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Chordin and noggin promote organizing centers of forebrain development in the mouse

¹ Department of Cell Biology, Duke University Medical Center, Durham, NC 27710-3709, USA
² Victor Goodhill Ear Center, Head and Neck Surgery Division, University of California, Los Angeles, CA 90095-1794, USA

View larger version (51K): [in a new window]   Fig. 1. Spectrum of head defects in Chrd-/-;Nog+/- neonates. External anatomy of (A) wild type, (B) single nostril (arrow) with narrow proboscis, (C) anterior truncation, (D) agnathia (arrow) with two distinct nostrils, (E) mid-face hypoplasia with loss of philtrum (arrow) and (F) bilateral cleft lip. Skeletal preparations of animals are shown of A-C: (A') wild type, (B') cebocephaly with cyclopic eye (arrow) beneath proboscis and (C') anterior truncation. Skeletal structures caudal to the head are normal. Scale bar: 2 mm.

View larger version (85K): [in a new window]   Fig. 4. Loss of rostral SHH and FGF8 expression and signaling. (A,B,E,F,K,O) Lateral aspect, (C,D,G,H) ventral aspect and (I,J,M,N) frontal aspect of cephalic region. Wild-type expression of Shh (A,B), Ptch1 (C), Nkx2.1 (D). (E) E8.5 mutant (approx. eight to 10 somites) lacking Shh in RVNM and PrCP (asterisks; n=2). (F) E9.5 (20-25 somites) mutant lacking expression in RVNM (asterisk; n=5). Note loss of Shh expression in first branchial arch (lower asterisk, ba1 in B). (G) Midline mutant lacking rostral Ptch1 expression (asterisk; n=4). (H) Midline mutant lacking rostral Nkx2.1 expression (asterisk; n=2). Wild-type expression of Fgf8 (I,J) and Foxg1 (formerly known as BF-1; K). (M) E8.25 (approx. five somites) mutant with diminished expression of Fgf8 in the ANR which is confined to the midline (asterisk; n=2). (N) E9.5 mutant with diminished Fgf8 expression in the commissural plate and olfactory placodes (asterisk; n=4). (O) Reduced Foxg1 expression (asterisk) in E9.0 (17 somites) mutant. (L,P) Midsagittal sections of E11.5 embryos. (L) In wild type, infundibulum (inf) and Rathke's pouch (rp) interact to form mature pituitary. (P) In midline mutant, infundibulum does not develop (asterisk) and Rathke's pouch is rudimentary. Note cyclopic eye (ey). anr, anterior neural ridge; prcp, prechordal plate; rvnm, rostral ventral neural midline; cp, commissural plate; op, olfactory placodes; fnm, frontonasal mass; md, mandible; ov, optic vesicle. Scale bar: 0.2 mm.

View larger version (80K): [in a new window]   Fig. 2. Midline and rostral neural defects. (A-C) Neonate transverse sections. (A) Wild type showing anterior commissure (ac) and bilateral cerebral hemispheres (ch). (B) Midline mutant lacking anterior commissure (asterisk) and showing cerebral hemispheres fused into a holosphere (hs). (C) Truncation mutant lacking CNS rostral to hindbrain. (D,E) E13.5 transverse sections. (D) Wild type showing medial falx cerebri (fc). (E) Midline mutant shows a single rostral ventricle (asterisk). (F,G) E9.5 embryos. (F) Wild-type embryo shows lamina terminalis (lt) marked by Vax1 expression. (G) Midline mutant lacks lamina terminalis (asterisk). (H-J) Neonatal brains. (H) Wild type showing olfactory bulbs (ob). (I,J) Mild truncation mutant showing hypoplastic, fused olfactory bulbs (I) or lacking olfactory bulbs (J). (K,L) E10.5 embryos. (K) Wild type showing telencephalic vesicles (tv). (L) Midline mutant has single holospheric vesicle (hs). (M) Moderate truncation mutant lacks telencephalon (asterisk). (N) Severe truncation mutant lacks both forebrain and midbrain. mo, medulla oblongata; ov, optic vesicle; p, pons; cbl, cerebellum; m/h, midbrain/hindbrain boundary. Scale bars: 2 mm in A-C; 0.5 mm in D,E; 0.2 mm in F,G; 1 mm in H-N.

View larger version (84K): [in a new window]   Fig. 3. Wild-type expression of Chrd, Nog and Bmps. (A-H) Frontal aspect of headfold stage (E7.75) embryos. Bracketed area represents approximate extent of Gsc expression, a prechordal plate (PrCP) marker. (A) Chrd is not expressed in the rostral-most PrCP (asterisk). (B) Shh and (C) Foxa2 are expressed in all midline mesendoderm, including rostral PrCP (double arrows). (D) Gsc is expressed throughout the PrCP. (E) Nog is not expressed in the rostral PrCP (asterisk), but is expressed in the ANR (arrows). (F-H) BMPs antagonized by CHRD and NOG are expressed in surface ectoderm (se), rostral paraxial mesoderm (arrowheads), node (n) and midline mesendoderm (mse). Bmp7 is not expressed in the rostral PrCP (H, asterisk). (I-K) Lateral aspect of bisected three-somite stage embryos. Neither Chrd (I) nor Nog (J) is detected in rostral PrCP at this stage by in situ hybridization, although Shh (K) is present (arrows). Chrd (L) and Nog (M) are both expressed throughout the rostral axial mesendoderm by the five-somite stage (arrows) and in head mesenchyme (arrowheads). Scale bar: 0.2 mm. (N) RT-PCR analysis of Chrd and Nog expression. Chrd and Nog are detected in the midline and trunk after 30 cycles of PCR (lanes 1,3). Nog is evident in the ANR (lane 4) after 30 cycles, and Chrd after additional amplification.

View larger version (84K): [in a new window]   Fig. 5. Analysis of apoptosis and cell proliferation in Chrd-/-;Nog+/- mutants. (A-D) TUNEL/proliferation assay on sections of wild-type (A,C) and Chrd-/-;Nog+/- mutant (B,D) embryos. E8.5 Chrd-/-;Nog+/- mutant (B) shows a 36% decrease in proliferation (labeled in red, P=0.018), while differences in apoptosis (labeled in green) were not observed in section. E9.5 mutant (D) shows a nearly 10-fold increase in apoptotic cells (947%; P=0.019) in the neural ectoderm rostral to the optic vesicles (ov) Increased apoptosis is also seen in rostral mesenchyme (mc) and trigeminal ganglion (V). Proliferation at E9.5 is not significantly different from wild type. Similar results were seen in three or more independent experiments. (E-H). Nile Blue Sulfate staining of wild-type (E,G) and Chrd-/-;Nog+/- mutant (F,H) embryos. (F) Lateral aspect of eight- to 10-somite mutant showing expansion of apoptosis in dorsal domains (arrowheads), and loss from the ventral midline (arrow; n=3). (H) Frontal aspect of 20-25 somite mutant showing expanded apoptosis in the dorsal midline (arrows) and frontonasal mass (n=5). (I-L) Rostral expression of Msx1 in wild-type (I,K) and Chrd-/-;Nog+/- mutants (J,L). (J) In severely affected mutants, rostral domains of Msx1 are expanded at eight to 10 somites (n=2). At E9.5 Chrd-/-;Nog+/- expression domains have shifted toward the midline (L, arrow) though expression levels are comparable with those in wild type (n=3). Scale bars: 0.2 mm.

View larger version (101K): [in a new window]   Fig. 6. Ectopic application of BMP2 to forebrain explants. (A-D) Expression of Msx1 in cultured cephalic explants. Protein-soaked beads (represented by asterisks) were placed between rostral neural folds of five- to seven-somite wild-type cephalic explants in type 1 explants (A-C) and on the midline in type 2 explants (D). (A) BMP2 induces strong Msx1 expression in the ANR (arrow) after 6 hours. Msx1 is induced at large distances from the BMP2 source. (B,C) Msx1 induction is transient and is diminished after 9 (B) and 12 (C) hours. (D) BMP2 induces Msx1 adjacent to the caudal but not the rostral half of the bead (arrowhead). However, Msx1 is induced in the ANR (arrow). (E,F) Expression of Fgf8 and Shh (two-probe in situ hybridization) in type 1 (E) and type 2 (F) explants. (E) BMP2 suppresses the expression of Fgf8 in the ANR (arrow) and Shh in the rostral ventral neural midline (arrowhead) in both explant types, whereas Fgf8 in the isthmic organizer (io) is not affected. BSA beads had no effect in any explants. (G,H) Hypersensitivity of Chrd;Nog mutant explants to BMP signaling. BMP2-soaked beads were placed adjacent to both rostral and lateral neural folds in type 1+ explants. (G) At a concentration of 10 µg/ml, Msx1 was induced adjacent to both beads in wild-type explants (left), but induction was more robust in Chrd-/-;Nog+/- explants (right). (H) At a concentration of 0.1 µg/ml, Msx1 was not induced in wild-type explants (left), but was induced in Chrd-/-;Nog+/- explants (right). anr, anterior neural ridge; rvnm, rostral ventral neural midline.

View larger version (44K): [in a new window]   Fig. 7. Regulation of BMP activity in rostral neural patterning. (A) Schematic representation of headfold/early somite stage neural plate with surrounding non-neural ectoderm and adjacent axial mesendoderm (after Rubenstein and Beachy, 1998). (B) In wild type, Chrd and Nog (blue crosses) are co-expressed with Fgf8 (yellow) in the anterior neural ridge (ANR). Here, CHRD and NOG antagonize (represented by blunted blue arrow) the activity of BMPs (red arrows) secreted from the non-neural ectoderm abutting the neural plate. This promotes expression of Fgf8, and its function (yellow arrow). Chrd and Nog are also co-expressed with Shh (green) in the caudal prechordal plate (PrCP) and notochord. Here, CHRD and NOG antagonize BMPs expressed in axial mesendoderm and adjacent paraxial mesoderm, permitting SHH signaling from the rostral PrCP (green arrows). (C) In affected Chrd-/-;Nog+/- mutants, BMP antagonist expression is reduced in the ANR and the PrCP (fewer blue crosses), and thus, BMP antagonism is reduced (smaller blunted arrows). In turn, expression of Fgf8 is repressed by BMPs in the ANR, reducing its activity (smaller yellow arrow) in adjacent tissues. Similarly, BMPs repress expression of Shh in the rostral PrCP, reducing its activity in surrounding tissues (smaller green arrows). In combination, these defects result in diminished growth and incorrect patterning of the rostral neural plate.