her1, a zebrafish pair-rule like gene, acts downstream of notch signalling to control somite development

Christina Takke Jos A. Campos-Ortega - control somite development SUMMARY During vertebrate embryonic development, the paraxial mesoderm becomes subdivided into metameric units known as somites. In the zebrafish embryo, genes encoding homologues of the proteins of the Drosophila NOTCH signalling pathway are expressed in the presomitic mesoderm and expression is maintained in a segmental pattern during somitogenesis. This expression pattern suggests a role for these genes during somite development. We misexpressed various zebrafish genes of this group by injecting mRNA into early embryos. RNA encoding a constitutively active form of NOTCH1a (notch1a-intra) and a truncated variant of deltaD [deltaD(Pst)], as well as transcripts of deltaC and deltaD, the hairy-E(spl) homologues her1 and her4, and groucho2 were tested for their effects on somite formation, myogenesis and on the pattern of transcription of putative downstream genes. In embryos injected with any of these RNAs, with the In Drosophila, the NOTCH signalling pathway has been studied chiefly from the point of view of neurogenesis, in particular with respect to its participation in the selection of neural progenitor cells (Campos-Ortega, 1993). However, in Drosophila, this regulatory pathway is also involved in several other developmental processes, including oogenesis (Ruohola et al., 1991; Xu et al., 1992), myogenesis (Corbin et al., 1991; Carmena et al., 1995), gut and heart development (Tepass and Hartenstein, 1994). Recent data indicate that homologues of the genes of the NOTCH pathway play crucial roles in vertebrate development as well. Besides their role in primary neurogenesis (Chitnis et al., 1995; Chitnis and Kintner, 1996; Dornseifer et al., 1997; Appel and Eisen, 1998; Haddon et al, 1998; Takke et al., 1999), there is growing evidence that a similar regulatory network is also involved in segmentation of the paraxial mesoderm. However, the situation in vertebrates is more complex, in part due to the presence of multiple Notch and Delta genes (Del Amo et al., 1992; Lardelli and Lendhal, 1993; Bierkamp and Campos-Ortega, 1993; Westin and Lardelli, 1997; Dornseifer et al., 1997; Appel and Eisen, 1998; Haddon et al., 1998). Mouse embryos deficient for Notch1 or for the Delta homologue Dll1 show severe somitic defects exception of groucho2 RNA, the paraxial mesoderm differentiated normally into somitic tissue, but failed to segment correctly. Activation of NOTCH results in ectopic activation of her1 and her4. This misregulation of the expression of her genes might be causally related to the observed mesodermal defects, as her1 and her4 mRNA injections led to effects similar to those seen with notch1aintra. deltaC and deltaD seem to function after subdivision of the presomitic mesoderm, since the her gene transcription pattern in the presomitic mesoderm remains essentially normal after misexpression of delta genes. Whereas NOTCH signalling alone apparently does not affect myogenesis, zebrafish groucho2 is involved in differentiation of mesodermal derivatives. (Conlon et al., 1995; Hrabe de Angelis et al., 1997). In Notch1D 1 mutant embryos, condensed unsegmented mesoderm was often present near the presomitic mesoderm. Later on, epithelialization sometimes appeared incomplete, and most of the newly formed somites were not as tightly packed as in the wild type (Conlon et al., 1995). In Dll1 mutant embryos, craniocaudal segment polarity appeared to be lost, no epithelialized somites were formed and myoblasts extended across the segment borders, suggesting that Dll1 is involved in compartmentalization of the somites and that muscle cell differentiation itself is an independent process (Hrabe de Angelis et al., 1997). From studies of the expression pattern of Dll1 and Dll3, Dunwoodie et al. (1997) concluded that the different members of this regulatory network act in a coordinate manner in establishing the intersomite boundaries. Similar somite defects to those described for the Dll1 and Notch1 mutants have been observed in mice that lack RBP-Jk (Oka et al., 1995; de la Pompa et al., 1997). This vertebrate homologue of the Drosophila Suppressor of Hairless product (Bailey and Posakony, 1995; Lecourtois and Schweisguth, 1995) thus appears to be required for at least some aspects of NOTCH signalling (Wettstein et al., 1997; Jen et al., 1997). While these observations suggest that the NOTCH pathway is required for segmentation of the paraxial mesoderm, it is not known how NOTCH performs this function, nor upon which step during segmentation it acts. In addition to being involved in segmentation, studies in vertebrates and invertebrates suggest that the LIN12/NOTCH receptors inhibit cell differentiation when activated by members of the DSL (for DELTA, SERRATE, Lag2) ligand family. Thus, the NOTCH signalling pathway appears to play a role in myogenesis as well. Both HES-1, a mouse homologue of E(SPL), and the intracellular domain of murine NOTCH are able to block myogenesis in vitro (Sasai et al., 1992; Nye et al., 1994; Kopan et al., 1994). However, the data suggest that inhibition of myogenesis by NOTCH is mediated by an RBPJk /CBF1-independent pathway (Shawber et al., 1996). Here we discuss some aspects of the function of the NOTCH signalling pathway in zebrafish segmentation and muscle development. Our results suggest that one of the zebrafish Notch genes, notch1 (Bierkamp and Campos-Ortega, 1993, corresponding to notch1a of Westin and Lardelli, 1997), acts during segmental prepatterning in the presomitic mesoderm. This function seems to be mediated by her1 and her4, both of which encode bHLH proteins of the HAIRY-E(SPL) family (Mller et al., 1996; Takke et al., 1999) and are misregulated following ectopic activation of NOTCH. Misexpression of deltaC and deltaD variants also causes somitic defects. However, neither delta gene affects the transcription pattern of her1 or her4; the delta genes thus appear to act on the establishment and/or maintenance of somite boundaries rather than on segmentation itself. Whereas NOTCH activation alone does not affect myogenesis, groucho2 is essential for the differentiation of mesodermal cells. MATERIALS AND METHODS Zebrafish embryos were obtained from spontaneous spawnings. Adult fish were kept at 28.5C on a 14 hour light/10 hour dark cycle. The embryos were staged according to Warga and Kimmel (1990) and Kimmel et al. (1995). mRNA injections Capped RNA was prepared from cDNAs cloned into the pCS2+ expression vector (Turner and Weintraub, 1994), as described in Takke et al. (1999), using a Message Kit from Ambion. 1-2 ng of the RNA were injected in a volume of 5 nl into one of the first two blastomeres. A 2976 bp cDNA clone (2/1 d) of deltaC (Haddon et al., 1998) was kindly provided by Julian Lewis (ICRF, London). PCR was used to obtain a full-length cDNA of deltaC from a randomly primed cDNA library made with RNA from 3-15 h (hour) zebrafish embryos. To facilitate clo (...truncated)