What's retinoic acid got to do with it? Retinoic acid regulation of the neural crest in craniofacial and ocular development

Retinoic acid (RA), the active derivative of vitamin A (retinol), is an essential morphogen signaling molecule and major regulator of embryonic development. The dysregulation of RA levels during embryogenesis has been associated with numerous congenital anomalies, including craniofacial, auditory, and ocular defects. These anomalies result from disruptions in the cranial neural crest, a vertebrate‐specific transient population of stem cells that contribute to the formation of diverse cell lineages and embryonic structures during development. In this review, we summarize our current knowledge of the RA‐mediated regulation of cranial neural crest induction at the edge of the neural tube and the migration of these cells into the craniofacial region. Further, we discuss the role of RA in the regulation of cranial neural crest cells found within the frontonasal process, periocular mesenchyme, and pharyngeal arches, which eventually form the bones and connective tissues of the head and neck and contribute to structures in the anterior segment of the eye. We then review our understanding of the mechanisms underlying congenital craniofacial and ocular diseases caused by either the genetic or toxic disruption of RA signaling. Finally, we discuss the role of RA in maintaining neural crest‐derived structures in postembryonic tissues and the implications of these studies in creating new treatments for degenerative craniofacial and ocular diseases.     

Retinoic acid-dependent eye morphogenesis is orchestrated by neural crest cells

Using genetic approaches in the mouse, we show that the primary target tissue of retinoic acid (RA) action during eye morphogenesis is not the retina nor the corneal ectoderm, which both express RA-synthesizing retinaldehyde dehydrogenases (RALDH1 and RALDH3), but the neural crest cell-derived periocular mesenchyme (POM), which is devoid of RALDH. In POM, the effects of the paracrine RA signal are mediated by the nuclear RA receptors heterodimers RXRalpha/RARbeta and RXRalpha/RARgamma. These heterodimers appear to control: (1) the remodeling of the POM through activation of Eya2-related apoptosis; (2) the expression of Foxc1 and Pitx2, which play crucial roles in anterior eye segment development; and (3) the growth of the ventral retina. We additionally show that RALDH1 and RALDH3 are the only enzymes that are required for RA synthesis in the eye region from E10.5 to E13.5, and that patterning of the dorsoventral axis of the retina does not require RA.     

Expression of retinoic acid receptor genes in neural crest-derived cells during mouse facial development

Retinoic acid (RA) is known as a teratogen that induces abnormalities in facial structures which are made up mainly of neural crest-derived mesenchyme. We investigated expression patterns of RA receptor (RAR) genes (subtypes alpha, beta, gamma) during mouse facial development. The expression of the RAR beta gene is specific for the mesenchyme around developing eyes and nose, whereas the RAR gamma gene is expressed in the mesenchyme differentiating to facial cartilages and bones. In contrast, the RAR alpha gene is expressed weakly and uniformly over the facial region. These results suggest that crucial roles of endogenous RA in facial development depend on differential functions of the RAR subtypes.     

Retinoids and nonvertebrate chordate development

Retinoic acid (RA) is required for the differentiation and morphogenesis of chordate-specific features, such as the antero-posterior regionalization of the dorsal hollow nerve cord and neural crest cells. RA receptors (RARs) have been reported exclusively in chordates, suggesting that the acquisition of the RAR gene was important for chordate evolution. A scenario is presented here for the establishment of an RAR-mediated developmental regulatory system during the course of chordate evolution. In the common chordate ancestor, RAR came to control the spatial expression pattern of Hox genes in the ectoderm and endoderm along the antero-posterior axis. In these germ layers, RA was required for the differentiation of epidermal sensory neurons and the morphogenesis of pharyngeal gill slits, respectively. As the diffuse epidermal nerve net in the chordate ancestor became centralized to form the dorsal nerve cord, the epidermal Hox expression pattern was carried into the central nervous system. Because the Hox code here came to specify neuronal identity along the antero-posterior axis, RA became inextricably linked to the antero-posterior patterning of the chordate central nervous system.     

Thyroid hormone and retinoic acid interact to regulate zebrafish craniofacial neural crest development

Craniofacial and ocular morphogenesis require proper regulation of cranial neural crest migration, proliferation, survival and differentiation. Although alterations in maternal thyroid hormone (TH) are associated with congenital craniofacial anomalies, the role of TH on the neural crest has not been previously described. Using zebrafish, we demonstrate that pharmacologic and genetic alterations in TH signaling disrupt cranial neural crest migration, proliferation, and survival, leading to craniofacial, extraocular muscle, and ocular developmental abnormalities. In the rostral cranial neural crest that gives rise to the periocular mesenchyme and the frontonasal process, retinoic acid (RA) rescued migratory defects induced by decreased TH signaling. In the caudal cranial neural crest, TH and RA had reciprocal effects on anterior and posterior pharyngeal arch development. The interactions between TH and RA signaling were partially mediated by the retinoid X receptor. We conclude that TH regulates both rostral and caudal cranial neural crest. Further, coordinated interactions of TH and RA are required for proper craniofacial and ocular development.     

Retinoic acid guides eye morphogenetic movements via paracrine signaling but is unnecessary for retinal dorsoventral patterning

Retinoic acid (RA) is required for patterning of the posterior nervous system, but its role in the retina remains unclear. RA is synthesized in discrete regions of the embryonic eye by three retinaldehyde dehydrogenases (RALDHs) displaying distinct expression patterns. Overlapping functions of these enzymes have hampered genetic efforts to elucidate RA function in the eye. Here, we report Raldh1, Raldh2 and Raldh3 single, double and triple null mice exhibiting progressively less or no RA synthesis in the eye. Our genetic studies indicate that RA signaling is not required for the establishment or maintenance of dorsoventral patterning in the retina, as we observe normal expression of Tbx5 and ephrin B2 (Efnb2) dorsally, plus Vax2 and Ephb2 ventrally. Instead, RA is required for the morphogenetic movements needed to shape the developing retina and surrounding mesenchyme. At early stages, Raldh2 expressed in mesenchyme and Raldh3 expressed in the retinal pigmented epithelium generate RA that delivers an essential signal to the neural retina required for morphogenetic movements that lead to ventral invagination of the optic cup. At later stages, Raldh1 expressed in dorsal neural retina and Raldh3 expressed in ventral neural retina (plus weaker expression of each in lens/corneal ectoderm) generates RA that travels to surrounding mesenchyme, where it is needed to limit the anterior invasion of perioptic mesenchyme during the formation of corneal mesenchyme and eyelids. At all stages, RA target tissues are distinct from locations of RA synthesis, indicating that RALDHs function cell-nonautonomously to generate paracrine RA signals that guide morphogenetic movements in neighboring cells.     

Expression and function of a retinoic acid receptor in budding ascidians

 Retinoic acid is thought to induce transdifferentiation of multipotent epithelial stem cells in the developing buds of the ascidian Polyandrocarpa misakiensis. We isolated a cDNA clone from this species, named PmRAR, encoding a retinoic acid receptor (RAR) homologue. PmRAR clusters with other RARs on phylogenetic trees constructed by three different methods. Within the cluster, PmRAR is on a separate branch from all the subtypes of RARs, suggesting that RAR subtypes arose in the ancestral vertebrates after divergence of vertebrates and urochordates. The embryos of another ascidian species Ciona intestinalis were co-electroporated with a mixture of a PmRAR expression vector and a lacZ reporter plasmid containing vertebrate-type retinoic acid response elements. The expression of lacZ depended on the presence of both retinoic acid and PmRAR, suggesting that PmRAR is a functional receptor. PmRAR mRNA is expressed in the epidermis and mesenchyme cells of the Polyandrocarpa developing bud. The mRNA is not detectable in the mesenchyme cells in the adult body wall, but its expression can be induced by retinoic acid in vitro. These results suggest that the PmRAR is a mediator of retinoic acid signalling in transdifferentiation during asexual reproduction of protochordates. Received: 6 April 1998 / Accepted: 27 July 1998     

Semaphorin3A/neuropilin-1 signaling acts as a molecular switch regulating neural crest migration during cornea development

Cranial neural crest cells migrate into the periocular region and later contribute to various ocular tissues including the cornea, ciliary body and iris. After reaching the eye, they initially pause before migrating over the lens to form the cornea. Interestingly, removal of the lens leads to premature invasion and abnormal differentiation of the cornea. In exploring the molecular mechanisms underlying this effect, we find that semaphorin3A (Sema3A) is expressed in the lens placode and epithelium continuously throughout eye development. Interestingly, neuropilin-1 (Npn-1) is expressed by periocular neural crest but down-regulated, in a manner independent of the lens, by the subpopulation that migrates into the eye and gives rise to the cornea endothelium and stroma. In contrast, Npn-1 expressing neural crest cells remain in the periocular region and contribute to the anterior uvea and ocular blood vessels. Introduction of a peptide that inhibits Sema3A/Npn-1 signaling results in premature entry of neural crest cells over the lens that phenocopies lens ablation. Furthermore, Sema3A inhibits periocular neural crest migration in vitro. Taken together, our data reveal a novel and essential role of Sema3A/Npn-1 signaling in coordinating periocular neural crest migration that is vital for proper ocular development.     

Phosphorylation of activation functions AF-1 and AF-2 of RAR alpha and RAR gamma is indispensable for differentiation of F9 cells upon retinoic acid and cAMP treatment.

The role of RAR alpha 1 and RAR gamma 2 AF-1 and AF-2 activation functions and of their phosphorylation was investigated during RA-induced primitive and parietal differentiation of F9 cells. We found that: (i) primitive endodermal differentiation requires RAR gamma 2, whereas parietal endodermal differentiation requires both RAR gamma 2 and RAR alpha 1, and in all cases AF-1 and AF-2 must synergize; (ii) primitive endodermal differentiation requires the proline-directed kinase site of RAR gamma 2-AF-1, whereas parietal endodermal differentiation additionally requires that of RAR alpha 1-AF-1; (iii) the cAMP-induced parietal endodermal differentiation also requires the protein kinase A site of RAR alpha-AF-2, but not that of RAR gamma; and (iv) the AF-1-AF-2 synergism and AF-1 phosphorylation site requirements for RA-responsive gene induction are promoter context-dependent. Thus, AF-1 and AF-2 of distinct RARs exert specific cellular and molecular functions in a cell-autonomous system mimicking physiological situations, and their phosphorylation by kinases belonging to two main signalling pathways is required to enable RARs to transduce the RA signal during F9 cell differentiation.     

Establishment of the scleral cartilage in the chick.

This study was undertaken to investigate the establishment of the scleral cartilage in the chick embryo. Johnston et al. (1974) has demonstrated that most of the cells of the scleral cartilage originate in the cranial neural crest. By means of a series of chorioallantoic grafts of pigmented retina, and its adherent periocular mesenchyme from stage 11 to 25, the present experiments show that the cranial neural crest cells arrive at the eye in sufficient numbers to form cartilage by stage 14. Pigmented retina, denuded of mesenchyme, from stage 16 embryos implanted into the head of stage 13 embryos induces cartilage formation in head mesenchyme. However, neither pigmented retina nor spinal cord could induce cartilage formation in chorioallantoic mesenchyme. Combination grafts of cranial neural crest and presumptive optic vesicle developed neural tissue, pigmented retina, and in some cases sclera-like cartilage. Thus, periorbital mesenchyme, derived largely from cranial neural crest, at about stage 14 develops the scleral cartilage in response to induction by the pigmented retina.     

Combinatorial roles for zebrafish retinoic acid receptors in the hindbrain, limbs and pharyngeal arches

Retinoic acid (RA) signaling regulates multiple aspects of vertebrate embryonic development and tissue patterning, in part through the local availability of nuclear hormone receptors called retinoic acid receptors (RARs) and retinoid receptors (RXRs). RAR/RXR heterodimers transduce the RA signal, and loss-of-function studies in mice have demonstrated requirements for distinct receptor combinations at different stages of embryogenesis. However, the tissue-specific functions of each receptor and their individual contributions to RA signaling in vivo are only partially understood. Here we use morpholino oligonucleotides to deplete the four known zebrafish RARs (raraa, rarab, rarga, and rargb). We show that while all four are required for anterior-posterior patterning of rhombomeres in the hindbrain, there are unique requirements for rarga in the cranial mesoderm for hindbrain patterning, and rarab in lateral plate mesoderm for specification of the pectoral fins. In addition, the alpha subclass (raraa, rarab) is RA inducible, and of these only raraa expression is RA-dependent, suggesting that these receptors establish a region of particularly high RA signaling through positive-feedback. These studies reveal novel tissue-specific roles for RARs in controlling the competence and sensitivity of cells to respond to RA.     

Altered expression of retinoic acid (RA) receptor mRNAs in the fetal mouse secondary palate by all-trans and 13-cis RAs: implications for RA-induced teratogenesis

Retinoic acid (RA) is mandatory for various biological processes and normal embryonic development but is teratogenic at high concentrations. In rodents, one of the major malformations induced by RA is cleft palate (CP). RA mediates its effects by RA receptors (RARs), but the expression patterns of RARs in the developing palate are still unclear. We investigated the normal expression of RAR alpha, beta, and gamma messenger RNAs (mRNAs) in the fetal mouse secondary palate and the effects of all-trans and 13-cis RAs on the expression of RAR mRNAs by Northern blot analysis. RAR alpha (2.8, 3.8 kb), RAR beta (3.3 kb), and RAR gamma (3.7 kb) mRNAs were detected in the fetal palate on gestational days (GD) 12.5-14.5. The expression of RAR alpha and gamma mRNAs did not show apparent sequential changes, but that of RAR beta mRNA increased at GD 13.5. Treatment of pregnant mice with 100 mg/kg all-trans RA induced CP in 94% of the fetuses and elevated the levels of RAR beta and gamma mRNAs in the fetal palate. The up-regulation of RAR beta mRNA by all-trans RA was more marked than that of RAR gamma mRNA. Treatment with 100 mg/kg 13-cis RA induced CP in only 19% of the fetuses. Although 13-cis RA elevated the RAR beta and gamma mRNA levels in fetal palates, its up-regulation was slower and less marked than that induced by all-trans RA. These findings indicate that the induction of RAR beta mRNA in the fetal palate correlates well with the tissue concentration of all-trans RA after RA treatment, and RAR beta may be one of the most influential candidate molecules for RA-induced teratogenesis.     

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

Retinoids, and in particular retinoic acid (RA), are known to induce posterior fates in neural tissue. However, alterations in retinoid signalling dramatically affect anterior development. Previous reports have demonstrated a late role for retinoids in patterning craniofacial and forebrain structures, but an earlier role in anterior patterning is not well understood. We show that enzymes involved in synthesizing retinoids are expressed in the avian hypoblast and in tissues directly involved in head patterning, such as anterior definitive endoderm and prechordal mesendoderm. We found that in the vitamin A-deficient (VAD) quail model, which lacks biologically active RA from the first stages of development, anterior endodermal markers such as Bmp2, Bmp7, Hex and the Wnt antagonist crescent are affected during early gastrulation. Furthermore, prechordal mesendodermal and prospective ventral telencephalic markers are expanded posteriorly, Shh expression in the axial mesoderm is reduced, and Bmp2 and Bmp7 are abnormally expressed in the ventral midline of the neural tube. At early somite stages, VAD embryos have increased cell death in ventral neuroectoderm and foregut endoderm, but normal cranial neural crest production, whereas at later stages extensive apoptosis occurs in head mesenchyme and ventral neuroectoderm. As a result, VAD embryos end up with a single and reduced telencephalic vesicle and an abnormally patterned diencephalon. Therefore, we propose that retinoids have a dual role in patterning the anterior forebrain during development. During early gastrulation, RA acts in anterior endodermal cells to modulate the anteroposterior (AP) positional identity of prechordal mesendodermal inductive signals to the overlying neuroectoderm. Later on, at neural pore closure, RA is required for patterning of the mesenchyme of the frontonasal process and the forebrain by modulating signalling molecules involved in craniofacial morphogenesis.     

Distinct stages in adipogenesis revealed by retinoid inhibition of differentiation after induction of PPARgamma.

Retinoic acid (RA) inhibits adipocyte differentiation of 3T3-L1 preadipocytes but is effective only early in adipogenesis. RA prevented induction of the adipogenic factors PPARgamma and C/EBPalpha. Using receptor-specific ligands, we determined that the effects of RA were mediated by liganded RA receptors (RARs) rather than retinoid X receptors. Preadipocytes expressed primarily RARalpha and RARgamma; during adipocyte differentiation, RARalpha gene expression was nearly constant, whereas RARgamma1 mRNA and protein levels dramatically decreased. Ectopic expression of RARgamma1 extended the period of effectiveness of RA by 24 to 48h; RARalpha expression had a similar effect, suggesting functional redundancy of RAR subtypes. Remarkably, RA inhibited differentiation when added after PPARgamma1 and PPARgamma2 proteins had already been expressed and resulted in the loss of PPARgamma proteins from cells. By 72 to 96 h after the induction of differentiation, RA failed to prevent differentiation of even ectopic-RAR-expressing cells. Thus, the unresponsiveness of 3T3-L1 preadipocytes to RA after the induction of differentiation is initially due to the reduction in cellular RAR concentration rather than to the induction of PPARgamma. At later times cells continue along the differentiation pathway in a manner which is RA and RAR independent.