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.

Fig. 1

Met induces apoptosis in a NTR dependent manner. (A) Schematic of construct used to drive ubiquitous expression of NTR. Construct includes two arms from the Tol2 transposable element (black triangles), the EF1α promoter and intronic sequence from the rabbit β-globin gene (Kawakami, 2004). EF1α drives ubiquitous expression of NTR. Embryos injected at 1 cell stage and incubated with or without Met were fixed at 48hpf and assayed for apoptosis using fluorescent TUNEL. (B) Negative control, mosaic for Ef1α:nfsB with no prodrug treatment. (C) Negative control, mosaic for Ef1α:eGFP incubated in Met (6 to 48hpf). (D) Mosaic embryos for Ef1α:nfsB, incubated in Met. (6 to 48hpf). Results demonstrate increased apoptosis when NTR is expressed in presence of Met.

Fig. 2

Insulin promoter reliably drives expression to the β cells. (A) Schematic of construct used including the 1kb promoter region of preproinsulin, and gene encoding eGFP, all within a Tol2 transposable element (black triangles). (B) Fluorescent image of a 3 day old ins:eGFP embryo showing expression within the islet of the pancreas. (C) Rendered composite of confocal images of a 10 day old ins:eGFP embryo following immunofluorescence to detect insulin, with overlaid 20x bright field. (D) Close up images of eGFP fluorescence and (E) immunofluorescence to detect Insulin and (F) merged image demonstrating co-localization of eGFP with Insulin. (G) 3 month ins-eGFP adult fish showing transcutaneous fluorescence. (H) Section through dissected pancreas showing immunofluorescence to detect Insulin (scale bar = 100μM). The principal islet can be seen in the anterior head of the pancreas (arrow). More posterior are small aggregatons of β cells (white bracket) and three secondary islets (arrowheads). (I) High magnification, the morphology of the smaller islets can be seen (black Targeted ablation of β cells bracket) surrounded by acinar tissue (arrow), by H&E staining (scale bar =10μM). Immunofluorescence detection for eGFP in (J), insulin in (K). (I) Merged image demonstrate co-localization in adult tissues (Hoescht staining for nuclei in blue). In (B to H) anterior is to the right and posterior to the left.

Fig. 3

The ins:nsfB-mCherry transgene leads to prodrug dependent loss of β cells. Schematic of constructs used to drive β cell expression of mCherry in (A) and the fusion protein NTR-mCherry in (B). The expression cassettes were cloned inside of a Tol2 transposable element (black triangles). In the experiment shown here, two groups of controls were used, ins:mCherry embryos incubated in Met (+Met, C, F, I) and ins:nfsB-mCherry without prodrug (no Met, D, G, J). Experimental groups consisted of ins:nfsB-mCherry embryos incubated for 24 hrs in Met (+Met, E, H, K). Two separate time points were chosen for addition of prodrug as shown, incubation starting at 24hpf and finished by 48hpf (48hpf, C to H) and starting at 56hpf and finishing at 80hpf (80hpf, I to K). Embryos were photographed by either epi-fluorescence in (C) to (E), or by confocal microscopy on isolated pancreata plus adjacent endoderm in (F) to (M). Red fluorescence indicated presence of mCherry (C, F, I) or NTR-mCherry fusion protein (D, E, G, H, J, K, L and M). eGFP marks the exocrine pancreas with green fluorescence and nuclei are stained blue (Hoescht). (L) Example of fragmented nucleus indicative of apoptosis (arrowhead) and cell debris inside and outside of islet in (M) (arrows). Orientation as indicated, d = dorsal, v = ventral, a = anterior, p = posterior, m = medial and l = lateral.

Fig. 4

Expression of insulin but not glucagon or Somatostatin, is lost following prodrug dependent β cell ablation. (A – C) Marker analysis on ins:nfsB-mCherry untreated control embryos or embryos treated with Met from 56hpf to 80hpf (D to F). In situ hybridization with riboprobes to detect the following transcripts: insulin (ins) (A, D); glucagon (gcg) (B, E). Immunofluorescence detection of Somatostatin protein, using confocal microscopy, (C, F). Presence of the NTR-mCherry fusion protein can be seen in the control embryo (C) as red fluorescence, but is lost following ablation (F).

Fig. 5

β cell recovery following ablation. (A) Schematic timeline of prodrug treatment. (B – D) Untreated ins:nfsB-mCherry embryos at stages indicated. Ins:nfsB-mCherry embryos before prodrug incubation (E), following 24hrs of ablation with prodrug (F) and following 36hrs of recovery in absence of prodrug (G). Return of β cells is indicated by the reoccurrence of NTR- mCherry. Dissected pancreas and adjacent endoderm from a untreated ins:nfsB-mCherry, 84hpf embryo (H) and treated and recovered 84hpf embryo (K). Nuclei are blue due to Hoescht counter staining. Images generated from ins:nfsB-mCherry; ptf1a:eGFP transgenics, hence exocrine pancreas appears green. (H, K) In comparison to controls, treated pancreata look morphologically normal. Close up images of an islet following treatment and recovery in (L), show presence of β cells with a normal appearance but in a reduced number compared to untreated controls (I). (J) Untreated ins:nfsB-mCherry in a fry, 8 days post fertilization (dpf), and following prodrug treatment described above (M).

Fig. 6

β cell recovery is independent of the exocrine pancreas. (A, E) Ins:nfsB-mCherry embryos before prodrug incubation. (B,F) Ablation following 24hrs of prodrug incubation. (C, D, G, H) Following 36hrs of prodrug removal, recovery can be detected. Control embryos (A – D), show normal exocrine pancreas as demonstrated by CPA immunofluorescent detection (pseudo color image D). Ptf1a morphant embryos shown in E to H lack an exocrine pancreas as demonstrated by absence of CPA (H), but still recover β cells (G).

Fig. 7

Following ablation, both continued maturation and proliferation may contribute to recovery in β cell numbers. (A) Construct used to generate ins:Kaede transgenics, made within a transposable element (black triangles). (B) Model of Kaede labeling, a green β cell is photoconverted at 48hpf (1). Subsequent differentiation of progenitors gives rise to β cells that possess only green Kaede (2) whereas division of β cells already present during photoconversion inherit red Kaede, produce green kaede and hence appears yellow (3). A live ins:Kaede F1 embryo viewed before (C) and after photoconversion (D). The β cells of the islet can be detected (arrowhead). (E) Later in development, at 120hpf, fluorescence in both channels is apparent and via confocal, it is clear that there are less cells fluorescing red in (F) than green in (G). (H) In a merged image β cells that fluoresce only green can be detected indicating that these cells have arisen from non-β cells. (I) Schematic of the experiment to test for proliferation following β cell ablation in ins:nfsB-mCherry embryos. (J, K) 1μM confocal sections through islet showing BrdU labelling (green nuclei) and β cells (red ). In both controls (J) and islets recovering from β cell ablation (K), proliferation of β cells can be detected as double labeled cells (arrows).