Valérie Fraulob

Retinaldehyde dehydrogenase 2 (RALDH2)- independent patterns of retinoic acid synthesis in the mouse embryo

Knockout of the murine retinoic acid (RA)-synthesizing enzyme retinaldehyde dehydrogenase 2 (RALDH2) gene leads to early morphogenetic defects and embryonic lethality. Using a RA-responsive reporter transgene, we have looked for RA-generating activities in Raldh2-null mouse embryos and investigated whether these activities could be ascribed to the other known RALDH enzymes (RALDH1 and RALDH3). To this end, the early defects of Raldh2−/− embryos were rescued through maternal dietary RA supplementation under conditions that do not interfere with the activity of the reporter transgene in WT embryos. We show that RALDH2 is responsible for most of the patterns of reporter transgene activity in the spinal cord and trunk mesodermal derivatives. However, reporter transgene activity was selectively detected in Raldh2−/− embryos within the mesonephric area that expresses RALDH3 and in medial-ventral cells of the spinal cord and posterior hindbrain, up to the level of the fifth rhombomere. The craniofacial patterns of RA-reporter activity were unaltered in Raldh2−/− mutants. Although these patterns correlated with the presence of Raldh1 and/or Raldh3 transcripts in eye, nasal, and inner ear epithelia, no such correlation was found within forebrain neuroepithelium. These data suggest the existence of additional RA-generating activities in the differentiating forebrain, hindbrain, and spinal cord, which, along with RALDH1 and RALDH3, may account for the development of Raldh2−/− mutants once these have been rescued for early lethality.

Dynamic expression of retinoic acid‐synthesizing and ‐metabolizing enzymes in the developing mouse inner ear

Retinoic acid signaling plays essential roles in morphogenesis and neural development through transcriptional regulation of downstream target genes. It is believed that the balance between the activities of synthesizing and metabolizing enzymes determines the amount of active retinoic acid to which a developing tissue is exposed. In this study, we investigated spatiotemporal expression patterns of four synthesizing enzymes, the retinaldehyde dehydrogenases 1, 2, 3, and 4 (Raldh1, Raldh2, Raldh3, and Raldh4) and two metabolizing enzymes (Cyp26A1 and Cyp26B1) in the embryonic and postnatal mouse inner ear by using quantitative reverse transcriptase polymerase chain reaction (RT‐PCR), in situ hybridization, and Western blot analysis. Quantitative RT‐PCR analysis and Western blot data revealed that the expression of CYP26s was much higher than that of Raldhs at early embryonic ages but that Cyp26 expression was downregulated during embryonic development. Conversely, the expression levels of Raldh2 and ‐3 increased during development and were significantly higher than the Cyp26 levels at postnatal day 20. At this age, Raldh3 was expressed predominantly in the cochlea, whereas Raldh2 was present in the vestibular end organ. At early embryonic stages, as observed by in situ hybridization, the synthesizing enzymes were expressed only in the dorsoventral epithelium of the otocyst, whereas the metabolizing enzymes were present mainly in mesenchymal cells surrounding the otic epithelium. At later stages, Raldh2, Raldh3, and Cyp26B1 were confined to the stria vascularis, spiral ganglion, and supporting cells in the cochlear and vestibular epithelia, respectively. The downregulation of Cyp26s and the upregulation of Raldhs after birth during inner ear maturation suggest tissue changes in the sensitivity to retinoic acid concentrations. J. Comp. Neurol. 496:643–654, 2006.

The oxidizing enzyme CYP26a1 tightly regulates the availability of retinoic acid in the gastrulating mouse embryo to ensure proper head development and vasculogenesis.

Retinoic acid (RA) has been implicated as one of the signals providing a posterior character to the developing vertebrate central nervous system. Embryonic RA first appears in the posterior region of the gastrulating embryo up to the node level, where it may signal within the adjacent epiblast and/or newly induced neural plate to induce a hindbrain and spinal cord fate. Conversely, rostral head development requires forebrain‐inducing signals produced by the anterior visceral endoderm and/or prechordal mesoderm, and there is evidence that RA receptors must be in an unliganded state to ensure proper head development. As RA is a diffusible lipophilic molecule, some mechanism(s) must therefore have evolved to prevent activation of RA targets in anterior regions of the embryo. This might result from RA catabolism mediated by the CYP26A1 oxidizing enzyme, which is transiently expressed in anteriormost embryonic tissues; however, previous analysis of Cyp26a1−/− mouse mutants did not clearly support this hypothesis. Here we show that Cyp26a1−/− null mutants undergo head truncations when exposed to maternally‐derived RA, at doses that do not affect wild‐type head development. These anomalies are linked to a widespread ectopic RA signaling activity in rostral head tissues of CYP26A1‐deficient embryos. Thus, CYP26A1 is required in the anterior region of the gastrulating mouse embryo to prevent teratological effects that may result from RA signaling. We also report a novel role of CYP26A1 during early development of the intra‐ and extra‐embryonic vascular networks. Developmental Dynamics 236:644–653, 2007.

Fate of retinoic acid–activated embryonic cell lineages

Retinoic acid (RA), a vitamin A derivative, is synthesized by specific cell populations and acts as a diffusible embryonic signal activating ligand‐inducible transcription factors, the RA receptors (RARs). RA‐activatable transgenic systems have revealed many discrete, transient sites of RA action during development. However, there has been no attempt to permanently label the RA‐activated cell lineages during mouse ontogenesis. We describe the characterization of a RA‐activatable Cre transgene, which through crosses with a conditional reporter strain (the ROSA26R lacZ reporter), leads to a stable labeling of the cell populations experiencing RA signaling during embryogenesis. RA response‐element (RARE) ‐driven Cre activity mimics at early stages the known activity of the corresponding RARE‐lacZ transgene (Rossant et al., 1991 ). Stable labeling of the Cre‐excised cell populations allows to trace the distribution of the RA‐activated cell lineages at later stages. These are described in relationship with current models of RA activity in various developmental systems, including the embryonic caudal region, limb buds, hindbrain, sensory organs, and heart. Developmental Dynamics 239:3260–3274, 2010.

Retinoic acid controls early neurogenesis in the developing mouse cerebral cortex

A tight regulation of neuron production is required to generate a functional cerebral cortex and is achieved by a proper balance between proliferation and differentiation of progenitor cells. Though the vitamin A (retinol) active derivative retinoic acid (RA) has been implicated as one of the signals acting during mammalian forebrain neurogenesis, its function at the onset of neurogenesis as well as during establishment of cortical layers and neuronal subtypes remains elusive. One limitation is that murine mutants for genes encoding key enzymes involved in RA synthesis die during early embryonic development. We analysed corticogenesis in Rdh10 null mutants, in which an RA deficiency is generated as the intracellular retinol to retinaldehyde conversion is abolished. When analysed at the latest stage before lethality occurs (embryonic day [E]13.5), the mutants show smaller telencephalic vesicles and the thickness of their cortical plate is strongly reduced. The first progenitors formed in the cortical plate are radial glial (RG) cells which generate neurons either directly, or through an indirect mechanism involving the production of intermediate neuronal progenitors (INPs) which then give rise to neurons. We show that in absence of RA, the RG progenitors proliferate less and prematurely produce neurons, leading to their depletion at E11.5. Furthermore, we could demonstrate that lack of RA impairs the generation of INPs at E13.5 and affects the cell cycle exit of progenitor cells during corticogenesis, altogether leading to a deficit in projection neurons and to microcephaly.