Mikihito Shibata

MicroRNA-9 Regulates Neurogenesis in Mouse Telencephalon by Targeting Multiple Transcription Factors

microRNA-9-2 and microRNA-9-3 double-mutant mice demonstrate that microRNA-9 (miR-9) controls neural progenitor proliferation and differentiation in the developing telencephalon by regulating the expression of multiple transcription factors. As suggested by our previous study, the Foxg1 expression was elevated, and the production of Cajal-Retzius cells and early-born neurons was suppressed in the miR-9-2/3 double-mutant pallium. At embryonic day 16.5 (E16.5), however, the Foxg1 expression was no longer elevated. The expression of an AU-rich RNA-binding protein Elavl2 increased at E16.5, Elav2 associated with Foxg1 3′ untranslated region (UTR), and it countered the Foxg1 suppression by miR-9. Later, progenitor proliferation was reduced in the miR-9-2/3 double-mutant pallium with the decrease in Nr2e1 and Pax6 expression and the increase in Meis2 expression. The analyses suggest that microRNA-9 indirectly inhibits Pax6 expression by suppressing Meis2 expression. In contrast, together with Elavl1 and Msi1, microRNA-9 targets Nr2e1 mRNA 3′ UTR to enhance the expression. Concomitantly, cortical layers were reduced, each cortical projection was malformed, and the tangential migration of interneurons into the pallium was impaired in the miR-9-2/3 double mutants. miR-9 also targets Gsh2 3′ UTR, and Gsh2, as well as Foxg1, expression was elevated in the miR-9-2/3 double-mutant subpallium. The subpallium progenitor proliferation was enhanced, and the development of basal ganglia including striatum and globus pallidus was suppressed. Pallial/subpallial boundary shifted dorsally, and the ventral pallium was lost. Corridor was malformed, and thalamocortical and corticofugal axons were misrouted in the miR-9-2/3 double mutants.

MicroRNA-9 Modulates Cajal–Retzius Cell Differentiation by Suppressing Foxg1 Expression in Mouse Medial Pallium

Vertebrate brain hosts a diverse collection of microRNAs, but little is known about their functions in vivo. Here we propose that mouse microRNA-9 (miR-9) targets Foxg1 mRNAs for proper generation of Cajal–Retzius cells in the medial pallium. miR-9 expression is mediolaterally graded, being most intense in the cortical hem; it contrasts with the Foxg1 expression in a reciprocal gradient. The 3′ untranslated regions of tetrapod, but not of teleost, Foxg1 mRNAs conserve miR-9 target sequences and are regulated by miR-9. Gain- and loss-of-function analyses of miR-9 showed that miR-9 negatively regulates endogenous Foxg1 protein level. Moreover, miR-9 overexpression in developing telencephalon at E11.5 by electroporation resulted in ectopic Reelin-positive cells over the cortex beyond the marginal zone. In addition, inhibition of endogenous miR-9 function by antisense oligonucleotides caused the regression of Wnt3a-positive cortical hem and reduction of reelin-, p73-, and NeuroD1-positive cells.

Xenopus crescent encoding a Frizzled-like domain is expressed in the Spemann organizer and pronephros

The Spemann organizer can be subdivided into head- and trunk-inducing tissues along the anteroposterior axis (Mangold, 1933. Naturwiisenschaften 43, 761-766; Spemann, 1931. Wilhelm Roux Arch. Entwicklungsmech. Org. 123, 389-517). Recent studies have suggested that head formation is brought about by repression of both Wnt and BMP signalling (Glinka et al., 1998. Nature 391, 357-362; Glinka et al., 1997. Nature 389, 517-519). Several Wnt inhibitors secreted from the head organizer region have been identified in Xenopus, such as Cerberus (Bouwmeester et al., 1996. Nature 382, 595-601), Frzb-1 (Leyns et al., 1997. Cell 88, 747-756; Lin et al., 1997. Proc. Natl. Acad. Sci. USA 94, 11196-11200), and Dkk-1 (Glinka et al., 1998. Nature 391, 357-362), supporting this two-inhibitor model. To isolate genes expressed in the head organizer, we screened a prechordal plate cDNA library by sequencing and expression pattern, and isolated the Xenopus ortholog of chick crescent encoding a Frizzled-like domain that is related to Wnt-binding regions of the Frizzled-family proteins. Expression of Xenopus crescent was first detected in the Spemann organizer region at the early gastrula stage and later in prechordal plate cells lining the boundary of mesoderm and ectoderm layers and in the anterior endoderm. At tailbud stages, the expression in the endomesoderm region was diminished, while expression in the pronephros became detectable. In animal cap assays, crescent gene was synergistically upregulated by coexpression of Xlim1, Ldb1, and Siamois, but not by Activin treatment.

Role of crescent in convergent extension movements by modulating Wnt signaling in early Xenopus embryogenesis

The Xenopus gene crescent encodes a member of the secreted Frizzled-related protein (sFRP) family and is expressed in the head organizer region. However, the target and function of Crescent in early development are not well understood. Here, we describe a role of Crescent in the regulation of convergent extension movements (CEMs) during gastrulation and neurulation. We show that overexpression of Crescent in whole embryos or animal caps inhibits CEMs without affecting tissue specification. Consistent with this, Crescent efficiently forms complexes with Xwnt11 and Xwnt5a, in contrast to another sFRP, Frzb1. As expected, the inhibitory effect of Crescent or Xwnt11 on CEMs is cancelled when both proteins are coexpressed in the neuroectoderm. Interestingly, when coexpressed in the dorsal mesoderm, the activity of Xwnt11 is rather enhanced by Crescent. Supporting this finding, the inhibition of CEMs by Crescent in mesodermalized but not neuralized animal caps is reversed by the dominant-negative form of Cdc42, a putative mediator of Wnt/Ca2+ pathway. Antisense morpholino oligos for Crescent impair neural plate closure and elicit microcephalic embryos with a shortened trunk without affecting early tissue specification. These data suggest a potential role for Crescent in head formation by regulating a non-canonical Wnt pathway positively in the adjacent posterior mesoderm and negatively in the overlying anterior neuroectoderm.

Gene expression pattern analysis of the tight junction protein, Claudin, in the early morphogenesis of Xenopus embryos

To study how epithelial layers are formed during early development in Xenopus embryos, we have focused on Claudin, the major component of the tight junction. So far, 19 claudin genes have been found in the mouse, expressed in different epithelial tissues. However, though a number of cytological studies have been done for the roles of Claudins, their expression patterns and functions during early embryogenesis are largely unknown. We found three novel Xenopus claudin genes, which are referred to as claudin-4L1, -4L2, and -7L1. At the early gastrula stage, claudin-4L1, -4L2, and -7L1 mRNAs were detected in the ectoderm and in the mesoderm. At the late gastrula stage, claudin mRNAs were detected in the ectoderm through the involuting archenteron roof. At the neurula stage, claudin-4L1/4L2 and -7L1 mRNAs were differentially expressed in the neural groove and the epidermal ectoderm. At the tailbud stage, the claudin mRNAs were found in the branchial arches, the otic vesicles, the sensorial layer of the epidermis, and along the dorsal midline of the neural tube. In addition, claudin-4L1/4L2 mRNAs were detected in the pronephros and the endoderm, whereas claudin-7L1 mRNA was observed in the epithelial layer of the epidermis.

RETRACTED: Gene expression pattern analysis of the tight junction protein, Claudin, in the early morphogenesis of Xenopus embryos

To study how epithelial layers are formed during early development in Xenopus embryos, we have focused on Claudin, the major component of the tight junction. So far, 19 claudin genes have been found in the mouse, expressed in different epithelial tissues. However, though a number of cytological studies have been done for the roles of Claudins, their expression patterns and functions during early embryogenesis are largely unknown. We found three novel Xenopus claudin genes, which are referred to as claudin-4L1, -4L2, and -7L1. At the early gastrula stage, claudin-4L1, -4L2, and -7L1 mRNAs were detected in the ectoderm and in the mesoderm. At the late gastrula stage, claudin mRNAs were detected in the ectoderm through the involuting archenteron roof. At the neurula stage, claudin-4L1/4L2 and -7L1 mRNAs were differentially expressed in the neural groove and the epidermal ectoderm. At the tailbud stage, the claudin mRNAs were found in the branchial arches, the otic vesicles, the sensorial layer of the epidermis, and along the dorsal midline of the neural tube. In addition, claudin-4L1/4L2 mRNAs were detected in the pronephros and the endoderm, whereas claudin-7L1 mRNA was observed in the epithelial layer of the epidermis.

01-P006 MicroRNA-9 regulates early neurogenesis and morphogenesis in mouse telencephalon

revealed a marked reduction in radial thickness starting at E13.5, and defective postnatal cortical layering. Whereas the former was due to neuronal apoptosis starting at E12.5, which was the earliest detectable phenotype, the latter reflected dramatic impairment of neuronal differentiation. Remarkably, the primary target cells of Dicer ablation, the neuroepithelial cells, and the neurogenic progenitors derived from them, were unaffected by miRNA depletion with regard to cell cycle progression, cell division, differentiation and viability during the early stage of neurogenesis, and only underwent apoptosis starting at E14.5. Our results support the emerging concept that progenitors are less dependent on miRNAs than their differentiated progeny. In order to identify miRNAs targets, we extracted total RNA from E13.5 cortices of Dicer-ablated and of control littermates, and mRNAs were subjected to microarray analysis. Our results show that upon miRNA depletion the number of upregulated mRNAs is higher than that of downregulated ones, suggesting that identified mRNAs might represent primary miRNAs targets rather than mRNAs indirectly affected by miRNAs depletion.

The developing Xenopus embryo as a complex system: Maternal and zygotic contribution of gene products in nucleo-cytoplasmic and cell-to-cell interactions

The developing animal embryo constitutes a complex system in which various nucleo-cytoplasmic (N-C) and cell-to-cell (C-C) interactions take place. In that sense, it is possible to define early embryogenesis as a function of these interactions, as for instance is expressed by a formula “Development = f (N-C, C-C)”. We present here our recent studies on temporal and spatial control of the expression of genes in zygotic nucleus and of genes exogenously introduced in Xenopus embryos. For zygotic gene expression, our studies revealed that the syntheses of mRNA, tRNA and rRNA are initiated at the cleavage stage, the stage of midblastula transition (MBT) and late blastula stage, respectively. For exogenously-injected genes, we summarize their expression pattern which is controlled by the promoter they carry in addition to the cytological effects of the injection. We also briefly present our recent results obtained with embryos which had been injected with in vitro-transcribed mRNAs.