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Local retinoid signaling coordinates forebrain and facial morphogenesis by maintaining FGF8 and SHH

Richard A. Schneider^1,*, Diane Hu^1,*, John L. R. Rubenstein², Malcolm Maden³ and Jill A. Helms^1,

¹ Department of Orthopaedic Surgery, 533 Parnassus Avenue, Suite U-453, University of California at San Francisco, San Francisco, CA 94143-0514, USA
² Department of Psychiatry, Nina Ireland Lab of Developmental Neurobiology, University of California at San Francisco, San Francisco, CA 94143, USA
³ MRC Centre for Developmental Neurobiology, King’s College London, Guy’s Campus, London Bridge, London SE1 9RT, UK
^* These authors contributed equally to this work

View larger version (65K): [in a new window]   Fig. 1. Retinoid signaling is required for forebrain and FNP morphogenesis. (A) ALDH6, which synthesizes RA, is detected in ventral ectoderm (ec; arrow) of the presumptive FNP adjacent to the forebrain (f) of chick embryos at stage 10 (sagittal section, rostral towards right, dorsal on top). (B) RXR is expressed in neural crest cells (arrows) that migrate out of the rostral neural tube of stage 10 embryos (dorsal view). (C) RARß is expressed in neural crest cells (arrows) that migrate between forebrain (f) neuroepithelium and overlying ectoderm (ec) of stage 10 embryos (sagittal section, rostral towards right, dorsal on top). Relative to cell density (blue nuclear stain), the neural crest shows higher expression levels (white dots) when compared with the neuroepithelium. (D) Ion exchange beads (asterisks) were soaked in RAR and RXR antagonists (100 µg/ml) and placed along the rostral margins of the forebrain (f) of stage 10 embryos. Midbrain (m), hindbrain (h). (E) DMSO control; (F) RAR/RXR antagonist-treated embryos at stage 36 showing the effects of disrupting retinoid signaling. Antagonist-treated embryos lack forebrain tissues, fail to form an FNP (fn), and have fused eyes (n=18, i.e. number of embryos examined with this phenotype). These results are further illustrated by sagittal histological sections of control embryos (G) and treated embryos (H). In treated embryos, the maxillary (mx) and mandibular (ma) processes are unaffected whereas the FNP (asterisk) and forebrain are absent. Eye (e), ear (ot), midbrain (m) and hindbrain (h). Two additional controls confirm that these defects are due to disruptions in retinoid signaling specifically. First, we treated embryos at stage 10 with beads soaked citral (I), which is an inhibitor of RA biosynthesis. These embryos also lack forebrain tissues and an FNP (asterisk) while the maxillary (mx) and mandibular (ma) processes are unaffected (n=22). Second, we used beads soaked concomitantly in all-trans RA and RAR/RXR antagonists (K). (J) Control embryos treated with RA alone have severe forebrain and FNP hypoplasia (asterisk; n=7), whereas embryos exposed to RA and RAR/RXR antagonists simultaneously (K) are relatively normal with a slightly shortened FNP (n=10). Scale bars: 100 µm in A; 200µm in B,D; 30 µm in C; 1 mm in E-H; 3 mm in I; 2 mm in J,K.

View larger version (61K): [in a new window]   Fig. 2. Neural crest cells migrate into the presumptive FNP despite exposure to RAR/RXR antagonists. (A) Quail donor neural crest cells from the forebrain (f) and midbrain (m), when transplanted orthotopically to chick host embryos between stage 9 and stage 10–, give rise to components of the FNP (fn), as shown schematically (B) in a lateral view of the avian head skeleton (based on similar drawings by Noden, 1987). (C) In sagittal sections of chimeric embryos at stage 36, quail donor neural crest cells are found throughout chick host FNP-derived tissues, but not in the maxillary (mx) or mandibular (ma) processes (n=6). In an area of nasal capsule cartilage (boxed, shown at higher magnification in D), quail neural crest-derived cells appear black and are completely integrated into the host structures. Cartilage of mixed chick host and quail donor origin can be observed (arrows). Quail donor neural crest cells were transplanted into chick host embryos between stage 9 and stage 10–, and then exposed 2-4 hours later at stage 10 to DMSO control beads (E, asterisk) or RAR/RXR antagonist beads (F, asterisk). After 24 hours, transplanted cells are integrated into the neuroepithelium above the lumen of the forebrain (f) and also have migrated into the FNP (fn; arrows) as shown in sagittal sections (n=7). Embryos were also exposed to RAR/RXR antagonists at stage 10, and immediately thereafter neural crest cells from the forebrain (f) and midbrain (m) were labeled with DiI. Twenty-four hours later, we observe DiI labeled cells in the presumptive FNP of control (G) and treated (H) embryos, which demonstrates that the neural crest still migrates into the region despite treatment with RAR/RXR antagonists (n=7). Scale bars: 1 mm in C; 100 µm in D; 200 µm in E-H.

View larger version (75K): [in a new window]   Fig. 3. Inhibition of retinoid signaling with RAR/RXR antagonists causes a loss of FGF8 and SHH in the forebrain and FNP. Whole-mount in situ hybridization 48 hours after bead implantation at stage 10 demonstrates that control embryos (A) express FGF8 in the rostromedial forebrain, which appears as a midline horseshoe-shape (arrow), and in the ectoderm covering the FNP (fn), as well as in the maxillary (mx) and mandibular (ma) processes. (C) Treated embryos lose FGF8 in the forebrain and FNP ectoderm (asterisk; n=32). Note normal expression of FGF8 in the maxillary and mandibular processes. (B) Control embryos express SHH in the ectoderm of the FNP (fn; arrow) and forebrain (f), whereas treated embryos (D) show a downregulation of SHH (asterisk; n=35). Even at this early stage, the facial midline of treated embryos is hypoplastic, causing the eyes (e) to approximate one another. The lack of a forebrain is evidenced by a depression in the upper face. (E,F) Embryos treated with citral, which is an inhibitor of RA biosynthesis, lack FGF8 in the forebrain and ectoderm covering the FNP (E, asterisk; n=4) and SHH in the ectoderm of the FNP (F, asterisk) and forebrain (n=5). This result demonstrates that inhibiting retinoid signaling either at the point of RA biosynthesis or at the level of the receptors has similar downstream molecular and morphological consequences in the forebrain and FNP. Scale bars: 200 µm.

View larger version (54K): [in a new window]   Fig. 4. Retinoid signaling is required for maintenance of FGF8 and SHH in the forebrain and FNP ectoderm. In situ hybridization of midline sagittal sections 4 hours after bead implantation at stage 10 show that control embryos (A) express FGF8 (red) in the rostrodorsal forebrain (f) and ectoderm of the presumptive FNP (arrows). (E) SHH (yellow) is strongly expressed in the ventral forebrain (f) and notochord (n). (B) Exposure to RAR/RXR antagonists results in a downregulation of FGF8 in the forebrain (asterisk), but not in the isthmus (i), where expression is normal (n=15). (F) At this early stage, SHH expression does not appear to be downregulated (n=15). The change in FGF8 expression precedes any evidence of an increase in programmed cell death. Four hours after bead implantation at stage 10, the amount of programmed cell death (bright green) is equivalent in control (I) and RAR/RXR antagonist-treated (J) embryos (arrows), as determined with a TUNEL assay (n=10). Sections 6 hours after bead implantation at stage 10 show that control embryos continue to express FGF8 (C) and SHH (G) in the ventral forebrain (arrow). (D) RAR/RXR antagonist-treated embryos lack FGF8 in the forebrain and FNP ectoderm (asterisk; n=15). (H) SHH is downregulated along the ventral forebrain (asterisk; n=15). Six hours after bead implantation at stage 10, the amount of programmed cell death is nominally increased in the mesenchyme (asterisk) of RAR/RXR antagonist-treated embryos (L, n=7), compared with that found in controls (K, n=6). Other labeled structures are the hindbrain (h) and midbrain (m). Scale bars: 200 µm.

View larger version (93K): [in a new window]   Fig. 5. A loss of retinoid signaling causes a downregulation of FGF8 and SHH in the forebrain and FNP ectoderm, as well as an increase in programmed cell death and a decrease in cell proliferation in the FNP mesenchyme. In situ hybridization of midline sagittal sections 12 hours after bead implantation at stage 10 show that control embryos (A) express FGF8 (red) in the forebrain (f), FNP ectoderm and isthmus (i); (E) SHH (yellow) is strongly expressed in the ventral forebrain (f). (B) RAR/RXR antagonist-treated embryos lose FGF8 in the remnant of the forebrain (f; asterisk), but not in the isthmus (i; n=25). (F) SHH is lost in the remaining forebrain tissue (asterisk) but is present in the floorplate of the midbrain (n=25). (I,J) Twelve hours after bead implantation, the amount of programmed cell death (bright green) found in the FNP mesenchyme (arrows) is much greater in RAR/RXR antagonist-treated embryos (J, asterisk; n=5), compared with controls (I, n=6), as determined by a TUNEL assay. (M,N) The amount of cell proliferation is reduced in the forebrain and FNP mesenchyme of RAR/RXR antagonist-treated embryos (N, asterisks; n=23), compared with controls (M, n=6), as determined with BrdU labeling (black cells). (C,G) Sections 24 hours after bead implantation at stage 10 show that control embryos (C,G) express FGF8 and SHH. (D) RAR/RXR antagonist-treated embryos lack FGF8 in the forebrain and FNP ectoderm (asterisk) but still maintain expression in the mandible (ma) and eye (e; n=25). (H) SHH is lost along the forebrain floor (asterisk; n=25). (K,L) Twenty-four hours after bead implantation at stage 10, the amount of programmed cell death found in the FNP mesenchyme is much greater in RAR/RXR antagonist-treated embryos (L, asterisk; n=6), compared with controls (K, arrow; n=4). (P,O) Cell proliferation is much less in the forebrain and FNP mesenchyme of RAR/RXR antagonist-treated embryos (P, asterisks; n=35), compared with controls (O, n=6). Scale bar: 200 µm.

View larger version (122K): [in a new window]   Fig. 6. Inhibition of retinoid signaling alters expression of regulatory genes in the forebrain and FNP as shown by in situ hybridization on parasagittal sections 72 hours after bead implantation at stage 10. (A) FGF8 is normally expressed in the rostral neural tube, including the optic recess (o) and isthmus (i). (E) In antagonist-treated embryos, FGF8 expression is lost in the rostral neural tube, although low levels can be detected in the truncated diencephalon (d). (B) Normal BF1 expression marks most of the telencephalon (t) and hypothalamus (ht). (F) In antagonist-treated embryos, expression of BF1 is not detected in the remnant of the forebrain except some expression remains in the malformed hypothalamus. (C) SHH is normally expressed in ventral neural tissues, including the hypothalamus and basal telencephalon. (G) In antagonist-treated embryos, SHH expression is lost in the diencephalic remnant. (D) Normal PAX6 expression marks dorsal forebrain structures up to the midbrain (m) and the eye (not shown). (H) In antagonist-treated embryos, PAX6 expression is expanded into the remaining diencephalon (d) and can also be detected in the eye (e) and hypothalamic remnant. (I) Normally, OTX2 is broadly expressed in the forebrain and midbrain. (M) In antagonist-treated embryos, OTX2 expression is maintained in these domains. The expression of PAX6 and OTX2 serves as an important control, demonstrating that loss of expression is due to RAR/RXR antagonist-induced misregulation of target genes, rather than a general inability of remaining tissues to synthesize mRNA transcripts. (J) Normally, NKX2.1 marks most of the hypothalamus and basal telencephalon. (N) In antagonist-treated embryos, residual NKX2.1 expression remains in the hypothalamic remnant. (K) Normally, NKX6.1 marks the basal plate. (O) In antagonist-treated embryos, NKX6.1 expression persists in the basal plate of the midbrain but not in the residual forebrain. (L) Normally, DLX2 expression marks part of the hypothalamus, all of the basal telencephalon, and mandibular arch (ma) neural crest. (P) In antagonist-treated embryos, DLX2 expression persists in the hypothalamic remnant and in the mandibular arch. Scale bars: 100 µm.

View larger version (82K): [in a new window]   Fig. 7. Forebrain and FNP defects can be ‘rescued’ by local application of either all-trans RA, or recombinant FGF2 and SHH proteins. Embryos were exposed to RAR/RXR antagonists at stage 10 and subsequently treated 8-10 hours later with either RA, or with FGF2 and SHH. Control embryos were treated at stage 10 with DMSO-soaked beads or RAR/RXR antagonist-soaked beads, which were removed after 8-10 hours. (A,B) By stage 19, DMSO-treated control embryos (A) exhibit normal SHH expression in the FNP ectoderm (fn), maxillary process (mx), and hyoid arch (hy), whereas RAR/RXR antagonist-treated embryos (B) lose SHH expression in the FNP ectoderm (asterisk), fail to develop a forebrain, and have eyes (e) that approximate one another. SHH expression remains unaffected in the maxillary process (mx) and hyoid (hy) arch. This control demonstrates that RAR/RXR antagonists elicit their effects between stage 10 and stage 12, as the RAR/RXR antagonist-soaked beads had been removed after 8-10 hours. Embryos exposed to RAR/RXR antagonists at stage 10 had their beads removed after 8-10 hours and then were treated with either RA, or with FGF2 and SHH. (C) By stage 19, expression of SHH is restored in the forebrain (f) and FNP (fn; arrow; n=15). SHH expression in the maxillary process (mx) and hyoid arch (hy) is unaffected. (D) DMSO-treated control embryos exhibit normal FGF8 expression in FNP ectoderm adjacent to the nasal pits (arrow) whereas RAR/RXR antagonist-treated embryos (E) lose this domain of FGF8 expression and show a collapse of the facial midline (asterisk). FGF8 expression remains unaffected in the maxillary (mx) and mandibular (ma) processes. (F) Embryos exposed to RAR/RXR antagonists at stage 10 had their beads removed after 8-10 hours and then were treated with either RA, or with FGF2 and SHH. Expression of FGF8 is restored adjacent to the nasal pits (arrow; n=11). FGF8 expression in the maxillary (mx) and mandibular processes (ma) is unaffected. By stage 36, embryos rescued with RA (G) or with FGF2 and SHH (H) have a well-developed FNP and forebrain tissues (n=27). Scale bars: 2 mm in A-F; 1 mm in G,H.

View larger version (49K): [in a new window]   Fig. 8. A proposed model for the molecular regulation of forebrain and FNP morphogenesis via epithelial-mesenchymal signaling interactions. (A) Spatial relations of tissues and expression domains in the rostral head are shown in a schematic sagittal section through a stage 11 embryo. FGF8 (pink) and SHH (yellow) are expressed in the neuroepithelium and FNP ectoderm. RARß and RXR (blue) are detected in neural crest mesenchyme, and ALDH6 (green) is localized to ventral FNP ectoderm. The black dashed box indicates area drawn at higher magnification in B, where we propose the retinoid-mediated signaling events occur. (Step 1) Between stage 10 and stage 12, RA is synthesized in FNP ectoderm (based on expression of ALDH6), and signals through receptors in neural crest cells that populate the FNP (based on expression of RARß and RXR). Blocking this step between stage 10 and stage 12 either by citral inhibition of RA biosynthesis or by antagonizing the receptors, has similar downstream consequences. (Step 2) We hypothesize that a retinoid-dependent signal (currently unidentified) emanates from the neural crest mesenchyme and signals to the forebrain and FNP epithelia, maintaining expression of FGF8 and SHH. Alternatively, if retinoid receptors other than the ones we examined are present in the forebrain and FNP epithelia, then RA might also signal through them and maintain expression of FGF8 and SHH. (Step 3) The maintenance of FGF8 and SHH expression is required for survival of the neural crest mesenchyme. A loss of gene expression at this step leads to increased programmed cell death and decreased proliferation. (C) Similar patterns of gene expression are observed in embryos through stage 20. (Step 4) The continued expression of FGF8 and SHH enables the forebrain and FNP to undergo their patterned outgrowth.