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Keywords:

  • procollagen IIA;
  • mouse forebrain;
  • sonic hedgehog;
  • anterior mesendoderm

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

Morphogenesis of the mammalian forebrain is influenced by the patterning activity of signals emanating from the anterior mesendoderm. In this study, we show that procollagen IIA (IIA), an isoform of the cartilage extracellular matrix protein encoded by an alternatively spliced transcript of Col2a1, is expressed in the prechordal plate and the anterior definitive endoderm. In the absence of IIA activity, the null mutants displayed a partially penetrant phenotype of loss of head tissues, holoprosencephaly, and loss of mid-facial structures, which is associated with reduced sonic hedgehog (Shh) expression in the prechordal mesoderm. Genetic interaction studies reveal that IIA function in forebrain and face development does not involve bone morphogenetic protein receptor 1A (BMPR1A)- or NODAL-mediated signaling activity. Developmental Dynamics 239:2319–2329. © 2010 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

Type II collagen, encoded by Col2a1, is a major extracellular matrix (ECM) component in cartilages. The expression and deposition of collagen II at the epithelial-mesenchymal interface of ectodermal tissues (e.g., neuroepithelium and cranial placodes) in the embryonic head marks the site of initiation of chondrogenesis leading to the formation of the cartilaginous tissues that encapsulate the brain and the sensory organs (Thorogood et al.,1986; Fitch et al.,1989; Cheah et al.,1991; Wood et al.,1991). Type II collagen is synthesized initially as procollagen II, which is processed by proteolytic removal of the globular extensions at the amino and carboxyl termini before the formation of the triple helical chain. Alternative splicing of the Col2a1 mRNAs produces different transcripts which contain exon 2 (the IIA mRNA) or with this exon spliced out (the IIB mRNA) (Ryan and Sandell,1990). The IIA, rather than the IIB, isoform is preferentially expressed in nonchondrogenic embryonic tissues suggesting an isoform-specific function during mouse embryogenesis (Cheah et al.,1991; Ng et al.,1993; Sandell et al.,1994; Lui et al.,1995). Exon 2 of the IIA transcript encodes a 69 amino acid von Willebrand factor C-like cysteine-rich (CR) domain in the amino-propeptide. CR domains are also present in CHORDIN (CHRD), which binds the bone morphogenetic proteins (BMPs) and negatively regulates their signalling activity (Garcia et al.,2002). In Xenopus embryos, enforced expression of CHRD and NOGGIN (NOG), another extracellular BMP antagonist, induces an ectopic axis and activates expression of dorsal mesoderm gene in explants of ventral marginal tissues (Larrain et al.,2000). Ectopic expression of procollagen IIA in Xenopus embryos elicits a similar effect, suggesting procollagen IIA may act as an antagonist to BMP signaling during embryogenesis.

Patterning of the embryonic forebrain is influenced by the morphogenetic cues from the anterior mesendoderm (AME) and the brain organizers, e.g., the anterior neural ridge and the isthmus, that are mediated by a multitude of signaling factors including BMP, WNT, FGF, and SHH (Robb and Tam,2004). Mutant studies have shown that loss of BMP antagonists (CHRD & NOG) and WNT antagonist (DICKKOPF1, DKK1) are associated with truncation of head structures including the loss or reduction of the forebrain (Mukhopadhyay et al.,2001; Anderson et al.,2002; del Barco Barrantes et al.,2003). The AME comprises of the prechordal plate (Prcp) and the head process (anterior notochord) that are derived from the gastrula organizer (Tam and Steiner,1999; Kinder et al.,2001). Microsurgical ablation of the AME and the adjacent floor plate of the cephalic neural plate lead to head truncation. Deletion of the anterior segment of the AME, containing the prechordal plate and part of the head process underneath the fore- and midbrain, also results in head truncation, but there is an up-regulation of Gsc and Shh expression in the remaining AME and expression of ventral forebrain markers (Six3 and Nkx2.1) in the rostral part of the truncated neural tube (Camus et al.,2000). Consistent with this finding, loss of SHH is associated with the loss of ventral telencephalic structures, accompanied by expansion of dorsal structures (Chiang et al.,1996); and provision of SHH to neural plate explants that lack AME could reconstitute expression of Nkx2.1, indicating the induction of ventral forebrain characteristics (Shimamura and Rubenstein,1997). Genetic study of Ssdp1 function in the headshrinker mutant (Nishioka et al.,2005) and the interaction of Dkk1 and Gsc in the compound mutant (Lewis et al.,2007) shows that loss of these gene activities in the prechordal part of the AME has a significant impact on head formation. Further embryological and genetic studies reveal that maintenance of the morphogenetic function of the prechordal segment of the AME requires the presence of the head process mesoderm. Ablation of the AME caudal to the prechordal plate results in down-regulation of Gsc and Shh expression in the remaining AME tissue and down-regulation of ventral forebrain markers (Camus et al.,2000). The findings of these embryological and genetic experiments suggest that the anterior segment of the AME is required for the formation of the head and the patterning of the ventral forebrain and its maintenance depends on the presence of the posterior segment of the AME.

Presently, the molecular activity associated with the maintenance of the prechordal component of the AME is not known. While combined loss of CHRD and NOG which are expressed in the posterior segment of the AME also leads to a head truncation (Bachiller et al.,2000), it is unclear if the loss of BMP antagonistic activity has any direct impact on the maintenance of the AME for head formation. In view of the potential role of procollagen IIA in counteracting BMP signaling activity, it is possible that IIA function may interface with that of other BMP antagonists in head development. In the present study, we investigated the role of IIA and its interaction with signaling pathways in head development by analyzing the phenotype of IIA-null mutant mice. Procollagen IIA-deficient mice were found to display variable and partially penetrant phenotype of malformations of the telencephalon and hypoplasia of the mid-facial structures. The concomitant loss of IIA affects Shh expression in the prechordal AME and the patterning of the ventral embryonic forebrain. Surprisingly, we found no evidence for IIA in antagonizing BMPR1A-mediated signaling or modulating activity of NODAL, another transforming growth factor-beta (TGFβ) protein, suggesting IIA function may not be connected with the signaling activity of these two TGFβ superfamily molecules in mouse forebrain development.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

IIA mRNA Is Expressed in the Anterior Mesendoderm and the Protein Is Localized to the Adjacent Intercellular Space

The expression of IIA transcript in wild-type embryos was examined by whole-mount in situ hybridization (WISH) using IIA (exon 2)-specific and Col2a1 riboprobes. No IIA transcript was detected in mid- to late-streak-stage embryo (Supp. Fig. S1A, which is available online). However, WISH using the Col2a1 riboprobe (capable of detecting both IIA and IIB transcripts) revealed robust hybridization signals in the anterior mesendoderm (Supp. Fig. S1B), suggesting that IIB transcript was expressed. IIA transcript was detected in the AME of the neural-plate stage (embryonic day [E]7.75) embryo (Fig. 1A-i). In the early headfold-stage (E8.0) embryo, IIA transcript was found in the prechordal plate, the head process and the anterior definitive endoderm in the adjacent paraxial region (Fig. 1B-ii,iii), overlapping with Dkk1 expression domain in the midline mesendoderm (Fig. 1C-iv) and the anterior definitive endoderm (Fig. 1C-v).

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Figure 1. Expression pattern of IIA during neurulation. A,B: Whole-mount in situ hybridization using IIA riboprobe of neural plate-stage (A) and early headfold stage (B) wild-type embryos. Lateral views. C:Dkk1 expression in the early headfold stage embryo. Frontal view. (i–v) Transverse sections showing expression domains of IIA in (i, ii) the prechordal tissue and (iii) the adjacent anterior definitive endoderm which overlaps with that of Dkk1 (iv,v). D: Wild-type neural plate stage embryo for orientation of planes of sectioning (left panel). Immunostaining using IIA antibody (right panels). Boxed regions (vi, vii; also shown in magnified views) reveal the localization of IIA protein at the interface of the prechordal plate and the ventral neurepithelium (vi) but is not detected in more posterior regions (vii). E: IIA immunostaining of wild-type early somite stage embryo showing the protein localized in the extracellular space between the floor plate and the axial mesendoderm tissue (arrow, viii) and along the basement membrane of the ventral neuroepithelium of the anterior neural plate (arrowheads). al, allantois; de, definitive endoderm; fp, floorplate; ne, neurectoderm. Scale bars = 0.1 mm.

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IIA protein was detected in the extracellular matrix (Zhu et al.,1999) on the basal side of the anterior definitive endoderm and the neural plate, and in the space between the neural groove and the prechordal plate of the early headfold-stage (embryonic day [E] 8.0) embryo (Fig. 1D-vi). No staining reaction was found in more posterior part of the head folds (Fig. 1D-vii), consistent with the absence of IIA hybridization signal in this region (Fig. 1A). In the early somite stage embryo, strong IIA immunostaining reaction was found in the matrix between the ventral region of the forebrain and the foregut endoderm (Fig. 1E-viii).

Loss of IIA Results in Midface Defect and Holoprosencephaly

We generated a mouse mutant lacking exon 2 of Col2a1 by gene targeting (see the Experimental Procedures section; Supp. Fig. S2A,B). Analysis by reverse transcriptase-polymerase chain reaction (RT-PCR) of total RNAs isolated from E9.5 homozygous (IIA−/−) embryos revealed the absence of IIA transcript but IIB expression persisted (Supp. Fig. S2C), confirming that the mutation led to deficiency of the IIA isoform of procollagen II. At E13.5–E16.5, 6/10 (60%) of the IIA−/− mutants displayed abnormal head structures (Table 1): foreshortened and tapered snout, retrognathia, reduced size of the frontal region of head (Fig. 2A,B) and a narrower transverse dimension of the forebrain (Fig. 2B:iv,v). Histological examination of the head of E13.5 IIA−/− embryos revealed a single unpartitioned prosencephalic vesicle (Fig. 2A-i,ii,B-iv,v) and absence of nasal septum and nasal cavity (Fig. 2A-iii,B-vi), which are signature features of holoprosencephaly. Other abnormalities include dysmorphogenesis of the diencephalon and ectopic location of the eye primordium and cranial nerve ganglia. IIA−/− embryos developed to full term, but 4 of 11 of the newborn (36.4%, Table 1) displayed severe craniofacial abnormalities: reduced size of frontonasal, premaxillary, and maxillary skeleton, hypoplasia of the mid-face, enophthalmia (Fig. 2C–H), and, in an extreme case, cyclopia with proboscis (data not shown). Some IIA+/− mutants at E9.5–E10.5 showed lack of separation of the forebrain vesicles (Fig. 3A–D) and head truncation (Fig. 3E–H). At E13.5–16.5, IIA+/− displayed craniofacial abnormalities including truncated frontonasal structures and hypoplasia of the mid-face tissues, less frequently than IIA−/− embryos at these stages (2/26 IIA+/− vs. 6/10 IIA−/−: P < 0.05 by χ2 test, Table 1) but not at birth (2/22 IIA+/− vs. 4/11 IIA−/−, Table 1). The haploinsufficiency effect and the partial penetrance of IIA mutation suggest that IIA functions in a dosage-dependent manner.

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Figure 2. Mutant phenotype of IIA−/− embryos and newborns. A,B: Embryonic day (E) 13.5 wild-type (A) and IIA−/− (B) embryos: absence of the lateral ganglionic eminence (lg; iv) and lateral ventricle (lv) in the forebrain (asterisk in v), rudimentary eyes (ey) localized ectopically and loss of nasal cavity as noted by the absence of nasal septum (ns) [asterisk in (vi)]. C,D,F,G: Craniofacial morphology of wild-type (C,D) and IIA−/− (F,G) newborn. E,H: Skeletal staining of wild-type (E) and IIA−/− (H) newborn: IIA−/− newborn displayed a shortened mandible (asterisks) and hypoplasia of the nasal bones (nb), premaxilla (pm) and maxilla (mx). di, diencephalons; fc, falx cerebri; fn, frontal bone; mb, midbrain; mo, medulla oblongata; ps, pons; sc, spinal cord; tn, tongue. Scale bars = 1 mm.

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Figure 3. Haploinsufficiency effect of IIA mutation. A–D: Embryonic day (E) 10.5 IIA+/− embryo displays single prosencephalic vesicle and small optic primordium. A–D: Lateral (A,B) and frontal (C,D) views of wild-type (A,C) and IIA+/− (B,D) embryos. E:Foxa2 expression in E9.5 wild-type embryo. F: The E9.5 wild-type embryo. G,H: Absence of optic vesicles (asterisk in wild-type, E) and deficiency of head tissues revealed by the truncated Foxa2 expression domain (arrows) in IIA+/− embryos.

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Table 1. Frequency of Head Defects and Genotype Distribution of IIA and IIA;Bmpr1a Mutants
Crosses% Embryos or mice displaying head defects
  • *

    Other defects: enophthalmia: IIA+/− 1/20, IIA+/−;Bmpr1a+/− 1/15; tail and neural tube deformity: IIA−/− 1/5, Bmpr1a+/− 1/11, IIA+/−;Bmpr1a+/− 2/15.

  • #

    IIA−/− and IIA−/−;Bmpr1a+/− offspring were under-represented at birth and during embryonic stages (expected: 12.5%) but frequency for IIA+/− and IIA+/−;Bmpr1a+/− was normal (expected: 25%).

  • +Resorptions. ++ dead before weaning, no craniofacial defect observed.

A. IIA+/−X IIA+/−WTIIA+/−IIA−/−
E13.5–16.50% (0/10)7.7% (2/26)60% (6/10)
Postnatal Day 10% (0/8)9.1% (2/22)36.4% (4/11)
B. IIA+/−X IIA+/−;Bmpr1a+/−WTIIA+/−IIA−/−Bmpr1a+/−IIA+/−; Bmpr1a+/−IIA−/−; Bmpr1a+/−
Reduced head size, midface hypoplasia and cyclopia *0/43/202/50/110/151/1
 Genotype distribution
C. IIA+/−X Bmpr1a+/−WTIIA+/−Bmpr1a+/−IIA+/−;Bmpr1a+/−
Postnatal Week 6 #25.3% (23/91)18.7% (17/91)35.2% (32/91)20.9% (19/91)
D. IIA+/−X IIA+/−Bmpr1a+/−WTIIA+/−IIA−/−Bmpr1a +/−IIA+/−; Bmpr1a+/−IIA−/−; Bmpr1a+/−not genotyped
E9.5–18.5 #10.5% (8/76)26.3% (20/76)6.6% (5/76)14.5% (11/76)19.7% (15/76)3.9% (3/76)18.4% (14/76)+
Postnatal Week 6 #15.1% (16/103)25.5% (27/103)4.7% (5/104)20.8% (22/103)29.3% (31/103)2.8% (3/103)1.9% (2/103)++

Loss of IIA Disrupts Shh Expression in the Prechordal AME

Morphogenetic activity of prechordal tissue and anterior mesendoderm, mediated by SHH signaling is reputed to play a key role in the development of the embryonic forebrain (Chiang et al.,1996). In the headfold to four-somite stage IIA−/− embryo, Shh expression was reduced in the prechordal AME (n = 5/8, Fig. 4A). TUNEL (terminal deoxynucleotidyl transferase–mediated deoxyuridinetriphosphate nick end-labeling) signals indicative of cell death were seen in the prechordal plate of IIA+/+ and IIA−/− embryos (Fig. 4B), and there was no difference (χ2 tests, P > 0.05) in the number of wild-type (n = 4/9), IIA+/− (n = 10/15) and IIA−/− (n = 4/16) embryos that displayed positive TUNEL reaction. The reduced expression domain of Shh in the prechordal plate cannot therefore be accounted for by excessive cell death in this tissue. Cell proliferative activity also did not change: a similar frequency of phospho-histone H3 (PHH3) stained nuclei was found in the forebrain of E9.5 wild-type and IIA−/− embryos (data not shown). In the early somite stage IIA−/− embryo with smaller head folds, Shh is expressed in the remaining AME (2/3 embryos; Fig 4C, i-iv). In support of defective prechordal plate development and signaling, Foxa2 expression is reduced in the prechordal tissue (n = 1/3) and the ventral forebrain (n = 3/3), but not the anterior notochord and floor plate, of the IIA−/− embryo (Fig. 4D). In neural plate stage IIA−/− embryos, expression of brachyury (T) is detected strongly in more posterior axial mesoderm (Fig. 4E). Loss of IIA therefore affects primarily the prechordal plate but not other parts of the AME.

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Figure 4. Molecular defects of the axial mesendoderm of IIA−/− embryos. A:Shh expression in IIA−/− embryos at 0-somite (0s) and 4-somite (4s) stages. Shh expression is reduced in the prechordal tissues (bracket) of IIA−/− embryos. B:Msx2-lacZ expression and fluorescent TUNEL signals in embryonic day (E) 8.0 wild-type and IIA−/− embryos. C: Reduced Shh expression domain of E8.5 IIA−/− embryos in the prechordal plate (arrows in i, iii) but normal in the anterior notochord (ii, iv). D:Foxa2 expression in E8.5 IIA+/− and IIA−/− embryos. Intact Foxa2 expression was found in the anterior notochord (arrowhead, vii) and the floorplate but was reduced in the prechordal plate (dash box, viii) and ventral forebrain (arrow, viii) tissues. E:T expression in IIA+/− and IIA−/− embryos. Scale bar = 0.1 mm.

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Loss of Shh Expression Is Accompanied by Early Head Defects

Consistent with the loss of SHH activity in the prechordal plate, E9.5 IIA−/− embryos displayed reduced head size and retarded development of the prosencephalon, particularly the optic evagination (data not shown). The embryonic forebrain showed diminished expression of Fgf8 in the anterior neural ridge (Fig. 5A,B; Shimamura and Rubenstein,1997), and Pax2 in the rostral forebrain (Fig 5C,D; Nornes et al.,1990). Pax6, another homeodomain transcription factor whose expression demarcating the dorsal forebrain, was expressed in both wild-type and IIA−/− mutants but domain of its expression was drastically reduced in the mutant (Supp. Fig. S3). Apparently normal expression of Fgf8 and Pax2 was found in the isthmus at the mid-hindbrain junction and the midbrain respectively, highlighting that the loss of IIA impacts mainly on the development of the forebrain. Six3 activity is required for forebrain differentiation and mutations of which have been implicated in holoprosencephaly pathogenesis (Geng et al.,2008; Geng and Oliver,2009). Six3 was expressed appropriately in the neural precursor tissues in E7.5 IIA−/− embryos (n = 3/3;Fig. 5E,F). However, Six3-expression domain in the head folds was reduced progressively at E8.0 (n = 4/5; Fig. 5G,H) to E8.25 (n = 4/4; Fig. 5I,J), raising the possibility that the maintenance of the forebrain precursor has been compromised (Oliver et al.,1995).

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Figure 5. Molecular defects of the forebrain of IIA−/− embryos. A–D: Reduction of Fgf8 expression in the anterior neural ridge (arrowhead, A,B) and Pax2 expression in the primordium of the optic evagination (arrowhead, C,D) of the forebrain in IIA−/− embryos (B,D) and wild-type +/+ (A,C) embryos. E–J:Six3 expression in the neural plate-stage (E,F), early headfold-stage (G,H), and early somite stage (I,J) embryos (E,G,I: wild-type +/+ embryos; F,H,J: IIA−/− embryos), showing progressive reduction in the expression domain (brackets) in the mutant embryos.

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IIA Functions Independently of BMPR1A-Mediated Signaling Activity

IIA is reputed to induce axis development of Xenopus embryos by binding to BMP ligand, thereby enhancing the inhibition of BMP activity by other antagonist such as CHRD (Zhu et al.,1999; Larrain et al.,2000). In the mouse embryo, IIA expression was initiated earlier than that of Chrd and Nog (Fig. 1C,D) and overlapped with that of Chrd and Nog only in the anterior region of AME by the early somite stage (Fig. 6; Yang and Klingensmith,2006). Collectively, these three factors may provide a complete complement AME-mediated BMP antagonistic activity. However, our results showed that, there is no change in the population of cells displaying nuclear localization of BMP-regulated phospho-SMAD1/5/8 in the AME and the neural plate tissues of the IIA−/− mutant (Supp. Fig. S4; Table 2) that might be indicative of an elevated level of BMP signaling that accompanies head development. There was also no discernible change in the expression of Msx2-lacZ (a read-out for BMP signaling activity; Brugger et al.,2004; Yang and Klingensmith,2006) in the anterior ectoderm of the E8.0–E8.5 IIA−/− embryos (Fig. 4B, and data not shown). To test if modulating the level of BMP signaling activity may influence the manifestation of the forebrain defects of IIA−/− embryo, a genetic interaction study was undertaken by analyzing the compound IIA;Bmpr1a mutant. It is anticipated that if IIA acts to antagonize BMP signaling activity, loss of IIA will lead to enhance signaling, which may be offset by minimizing the signaling activity by reducing BMPR1A function. Conversely, if IIA acts as an agonists, additional loss of BMP signaling activity will lead to head defects in the IIA+/−;Bmpr1a+/− embryos. Our results revealed no consistent changes in the head phenotype between the IIA+/−;Bmpr1a+/− and IIA+/− mutant or that of IIA−/−;Bmpr1a+/− and IIA−/− mutant (Table 1). Overall, the findings suggest that loss of IIA has no relationship with the BMPR1A-mediated signaling during head formation.

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Figure 6. Expression of IIA, Chrd, and Nog in the anterior tissues of two- to four-somite stage wild-type mouse embryos. IIA is strongly expressed in the prechordal plate (arrows) and the heart mesoderm (arrowhead) and in the anterior segment of the head process (asterisks) where its expression overlaps with that of Chrd and Nog. Expression of Chrd and Nog is absent from the prechordal plate (arrow).

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Table 2. Quantification of Phospho-SMAD 1/5/8 Cell Population Showing Positive Nuclear Immunostaining in Late Headfold to Early Somite (4s) Stage Embryosa
 IIA+/+ EmbryosIIA−/− Embryos
% Positive score/total cell count (n=5)% Positive score/total cell count (n=6)
AME+FPCRMNEAME+FPCRMNE
  • a

    AME+FP, anterior mesendoderm and floor plate; CRM, cranial mesenchyme; NE, neuroepithelium.

Mean14.772.404.5730.578.926.88
Standard deviation8.112.946.5517.439.495.52
IIA+/+ vs IIA−/− by Mann-Whitney testP>0.05P = 0.1P>0.1   

NODAL Activity May Modulate Gene Expression in the IIA-Null Forebrain

In view of the similarity of head phenotype of IIA−/− mutant and other mutants that are deficient in NODAL signals (Vincent et al.,2003; Chu et al.,2005; Andersson et al.,2006), we tested if reduction of NODAL activity might enhance the effect of loss of IIA on forebrain development. We examined the phenotype of compound IIA;Nodal mutant, in which the function of IIA and NODAL has been disrupted. IIA+/−Nodal+/lacZ newborns were viable and present at a similar frequency as IIA+/− newborns (data not shown). The frequency of head defects among the compound mutant is not different from that of the corresponding IIA mutants at E9.5 (IIA−/−Nodal+/lacZ: 13/14 vs. IIA−/−: 8/12, P > 0.05 and IIA+/−: 2/24 vs. IIA+/−Nodal+/lacZ: 3/28, P > 0.05 by χ2 test; Table 3). However, more IIA−/−Nodal+/lacZ mutant embryos showed loss of Shh expression (n = 5/6) in the ventral forebrain (Fig. 7) than IIA−/− embryos (1/5; Shh was expressed in 3 of 3 wild-type and 3 of 3 Nodal+/lacZ embryos). Based on the outcome of Shh expression, it seems that reducing NODAL activity in addition to the complete loss of IIA function has an impact on Shh expression and the morphogenesis of forebrain of some IIA−/−Nodal+/lacZ embryos (Fig. 7). However, the lack of a consistent enhancement of the mutant phenotype suggests that the primary action of IIA is not directed through NODAL signaling during forebrain development.

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Figure 7. Reduced domains of Shh expression in the ventral forebrain of IIA−/− embryos. Whole-mount in situ hybridization showing Shh expression in wild-type (+/+), IIA−/−, and IIA−/−;Nodal+/lacZ embryos. The expression domain is marked by brackets in the wild-type (+/+) and IIA−/− embryos and by arrowheads in the IIA−/−;Nodal+/lacZ embryos. Shh expression is drastically curtailed in the IIA−/−; Nodal+/lacZ embryo with severe head truncation (asterisk).

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Table 3. Genotype Distribution and Frequency of Head Defects of E9.5 IIA and IIA;Nodal Mutant Embryos
 Genotype distribution (11 litters, 100 embryos & 10 resorptions)
WTIIA+/−IIA−/−NodallacZ/+IIA+/−;NodallacZ/+IIA−/−;NodallacZ/+
No. embryos Reduced forebrain size and truncation102412122814
02 (8.3%)8 (66.6%)03 (10.7%)13 (92.8%)

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

Inductive signals from AME have been shown to be essential for the development of the cephalic neural tube. AME expresses signaling ligands such as SHH (Shimamura and Rubenstein,1997; Gunhaga et al.,2000) and BMP7 (Furuta et al.,1997), and modulators of signaling activity including CHRD, NOG, CERBERUS, and DKK1 (Biben et al.,1998; Belo et al.,2000; Anderson et al.,2002; Kemp et al.,2005) which act synergistically to regulate the temporal–spatial activity of signaling pathways for forebrain induction and patterning (Dale et al.,1997,1999; del Barco Barrantes et al.,2003). We have shown that deficiency of IIA in mice results in loss of expression of Shh in the prechordal plate and reduced Six3 and Pax2 expression in the forebrain, and phenocopies the malformation of the prosencephalon and mid-facial structures of the Shh-null mutant (Chiang et al.,1996). Although SHH activity is still maintained in the other segments of the AME, the morphogenesis of the forebrain is impaired in the IIA−/− mutant embryo.

Mouse embryos lacking the function of BMP antagonist, like those without IIA activity, also fail to develop forebrain structures (Bachiller et al.,2000). Our results, however, do not support the concept that IIA antagonizes BMPR1A-mediated signaling like other CR domain containing factors that inhibit BMP signaling in head formation. The IIA-deficient head phenotype is not affected by reducing the level of BMPR1A-mediated signaling activity (Davis et al.,2004) through the introduction of a loss of function Bmpr1a allele over IIA deficiency. However, the lack of genetic interaction between IIA and Bmpr1a and changes of pSMAD1/5/8 and Msx2-lacZ expression level in IIA mutant do not definitively rule out the possibility that IIA may antagonize (or facilitate) the signaling activity mediated by other BMP receptor subtypes or other TGFβ factors.

Our preliminary work has shown that the mouse IIA itself did not induce ectopic axis or supernumerary cement glands in the Xenopus embryo, although it may do so synergistically with the Xenopus CHRD homolog. However, the IIA activity is apparently not mediated by repression of BMP signaling as determined by the activity of a BMP-responsive luciferase reporter (XVent2-Luc) driven by the promoter of a direct BMP target XVent2 (Candia et al.,1997). Regulation of the axis inductive activity by IIA may involve the CR domain, because co-expression of IIB with Chrd has no inductive effect (Leung et al., unpublished observations). In this regard, the mouse IIA protein may have different functions in BMP signaling from its Xenopus counterpart (Larrain et al.,2000), a possibility highlighted by the divergent functional properties of other modulators of TGFβ signaling, e.g. CERBERUS and CERBERUS-LIKE factors (Belo et al.,2000).

In the zebrafish, NODAL-related protein activity in the prechordal mesoderm is implicated in direct regulation of Shh expression in the ventral forebrain (Sampath et al.,1998; Feldman et al.,1998; Gritsman et al.,1999). Although our results of the compound mutant study indicate that IIA and NODAL do not interact genetically in head development, there is striking resemblance of the pattern of gene expression in the forebrain of Smad2CA/CA (the epiblast conditional mutant of Smad2), Nodalmath image (a hypomorphic Nodal mutant; Vincent et al.,2003) and IIA−/− embryos. It is conceivable that NODAL-related factors may act with IIA to influence the patterning of the forebrain in the mouse as in other species (Muller et al.,2000). Signaling intensity of TGFβ family members GDF1 and GDF3 are modulated by binding partners such as LEFTY (Chen et al.,2006; Shen,2007) and GDF1/3 are implicated in a NODAL-like CRIPTO/ALK4/SMAD2 pathway during mouse anterior development (Wall et al.,2000; Andersson et al.,2006,2007). An important issue for future study is to determine if IIA can interact with the GDFs to elicit NODAL-like activity in the mesendoderm and the ventral forebrain during head formation (Constam and Robertson,2000; Wall et al.,2000; Chu et al.,2005; Andersson et al.,2006).

Holoprosencephaly (HPE; Cohen,2006; Geng and Oliver,2009) in humans is characterized by the presence of a single prosencephalic cavity or an incomplete fission of the cerebral hemispheres with remnants of interhemispheric fissure. Genetic investigations of HPE reveal that only approximately 15–20% of the cases have identifiable genetic causes. HPE disease genes have been identified as sequence variants of genes such as SHH, ZIC2, SIX3, TGIF, PTCH, GLI2, FOXH1, and TDGF1. Many of these genes encode either the ligands or regulators of SHH and NODAL pathways. Multi-hit model for HPE pathogenesis indicates that accumulated genetic alterations in NODAL pathway components may predispose to development of HPE (Roessler et al.,2009). Recently, combinations of HPE and congenital heart defects are shown to associate with mutations of components of NODAL pathway such as NODAL, CFC1, SMAD2, and FOXH1 (Roessler et al.,2008,2009). Mutations in the COL2A1 gene are associated with type II collagenopathies (Freisinger et al.,1996), which include a variable degree of midface hypoplasia (Snead and Yates,1999), one of the features of HPE. These observations raise the possibility that mutations of IIA might be an enhancing aetiological factor in NODAL- and SHH-related HPE.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

Mouse Lines

A targeted disruption of the exon 2 of the Col2a1 gene was generated by insertion of a neomycin (neo) cassette flanked by loxP sites to produce a IIAneo mutant allele (Supp. Fig. S2). The targeting vector contained a 1.2 kb 5′ homology arm corresponding to the intron 1 of the Col2a1 gene and a 4.2 kb 3′ homology arm encompassing the intron 2 to the exon 15 sequence. Correctly targeted 129Sv/J embryonic stem (ES) cell clones were identified by an external probe that distinguishes the endogenous (6.8 kb) and the targeted (2.1 kb) alleles on BamHI-digested genomic DNA by southern blotting (Supp. Fig. S2B). The IIAneo allele only codes for Col2a1 mRNA without exon 2 (i.e., IIB transcript). The neo gene was subsequently excised by Cre-mediated recombination by crossing heterozygous IIA+/neo mice to β-actin-Cre mice to generate the IIA+/− mice. IIA+/− mutant mice were crossed to C57BL/6 mice, and the offspring were inter-crossed to generate homozygous IIA−/− mutants.

For the genetic interaction study, IIA+/− mice were first crossed to Bmpr1a+/− (Mishina et al.,1995) mice maintained on a C57BL/6 background to produce IIA+/−;Bmpr1a+/− mice. IIA+/−;Bmpr1a+/− mice were then backcrossed with IIA+/− mice to generate compound IIA;Bmpr1a mutant embryos. A similar mating strategy was used for crossing Nodal+/lacZ (Collignon et al.,1996) with IIA+/− mice to generate compound IIA; NodallacZ mutant embryos.

Transgenic mice were generated by pronuclear DNA injection (Hogan et al.,1986) of the Δ4Msx2-hsp68-LacZ construct (gift of R. Maxson) into the C57/BL6 F1 oocytes. Two transgenic lines [Tg(Msx2-LacZ)13Kc and Tg(Msx2-LacZ)16Kc] were characterized and found to recapitulate the Msx2 expression pattern at E9.5–E12.5 (Kwang et al.,2002). Both lines were crossed with IIA+/− mice to produce IIA;Tg(Msx2-lacZ) embryos for assessing the expression of the Msx2 reporter as a readout for BMP signaling activity.

Analysis of Mutant Phenotype

Genotyping.

Genotyping was performed on yolk sac DNAs of embryos harvested between E7.5 and E18.5 by PCR using a common primer OYYK03 (5′-TCATCCTT TCAACTCCCAGA-3′) coupling with Col2-intron1 primer (5′-CCACCA TTC CCTAGCATTTG-3′) to detect the mutant allele or 5′exon2 primer (5′-TGT ATGGAAGCCCTCATCTTG-3′) to detect the wild-type alleles.

β-Galactosidase and cell death assays.

E8.0–E8.5 wild-type and mutant embryos were fixed briefly in 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS) and stained in the dark at 37°C with X-gal reagents prepared essentially as described (Yee and Rigby,1993). To assay cell death, X-gal stained embryos were post-fixed and permeabilized for 10 min in PBS buffer containing 0.1% Triton X-100, 0.1% Tween-20, and 0.1% sodium citrate. Embryos were incubated in the TUNEL reaction mixture prepared according to the manufacturer's instructions (In Situ Cell Death Detection Kit, Fluorescein, Roche) for 1 hr at 37°C in the dark. Fluorescent images on embryos were taken using Leica MZ FLIII fluorescence Stereomicroscope and SONY DXC-S500 color digital camera.

Skeletal Staining.

Newborn wild-type and IIA mutant mice were eviscerated, fixed in 100% ethanol and transferred to acetone solution. Specimens were rinsed with water and stained in Alcian blue/Alizarin red solution (McLeod,1980) for 10 days before clearing and storing in 100% glycerol for photography.

Immunohistochemistry.

Embryos were fixed in 4% PFA overnight at 4°C. For conventional histology, paraffin-embedded embryos were sectioned and counterstained with hematoxylin and eosin. For section immunohistochemistry (IHC), embryos were sectioned at 6 μm. Rabbit affinity purified IIA antibody raised against the peptide, PICPADLATASGRKL, of the IIA CR domain (Covalabs, Lyon, France) was used at 1:400 dilution to detect IIA protein in sections of E8.0–E10.5 embryos pretreated with 0.8% hyaluronidase. Phospho-Histone H3 (Ser10) antibody (#9701, Cell Signaling Technology) was used to detect proliferating cells in sections of E9.5 embryos. For whole-mount IHC, embryos were dehydrated and rehydrated in methanol series. H2O2-bleached embryos were immersed in acetone for 7 min at −20°C before incubating overnight at 4°C with phospho-Smad1/5/8 (1:200) antibody (Persson et al.,1998). Embryos were incubated with 1:200 goat anti-rabbit IgG-horseradish peroxidase secondary antibody (DAKO) and processed for color detection using DAB solution (DAKO).

WISH.

WISH was carried out with modifications on the standard protocol (Wilkinson,1992) using digoxigenin (DIG)-labeled riboprobes specific for transcripts of IIA (Ng et al.,1993), Col2a1 (Ng et al.,1993), Shh (Echelard et al.,1993), Foxa2 (Ang and Rossant,1994), Brachyury (Wilkinson et al.,1990), Dkk1 (Mukhopadhyay et al.,2001), Sox17 (Tam et al.,2004), Six3 (Oliver et al.,1995), Pax2 (Dressler et al.,1990), and Fgf8 (Crossley and Martin,1995). Anti-DIG-AP antibody and BM Purple (Roche) were used for signal detection.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

We thank Richard Behringer for valuable advice on the design of the Bmpr1a mutant experiment and for the Bmpr1a mutant mice, Liz Robertson for the Nodal mutant mice, Peter ten Dijke for pSMAD1/5/8 antibody, James Lau for pronuclear injection of the Msx2-LacZ construct for transgenesis, and Paul L.F. Tang for technical assistance in mouse husbandry. P.P.L.T. is a Senior Principal Research Fellow of the NHMRC of Australia. K.S.E.C. was funded by the Arthritis & Rheumatism Campaign (UK), and the Research Grants Council and University Grants Council of Hong Kong SAR and P.P.L.T. was funded by NHMRC of Australia.

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  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
DVDY_22366_sm_suppfig1.tif595KSupporting Figure 1.
DVDY_22366_sm_suppfig2.tif1598KSupporting Figure 2.
DVDY_22366_sm_suppfig3.tif391KSupporting Figure 3.
DVDY_22366_sm_suppfig4.tif3979KSupporting Figure 4.

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