Sogame A et al. (2003), Screening for novel pancreatic genes from in vi...

XB-ART-5288

Dev Growth Differ 2003 Apr 01;452:143-52. doi: 10.1034/j.1600-0854.2004.00683.x.

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Screening for novel pancreatic genes from in vitro-induced pancreas in Xenopus.

Sogame A , Hayata T , Asashima M .

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The processes of development and differentiation of the pancreas, an endoderm-derived vital organ that consists of both endocrine and exocrine cells, are highly conserved across most vertebrates. Recently, an in vitro system has been reported to induce embryonic pancreas using multipotent Xenopus ectodermal cells treated with activin and retinoic acid. In this study, this system was first modified to eliminate the mesoderm-derived pronephros. It was found that pronephros, which appeared with the use of low concentrations of activin, was eliminated at higher concentrations (400 ng/mL), while pancreas developed at a high frequency. Using this modified system, subtractive hybridization screening for novel pancreatic genes was done to better understand the molecular mechanisms of pancreas formation. Four novel genes were identified and characterized that were also found to be specifically expressed in the developing pancreas: carboxyl ester lipase, pancreatic elastase2, placental protein11 and protein disulfide isomerase A2 precursor. This in vitro pancreas-induction system may provide a useful model for analysis of the molecular mechanisms that function during pancreas development.

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Species referenced: Xenopus
Genes referenced: cel.2 cela1.2 cela1.6 cela2a cpa1 dnase1 endoul2 pdia2 pdx1

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	Fig. 1. Light micrographs of explants treated with activin and retinoic acid (RA). Ectoderm was cultured for 3 days (A–C) or 5 days (D,E) and examined histologically. (A) Untreated presumptive ectoderm cultured in Steinberg’s solution (SS) containing 0.1% bovine serum albumin (BSA). Ectoderm treated with (B) 100 ng/mL and (C) 400 ng/mL activin alone for 1 h. (D) Ectoderm first treated with activin 100 ng/mL for 1 h, kept in BSA–SS for 5 h and then treated with RA (10–4 M) for 1 h. (E) Ectoderm first treated with activin 400 ng/mL for 1 h, kept in BSA–SS for 5 h and then treated with RA (10–4 M) for 1 h. ae, atypical epidermis; int, intestine; mus, muscle; ne, neural tissue; no, notochord; pa, pancreas; pro, pronephros. Bar, 100 μm.
	Fig. 2. Expression of molecular markers in explants. Ectoderm isolated from Xenopus blastulae was treated and then cultured for 3 days. Total RNA was extracted and analyzed by reverse transcription–polymerase chain reaction. Lane 1, ectoderm treated with activin 100 ng/mL alone for 1 h; lanes 2, 3, 4, ectoderm treated with indicated concentration of activin for 1 h, kept in Steinberg’s solution (SS) containing 0.1% bovine serum albumin (BSA) for 5 h and then treated with RA (10–4 M) for 1 h; lane 5, untreated ectoderm; lane 6, negative control lacking reverse transcriptase (RT). XlHbox8 and carboxypeptidaseA (carbA) are pancreatic markers and XSMP-30 is a pronephric marker. EF-1 serves as a loading control.
	Fig. 3. Amino acid sequence alignment of Xenopus proteins with those of the human. The predicted amino acid sequences of Xenopus genes share the following similarity with those of human: (a) CEL (amino acid residue 318–440), 54%; (b) PE2 (20–122), 64%; (c) PP11 (99–190), 43%; (d) and (e) are two distinct fragments encoding PDIp, which showed 54 and 61% (119–159 and 460–498) similarity, respectively. Parentheses indicate the position of amino acid sequences in the human. Alignment was done using the Gene Works program (Oxford Molecular Group, San Diego, CA, USA).
	Fig. 4. Model for developmental anatomy of the Xenopus pancreas. Schematic diagrams of gut tubes from tadpoles are shown, truncated anteriorly at the junction between the stomach and esophagus. All associated endodermal organs except for the pancreas have been removed for clarity. Pancreas rudiments are indicated in blue. (A) Gut tube isolated from stage 40 embryo. Gastrointestinal tract undergoes dynamic movement and pancreas rudiments are repositioned. (B) Gut tube isolated at stage 42. Both rudiments have completely fused. (C) Gut tube isolated at stage 44. d, duodenum; int, intestine; st, stomach.
	Fig. 5. Expression pattern of pancreatic genes. Embryos at three stages were examined by in situ hybridization using specific probes for each clone. CEL was expressed in dorsal and ventral pancreas rudiments (arrowhead) at stage 40 and continued after stage 42 (A,B,C: stages 40, 42 and 44, respectively). PE2 (D,E,F: stages 40, 42 and 44, respectively), PDIp (J,K,L: stages 40, 42 and 44, respectively) and DNaseI (M,N,O: stages 40, 42 and 44, respectively) were detected as weak signals at stage 40 and strong signals at stage 42. PP11 (G,H,I: stages 40, 42 and 44, respectively) was barely detectable at stage 40 but expression increased by stage 42.
	cel (carboxyl ester lipase) gene expression in dissected Xenopus laevis gut, assayed via in situ hybridization, NF stage 42, lateral view, anterior left, dorsal up.
	dnase1 (deoxyribonuclease I) gene expression in dissected Xenopus laevis gut, assayed via in situ hybridization, NF stage 42, lateral view, anterior left, dorsal up.
	cela2a (chymotrypsin-like elastase family, member 2A) gene expression in dissected Xenopus laevis gut, assayed via in situ hybridization, NF stage 42, lateral view, anterior left, dorsal up.
	unnamed (26 serine protease) gene expression in dissected Xenopus laevis gut, assayed via in situ hybridization, NF stage 42, lateral view, anterior left, dorsal up.