title: Lnx2 ubiquitin ligase is essential for exocrine cell differentiation in the early zebrafish pancreas.
abstract: The gene encoding the E3 ubiquitin ligase Ligand of Numb protein-X (Lnx)2a is expressed in the ventral-anterior pancreatic bud of zebrafish embryos in addition to its expression in the brain. Knockdown of Lnx2a by using an exon 2/intron 2 splice morpholino resulted in specific inhibition of the differentiation of ventral bud derived exocrine cell types, with little effect on endocrine cell types. A frame shifting null mutation in lnx2a did not mimic this phenotype, but a mutation that removed the exon 2 splice donor site did. We found that Lnx2b functions in a redundant manner with its paralog Lnx2a. Inhibition of lnx2a exon 2/3 splicing causes exon 2 skipping and leads to the production of an N-truncated protein that acts as an interfering molecule. Thus, the phenotype characterized by inhibition of exocrine cell differentiation requires inactivation of both Lnx2a and Lnx2b. Human LNX1 is known to destabilize Numb, and we show that inhibition of Numb expression rescues the Lnx2a/b-deficient phenotype. Further, Lnx2a/b inhibition leads to a reduction in the number of Notch active cells in the pancreas. We suggest that Lnx2a/b function to fine tune the regulation of Notch through Numb in the differentiation of cell types in the early zebrafish pancreas. Further, the complex relationships among genotype, phenotype, and morpholino effect in this case may be instructive in the ongoing consideration of morpholino use.
authors.affiliation: Section on Developmental Biology, National Institute of Child Health and Human Development, Bethesda, MD 20892-2790.
authors.forename: Minho
authors.initials: M
authors.lastname: Won
authors.affiliation: Department of Biological Sciences, College of Bioscience and Biotechnology, Chungnam National University, Daejeon 305-764, Korea.
authors.forename: Hyunju
authors.initials: H
authors.lastname: Ro
authors.affiliation: Section on Developmental Biology, National Institute of Child Health and Human Development, Bethesda, MD 20892-2790, idawid@nih.gov.
authors.forename: Igor B
authors.initials: IB
authors.lastname: Dawid
mesh_terms: D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D002454:Cell Differentiation / Q000235:genetics*; D018507:Gene Expression Regulation, Developmental; D055785:Gene Knockdown Techniques; D057809:HEK293 Cells; D006801:Humans; D015151:Immunoblotting; D017403:In Situ Hybridization; D018613:Microscopy, Confocal; D060172:Morpholinos / Q000235:genetics; D009154:Mutation; D010179:Pancreas / Q000166:cytology / Q000196:embryology / Q000378:metabolism*; D046790:Pancreas, Exocrine / Q000166:cytology / Q000196:embryology / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D044767:Ubiquitin-Protein Ligases / Q000235:genetics* / Q000378:metabolism; D015027:Zebrafish / Q000196:embryology / Q000235:genetics / Q000378:metabolism; D029961:Zebrafish Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: D002352:Carrier Proteins; D060172:Morpholinos; D044767:Ubiquitin-Protein Ligases; D029961:Zebrafish Proteins; C570847:LNX2 protein, human; C554948:lnx2b protein, zebrafish
keywords: Notch; Numb; TALEN; morpholino; pancreas