Targeted ablation of beta cells in the embryonic zebrafish pancreas using E. coli nitroreductase

Abstract

In order to generate a zebrafish model of beta cell regeneration, we have expressed an Escherichia coli gene called nfsB in the beta cells of embryonic zebrafish. This bacterial gene encodes a nitroreductase (NTR) enzyme, which can convert prodrugs such as metronidazole (Met) to cytotoxins. By fusing nfsB to mCherry, we can simultaneously render beta cells susceptible to prodrug and visualize Met dependent cell ablation. We show that the neighboring alpha and delta cells are unaffected by prodrug treatment and that ablation is beta cell specific. Following drug removal and 36h of recovery, beta cells regenerate. Using ptf1a morphants, it is clear that this beta cell recovery occurs independently of the presence of the exocrine pancreas. Also, by using photoconvertible Kaede to cell lineage trace and BrdU incorporation to label proliferation, we investigate mechanisms for beta regeneration. Therefore, we have developed a unique resource for the study of beta cell regeneration in a living vertebrate organism, which will provide the opportunity to conduct large-scale screens for pharmacological and genetic modifiers of beta cell regeneration.

Ribosomal biogenesis genes play an essential and p53-independent role in zebrafish pancreas development

“…The following strains were used: Tg BAC ptf1a:eGFP jh1 (herein ptf1a:eGFP) (Godinho et al, 2007), Tg Tol2 ins:mCherry jh2 (herein ins:mCherry) (Pisharath et al, 2007), Tg (gcga:GFP) ia1 (herein gcga:GFP) (Pauls et al, 2007), Tg (T2KTp1hglob:hmgb1-mCherry) jh11 (herein Tp1:hmgb1-mCherry) (Parsons et al, 2009), Tg (kdrl:GRCFP) zn1 (Cross et al, 2003), tp53 zdf1 [M214K (Berghmans et al, 2005), obtained from the Zebrafish International Resource Center (ZIRC), Eugene, OR, USA], pes hi2Tg (ZIRC) and rpl3 hi2347 (ZIRC). Tail fins of adult fish were digested in low TE (10 mM Tris-HCl pH 7.5, 1 mM EDTA) plus proteinase K. PCR genotyping was performed to confirm the genotype of tp53 zdf1 homozygous embryos.…”

“…Finally, we evaluated the effect of Sbds ATG MO injection on the developing pancreas using a ptf1a:eGFP jh1 transgenic line to visualize early pancreatic progenitor cells and later differentiating acinar cells , in combination with an ins:mCherry jh2 transgenic line (Pisharath et al, 2007) to visualize beta cells in the principal islet. At 72 hpf, embryos injected with the Sbds ATG MO exhibited dramatic pancreatic hypoplasia, initially manifested by a marked decrease in the number of undifferentiated ptf1a-expressing pancreatic progenitor cells (Fig.…”

Targeted cell‐ablation in Xenopus embryos using the conditional, toxic viral protein M2(H37A)

“…The principal limitation with this system is the issue of subsequent diffusion of the toxic metabolites into neighboring cells (Helsby et al, 2004), but this has apparently been solved using a new prodrug, metronidazole. The new studies have produced targeted cell-ablations within the pancreas, liver, and the heart (Curado et al, 2007;Pisharath et al, 2007), although their cardiac cell "damage" phenotype does not appear as extensive as we report here. We believe it would be fascinating to compare directly the many attributes of M2(H37A) with those of the Kid and nitroreductase systems by producing equivalent cell-ablation effector (binary) transgenic lines that could be widely used by the frog and fish research communities.…”

Functional characterization of lmo2‐Cre transgenic zebrafish

“…In mouse, Cre transgenic lines have been widely used in the tissue-specific gene loss-offunction studies, lineage tracing, and cancerogenesis (Branda and Dymecki, 2004;Gaveriaux-Ruff and Kieffer, 2007). It can be anticipated that, in zebrafish, the tissue-specific Cre-loxP system, in combination with other newly established technologies such as targeted cell ablation (Pisharath et al, 2007) and GAL4-UAS system (Inbal et al, 2006), should make zebrafish a more powerful model for dissecting gene functions in a spatialtemporal context.…”