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/bdeficient 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.he pancreas is a vertebrate-specific bifunctional organ that is composed of exocrine tissue for secretion of digestive enzymes and endocrine tissue for production of hormones involved in regulating glucose homeostasis. Morphogenesis of the developing pancreas has been well characterized in amniotes and other vertebrates (1). The zebrafish has emerged as a useful model organism for studying pancreas formation. As in mammals, the zebrafish pancreas develops from two distinct pancreatic anlagen of the endoderm, the dorsal-posterior bud and the ventral-anterior bud, which subsequently fuse to form the definitive pancreas. In zebrafish, the dorsal bud gives rise to the primary islet, whereas the ventral bud gives rise to exocrine cells, the pancreatic duct, and secondary islets (2, 3).Pancreas development is regulated by a network of transcription factors and signal transduction pathways. The Pdx1 homeobox factor is of critical importance to pancreas formation in the mouse (4, 5) and is the earliest marker for cells specified as pancreatic precursors in all animals studied including the zebrafish (6). The basic helix-loop-helix factor Ptf1a is essential for the development of exocrine precursor cells in the mouse (7) and zebrafish (8, 9) and, furthermore, represents a valuable early marker for exocrine precursors. Among multiple signaling pathways that have a role in the specification and differentiation of the pancreas, the Notch pathway has received particular attention. Studies in the mouse showed that artificial activation of the Notch pathway prevents precursors from differentiating into functional pancre...

PCR artifact in testing for homologous recombination in genomic editing in zebrafish

“…In earlier studies we found that the lnx2a gene is required for the differentiation of exocrine cells in the pancreas of zebrafish embryos [19]. During these studies we felt that it would be desirable to delete almost the entire lnx2a gene, a segment of about 29 kb, and replace it with a donor cassette that could assist in future studies of the locus.…”

“…The donor cassette was flanked by homology arms of about 1 kb on the 5’ and 3’ sides, as suggested by Zu et al, 2013 [11]. We used the previously described TALEN pair, found to be efficient in generating deletions in the lnx2a gene [19], to create a double stranded break in the locus to stimulate recombination. In addition we introduced reagents designed to inhibit nonhomologous end joining (NHEJ) and to stimulate homologous recombination, as suggested by Qi et al and Panier and Boulton [22,23].…”

“…Please refer to our earlier publication [19] for most of the materials and methods used. See S1 and S2 Figs for sequences of Donor DNA and homology arms.…”

“…In our studies of lnx2a gene function in pancreas development in zebrafish [19] we attempted replacement of essentially the entire gene by a donor cassette. When assayed by PCR, it appeared that our attempts had been successful.…”

PCR artifact in testing for homologous recombination in genomic editing in zebrafish

“…The donor cassette was flanked by homology arms of about 1 kb on the 5' and 3' sides, as suggested by Zu et al, 2013 [11]. We used the previously described TALEN pair, found to be efficient in generating deletions in the lnx2a gene [19], to create a double stranded break in the locus to stimulate recombination. In addition we introduced reagents designed to inhibit nonhomologous end joining and to stimulate homologous recombination, as suggested by Qi et al and Panier and Boulton [22,23].…”