Onecut factors control development of the Locus Coeruleus and of the mesencephalic trigeminal nucleus.

The Locus Coeruleus (LC), the main noradrenergic nucleus in the vertebrate CNS, contributes to the regulation of several processes including arousal, sleep, adaptative behaviors and stress. Regulators controlling the formation of the LC have been identified but factors involved in its maintenance remain unknown. Here, we show that members of the Onecut (OC) family of transcription factors, namely HNF-6, OC-2 and OC-3, are required for maintenance of the LC phenotype. Indeed, in embryos lacking any OC proteins, LC neurons properly differentiate but abnormally migrate and eventually lose their noradrenergic characteristics. Surprisingly, the expression of Oc genes in these neurons is restricted to the earliest differentiation stages, suggesting that OC factors may regulate maintenance of the LC in a non cell-autonomous manner. Accordingly, the OC factors are present throughout development in a population directly adjacent to the LC, the rhombencephalic portion of the mesencephalic trigeminal nucleus (MTN). In the absence of OC factors, rhombencephalic MTN neurons fail to be generated, suggesting that OC proteins cell-autonomously control their production. Hence, we propose that OC factors are required at early developmental stages for differentiation of the MTN neurons that are in turn necessary for maintenance of the LC.

Dynamic expression of the Onecut transcription factors HNF-6, OC-2 and OC-3 during spinal motor neuron development.

The Onecut (OC) transcription factors, namely Hepatocyte nuclear factor 6 (HNF-6), OC-2 and OC-3, are transcriptional activators expressed in liver, pancreas and nervous system during development. Although their expression and roles in endoderm-derived tissues and in the trigeminal ganglia have been investigated, their expression in the CNS has not been described yet. In this study, we report a qualitative and quantitative expression profile of the OC factors in the developing spinal motor neurons (MN). We provide evidence that OC expression is initiated in newly-born MN. At later stages, they are differentially and dynamically expressed in subsets of differentiating motor neuron within the four motor columns. We also show that the expression profile of HNF-6 in spinal MN is conserved in chick embryos. Together, our data unveil a complex and dynamic expression profile of the OC proteins in spinal MN, which suggests that these factors may participate in regulatory networks that control different steps of motor neuron development.

[Control of bile duct and hepatic artery development by liver-specific transcription factors]. / Contrôle du développement des voies biliaires et de l'artère hépatique par des facteurs de transcription hépato-spécifiques.

During liver development, precursor cells called hepatoblasts differentiate into hepatocytes, which synthesize, degrade and store several molecules, or into cholangiocytes which form the bile ducts. The hepatic artery brings nutrients and oxygen to the hepatocytes and cholangiocytes. The aim of my work is to study the role of the transcription factor HNF-6 during liver development. In the course of this project, I made the first description of the development of the biliary tract and of the hepatic artery in the mouse. The analysis of transgenic mice in which the HNF-6 gene had been inactivated led me to the first identification of a genetic cascade that controls biliary development, a cascade wherein HNF-6 regulates the expression of the gene encoding the transcription factor HNF-1beta. Finally, I showed that biliary anomalies can cause perturbations in the development of the hepatic artery.

Absence of ventral cell populations in the developing brain in a rat model of the Smith-Lemli-Opitz syndrome.

The Smith-Lemli-Opitz syndrome (SLOS) is an autosomal recessive condition involving craniofacial and central nervous system malformations with occasional holoprosencephaly (HPE). It is caused by a defect in the 7-dehydrocholesterol (7-DHC) reductase, the enzyme catalyzing the last step of cholesterol biosynthesis. Treatment of pregnant rats with inhibitors of 7-DHC reductase, either AY9944 or BM15.766, has provided a valuable model to study the pathogenesis in SLOS. Recently, cholesterol has been shown to be involved in the post-translational activation of the signaling protein Sonic Hedgehog. To identify the early defects associated with HPE in a rat model of SLOS, and to compare the phenotype of the treated embryos with that of the Shh(-/-) mutants, we examined brain morphology and expression of three developmental genes (Shh, Otx2, and Pax6 ) in 23-somite stage embryos from AY9944-treated dams. We report clearly abnormal morphology of the developing brain, concerning primarily the ventral aspect of the neural tube. We observed a reduced or absent expression of Shh and Otx2 in their ventral domain associated with extended ventral expression of Pax6. The results suggest an absence of the midline ventral cell type at all levels of the cranial neural tube. They provide further evidence that cholesterol-deficiency-induced HPE originates from impaired Shh signaling activity in the ventral neural tube.

Targeted deficiency or cytosolic truncation of the VE-cadherin gene in mice impairs VEGF-mediated endothelial survival and angiogenesis.

Vascular endothelial cadherin, VE-cadherin, mediates adhesion between endothelial cells and may affect vascular morphogenesis via intracellular signaling, but the nature of these signals remains unknown. Here, targeted inactivation (VEC-/-) or truncation of the beta-catenin-binding cytosolic domain (VECdeltaC/deltaC) of the VE-cadherin gene was found not to affect assembly of endothelial cells in vascular plexi, but to impair their subsequent remodeling and maturation, causing lethality at 9.5 days of gestation. Deficiency or truncation of VE-cadherin induced endothelial apoptosis and abolished transmission of the endothelial survival signal by VEGF-A to Akt kinase and Bcl2 via reduced complex formation with VEGF receptor-2, beta-catenin, and phosphoinositide 3 (PI3)-kinase. Thus, VE-cadherin/ beta-catenin signaling controls endothelial survival.

All-trans-retinoic acid upregulates the expression of COUP-TFI in early-somite mouse embryos cultured in vitro.

Exposure of embryos to an excess of retinoic acid (RA) modifies the spatio-temporal pattern of expression of developmental genes. RA regulates the expression of target genes through binding of the retinoid nuclear receptors (RARs and RXRs), as heterodimers, to regulatory cis-acting elements. COUP-TF factors, which are able to dimerize with the RXRs and to compete with the retinoid receptors for their DNA binding sites, are suspected to modulate the retinoid signal transduction pathway. Therefore, COUP-TF factors may be involved in the regulation of the expression of developmental genes and/or in the modifications induced by an excess of RA in the expression of these genes. The aim of this work is to assess whether RA-induced modifications in the expression of Krox-20 and Hox genes correlate with alterations of the expression of COUP-TF genes. In addition to spatial modifications in the expression patterns of Krox-20 and Hox genes, we report here an upregulation of the expression level of COUP-TFI after RA exposure. However, this abnormality did not spatially overlap with the modifications observed in the expression of Krox-20 and Hox genes. These data suggest an involvement of COUP-TFI in the generation of RA-induced abnormalities, but do not support the hypothesis of an involvement of this factor in the regulation of the expression of Hox or Krox-20 genes.

Mice lacking factor VII develop normally but suffer fatal perinatal bleeding.

Blood coagulation in vivo is initiated by factor VII (FVII) binding to its cellular receptor tissue factor (TF). FVII is the only known ligand for TF, so it was expected that FVII-deficient embryos would have a similar phenotype to TF-deficient embryos, which have defective vitello-embryonic circulation and die around 9.5 days of gestation. Surprisingly, we find that FVII-deficient (FVII-/-) embryos developed normally. FVII-/- mice succumbed perinatally because of fatal haemorrhaging from normal blood vessels. At embryonic day 9.5, maternal-fetal transfer of FVII was undetectable and survival of embryos did not depend on TF-FVII-initiated fibrin formation. Thus, the TF-/- embryonic lethal and the FVII-/- survival-phenotypes suggest a role for TF during embryogenesis beyond fibrin formation.

Retinoic acid induces a tissue-specific deletion in the expression domain of Otx2.

The expression domain of Otx2, a gene essential for the development of the fore- and midbrain, has previously been shown to be affected by exposure to all-trans-retinoic acid (AT-RA). However, morphological abnormalities of the fore- and midbrain induced by exposure of early somite-stage embryos to AT-RA were not associated with abnormal Otx2 expression. To identify abnormal expression of developmental genes induced by exposure at early somite-stages, we performed a fine analysis of the expression domains of Otx2, Otx1, Emx2, and Pax-6 by combining in situ hybridization (ISH) with computer-assisted superpositions and three-dimensional reconstructions of these expression domains. No alteration in the relative location of the caudal boundaries of the expression domains of these genes was observed. The only abnormality was a deletion of the most cranial portion of the neural folds (NF).

Postimplantation mouse embryos cultured in vitro. Assessment with whole-mount immunostaining and in situ hybridization.

The postimplantation embryos of rodents have been particularly convenient to study in culture using the whole embryo culture (WEC) system developed by New. Two serious limitations of the method will be illustrated in the present paper and proposals will be made to improve the quality of the information. The first limitation is that the developmental period amenable to culture has not been significantly extended in recent years. In the present paper, we show that the culture of mouse presomitic stages for 48 h leads to poorly reproducible results and frequent dysmorphogenic embryos. We also show that early somite stages cultured for 54 h or less have a normal growth and differentiation. In contrast, the culture of these embryos for 72 h results in subtle abnormalities of the head and the first branchial arch. The second limitation is that the gross morphology and histology are often not informative enough to distinguish between overall toxicity and developmental toxicity. We suggest some improvements by the association of WEC with two specific techniques: 1) whole-mount immunostaining of sensory ganglia and nerves and 2) in situ hybridization on histological sections using molecular probes for some developmental genes. Embryos reaching about the 30 somite stage at the end of the culture were processed for whole-mount immunostaining of sensory ganglia and nerves. We show that these structures are very sensitive to the noxious effects of HgCl2 and valproate. Both developmental retardations and dysmorphogeneses of the cervical ganglia and nerves were observed. Embryos were also exposed in vitro to low concentrations of all-trans-retinoic acid (AT-RA) and processed for in situ hybridization with radiolabeled anti-sense RNA probes for the Hoxb-1 and Hoxb-2 developmental genes. Three-dimensional reconstructions of the expression domains were performed. The data show that AT-RA induces ectopic expression domains of Hoxb-1. Our experiments demonstrate that techniques such as immunostaining and in situ hybridization can significantly expand the information obtained from whole postimplantation embryo culture.

HNF-6 is expressed in endoderm derivatives and nervous system of the mouse embryo and participates to the cross-regulatory network of liver-enriched transcription factors.

Hepatocyte nuclear factor-6 (HNF-6) is a liver-enriched transcription factor that contains a single cut domain and a novel type of homeodomain. Here we have studied the developmental expression pattern of HNF-6 in the mouse. In situ hybridization experiments showed that HNF-6 mRNA is detected in the liver at embryonic day (E) 9, at the onset of liver differentiation. HNF-6 mRNA disappeared transiently from the liver between E12.5 and E15. In transfection experiments HNF-6 stimulated the expression of HNF-4 and of HNF-3 beta, two transcription factors known to be involved in liver development and differentiation. HNF-6 was detected in the pancreas from E10.5 onward, where it was restricted to the exocrine cells. HNF-6 was also detected in the developing nervous system. Both the brain and the spinal cord started to express HNF-6 at E9-9.5 in postmitotic neuroblasts. Later on, HNF-6 was restricted to brain nuclei, to the retina, to the ventral horn of the spinal cord, and to dorsal root ganglia. Our observations that HNF-6 contributes to the control of the expression of transcription factors and is expressed at early stages of liver, pancreas, and neuronal differentiation suggest that HNF-6 regulates several developmental programs.

Alterations of mouse embryonic branchial nerves and ganglia induced by ethanol.

An immunostaining technique using monoclonal antibodies to a neurofilament protein has allowed us to visualize defects in the development of cranial nerves and ganglia of 10 to 10.5 days mouse embryos following exposure to ethanol in whole embryo culture. Reference patterns for development of cranial nerves and ganglia of control mouse embryos explanted and examined when they had 25 to 34 pairs of somites were established. Additionally, control mouse embryos were grown in whole embryo culture for 48 h, with culture being initiated in embryos having 6 to 7 somite pairs. At the end of the culture period, only minor differences were observed between the control groups. An experimental group of embryos was cultured in the presence of increasing doses (1.6, 3.2, 4, and 4.8 g/l) of ethanol. Defects were observed in the development of the glossopharyngeal and vagus nerves. These abnormalities included absence of the dorsal root (superior ganglion) of IX, star-like shape of inferior ganglion IX, disorganization of the rootlets of nerve X and abnormal fibers between the two nerves and ganglia. These results suggest that the migration and patterning of neural crest cells derived from r6 and r7 may be particularly affected by ethanol. The results also demonstrate the usefulness of this approach in evaluating the susceptibility of the developing cranial nerves to toxicant exposure.