Endothelin receptor B (EDNRB) is a G-protein-coupled receptor with seven transmembrane domains which is required for the development of melanocytes and enteric neurons. Mice that are homozygous for a null mutation in the Ednrb gene are almost completely white and die as juveniles from megacolon. To determine when EDNRB signalling is required during embryogenesis, we have exploited the tetracycline-inducible system to generate strains of mice in which the endogenous Ednrb locus is under the control of the tetracycline-dependant transactivators tTa or rtTA. By using this system to express Ednrb at different stages of embryogenesis, we have determined that EDNRB is required during a restricted period of neural crest development between embryonic days 10 and 12.5. Moreover, our results imply that EDNRB is required for the migration of both melanoblasts and enteric neuroblasts.
Mutations in the genes encoding endothelin receptor-B (Ednrb) and its ligand endothelin-3 (Edn3) affect the development of two neural crest-derived cell types, melanocytes and enteric neurons. EDNRB signaling is exclusively required between E10.5 and E12.5 during the migratory phase of melanoblast and enteric neuroblast development. To determine the fate of Ednrb-expressing cells during this critical period, we generated a strain of mice with the bacterial beta-galactosidase (lacZ) gene inserted downstream of the endogenous Ednrb promoter. The expression of the lacZ gene was detected in melanoblasts and precursors of the enteric neuron system (ENS), as well as other neural crest cells and nonneural crest-derived lineages. By comparing Ednrb(lacZ)/+ and Ednrb(lacZ)/Ednrb(lacZ) embryos, we determined that the Ednrb pathway is not required for the initial specification and dispersal of melanoblasts and ENS precursors from the neural crest progenitors. Rather, the EDNRB-mediated signaling is required for the terminal migration of melanoblasts and ENS precursors, and this pathway is not required for the survival of the migratory cells.
A c/s-acting negative regulator of splicing (NRS) within the gag gene of RSV is involved in control of the relative levels of spliced and unspliced viral mRNAs. Insertion of the NRS into the intron of an adenovirus pre-mRNA resulted in inhibition of splicing in vitro before the first cleavage step. Analyses of spliceosome assembly with this substrate showed that it formed large RNP complexes that did not migrate like mature spliceosomes on native gels. Affinity selection of the RNP complexes formed on NRS-containing pre-mRNAs showed an association with U l l and U12 snRNPs, as well as with the spliceosomal snRNPs. Immunoprecipitation with antisera specific for U1 and U2 snRNPS showed binding of both snRNPs to NRS RNA. A 7-nucleotide missense mutation in the NRS that prevented binding of U l l and U12 snRNPs impaired NRS activity in vivo, suggesting a functional role for U l l and U12 snRNPS in the inhibition of splicing mediated by the RSV NRS RNA.[Key Words: Retrovirus; RNA splicing; snRNPs] Received June 23, 1993; revised version accepted July 28, 1993.Retroviral replication involves reverse transcription of the RNA genome into DNA, which integrates into the host genome and is transcribed by cellular RNA polymerase II (for review, see Coffin 1990). While the viral RNA transcripts are processed, transported, and translated by cellular machinery, they yield both spliced and unspliced cytoplasmic mRNAs, unlike cellular premRNAs. The majority of the viral transcripts are not spliced but are transported to the cytoplasm, where they become full-length mRNA for Gag and Gag-Pol polyproteins, as well as packaged virion RNA. In the case of Rous sarcoma virus (RSV), the remainder of the primary transcripts are alternatively spliced in the nucleus to yield mRNAs for the env and src gene products. Production of infectious virus requires a balance of spliced and unspliced mRNAs.Splicing of mRNA precursors occurs in a dynamic macromolecular complex called a spliceosome; the reaction requires ATP, functional splice site and branchpoint sequences, small nuclear ribonucleoproteins (snRNPs), heterogeneous ribonucleoproteins (hnRNPs), and essential non-snRNP splicing factors (for review, see Krainer and Maniatis 1988; Green 1991). The spliceosomal snRNP particles U1, U2, U4/U6, and U5 are abundant in animal cells (10s-10 6 copies per cell) and participate in both steps of pre-mRNA splicing (for review, see Steitz et al. 1988). Genetic experiments have confirmed the sig- nificance of RNA-RNA base-pairing between U1 snRNA and the 5' splice site (Zhuang and Weiner 1986) and U2 snRNA and the branchpoint (Wu and Manley 1989; Zhuang and Weiner 1989). The U4/U6 and U5 snRNPs, which appear to associate as a multi-snRNP complex (Konarska and Sharp 1987), have also been identified as components of the mature spliceosome (Frendewey and Keller 1985; Bindereif and Green 1987). Other less abundant snRNPs have been identified whose functions have not been defined (Kramer 1987; Reddy and Busch 1988). For example, Wassarman and Steitz (1992...
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