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doi: 10.1242/10.1242/dev.00423

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A novel role for retinoids in patterning the avian forebrain during presomite stages

¹ MRC Centre for Developmental Neurobiology, King's College London, Guy's Campus, London SE1 1UL, UK
² Department of Food Science and Human Nutrition, Michigan State University, East Lansing, MI 48824, USA
³ TIGEM, Telethon Institute of Genetics and Medicine, Naples 80131, Italy

View larger version (84K): [in a new window]   Fig. 1. Retinoids are synthesized in tissues involved in early anterior patterning. (A-G') Dorsal views and sections of stages 3-7+ whole-mount chick embryos hybridized with the markers indicated laterally. (A) In anterior regions, Raldh2 is expressed in the hypoblast (hy), and it remains confined (B,C) to the lower layer in anterior endoderm (ae in mid-sagittal section in inset). Asterisk in C indicates anterior endoderm. (D) At head process stages, anterior Raldh2 expression is mainly localized in prechordal mesendoderm (pme), as seen in transverse sections (D'). Lines (D) indicate the position of the transverse sections (D',D''). (E) At two-somite stages, Raldh2 is maintained in prechordal mesoderm and foregut endoderm with a sharp posterior boundary at the level of the notochord (blue arrow in mid-sagittal section in E', dorsal is left). (F,F') Expression of Raldh3 in the node (no) and in ingressing mesodermal cells (red arrowhead). Strong expression is also visible in the marginal zone surrounding the embryo. (G,G') Stage 4+ embryos hybridized with RXRg have a restricted expression in mesodermal cells in the node region (G', red arrowhead). Red arrows indicate the position of the node. Scale bar in A: 260 µm in A; 400 µm in B,C,D,F,G; 220 µm in E; 200 µm in D'-D''',E',F',G'; 270 µm in inset in C.

View larger version (138K): [in a new window]   Fig. 3. Altered specification of midline properties in the absence of retinoids. Dorsal views and transverse sections of normal and VAD whole-mount quail embryos hybridized with the markers indicated. (A-B') Compared with normal embryos where Raldh2 expression is localized in prechordal mesendoderm (pme) and at lower levels in anterior endoderm (ae), VAD embryos have patches of ectopic expression along the whole length of anterior axial mesoderm (B, arrowheads in inset and in B'). (C-D') Ectopic Gsc expression is detected in presumptive notochord (no) cells (arrowhead in D'). (E-F'') Axial mesodermal Crs expression is found more posteriorly in VAD embryos than in normal embryos (compare arrows in E,F). Transverse sections shown (E'-F'') indicate absence of Crs expression in anterior (en) (arrowhead in F') and lateral embryonic endoderm (le) (arrows in F'') of VAD embryos. (G-H') Similar to the previous markers, Bmp2 expression in prechordal mesoderm is posteriorly expanded in VAD embryos (arrow in H). In addition, ectopic expression is detected in ventral neuroectodermal cells (asterisk in H'). (I-J') The notochord marker chordin shows a posteriorly displaced anterior expression boundary in VAD embryos when compared with normal embryos (arrow in inset in J). Transverse sections at the level of presumptive notochord indicate diminished expression of chordin in VAD embryos (arrowhead in J'). (K-L') The axial mesodermal marker Shh is expressed at lower levels in the most anterior region (arrow in inset in L and arrowhead in L'). Coloured lines designate the AP position of the transverse sections shown laterally. Arrowheads in C,D,I,J indicate the position of the somites. Note that the embryo in H is slightly older than the embryo in G. Scale bar in A: 100 µm in A,B,E,F,G,H; 125 µm in C,D; 150 µm in I-L; 50 µm in A'-L'.

View larger version (115K): [in a new window]   Fig. 2. Endodermal properties are altered in VAD embryos. Anterior dorsal views and transverse sections of normal and VAD whole-mount quail embryos hybridized with the markers indicated. (A-D'') Anterior expression of Bmp2 and Bmp7 in stage 4/5 normal and VAD embryos. The panels show only expression anterior to the node (asterisks). Lines (A,C,E) indicate the position of the transverse sections shown at hypoblast (hy) (A',C',E') and anterior definitive endoderm (en) (A'',C'',E'') levels. Note that at stage 4/5 Bmp2 and Bmp7 expression is detected at higher levels in hypoblast cells of VAD embryos (asterisks in B,B',D,D') than of normal embryos (A,A',C,C'). Furthermore, VAD embryos have an expanded expression domain of Bmp2 and to a lesser extent of Bmp7, in anterior definitive endoderm (arrowheads in B,B'',D,D''). (E-F'') Anterior endodermal expression of Hex in stage 5 normal and VAD embryos. Note the dramatic downregulation of Hex expression in anterior endoderm (arrow in F), in particular in definitive endoderm (F''), whereas expression in hypoblast is only partially reduced (F'). Scale bar in A: 100 µm in A-F; 40 µm in A'-F''.

View larger version (47K): [in a new window]   Fig. 4. Posterior expansion of prospective telencephalic tissue. Dorsal views and sections of normal and VAD whole-mount quail embryos hybridized with the markers indicated. (A,B) The general fore- and midbrain marker Otx2 shows an expanded anterior expression in VAD embryos (see arrows), whereas (C,D) the Pax6-negative region indicates that the affected expanded territory corresponds to the future telencephalon (compare AP length of region marked with red lines in C,D). Note that the Pax-6-positive domain is not changed between normal and VAD embryos. (E-H) The markers Nkx2.1 and Bf1 confirm that the expansion of anterior territories corresponds to the presumptive ventral telencephalon, which is maintained until stage 11+ (arrow in F and red lines in G,H). Note that only the posterior Nkx2.1 expression border is shifted (arrowheads in mid-sagittal sections E',F'), whereas the anterior boundary has not changed. In VAD embryos, ectopic expression of Nkx2.1 is also detected in the ventral foregut (asterisk in F'). Arrows in insets in G,H indicate a caudal shift of Bf1 expression in horizontal section at ventral levels. The asterisks (A,B) indicate the position of the node. Note that the embryos in B and F are slightly older than the embryos in A and E. s, somite; t, telencephalon; di, diencephalon; mes, mesencephalon; e, eye. Scale bars: 100 µm in A,B; 200 µm in C-H.

View larger version (51K): [in a new window]   Fig. 5. VAD embryos show increased cell death in ventral neuroectoderm and head mesenchyme. Apoptotic cells in sections of normal (A-E) and VAD (F-J) quail embryos detected using the TUNEL assay (A-C,F-H), and neural crest cells detected using an HNK1 antibody (D,E,I,J). (A,F,D,I) Mid-sagittal and para-sagittal sections, (B,G,E,J; insets in A,F) frontal and (C,H) horizontal sections. Stage 10 VAD embryos show an increased rate of apoptotic cells in ventral neuroectoderm (arrow in F and arrowheads in inset) and foregut endoderm (asterisk in F and arrow in inset), but no change in the distribution of neural crest cells (D,I). VAD embryos (arrow in I) have an apparent difference of HNK1-positive cells because the section is more lateral. (B,G) In stage 13/14 VAD embryos, ventral neuroectodermal (arrows in G) and mesenchymal cells around the eye regions (arrowheads in G) show a dramatic increase of programmed cell death. (C,H,E,J) At stage 19/20 the dramatic increase in the rate of dying cells is still detected in neuroectodermal (arrows in H) and head mesenchymal tissues (arrowheads in H). Moreover, the absence of HNK1-positive cells in ventral regions (arrow in J) indicates a lack of neural crest-derived cells. Lines in A and F designate the level of the frontal section showed in the insets. Scale bars: 100 µm in A,F,D,I; 200 µm in B,C,E,G,H,J.

View larger version (100K): [in a new window]   Fig. 6. Abnormal forebrain morphogenesis in the absence of retinoids. Lateral (A,B) and frontal (C,D,E) views of normal (A,C) and severely affected VAD (B,D,E) whole-mount stage 20 embryos. VAD embryos develop a smaller and single telencephalic vesicle circled in (D,E), which can be located laterally (D) or centrally (E) instead of two telencephalic (t) vesicles (A,C). Note (B) the ectopic morphological borders within the diencephalon (black arrowheads) and pronounced constrictions (red arrows) between vesicles. (F,G) Frontal sections of normal (F) and VAD (G) quail embryos at the level of the telencephalon/nasal pit. Note the lack of a rostral midline (rm) and the presence of a thick neuroectoderm in the single anterior vesicle of VAD embryos (arrow in G). e, eye; n, nasal pit; ov, optic vesicle; h, heart. Scale bars: 200 µm (bar in D also applies to E).

View larger version (17K): [in a new window]   Fig. 7. RA action during presomite stages in anterior regions. (A) At early gastrula stages, RA signals from the anterior endoderm (ae) and node (n) to specify axial mesodermal cells emerging from the node (arrows 1 and 2). (B) During the stabilization step RA signals from the prechordal mesendoderm (pme) to restrict the extent of posterior pme (arrow 3); during this stage other signalling pathways pattern the overlying forebrain primordium (red arrows). (C) In the absence of RA, pme is extended posteriorly (yellow arrow) and altered signals are transferred to the overlying neuroectoderm (red dashed arrows). ae, anterior endoderm; pme, prechordal mesendoderm; a-me, axial mesoderm; pame, paraxial mesoderm; anp, anterior neural plate; pop, posterior neural plate; n, node.