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Distinct enhancers of ptf1a mediate specification and expansion of ventral pancreas in zebrafish.

ABSTRACT: Development of the pancreas and cerebellum require Pancreas-specific transcription factor-1a (Ptf1a), which encodes a subunit of the transcription factor complex PTF1. Ptf1a is required in succession for specification of the pancreas, proper allocation of pancreatic progenitors to endocrine and exocrine fates, and the production of digestive enzymes from the exocrine acini. In several neuronal structures, including the cerebellum, hindbrain, retina and spinal cord, Ptf1a is transiently expressed and promotes inhibitory neuron fates at the expense of excitatory fates. Transcription of Ptf1a in mouse is maintained in part by PTF1 acting on an upstream autoregulatory enhancer. However, the transcription factors and enhancers that initially activate Ptf1a expression in the pancreas and in certain structures of the nervous system have not yet been identified. Here we describe a zebrafish autoregulatory element, conserved among teleosts, with activity similar to that described in mouse. In addition, we performed a comprehensive survey of all non-coding sequences in a 67kb interval encompassing zebrafish ptf1a, and identified several neuronal enhancers, and an enhancer active in the ventral pancreas prior to activation of the autoregulatory enhancer. To test the requirement for autoregulatory control during pancreatic development, we restored ptf1a function through BAC transgenesis in ptf1a morphants, either with an intact BAC or one lacking the autoregulatory enhancer. We find that ptf1a autoregulation is required for development of the exocrine pancreas and full rescue of the ptf1a morphant phenotype. Similarly, we demonstrate that a ptf1a locus lacking the early enhancer region is also capable of rescue, but only supports formation of a hypoplastic exocrine pancreas. Through our dissection of the complex regulatory control of ptf1a, we identified separate cis-regulatory elements that underlie different aspects of its expression and function, and further demonstrated the requirement of maintained ptf1a expression for normal pancreatic morphogenesis. We also identified a novel enhancer that mediates initiation of ptf1a expression in the pancreas, through which the signals that specify the ventral pancreas are expected to exert their action.

SUBMITTER: Pashos E

PROVIDER: S-EPMC5367438 | biostudies-literature | 2013 Sep

REPOSITORIES: biostudies-literature

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Distinct enhancers of ptf1a mediate specification and expansion of ventral pancreas in zebrafish.

Pashos Evanthia E Park Joon Tae JT Leach Steven S Fisher Shannon S

Developmental biology 20130720 2

Development of the pancreas and cerebellum require Pancreas-specific transcription factor-1a (Ptf1a), which encodes a subunit of the transcription factor complex PTF1. Ptf1a is required in succession for specification of the pancreas, proper allocation of pancreatic progenitors to endocrine and exocrine fates, and the production of digestive enzymes from the exocrine acini. In several neuronal structures, including the cerebellum, hindbrain, retina and spinal cord, Ptf1a is transiently expressed ...[more]

PMID: 23876428

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Project description:Both endocrine and exocrine pancreatic cells arise from pancreatic-duodenal homeobox 1 (pdx1)-positive progenitors. The molecular mechanisms controlling cell fate determination and subsequent proliferation, however, are poorly understood. Unlike endocrine cells, less is known about exocrine cell specification. We report here the identification and characterization of a novel exocrine cell determinant gene, exocrine differentiation and proliferation factor (exdpf), which is highly expressed in the exocrine cell progenitors and differentiated cells of the developing pancreas in zebrafish. Knockdown of exdpf by antisense morpholino caused loss or significant reduction of exocrine cells due to lineage-specific cell cycle arrest but not apoptosis, whereas the endocrine cell mass appeared normal. Real-time PCR results demonstrated that the cell cycle arrest is mediated by up-regulation of cell cycle inhibitor genes p21(Cip), p27(Kip), and cyclin G1 in the exdpf morphants. Conversely, overexpression of exdpf resulted in an overgrowth of the exocrine pancreas and a severe reduction of the endocrine cell mass, suggesting an inhibitory role for exdpf in endocrine cell progenitors. We show that exdpf is a direct target gene of pancreas-specific transcription factor 1a (Ptf1a), a transcription factor critical for exocrine formation. Three consensus Ptf1a binding sites have been identified in the exdpf promoter region. Luciferase assay demonstrated that Ptf1a promotes transcription of the exdpf promoter. Furthermore, exdpf expression in the exocrine pancreas was lost in ptf1a morphants, and overexpression of exdpf successfully rescued exocrine formation in ptf1a-deficient embryos. Genetic evidence places expdf downstream of retinoic acid (RA), an instructive signal for pancreas development. Knocking down exdpf by morpholino abolished ectopic carboxypeptidase A (cpa) expression induced by RA. On the other hand, exdpf mRNA injection rescued endogenous cpa expression in embryos treated with diethylaminobenzaldehyde, an inhibitor of RA signaling. Moreover, exogenous RA treatment induced anterior ectopic expression of exdpf and trypsin in a similar pattern. Our study provides a new understanding of the molecular mechanisms controlling exocrine cell specification and proliferation by a novel gene, exdpf. Highly conserved in mammals, the expression level of exdpf appears elevated in several human tumors, suggesting a possible role in tumor pathogenesis.

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Distinct enhancers of ptf1a mediate specification and expansion of ventral pancreas in zebrafish.

Publications

Distinct enhancers of ptf1a mediate specification and expansion of ventral pancreas in zebrafish.

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