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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

Byline: Masayuki Uehara (a), Kenta Yashiro (a), Satoru Mamiya (a), Jinsuke Nishino (a), Pierre Chambon (b), Pascal Dolle (b), Yasuo Sakai (a) Keywords: Central nervous system; CYP26; Anteroposterior axis; Neural crest cells; Retinoic acid Abstract: The appropriate regulation of retinoic acid signaling is indispensable for patterning of the vertebrate central nervous system along the anteroposterior (A-P) axis. Although both CYP26A1 and CYP26C1, retinoic acid-degrading enzymes that are expressed at the anterior end of the gastrulating mouse embryo, have been thought to play an important role in central nervous system patterning, the detailed mechanism of their contribution has remained largely unknown. We have now analyzed CYP26A1 and CYP26C1 function by generating knockout mice. Loss of CYP26C1 did not appear to affect embryonic development, suggesting that CYP26A1 and CYP26C1 are functionally redundant. In contrast, mice lacking both CYP26A1 and CYP26C1 were found to manifest a pronounced anterior truncation of the brain associated with A-P patterning defects that reflect expansion of posterior identity at the expense of anterior identity. Furthermore, Cyp26a1 .sup.-/- Cyp26c1 .sup.-/- mice fail to produce migratory cranial neural crest cells in the forebrain and midbrain. These observations, together with a reevaluation of Cyp26a1 mutant mice, suggest that the activity of CYP26A1 and CYP26C1 is required for correct A-P patterning and production of migratory cranial neural crest cells in the developing mammalian brain. Author Affiliation: (a) Developmental Genetics Group, Graduate School of Frontier Biosciences, Osaka University, and CREST, Japan Science and Technology Corporation (JST), 1-3 Yamada-oka, Suita, Osaka 565-0871, Japan (b) Institut de Genetique et de Biologie Moleculaire et Cellulaire, CNRS/INSERM/ULP/College de France, BP10142, 67404 Illkirch Cedex, C.U. de Strasbourg, France Article History: Received 22 June 2006; Revised 25 September 2006; Accepted 27 September 2006

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2006.05.019 Byline: Zengxin Wang (a), Pascal Dolle (b), Wellington V. Cardoso (c), Karen Niederreither (a) Keywords: Retinoids; Lung; Stomach; Liver; Fgf10; Sonic hedgehog; Hox genes; Tbx genes Abstract: Retinoic acid (RA) is an embryonic signaling molecule regulating a wide array of target genes, thereby being a master regulator of patterning and differentiation in a variety of organs. Here we show that mouse embryos deficient for the RA-synthesizing enzyme retinaldehyde dehydrogenase 2 (RALDH2), if rescued from early lethality by maternal RA supplementation between E7.5 and E8.5, lack active RA signaling in the foregut region. The resulting mutants completely fail to develop lungs. Development of more posterior foregut derivatives (stomach and duodenum), as well as liver growth, is also severely affected. A primary lung bud is specified in the RA-deficient embryos, which fails to outgrow due to defective FGF10 signaling and lack of activation of FGF-target genes, such as Pea3 and Bmp4 in the epithelium. Specific Hox and Tbx genes may mediate these RA regulatory effects. Development of foregut derivatives can be partly restored in mutants by extending the RA supplementation until at least E10.5, but lung growth and branching remain defective and a hypoplastic lung develops on the right side only. Such conditions poorly restore FGF10 signaling in the lung buds. Explant culture of RALDH2-deficient foreguts show a capacity to undergo lung budding and early branching in the presence of RA or FGF10. Our data implicate RA as a regulator of gene expression in the early embryonic lung and stomach region upstream of Hox, Tbx and FGF10 signaling. Author Affiliation: (a) Departments of Medicine and Molecular and Cellular Biology, Center for Cardiovascular Development, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA (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) Pulmonary Center, Boston University School of Medicine, Boston, MA 02118, USA Article History: Received 21 February 2006; Revised 24 April 2006; Accepted 15 May 2006

Biochemical process which will regulate morphogenesis and patterning in mammals is examined.

Using a study on mice, the influence of signaling activity of retinoic acid receptors on initial lung development is examined.

During embryogenesis, the pancreas arises from dorsal and ventral pancreatic protrusions from the primitive gut endoderm upon induction by different stimuli from neighboring mesodermal tissues. Recent studies have shown that Retinoic Acid (RA) signaling is essential for the development of the pancreas in non-mammalian vertebrates. To investigate whether RA regulates mouse pancreas development, we have studied the phenotype of mice with a targeted deletion in the retinaldehyde dehydrogenase 2 (Raldh2) gene, encoding the enzyme required to synthesize RA in the embryo. We show that Raldh2 is expressed in the dorsal pancreatic mesenchyme at the early stage of pancreas specification. RA-responding cells have been detected in pancreatic endodermal and mesenchymal cells. Raldh2-deficient mice do not develop a dorsal pancreatic bud. Mutant embryos lack Pdx1 expression, an essential regulator of early pancreas development, in the dorsal but not the ventral endoderm. In contrast to Pdx1-deficient mice, the early glucagon-expressing cells do not develop in Raldh2 knockout embryos. Shh expression is, as in the wild-type embryo, excluded from the dorsal endodermal region at the site where the dorsal bud is expected to form, indicating that the dorsal bud defect is not related to a mis-expression of Shh. Mesenchymal expression of the LIM homeodolnain protein Is11, required for the formation of the dorsal mesenchyme, is altered in Raldh2-/- embryos. The homeobox gene Hlxb9, which is essential for the initiation of the pancreatic program in the dorsal foregut endoderm, is still expressed in Raldh2-/- dorsal epithelium but the number of HB9-expressing cells is severely reduced. Maternal supplementation of RA rescues early dorsal pancreas development and restores endodermal Pdx1 and mesenchymal Is11 expression as well as endocrine cell differentiation. These findings suggest that RA signaling is important for the proper differentiation of the dorsal mesenchyme and development of the dorsal endoderm. We conclude that RA synthesized in the mesenchyme is specifically required for the normal development of the dorsal pancreatic endoderm at a stage preceding Pdx1 function. Keywords: Pancreas; Raldh2; Retinoids; Foregut; Endoderm; Mesenchymc; Shh; Is11; H1xb9

Tbx20 is a member of the T-box transcription factor family expressed in the forming hearts of vertebrate and invertebrate embryos. We report here analysis of Tbx20 expression during murine cardiac development and assessment of DNA-binding and transcriptional properties of Tbx20 isoforms. Tbx20 was expressed in myocardium and endocardium, including high levels in endocardial cushions, cDNAs generated by alternative splicing encode at least four Tbx20 isoforms, and Tbx20a uniquely carried strong transactivation and transrepression domains in its C terminus. Isoforms with an intact T-box bound specifically to DNA sites resembling the consensus brachyury half site, although with less avidity compared with the related factor, Tbx5. Tbx20 physically interacted with cardiac transcription factors Nkx2-5, GATA4, and GATA5, collaborating to synergistically activate cardiac gene expression. Among cardiac GATA factors, there was preferential synergy with GATA5, implicated in endocardial differentiation. In Xenopus embryos, enforced expression of Tbx20a, but not Tbx20b, led to induction of mesodermal and endodermal lineage markers as well as cell migration, indicating that the long Tbx20a isoform uniquely bears functional domains that can alter gene expression and developmental behaviour in an in vivo context. We propose that Tbx20 plays an integrated role in the ancient myogenic program of the heart, and has been additionally coopted during evolution of vertebrates for endocardial cushion development. Keywords: T-box; Tbx20; Tbx5; Tbx genes; Nkx2-5; GATA; Heart development; Embryonic development; Transcription factor; Xenopus; Cell migration

The retinoic acid (RA)-metabolizing enzyme, CYP26A1, has been studied by disrupting the murine Cyp26A1 gene and found to be required for normal development. Exposure to RA during formation of the antero-posterior body axis results in homeotic transformations along the vertebral column. CYP26A1 likely is needed to maintain given embryonic areas in an RA-depleted state to protect them against harmful effects of ectopic RA signaling.

The active derivatives of vitamin A (the retinoids) play important and multiple roles in mammalian development and homeostasis. We have previously shown that specific retinoic acid receptors are expressed in the chorioallantoic placenta of the mouse and that among these, RXR[Alpha] is strongly expressed in the developing labyrinthine zone (Sapin, V., Ward, S. J., Bronner, S., Chambon, P., Dolle, P., Dev. Dyn. 208, 199-210, 1997). Here, we show that mouse fetuses with a targeted disruption of the RXR[Alpha] gene develop defects of the chorioallantoic placenta. Both morphological abnormalities and alterations in the expression of molecular markers were found, mostly confined to the labyrinthine zone of placentas from mid-late gestation mutants. This region exhibited edema, abnormal stasis of maternal blood, and signs of disruption of the endothelial layer of fetal vessels. We also detected a reduction in the number of lipid droplets in the trophoblastic layer and abnormal fibrin deposits in the junctional zone of the mutant placentas. These abnormalities most probably result in an impairment of the functional capacities of exchange between the maternal and fetal circulations in the mutant placentas. Thus, placental defects could represent an extraembryonic cause of lethality for RXR[Alpha] null mutant fetuses, in addition to the previously described embryonic cardiac defects. Key Words: gene knockout; mouse development; placenta; retinoids; trophoblast.

Disruption of the mu-opioid-receptor gene in mice by homologous recombination fails to cause a significant change in expression of other components of the opioid system or result in overt behavioral abnormalities. However, the lack of mu-receptors attenuates the analgesic effect of morphine, its place-preference activity and physical dependence. The mu-opioid-receptor gene product is an essential requirement of the opioid system for morphine action. The delta- and kappa-receptors fail to mediate biological actions of morphine in the absence of mu-receptors.

Segmentation of the hindbrain has been conserved throughout the vertebrate species and results in the transient formation of rhombomeres, which are lineage-restricted compartments. Studies on the molecular mechanisms underlying the segmentation process have revealed that rhombomeric boundaries coincide with the expression limits of several evolutionary conserved genes such as the zinc-finger transcription factor Krox-20 and homeobox genes which are expressed in a specific spatial and temporal order and have been shown to be important regulators of segmental identity. In addition to Krox-20 and Hox genes, several members of the Eph subfamily of receptor protein tyrosine kinase (RTK) genes are also expressed in a segment-restricted manner in the hindbrain, suggesting that these receptors may act in concert with Hox genes to establish regional identity. In the cascade of regulatory interactions leading to segmental identity, Krox-20 appears to act 'upstream' of Hox genes, but the identity of the 'downstream' effectors has not yet been identified. We report here the isolation of the zebrafish orthologue of the mouse RTK gene MDK1 which belongs to the Eph receptor subfamily and show that the major expression domains of the mouse and the zebrafish genes have been conserved through evolution. Since the coincident spatial and temporal expression of Hoxa-2 and MDK1 in the mouse hindbrain suggested a possible regulatory link between them, we analyzed the expression of the MDK1 in Hoxa-2 null mutant embryos. A selective lack of MDK1 expression in rhombomere 3 of Hoxa-2 mutant hindbrains together with an overall altered expression pattern in the other rhombomeres was observed, thus demonstrating that MDK1 lies downstream of Hoxa-2 in the morphogenetic signaling cascade.

This study reports a morphological, skeletal, and molecular characterization of the supernumerary limbs induced by systemic administration of all-trans retinoic acid to egg-cylinder stage mouse embryos. As initially described by Rutledge et al. (Proc. Natl. Acad. Sci. USA 91, 5436, 1994), we have found that oral administration of all-trans retinoic acid (70 mg/kg body weight) at 5.5 days postcoitum induced the formation of supernumerary limbs. Most often, these arose as a pair of extra buds located caudally and ventrally to the normal (orthotopic) hindlimb buds without duplication of the lower body axis. The resulting one or two supernumerary hindlimbs were connected to an imperfectly mirror-image-duplicated pelvic girdle. Variable truncations of the stylopodium and zeugopodium skeleton, as well as abnormal splitting of the distal skeleton, were frequently observed. The apical ectodermal ridge of the extra limb buds expressed expected growth factor genes. However, an ectopic anterior expression of Sonic hedgehog and Hoxd-13 was seen in the supernumerary buds, suggesting that these buds would incorporate potential polarizing cells of the hindlimb or genital field and generate an ectopic polarizing zone. This is consistent with the reverse orientation of most supernumerary limbs at later stages. Some of the buds did not express limb-specific markers and were thus expected to degenerate or form nonlimb structures, as observed in an adult specimen. Less frequently, extra limb buds with normal polarity were associated to a duplicated lower body axis. Retinoic acid also generated a novel type of duplication in which 'twin' hindlimbs with two parallel apical ectodermal ridges and zones of polarizing activity arose on one side of the embryo.

An efficient differential subtractive hybridization cloning strategy has been developed to systematically isolate mouse genes whose expression is induced in P19 cells at different times following exposure to retinoic acid (RA). Fifty different cDNA fragments corresponding to RA-induced genes were isolated, of which 40 represent novel genes. Ten of the isolated cDNAs correspond to known genes, of which four have previously been identified as RA-inducible. One of the novel RA-inducible genes (Stra1) has been studied further, and it appears to correspond to a ligand for Cek5, a receptor protein-tyrosine kinase.

Malformations in heterozygote mice, associated with fetal vitamin A deficiency syndrome, show that the mouse RXR-alpha gene is related to retinoid signalling processes. Targeted disruption in the mouse RXR-alpha gene produces null mutation, which results in retarded growth of the mouse. The death of null mutants caused ocular and myocardial deficiencies. RXR-alpha gene double mutants exhibit several physical malformations in the mouse, which are not caused by single mutants.

The disrupted Hoxa-2 gene of the Hoxa-2 mutant is transcribed in the mesenchymal of the second arch, and not the first as in normal embryos. A common skeleton program underlies the mesenchymal neural crest cell (NCC) segmentation, originating from the second and fourth rhombomeres. The existence of an atavistic reptilian pterygoquadrate element in the Hoxa-2 mutants points a ground pattern that is transitional between reptiles and mammals.

The last gene of complex Hoxd-13 induces local heterochrony due to abnormalities in limb morphogenesis. This is revealed by the method of gene disruption through homologous recombination in embryonic stem cells. These observations facilitate better comprehension of the growth and origin of the tetrapod limb. The existence of a general multiaxial patterning system is proved by skeletal changes in the body axes of mutant mice.

The disruption of the Hox gene in mice causes the occipital bones of the skull to develop abnormally but only in regions anterior to the gene's usual area of expression. The ectopic expression of Hox 4.2 induces a homeotic alteration of the occipital bones into a posterior phenotype similar in shape to the cervical vertebrae. However, no such change affects structures ordinarily controlled by Hox 4.2. Thus, it is manifest that posterior Hox genes can reconfigure anterior skeletal arrangements.