Hideta Fujii

Metabolic inactivation of retinoic acid by a novel P450 differentially expressed in developing mouse embryos

Retinoic acid (RA) is a physiological agent that has a wide range of biological activity and appears to regulate developmental programs of vertebrates. However, little is known about the molecular basis of its metabolism. Here we have identified a novel cytochrome P450 (P450RA) that specifically metabolizes RA. In vitro, P450RA converts all‐trans RA into 5,8‐epoxy all‐trans RA. P450RA metabolizes other biologically active RAs such as 9‐cis RA and 13‐cis RA, but fails to metabolize their precursors, retinol and retinal. Overexpression of P450RA in cell culture renders the cells hyposensitive to all‐trans RA. These functional tests in vitro and in vivo indicate that P450RA inactivates RA. The P450RA gene is not expressed uniformly but in a stage‐ and region‐specific fashion during mouse development. The major expression domains in developing embryos include the posterior neural plate and neural crest cells for cranial ganglia. The expression of P450RA, however, is not necessarily inducible by excess RA. These results suggest that P450RA regulates the intracellular level of RA and may be involved in setting up the uneven distribution of active RA in mammalian embryos.

cDNA cloning and tissue-specific expression of a novel basic helix-loop-helix/PAS factor (Arnt2) with close sequence similarity to the aryl hydrocarbon receptor nuclear translocator (Arnt).

We isolated mouse cDNA clones (Arnt2) that are highly similar to but distinct from the aryl hydrocarbon receptor (AhR) nuclear translocator (Arnt). The composite cDNA covered a 2,443-bp sequence consisting of a putative 2,136-bp open reading frame encoding a polypeptide of 712 amino acids. The predicted Arnt2 polypeptide carries a characteristic basic helix-loop-helix (bHLH)/PAS motif in its N-terminal region with close similarity (81% identity) to that of mouse Arnt and has an overall sequence identity of 57% with Arnt. Biochemical properties and interaction of Arnt2 with other bHLH/PAS proteins were investigated by coimmunoprecipitation assays, gel mobility shift assays, and the yeast two-hybrid system. Arnt2 interacted with AhR and mouse Sim as efficiently as Arnt, and the Arnt2-AhR complex recognized and bound specifically the xenobiotic responsive element (XRE) sequence. Expression of Arnt2 successfully rescued XRE-driven reporter gene activity in the Arnt-defective c4 mutant of Hepa-1 cells. RNA blot analysis revealed that expression of Arnt2 mRNA was restricted to the brains and kidneys of adult mice, while Arnt mRNA was expressed ubiquitously. In addition, whole-mount in situ hybridization of 9.5-day mouse embryos showed that Arnt2 mRNA was expressed in the dorsal neural tube and branchial arch 1, while Arnt transcripts were detected broadly in various tissues of mesodermal and endodermal origins. These results suggest that Arnt2 may play different roles from Arnt both in adult mice and in developing embryos. Finally, sequence comparison of the currently known bHLH/PAS proteins indicates a division into two phylogenetic groups: the Arnt group, containing Arnt, Arnt2, and Per, and the AhR group, consisting of AhR, Sim, and Hif-1alpha.

TWO NEW MEMBERS OF THE MURINE SIM GENE FAMILY ARE TRANSCRIPTIONAL REPRESSORS AND SHOW DIFFERENT EXPRESSION PATTERNS DURING MOUSE EMBRYOGENESIS

From a cDNA library of mouse skeletal muscle, we have isolated mouse Sim1 (mSim1) cDNA encoding a polypeptide of 765 amino acids with striking amino acid identify in basic helix-loop-helix (89% identify) and PAS (89 % identify) domains to previously identified mSim2, although the carboxy-terminal third of the molecule did not show any similarity to mSim2 or Drosophila Sim (dSim). Yeast two-hybrid analysis and coimmunoprecipitation experiments demonstrated that both of the mSim gene products interacted with Arnt even more efficiently than AhR, a natural partner of Arnt, suggesting a functional cooperativity with Arnt. In sharp contrast with dSim having transcriptional-enhancing activity in the carboxy-terminal region, the two mSims possessed a repressive activity toward Arnt in the heterodimer complex. This is the first example of bHLH-PAS proteins with transrepressor activity, although some genetic data suggest that dSim plays a repressive role in gene expression (Z. Chang, D. Price, S. Bockheim, M. J. Boedigheimer, R. Smith, and A. Laughon, Dev. Biol. 160:315-322, 1993; D. M. Mellerick and M. Nirenberg, Dev. Biol. 171:306-316, 1995). Whole-mount in situ hybridization showed restricted and characteristic expression patterns of the two mSim mRNAs in various tissues and organs during embryogenesis, such as those for the somite, the nephrogenic cord, and the mesencephalon (for mSim1) and those for the diencephalon, branchial arches, and limbs (for mSim2). From sequence similarity and chromosomal localization, it is concluded that mSim2 is an ortholog of hSim2, which is proposed to be a candidate gene responsible for Downs syndrome. The sites of mSim2 expression showed an overlap with the affected regions of the syndrome, further strengthening involvement of mSim2 in Downs syndrome.

Inhibition of Nodal signalling by Lefty mediated through interaction with common receptors and efficient diffusion

Background: Two TGFβ‐related proteins, Nodal and Lefty, are implicated in early embryonic patterning of vertebrates. Genetic data suggest that Nodal is a signalling molecule, while Lefty is an antagonist of Nodal, but their precise function remains unknown.

GFRα3, a Component of the Artemin Receptor, Is Required for Migration and Survival of the Superior Cervical Ganglion

Abstract GFRα3 is a component of the receptor for the neurotrophic factor artemin. The role of GFRα3 in nervous system development was examined by generating mice in which the Gfrα3 gene was disrupted. The Gfrα3 −/− mice exhibited severe defects in the superior cervical ganglion (SCG), whereas other ganglia appeared normal. SCG precursor cells in the mutant embryos failed to migrate to the correct position, and they subsequently failed to innervate the target organs. In wild-type embryos, Gfrα3 was expressed in migrating SCG precursors, and artemin was expressed in and near the SCG. After birth, SCG neurons in the mutant mice underwent progressive cell death. These observations suggest that GFRα3-mediated signaling is required both for the rostral migration of SCG precursors and for the survival of mature SCG neurons.

Meteorin: a secreted protein that regulates glial cell differentiation and promotes axonal extension

Glial cells are major components of the nervous system. The roles of these cells are not fully understood, however. We have now identified a secreted protein, designated Meteorin, that is expressed in undifferentiated neural progenitors and in the astrocyte lineage, including radial glia. Meteorin selectively promoted astrocyte formation from mouse cerebrocortical neurospheres in differentiation culture, whereas it induced cerebellar astrocytes to become radial glia. Meteorin also induced axonal extension in small and intermediate neurons of sensory ganglia by activating nearby satellite glia. These observations suggest that Meteorin plays important roles in both glial cell differentiation and axonal network formation during neurogenesis.

A CNS-specific POU transcription factor, Brn-2, is required for establishing mammalian neural cell lineages

The pluripotent embryonal carcinoma cell line P19 can differentiate in vitro into neurons and astrocytes. By employing this neural cell differentiation system, we have studied the expression and function of a POU transcription factor implicated in mammalian neurogenesis. When P19 cells differentiated to the neural cells, one of the CNS-specific POU genes (Brn-2) was selectively induced. The induction of Brn-2 was specific to the neural cell lineages and took place at the early stage of differentiation. When the Brn-2 induction was blocked/delayed by antisense RNA, such cells were unable to differentiate to neurons and astrocytes. Instead they differentiated to nonneural cells, including smooth and skeletal muscle cells. Furthermore, with reduced levels of antisense RNA, differentiation to neural cells occurred. These results indicate that Brn-2 is essential for the neural cell differentiation of P19 cells and suggest that Brn-2 is one of the genes required for establishing neural cell lineages in mammals.

Hybrid cell extinction and re-expression of Oct-3 function correlates with differentiation potential.

The Oct‐3 gene is expressed in highly undifferentiated cells and is implicated in mammalian early embryogenesis. We have generated a series of hybrid cells between pluripotent embryonal carcinoma cells (Oct‐3+) and fibroblasts (Oct‐3‐), and have studied the regulation and function of Oct‐3. Upon fusion, the hybrid cells differentiated to nestin+/Brn‐2+ cells resembling neuroepithelial stem cells. Expression of Oct‐3 was extinguished at the transcriptional level in all the hybrid cells examined. The Oct‐3 modulating activity required for the Oct‐3‐mediated enhancer activation was also extinguished. When the Oct‐3 transactivating function was introduced into the hybrid cells, they transformed into morphologically distinct nestin‐/Brn‐2‐ cells (‘revertants’). When the ‘revertant’ cells subsequently lost Oct‐3 expression, they differentiated back to nestin+/Brn‐2+ cells. The close correlation between the phenotypic changes and the gain/loss of Oct‐3 function indicates that Oct‐3 can induce dedifferentiation of the neural cells.

Identification of putative downstream genes of Oct-3, a pluripotent cell-specific transcription factor

Background:  Oct‐3, a pluripotent cell‐specific POU transcription factor, appears to be a key regulator in pluripotential early embryonic cells and germ cells. In order to study how pluripotency is maintained, it is essential to know what genes are regulated by Oct‐3

Preferential Differentiation of P19 Mouse Embryonal Carcinoma Cells Into Smooth Muscle Cells Use of Retinoic Acid and Antisense Against the Central Nervous System–Specific POU Transcription Factor Brn-2

Investigation of the molecular mechanisms that control smooth muscle cell (SMC) development and differentiation is a prerequisite in understanding the regulatory mechanisms of physiological and pathological SMC-associated vascular processes. The pluripotent murine embryonal carcinoma P19 cell, whose developmental potential resembles that of early embryonic cells, can develop into cell types derived from the neuroectoderm, mesoderm, and endoderm. In the present study, we have shown a unique strategy to enhance SMC differentiation in P19 cells. Under chemical induction of high concentrations of retinoic acid (1 micromol/L), P19 cells showed optimum differentiation into SMCs. Because the P19 cells thus induced also showed differentiation into neuronal cells, a strategy to block neuronal lineage differentiation was developed using a stable transformant antisense RNA construct against Brn-2, a neuronal lineage-specific POU-domain transcription factor; thus, by specifically inhibiting neuronal differentiation, enhanced SMC differentiation by P19 cells was attained. SMC expression was confirmed by immunohistochemical staining, RNA analysis (RNase protection assay), and protein analysis (Western blot) using SMC-specific markers (eg, SM1 and calponin) and alpha-smooth muscle actin. Our results show that the pathway of SMC differentiation may provide an in vitro system useful in the investigation of SMC regulatory mechanisms (eg, transcriptional regulation) and in the further understanding of SMC development and differentiation.