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To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2008.04.039 Byline: Nicolas Matt (c), Norbert B. Ghyselinck (b), Isabelle Pellerin (a), Valerie Dupe (a) Keywords: Retinoic acid receptor; Neural crest cells; Eye development; PITX2; Extraocular muscle Abbreviations: E(n), embryonic day; NCC, neural crest cell; POM, periocular mensenchyme; RA, retinoic acid; ALDH1A, retinaldehyde dehydrogenase; RAR, retinoic acid receptor; RPE, retinal pigmented epithelium; RXR, 9cis-RA receptor, VAD, vitamin A-deficient. Abstract: 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 RAR[alpha] 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. Author Affiliation: (a) Institut de Genetique et Developpement, CNRS UMR6061, Universite de Rennes 1, IFR140 GFAS, Faculte de Medecine, 2, avenue du Professeur Leon Bernard, Rennes 35043 Cedex, France (b) Institut de Genetique et de Biologie Moleculaire et Cellulaire (IGBMC), Institut Clinique de la Souris (ICS), CNRS/INSERM/ULP, College de France, Illkirch 67404, CU de Strasbourg, France (c) UPR9022 du CNRS, IBMC, 15 rues Descartes, 67084 Strasbourg, France Article History: Received 13 February 2008; Revised 7 April 2008; Accepted 30 April 2008

Byline: Aida Halilagic (a)(d), Vanessa Ribes (b), Norbert B. Ghyselinck (b), Maija H. Zile (c), Pascal Dolle (b), Michele Studer (a)(d) Keywords: Retinoids; Forebrain morphogenesis; Eye development; Raldh2; Raldh3; VAD quail; RPE; Neural retina; Signaling Abstract: We have previously shown that retinoic acid (RA) synthesized by the retinaldehyde dehydrogenase 2 (RALDH2) is required in forebrain development. Deficiency in RA due to inactivation of the mouse Raldh2 gene or to complete absence of retinoids in vitamin-A-deficient (VAD) quails, leads to abnormal morphogenesis of various forebrain derivatives. In this study we show that double Raldh2/Raldh3 mouse mutants have a more severe phenotype in the craniofacial region than single null mutants. In particular, the nasal processes are truncated and the eye abnormalities are exacerbated. It has been previously shown that retinoids act mainly on cell proliferation and survival in the ventral forebrain by regulating SHH and FGF8 signaling. Using the VAD quail model, which survives longer than the Raldh-deficient mouse embryos, we found that retinoids act in maintaining the correct position of anterior and dorsal boundaries in the forebrain by modulating FGF8 anteriorly and WNT signaling dorsally. Furthermore, BMP4 and FGF8 signaling are affected in the nasal region and BMP4 is ventrally expanded in the optic vesicle. At the optic cup stage, Pax6, Tbx5 and Bmp4 are ectopically expressed in the presumptive retinal pigmented epithelium (RPE), while Otx2 and Mitf are not induced, leading to a dorsal transdifferentiation of RPE to neural retina. Therefore, besides being required for survival of ventral structures, retinoids are involved in restricting anterior identity in the telencephalon and dorsal identity in the diencephalon and the retina. Author Affiliation: (a) MRC Centre for Developmental Neurobiology, King's College London, Guy's Campus, London SE1 1UL, UK (b) Institut de Genetique et de Biologie Moleculaire et Cellulaire, UMR 7104 du CNRS, U 596 de l'INSERM, Universite Louis Pasteur, BP 10142, 67404 Illkirch Cedex, CU de Strasbourg, France (c) Department of Food Science and Human Nutrition, Michigan State University, East Lansing, MI 48824, USA (d) TIGEM, Telethon Institute of Genetics and Medicine, Naples 80131, Italy Article History: Received 30 June 2006; Revised 1 September 2006; Accepted 14 November 2006

To investigate the relative contribution of retinoic acid receptor (RAR)[Beta] isoforms in conotruncal septation, RAR[Beta]1 and [Beta]3 were inactivated in the mouse. Mice lacking RAR[Beta]1 and [Beta]3 appear normal. Disruption of these isoforms in RAR[Alpha] or RAR[Gamma] null genetic backgrounds results in a high postpartum lethality. However, except for ocular defects found in RAR[Beta]1-3/RAR[Gamma] compound mutants, the double null mutants display only abnormalities seen in single null mutants. This probably reflects a functional redundancy with other RARs, most notably with RAR[Beta]2 which is five- to sixfold more abundant than RAR[Beta]1 and [Beta]3 and whose domain of expression is largely overlapping. The conotruncal ridges form normally in retinoid X receptor (RXR)[Alpha]/RAR[Beta] compound mutants but fail to fuse, apparently as a result of excessive apoptosis of mesenchymal cells. Additionally, many cardiomyocytes in the conotruncal wall of these mutants appear necrotic. Although RAR[Beta]1 and [Beta]3 are expressed specifically in the conotruncal ridges, failure of fusion of these structures is not more frequent in RXR[Alpha]/RAR[Beta]1-3 double null mutants than in RXR[Alpha] single null mutants. Similarly, the disruption of the sole RAR[Beta]2 isoform in a RXR[Alpha] null genetic background does not result in an increase of the frequency of conotruncal septum agenesis. However, this agenesis is fully penetrant in RXR[Alpha]/RAR[[Beta].sup.+/-] mutants, which reflects distinct roles of RXR[Alpha]:RAR[Beta]1 (and [Beta]3) and RXR[Alpha]:RAR[Beta]2 heterodimers in promoting the survival of conotruncal mesenchymal cells. Unexpectedly, we discovered that, in wild-type embryos, the conotruncal mesenchyme is a major site of morphogenetic cell death and that conotruncal myocytes are occasionally necrotic. Thus, excessive cell death in the conotruncus is a potential cause of ventricular septal defects in humans. Key Words: RAR; RXR; gene knock-out; genetic redundancy; mouse embryonic development; conotruncus.

In mammals, all-trans retinoic acid (ATRA) is instrumental to spermatogenesis. It is synthesized by two retinaldehyde dehydrogenases (RALDH) present in both Sertoli cells (SCs) and germ cells (GCs). In order to determine the relative contributions of each source of ATRA, we have generated mice lacking all RALDH activities in the seminiferous epithelium (SE). We show that both the SC- and GC-derived sources of ATRA cooperate to initiate and propagate spermatogenetic waves at puberty. In adults, they exert redundant functions and, against all expectations, the GC-derived source does not perform any specific roles despite contributing to two-thirds of the total amount of ATRA present in the testis. The production from SCs is sufficient to maintain the periodic expression of genes in SCs, as well and the cycle and wave of the SE, which account for the steady production of spermatozoa. The production from SCs is also specifically required for spermiation. Importantly, our study shows that spermatogonia differentiation depends upon the ATRA synthesized by RALDH inside the SE, whereas initiation of meiosis and expression of STRA8 by spermatocytes can occur without ATRA. [ABSTRACT FROM AUTHOR]