The midbrain-hindbrain boundary (MHB) is a long-lasting organizing center in the vertebrate neural tube that is both necessary and sufficient for the ordered development of midbrain and anterior hindbrain (midbrain-hindbrain domain, MH). The MHB also coincides with a pool of progenitor cells that contributes neurons to the entire MH. Here we show that the organizing activity and progenitor state of the MHB are co-regulated by a single microRNA, miR-9, during late embryonic development in zebrafish. Endogenous miR-9 expression, initiated at late stages, selectively spares the MHB. Gain- and loss-of-function studies, in silico predictions and sensor assays in vivo demonstrate that miR-9 targets several components of the Fgf signaling pathway, thereby delimiting the organizing activity of the MHB. In addition, miR-9 promotes progression of neurogenesis in the MH, defining the MHB progenitor pool. Together, these findings highlight a previously unknown mechanism by which a single microRNA fine-tunes late MHB coherence via its co-regulation of patterning activities and neurogenesis.

IntroductionWe recently identi®ed the 37-kDa laminin receptor precursor (LRP) as an interactor for the prion protein (PrP) (Rieger et al., 1997; for reviews see Rieger et al., 1999;Gauczynski et al., 2001a). Employing a series of neuronal and non-neuronal cells, we proved that the 37-kDa LRP/67-kDa high-af®nity laminin receptor (LR) acts as the receptor for the cellular PrP (Gauczynski et al., 2001b). In the present manuscript we used the yeast twohybrid system and cell-binding studies on neuronal as well as non-neuronal cells involving the Semliki Forest virus (SFV) system (for reviews see Liljestrom and Garoff, 1991;Tubulekas et al., 1997) to identify domains on the PrP and the LRP involved in the PrP±LRP interaction on the cell surface. We identi®ed two binding domains for LRP on PrP termed PrPLRPbd1 and PrPLRPbd2. The ®rst one binds directly to LRP, whereas the second one depends on the presence of heparan sulfate proteoglycans (HSPGs) on the cell surface. The yeast two-hybrid system and cell-binding assays on wild-type and mutant HSPGde®cient Chinese hamster ovary (CHO) cells also identi®ed two binding domains for PrP on LRP.The relationship between 37-kDa LRP and 67-kDa LR is not yet fully understood and has been explained with homodimerization of 37-kDa LRP (Landowski et al., 1995) or an additional factor, such as a polypeptide (Castronovo et al., 1991), which might bind to 37-kDa LRP to form the 67-kDa form of the receptor. The 67-kDa heterodimer might be stabilized by hydrophobic interactions mediated by fatty acids such as palmitate, oleate and stearate bound to 37-kDa LRP and to a galectin-3 (gal-3) cross reacting polypeptide (Landowski et al., 1995;Buto et al., 1998). However, we recently proved that the b-galactoside lectin gal-3 is not present on the surface of neuronal or non-neuronal cells used for PrP-binding/ internalization studies (Gauczynski et al., 2001b) and antigal-3 antibodies failed to compete for the 37-kDa LRP/67-kDa LR-mediated binding and internalization of the cellular PrP (Gauczynski et al., 2001b), suggesting that gal-3 is not a partner of the 37-kDa LRP in this context. In this study we investigated by a yeast two-hybrid system analysis whether gal-3 interacts with 37-kDa LRP and/or the cellular PrP. In addition, we investigated whether 37-kDa LRP interacts with itself in the yeast two-hybrid and analysed the monomer/dimer status of the receptor by sizeexclusion chromatography. Both PrP (Gabizon et al., 1993;Caughey et al., 1994;Chen,S.G. et al., 1995;Brimacombe et al., 1999) and the 37-kDa/67-kDa LR (Guo et al., 1992;Kazmin et al., 2000) bind to heparan sulfates. HSPGs are required for the binding of the ®broblast growth factor (FGF) to its FGFR receptor (Yayon et al., 1991;Spivak et al., 1994;Venkataraman et al., 1999) and act as initial attachment receptors for bacteria (Chen,T. et al., 1995) and viruses including alphaviruses (Byrnes and Grif®n, 1998), human immunode®ciency virus (HIV) type 1 (Mondor et al., 1998) and vaccinia virus (Chung et al., 1998). Heparan sulfates are...

Current models of vertebrate adult neural stem cells are largely restricted to the rodent forebrain. To extract the general mechanisms of neural stem cell biology, we sought to identify new adult stem cell populations, in other model systems and/or brain areas. The teleost zebrafish appears to be an ideal system, as cell proliferation in the adult zebrafish brain is found in many more niches than in the mammalian brain. As a starting point towards identifying stem cell populations in this system, we used an embryonic neural stem cell marker, the E(spl) bHLH transcription factor Her5. We demonstrate that her5 expression is not restricted to embryonic neural progenitors, but also defines in the adult zebrafish brain a new proliferation zone at the junction between the mid-and hindbrain. We show that adult her5-expressing cells proliferate slowly, self-renew and express neural stem cell markers. Finally, using in vivo lineage tracing in her5:gfp transgenic animals, we demonstrate that the her5-positive population is multipotent, giving rise in situ to differentiated neurons and glia that populate the basal midbrain. Our findings conclusively identify a new population of adult neural stem cells, as well as their fate and their endogenous environment, in the intact vertebrate brain. This cell population, located outside the forebrain, provides a powerful model to assess the general mechanisms of vertebrate neural stem cell biology. In addition, the first transcription factor characteristic of this cell population, Her5, points to the E(Spl) as a promising family of candidate adult neural stem cell regulators.

The accumulation of PrPSc in scrapie‐infected neuronal cells has been prevented by three approaches: (i) transfection of ScMNB cells with an antisense laminin receptor precursor (LRP) RNA‐expression plasmid, (ii) transfection of ScN2a cells and ScGT1 cells with small interfering RNAs (siRNAs) specific for the LRP mRNA, and (iii) incubation of ScN2a cells with an anti‐LRP/LR antibody. LRP antisense RNA and LRP siRNAs reduced LRP/LR expression and inhibited the accumulation of PrPSc in these cells. The treatments also reduced PrPc levels. The anti‐LRP/LR antibody, W3, abolished PrPSc accumulation and reduced PrPc levels after seven days of incubation. Cells remained free of PrPSc after being cultured for 14 additional days without the antibody, whereas the PrPc level was restored. Our results demonstrate the necessity of the laminin receptor (LRP/LR) for PrPSc propagation in cultured cells and suggest that LRP/LR‐specific antibodies could be used as powerful therapeutic tools in the treatment of transmissible spongiform encephalopathies.

We implemented a noninvasive optical method for the fast control of Cre recombinase in single cells of a live zebrafish embryo. Optical uncaging of the caged precursor of a nonendogeneous steroid by one-or two-photon illumination was used to restore Cre activity of the CreER T2 fusion protein in specific target cells. This method labels single cells irreversibly by inducing recombination in an appropriate reporter transgenic animal and thereby can achieve high spatiotemporal resolution in the control of gene expression. This technique could be used more generally to investigate important physiological processes (e.g., in embryogenesis, organ regeneration, or carcinogenesis) with high spatiotemporal resolution (single cell and 10-min scales).