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Coordinated development of muscles, tendons, and their attachment sites ensures emergence of functional musculoskeletal units that are adapted to diverse anatomical demands among different species. How these different tissues are patterned and functionally assembled during embryogenesis is poorly understood. Here, we investigated the morphogenesis of extraocular muscles (EOMs), an evolutionary conserved cranial muscle group that is crucial for the coordinated movement of the eyeballs and for visual acuity. By means of lineage analysis, we redefined the cellular origins of periocular connective tissues interacting with the EOMs, which do not arise exclusively from neural crest mesenchyme as previously thought. Using 3D imaging approaches, we established an integrative blueprint for the EOM functional unit. By doing so, we identified a developmental time window in which individual EOMs emerge from a unique muscle anlage and establish insertions in the sclera, which sets these muscles apart from classical muscle-to-bone type of insertions. Further, we demonstrate that the eyeballs are a source of diffusible all-trans retinoic acid (ATRA) that allow their targeting by the EOMs in a temporal and dose-dependent manner. Using genetically modified mice and inhibitor treatments, we find that endogenous local variations in the concentration of retinoids contribute to the establishment of tendon condensations and attachment sites that precede the initiation of muscle patterning. Collectively, our results highlight how global and site-specific programs are deployed for the assembly of muscle functional units with precise definition of muscle shapes and topographical wiring of their tendon attachments., Competing Interests: The authors have declared that no competing interests exist.

Retinoic acid (RA) is known to be required at various levels of eye patterning via Retinoic Acid Receptors (RAR); however the molecular and cellular mechanisms triggered by these nuclear receptors are still obscure. The genetic studies performed here enable us to present a new model to study RA action during eye development. By inactivating the three RARs, specifically in the periocular mesenchyme, we discriminate the individual contribution of each RAR during eye development and describe a new function for RARs during the formation of the optic nerve. We demonstrate that RARalpha is the only receptor that mediates RA signalling in the neurectoderm during ocular development. Surprisingly, and despite a sophisticated pattern of RA-activity in the developing retina, we observed that RA signalling is not autonomously required in this tissue for eye formation. We show that the action of RA during eye morphogenesis is occurring specifically in neural crest-derived periocular mesenchyme and is mediated by all three RARs. Furthermore, we point out that Pitx2, which encodes a homeodomain transcription factor, is a key RA-responsive gene in neural crest cells during eye development. Interestingly, we observed that RA is required in the neural crest cells for normal position of the extraocular muscle.

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.

The retinoic acid (RA) signal, produced locally from vitamin A by retinaldehyde dehydrogenase (Raldh) and transduced by the nuclear receptors for retinoids (RA receptor and 9-cis-RA receptor), is indispensable for ontogenesis and homeostasis of numerous tissues. We demonstrate that Raldh3 knockout in mouse suppresses RA synthesis and causes malformations restricted to ocular and nasal regions, which are similar to those observed in vitamin A-deficient fetuses and/or in retinoid receptor mutants. Raldh3 knockout notably causes choanal atresia (CA), which is responsible for respiratory distress and death of Raldh3-null mutants at birth. CA is due to persistence of nasal fins, whose rupture normally allows the communication between nasal and oral cavities. This malformation, which is similar to isolated congenital CA in humans and may result from impaired RA-controlled down-regulation of Fgf8 expression in nasal fins, can be prevented by a simple maternal treatment with RA.

Several recent studies have shown that retinoic acid signalling is required for correct patterning of the hindbrain. However, the data from these studies are disparate and the precise role of retinoic acid signalling in patterning the anteroposterior axis of the neural tube remains uncertain. To help clarify this issue, we have cultured a staged series of chick embryos in the presence of an antagonist to the all three retinoic acid receptors. Our data indicate that retinoic acid is the transforming signal involved in the expansion of posterior hindbrain structures. We find that the hindbrain region of the neural tube down to the level of the sixth somite acquires the identity of rhombomere 4 when retinoic acid signalling is blocked. Specification of future rhombomere boundaries has a retinoic acid dependency between stage 5 and stage 10(+) that is lost progressively in an anterior-to-posterior sequence. Furthermore, the application of various concentrations of antagonist shows that successively more posterior rhombomere boundaries require progressively higher concentration of endogenous retinoic acid for their correct positioning, a result that strengthens the hypothesis that a complex retinoid gradient acts to pattern the posterior hindbrain. Our dissection of early retinoic acid functions allows us to re-interpret the wide disparity of hindbrain phenotypes previously observed in various models of retinoic acid deficiency.

We previously reported that mice lacking the RARgamma gene and one or both alleles of the RARbeta gene (i.e., RARbeta+/-/RARgamma-/- and RARbeta-/-/RARgamma-/- mutants) display a severe and fully penetrant interdigital webbing (soft tissue syndactyly), caused by the persistence of the fetal interdigital mesenchyme (Ghyselinck et al., 1997, Int. J. Dev. Biol. 41, 425-447). In the present study, these compound mutants were used to investigate the cellular and molecular mechanisms involved in retinoic acid (RA)-dependent formation of the interdigital necrotic zones (INZs). The mutant INZs show a marked decrease in the number of apoptotic cells accompanied by an increase of cell proliferation. This marked decrease was not paralleled by a reduction of the number of macrophages, indicating that the chemotactic cues which normally attract these cells into the INZs were not affected. The expression of a number of genes known to be involved in the establishment of the INZs, the patterning of the autopod, and/or the initiation of apoptosis was also unaffected. These genes included BMP-2, BMP-4, Msx-1, Msx-2, 5' members of Hox complexes, Bcl2, Bax, and p53. In contrast, the mutant INZs displayed a specific, graded, down-regulation of tissue transglutaminase (tTG) promoter activity and of stromelysin-3 expression upon the removal of one or both alleles of the RARbeta gene from the RARgamma null genetic background. As retinoic acid response elements are present in the promoter regions of both tTG and stromelysin-3 genes, we propose that RA might increase the amount of cell death in the INZs through a direct modulation of tTG expression and that it also contributes to the process of tissue remodeling, which accompanies cell death, through an up-regulation of stromelysin-3 expression in the INZs. Approximately 10% of the RARbeta-/- /RARgamma-/- mutants displayed a supernumerary preaxial digit on hindfeet, which is also a feature of the BMP-7 null phenotype (Dudley et al., 1995, Genes Dev. 9, 2795-2807; Luo et al., 1995, Genes Dev. 9, 2808-2820). BMP-7 was globally down-regulated at an early stage in the autopods of these RAR double null mutants, prior to the appearance of the digital rays. Therefore, RA may exert some of its effects on anteroposterior autopod patterning through controlling BMP-7 expression., (Copyright 1999 Academic Press.)

The RAR and RXR families of retinoid nuclear receptors each comprise three isotypes (alpha, beta and gamma). In vitro, RARs bind to their cognate DNA response elements as heterodimers with RXRs. Null mutations of all six isotypes have been generated. The defects displayed by RAR alpha, beta and gamma single null mutant mice are confined to a small subset of the tissues normally expressing these receptors. This discrepancy reflects the existence of a functional redundancy, since RAR double null mutants exhibit congenital malformations in almost every organ system. In particular, most of the structures derived from the mesectoderm are severely affected. Analysis of mutant mice lacking both RARs and RXRs indicates that RXR alpha:RAR gamma heterodimers are instrumental in the patterning of craniofacial skeletal elements, whereas RXR alpha:RAR alpha heterodimers may be preferentially involved in the generation of neural crest cell-derived arterial smooth muscle cells. Both RXR alpha:RAR beta and RXR alpha:RAR gamma heterodimers appear to function during the development of the ocular mesenchyme. Moreover, atavistic reptilian cranial structures are generated in RAR mutants, suggesting that the RA signal has been implicated in the modification of developmental programs in the mesectoderm during evolution.

Two families of nuclear receptors for retinoic acid (RA) have been characterized. Members of the RAR family (types alpha, beta and gamma and their isoforms alpha 1, alpha 2, beta 1 to beta 4, and gamma 1 and gamma 2) are activated by most physiologically occurring retinoids (all-trans RA, 9-cis RA, 4oxo RA and 3,4 dihyroRA). In contrast, members of the RXR family (types alpha, beta and gamma and their isoforms) are activated by 9cis-RA only. In addition to the multiplicity of receptors, the complexity of retinoid signalling is further increased by the fact that, at least in vitro, RARs bind to their cognate response elements as heterodimers with RXRs. Moreover, RXRs can also bind, in vitro, to some DNA elements as homodimers, and are heterodimeric partners for other nuclear receptors, including TRs, VDR, PPARs and a number of orphan nuclear receptors. To evaluate the functions of the different RARs and RXRs types and isoforms, we have generated null mutant mice by targeted gene disruption in ES cells. As to the functions of RARs, we found that RAR alpha 1 and RAR gamma 2 null mutant mice are apparently normal. Mice deficient in RAR alpha or RAR gamma (i.e., all alpha or gamma isoforms disrupted) show aspects of the post-natal vitamin A deficiency (VAD) syndrome which can be cured or prevented by RA, including post-natal lethality, poor weight gain and male sterility. RAR beta 2 (and RAR beta) null mutants display a retrolenticular membrane which represents the most frequent defect of the fetal VAD syndrome. That these abnormalities were restricted to a small subset of the tissues normally expressing these receptors suggested that some degree of functional redundancy should exist in the RAR family. To test this hypothesis we then generated RAR double null mutants. RAR alpha beta, RAR alpha gamma and RAR beta gamma compound mutants exhibit all the malformations of the fetal VAD syndrome, thus demonstrating that RA is the vitamin A derivative which plays a crucial role at many different stages and in different structures during organogenesis. Interestingly, almost all the structures derived from mesenchymal neural crests cells (NCC) are affected in RAR compound mutants. As to the functions of RXRs, RXR gamma null mutants are viable, fertile and morphologically normal. In contrast, RXR alpha null fetuses display a thin ventricular wall and die in utero from cardiac failure. A myocardial hypoplasia has also been observed in some RAR compound mutants as well as in VAD fetuses. Thus, RXR alpha seems to act as an inhibitor of ventricular cardiocyte differentiation and/or as a positive regulator of their proliferation, and these functions might involve heterodimerization with RARs and activation by RA. RXR beta null mutants are viable but the males are sterile, most probably because of an abnormal lipid metabolism in the Sertoli cells. New abnormalities, absent in RXR alpha mutants, are generated in RXR alpha/RAR (alpha, beta or gamma) compound mutants. All these abnormalities are also seen in RAR double mutants as well as in VAD fetuses. In contrast, such manifestations of synergism are not observed between the RXR beta or RXR gamma and the RAR (alpha, beta or gamma) null mutations. These data strongly support the conclusion that RXR alpha/RAR heterodimers represent the main functional units of the RA signalling pathway during embryonic development. Moreover, since RXR gamma-/-/RXR beta-/-/RXR alpha +/-mutants are viable, a single allele of RXR alpha can perform most of the developmental RXR functions.

Retinoids are essential for normal development and both deficiency and excess of retinoic acid (RA) are teratogenic. Retinoic acid response elements (RAREs) have been identified in Hox gene promoters suggesting that endogenous retinoids may be involved in the direct control of Hox gene patterning functions. In order to test this hypothesis, we have mutated the Hoxa-1 3'RARE using the Cre-loxP targeting strategy, and studied its functional role during mouse development. We find that this enhancer plays an important role in the early establishment of the Hoxa-1 anterior expression boundary in the neural plate. This early disturbance in Hoxa-1 activation results in rhombomere and cranial nerve abnormalities reminiscent of those obtained in the Hoxa-1 total knockout, although their severity and penetrance are lower, thus providing strong evidence for direct control of Hox gene function by retinoids during normal development. Interestingly, we also find that the Hoxa-1 expression response to RA treatment is not entirely controlled by the RARE, suggesting the existence of other retinoid-induced factors mediating the Hoxa-1 response to RA and/or the presence of additional RAREs. Interestingly, although the RARE is not required for the spatiotemporal control of colinear expression of the Hoxa genes, it is absolutely required for correct Hoxa-2 expression in rhombomere 5.