platypus in the second week of post-hatching life; and of the hypothalamus of a platypus in week 5 (F,G). The hypothalamic neuroepithelium is thinning rapidly in the second post-hatching week and all the major tracts associated with the mature hypothalamus are visible. The posterior pituitary retains a projection of the neuroepithelium of the infundibular recess (asterisk in E) and pituicytes are visible. By the 5th week, the structure of the hypothalamus is mature. ac, anterior commissure; BST, bed nuclei of the stria terminalis; LM, lateral mammillary nucleus; ME, median eminence; ML, lateral part of medial mammillary nucleus; MM, medial part of medial mammillary nucleus; nehy, hypothalamo-neurohypophyseal tract; och, optic chiasm; opt, optic tract; Pe, periventricular nucleus of hypothalamus; RM, retromammillary nucleus; rmd, retromammillary decussation; TM, tuberomammillary nucleus; VTA, ventral tegmental area. All other abbreviations are the same as for previous figures. Scale bar in (C) also applies to (A) and (B). Scale bars in (D) and (F) apply to (E) and (G), respectively.

Download figure to PowerPoint

The morphology of the hypothalamus is essentially mature by about the fifth week of post-hatching life (Fig. 7F,G) in that nuclei throughout lateral, medial and periventricular zones of the hypothalamus are all visible.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References

The use of archived material

The present findings are based on analysis of specimens collected during the late 19th and early 20th centuries and processed according to the then current, best-practice histological techniques. It is not recorded how many monotremes (adult and young) were killed in the process, but it is likely to have been several hundred, a practice that would be unacceptable to the modern Australian public. It would therefore be impossible to make such a collection in the modern world.

The embryological collections at the Museum für Naturkunde in Berlin are scientifically important, but one must recognise their limitations. Most of the incubation phase material had been sectioned at a thickness of 10 μm and post-hatching material was even thicker, so detailed cytological analysis of mitotic activity in proliferative regions, the migration of hypothalamic neurons and the formation of fibre tracts is not possible. Furthermore, no unstained sections are available for immunostaining for modern markers of neurotransmitter, hormone receptor or enzyme development, so analysis of the maturation of hypothalamic neurons is dependent solely on cytoarchitecture. Despite these limitations, the material does allow the analysis of major morphological events in hypothalamic and pituitary development and the interpretation of those findings in the context of modern concepts of brain development.

The functional significance of hypothalamic and pituitary development in monotremes

Hypothalamic development in the monotreme incubation phase (summarised in Fig. 8) proceeds from closure of the prosencephalon, through segmentation of the hypothalamic neuroepithelium and the onset of neurogenesis of lateral zone neurons, to formation of the putative medial forebrain bundle and early differentiation of the lateral zone nuclei. Pituitary development during the same phase of monotreme development proceeds from formation of Rathke’s pouch and the infundibular stalk to the first vascularisation and expansion of the pars anterior and the first expansion of the posterior pituitary. Significantly, the newly hatched monotreme does not have the full complement of hypothalamic neurons; in particular some medial zone and most (if not all) periventricular zone nuclei (e.g. paraventricular and arcuate) are still to be generated. Furthermore, several key fibre tracts (e.g. fornix, hypothalamo-neurohypophyseal tract) are not present in the peri-hatching period.

image

Figure 8.  Summary of the sequence of major events in the development of the hypothalamus and pituitary of monotremes relative to CRL and estimated age. Hypothalamic neurogenesis starts at 7.0–8.0 mm CRL (late Ina, early Inb) but settling of the last hypothalamic neurons into periventricular zone nuclei is not complete until 50–60 mm CRL (lactational phase). H-x indicates x days before hatching; PH, days post-hatching.

Download figure to PowerPoint

Neurogenesis of the monotreme hypothalamus is still in progress at the time of hatching, so the newly emergent monotreme must be dependent on the available circuitry represented by the lateral hypothalamic zone and the associated medial forebrain bundle and marginal zone fibre tracts. Once it has broken through the egg membranes, the newly hatched monotreme faces several challenges: (i) repeatedly finding the teat-less mammary areolae; (ii) stimulating milk ejection, and (iii) controlling several fundamental homeostatic mechanisms, e.g. lung ventilation, cardiovascular and gastrointestinal function.

There are no data available concerning the connections of the adult monotreme hypothalamus with other parts of the neuraxis but if we assume these are conserved in mammals, then we can deduce those hypothalamic systems that may be available at hatching and those which are unlikely to be present. The lateral hypothalamus is known to function as a feeding regulatory centre in therians (Saper et al. 1979; Simerly, 2004) and has connections with the brainstem, periaqueductal grey, parabrachial nuclei and spinal cord (reviewed in Simerly, 2004). The early differentiation of the lateral hypothalamic zone in newly hatched monotremes is therefore consistent with a potential role in early regulation of feeding and rudimentary cardiorespiratory control. If monotremes are like marsupials, more sophisticated control of respiratory function in response to fluctuations in environmental O2 and CO2 are unlikely to be available until after the end of the first week (Baudinette et al. 1988). In contrast to the lateral zone of the hypothalamus, those medial and periventricular zone nuclei likely to be involved in the circuitry mediating the effects of accessory olfactory and limbic system input on reproductive behaviour and physiology are clearly undeveloped at hatching. Similarly, preoptic and paraventricular nuclei involved in mediating adaptive responses to stressful environmental stimuli (Simerly, 2004) are unlikely to be functionally active in the young monotreme until several weeks after birth.

Similarities and differences between monotremes and therians in hypothalamic development

Both marsupial and monotreme young emerge into the external environment in an apparently similar state of external physical immaturity. It is natural then to compare the development of the hypothalamus and pituitary in the two groups. In newborns of a representative diprotodont marsupial (e.g. the tammar wallaby), the hypothalamic neuroepithelium exhibits similar compartmentation to that seen in the monotremes (Cheng et al. 2002). The progression of neurogenesis at birth in the tammar is also similar to that seen in monotremes, in that neurons of the lateral zone have already settled in the mantle layer and neurons of the medial zone are in the process of settling (Cheng et al. 2002). Finally, the extent of development of hypothalamic fibre bundles is similarly restricted to the putative medial forebrain bundle in the mantle layer and marginal layer tracts. In both the monotremes and the tammar wallaby, the fornix does not develop until the second week after hatching or birth (present study; Cheng et al. 2002). Development of the pituitary in the newborn tammar (Cheng et al. 2002) and representative didelphids such as the short-tailed opossum (Gasse & Meyer, 1995) is also quite similar to that described for the two monotremes at hatching, in that Rathke’s pouch of the newborn marsupial presents a similar transverse slit, the epithelial cells of the pars anterior are similarly limited in differentiation and the posterior pituitary is small and does not receive a hypothalamo-neurohypophyseal tract.

In the laboratory rat (Rodentia), the first hypothalamic neurons projecting to the posterior pituitary are in the supraoptic nucleus (at embryonic day E15) with the paraventricular nucleus providing axons to the posterior pituitary at E17 (Makarenko et al. 2000). In the tammar wallaby (Cheng et al. 2002), the supraoptic nucleus connects with the posterior pituitary by postnatal day (P)5, with the paraventricular nucleus connecting by P10. In the present material, establishing the timing of connections is confined to identification of the respective tract, but the appearance of the putative hypothalamo-neurohypophyseal tract of the platypus at the end of the second post-hatching week (Fig. 8) is consistent with monotreme pituitary development following a similar developmental trajectory to that seen in marsupials.

Conclusion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References

Although the development of the monotreme hypothalamus and pituitary follows a similar sequence of events to that seen in placentals, both structures are morphologically immature at the time of hatching, with hypothalamic neurogenesis and tract formation still in process. In many respects, the extent of differentiation of the hypothalamus in the newborn monotreme is similar to that seen in newborn marsupials. In both marsupials and monotremes, whatever regulatory functions are required of the newly emergent young must be achieved with a partially formed (lateral) hypothalamus and probably without a functional hypothalamic-pituitary axis. The neural machinery for ventilation and cardiovascular regulation in the newborn marsupial or monotreme must be provided by the recently settled neurons of the lateral hypothalamus and the medial forebrain bundle, projecting to the brainstem and spinal cord.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References

The Alexander von Humboldt Foundation generously provided financial support for the analysis of material in the Museum für Naturkunde (MfN) embryological collections. I am very grateful to Dr Peter Giere of the MfN for his help. I could not have asked for a more obliging host during my stay in Berlin. I would like to thank Professor Ulrich Zeller of the MfN for access to his collection of sectioned platypus and echidna heads. The author reports no conflict of interest.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References
  • Altman J, Bayer SA (1986) The development of the rat hypothalamus. Adv Anat Embryol Cell Biol 100, 1178.
  • Ashwell KW, Lajevardi SE, Cheng G, et al. (2006) The hypothalamic supraoptic and paraventricular nuclei of the echidna and platypus. Brain Behav Evol 68, 197217.
  • Baudinette RV, Runciman SIC, Frappell PF, et al. (1988) Development of the marsupial cardiorespiratory system. In: The Developing Marsupial: Models for Biomedical Research (eds Tyndale-Biscoe CH, Janssens PA), pp. 132147. Berlin: Springer.
  • Cheng G, Marotte LR, Mai JK, et al. (2002) Early development of the hypothalamus of a wallaby (Macropus eugenii). J Comp Neurol 453, 199215.
  • Gasse H, Meyer W (1995) Neuron-specific enolase as a marker of hypothalamo-neurohypophyseal development in postnatal Monodelphis domestica (Marsupialia). Neurosci Lett 189, 5456.
  • Griffiths M (1940) The relationship between the secretory cells of the pars nervosa of the hypophysis and classical neuroglia. Endocrinology 26, 10321041.
  • Griffiths M (1968) Echidnas. London: Pergamon.
  • Griffiths M (1978) The Biology of the Monotremes. New York: Academic.
  • Griffiths M, McIntosh DL, Coles REA (1969) The mammary gland of the echidna, Tachyglossus aculeatus, with observations on the incubation of the egg and on the newly hatched young. J Zool (Lond) 158, 371386.
  • Hawkins M, Battaglia A (2009) Breeding behaviour of the platypus (Ornithorhynchus anatinus) in captivity. Aust J Zool 57, 283293.
  • Holland N, Jackson SM (2002) Reproductive behaviour and food consumption associated with the captive breeding of platypus (Ornithorhynchus anatinus). J Zool (Lond) 256, 279288.
  • Hughes RL, Hall LS (1998) Early development and embryology of the platypus. Philos Trans R Soc Lond B Biol Sci 353, 11011114.
  • Makarenko IG, Ugrumov MV, Derer P, et al. (2000) Projections from the hypothalamus to the posterior lobe in rats during ontogenesis:1,1’dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine perchlorate tracing study. J Comp Neurol 422, 327337.
  • Manger PR, Hall LS, Pettigrew JD (1998) The development of the external features of the platypus (Ornithorhynchus anatinus). Philos Trans R Soc Lond B Biol Sci 353, 11151125.
  • Puelles L, Rubenstein JLR (1993) Expression patterns of homeobox and other putative regulatory genes in the embryonic mouse forebrain suggest a neuromeric organization. Trends Neurosci 16, 472479.
  • Puelles L, Martínez S, Martínez-de-la-Torre M, et al. (2004) Gene maps and related histogenetic domains in the forebrain and midbrain. In: The Rat Nervous System, 3rd edn (ed. Paxinos G), pp. 325. San Diego: Elsevier Academic.
  • Renfree MB, Papenfuss AT, Shaw G, et al. (2009) Eggs, embryos and the evolution of imprinting: insights from the platypus genome. Reprod Fertil Dev 21, 935942.
  • Rismiller PD, McKelvey MW (2003) Body mass, age and sexual maturity in short-beaked echidnas, Tachyglossus aculeatus. Comp Biochem Physiol A Mol Integr Physiol 136, 851865.
  • Saper CB, Swanson LW, Cowan WM (1979) An autoradiographic study of the efferent connections of the lateral hypothalamic area in the rat. J Comp Neurol 183, 689706.
  • Simerly RB (2004) Anatomical substrates of hypothalamic integration. In: The Rat Nervous System, 3rd edn (ed Paxinos G), pp. 335368. San Diego: Academic Elsevier.
  • Temple-Smith P, Grant T (2001) Uncertain breeding: a short history of reproduction in monotremes. Reprod Fertil Dev 13, 487497.
  • Werneburg I, Sánchez-Villagra MR (2011) The early development of the echidna, Tachyglossus aculeatus (Mammalia: Monotremata), and patterns of mammalian development. Acta Zool 92, 7588.
  • Zeller U (1989) Die Entwicklung und Morphologie des Schadels von Ornithorhynchus anatinus: (Mammalia, Prototheria, Monotremata). Abhandlungen der Senckenbergischen Naturforschenden Gesellschaft.