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Graded Levels of Ptf1a Differentially Regulate Endocrine and Exocrine Fates in the Developing Pancreas

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Graded Levels of Ptf1a Differentially Regulate Endocrine and Exocrine Fates in the Developing Pancreas Downloaded from genesdev.cshlp.org on October 8, 2021 - Published by Cold Spring Harbor Laboratory Press RESEARCH COMMUNICATION factor Ptf1a is required for exocrine differentiation Graded levels of Ptf1a (Krapp et al. 1998). Ptf1a lineage tracing has also revealed differentially regulate an earlier role for Ptf1a in allocating foregut endodermal cells to the pancreas, implicating Ptf1a in pancreas speci- endocrine and exocrine fates fication. This finding is consistent with the observation in the developing pancreas that Ptf1a is expressed in precursors of all pancreatic cell types, including endocrine cells (Kawaguchi et al. 2002). P. Duc Si Dong,1,4 Elayne Provost,2 Importantly, it is not specifically known how Ptf1a regu- 2 1,3 lates endocrine development. The PTF1 complex binds Steven D. Leach, and Didier Y.R. Stainier directly to the promoters of terminal exocrine differen- 1Department of Biochemistry and Biophysics, University of tiation genes such as Trypsin and Elastase, indicating California, San Francisco, San Francisco, California 94158, that Ptf1a is also involved in exocrine cell differentiation USA; 2Department of Surgery, Johns Hopkins Medical (Cockell et al. 1989; Krapp et al. 1996). Furthermore, Institutions, Baltimore, Maryland 21287, USA Ptf1a interacts differentially with the vertebrate Sup- pressor of Hairless homologs, Rbpj and Rbpjl, to regulate The mechanisms regulating pancreatic endocrine versus pancreas specification and exocrine differentiation, re- exocrine fate are not well defined. By analyzing the ef- spectively (Masui et al. 2007). Lineage tracing of cells fects of Ptf1a partial loss of function, we uncovered novel expressing Carboxypeptidase A1, a Ptf1a target, has shown that endocrine and exocrine cells can originate roles for this transcription factor in determining pancre- from a common progenitor in the specified pancreas ptf1a atic fates. In a newly identified hypomorphic mu- (Zhou et al. 2007). Therefore, it will be necessary to ana- tant, pancreatic cells that would normally express ptf1a lyze the post-pancreas specification role of Ptf1a to de- and become exocrine cells, express the endocrine marker termine whether it conveys other functions, such as de- Isl1, indicating a cell fate switch. Surprisingly, a milder termining the balance of endocrine versus exocrine cell reduction of Ptf1a leads to an even greater increase of fates. ectopic endocrine cells, suggesting that Ptf1a also plays a role in promoting endocrine development. We propose Results and Discussion that low levels of Ptf1a promote endocrine fate, whereas To search for mutants with pancreas defects, we high levels repress endocrine fate and promote exocrine screened ENU mutagenized Tg(gutGFP)s854 zebrafish fate. (Ober et al. 2006), which express GFP in many endoder- mal organs, including the pancreas. akreass466 is a reces- Supplemental material is available at http://www.genesdev.org. sive mutant that exhibits a small ventral pancreas (Fig. Received February 14, 2008; revised version accepted April 4, 1A,B). The principal islet, which consists of endocrine 2008. cells that are initially derived from the dorsal pancreas (Field et al. 2003b), is present in akreas mutants, indi- cating that the dorsal pancreas does develop. Expression Loss of the insulin-producing ␤-cells from the endocrine of trypsin (data not shown) and Tg(elastase:GFP)gz2, pancreas occurs in diabetes, a disease that afflicts >125 markers of the exocrine pancreas, is lost in akreas mu- million people worldwide. Thus, endocrine cell replace- tants (Fig. 1C,D), indicating the absence of differentiated ment is likely to be an integral step in the treatment of exocrine cells. this disease (Donath and Halban 2004; Bertuzzi et al. The akreas mutation maps to chromosome 2, within a 2006). A major challenge lies in developing practical 2 cM region that contains ptf1a (data not shown). Like strategies for ␤-cell replacement, a task that will be fa- akreas mutants, Ptf1a mutant mice (Krapp et al. 1998) cilitated by a more profound understanding of the and zebrafish embryos injected with antisense ptf1a mechanisms underlying the specification and differen- morpholino oligonucleotides (MOs; Lin et al. 2004; Zec- tiation of pancreatic endocrine cells. chin et al. 2004) develop pancreatic endocrine cells but The pancreas is composed of digestive enzyme-produc- no exocrine cells, prompting us to sequence the ptf1a ing exocrine cells, ductal cells, and hormone-producing gene of akreas mutants. We identified a transversion in endocrine cells. Although many genes are known to be the coding region of ptf1a (Fig. 1E), causing an isoleucine required for the differentiation of endocrine and exocrine 142-to-asparagine (I142N) substitution in the first helix cells, factors regulating the endocrine versus exocrine of the highly conserved bHLH domain (Fig. 1F). This iso- fate decision remain elusive (Jensen 2004). The Atonal- leucine is present in the bHLH domain of diverse pro- related basic helix–loop–helix (bHLH) protein Neuro- teins in organisms from flies to humans (Krapp et al. genin3, and Lim domain protein Islet1 (Isl1) are tran- 1996; Lin et al. 2004; Zecchin et al. 2004). The genetic scription factors required for the specification and differ- linkage to ptf1a, expression of ptf1a in the affected tis- entiation of pancreatic endocrine cells (Ahlgren et al. sue, phenocopy of the ptf1a loss of function, and a ge- 1997; Gradwohl et al. 2000). The bHLH transcription netic lesion in a highly conserved residue of Ptf1a indi- cate that akreas is a mutation in ptf1a. To better understand the effect of the akreas mutation [Keywords: Ptf1a; diabetes; endocrine; exocrine; pancreas] on Ptf1a function, we examined the ability of the mutant Corresponding authors. protein to activate a modified rat Chymotrypsinogen B 3E-MAIL [email protected]; FAX (415) 476-3892. 4E-MAIL [email protected]; FAX (415) 476-3892. promoter. Both wild-type zebrafish and rat Ptf1a, in com- Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.1663208. bination with the cofactor E47, effectively activated a GENES & DEVELOPMENT 22:1445–1450 © 2008 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/08; www.genesdev.org 1445 Downloaded from genesdev.cshlp.org on October 8, 2021 - Published by Cold Spring Harbor Laboratory Press Dong et al. mutants (Fig. 1H,I), we hypothesized the presence of a low level of Ptf1a function. To test this hypothesis, we used a ptf1a MO (ptf1aMO1), which knocks down trans- lation of ptf1a but not Tg(ptf1a:eGFP)jh1 (Supplemental Fig. S1C; Supplemental Material), in the akreas mutant background. In ∼20% of ptf1aMO1 injected embryos (36 of 179) from an incross of akreas heterozygotes, we found a disorganization of retinal Tg(ptf1a:eGFP)-ex- pressing cells similar to what is observed in the Ptf1a- null mice. Particularly, we found a reduction of Tg(ptf1a:eGFP)-expressing cells in the amacrine layer and ectopic Tg(ptf1a:eGFP) expression in scattered cells in the ganglion layer, as marked by Isl1 (Fig. 1J,K; Fischer et al. 2002) and Alcam (Weiner et al. 2004) expression. Furthermore, many of the Tg(ptf1a:eGFP)-expressing cells in the ganglion layer coexpress Isl1 and Alcam (Supplemental Fig. S1D–O), suggesting that presumptive amacrine cells are being transfated toward ganglion cells, a phenotype similar to that seen in Ptf1a-null mice. We also observed that the Tg(ptf1a:eGFP)-expressing hori- zontal cells, which in wild-type embryos coexpress Isl1, lack Isl1 expression and are severely reduced in number. In addition, we found Tg(ptf1a:eGFP) expression in the adjacent photoreceptor cells, suggesting that horizontal cells are being transfated into photoreceptor cells, a phe- notype not previously described. These phenotypes were not observed in uninjected akreas mutants or in ptf1aMO1-injected wild-type embryos. Therefore, a more severe ptf1a loss-of-function phenotype can be Figure 1. akreas is a hypomorphic allele of ptf1a.(A,B) Three-di- achieved by a ptf1a morpholino/mutant combination. mensional rendering of Tg(gutGFP) (green) wild-type (A) and akreas Based on these in vivo data, as well as the transactivation mutant (B) embryos at 56 hpf stained for Isl1 (red) and Insulin (Ins; and gel shift results, we conclude that the akreas muta- blue) showing a smaller ventral pancreas (arrows) in akreas mutants. (C,D) Tg(elastase:GFP); Tg(lfabp:dsRed) wild-type (C) and akreas tion does not completely abolish Ptf1a function. Further- mutant (D) at 100 hpf stained for pan-Cadherin showing the absence more, the role of ptf1a in retinal cell fate decision of Tg(elastase:GFP) expression (green) in akreas mutants. Asterisks prompted us to examine whether ptf1a might also have mark the EPD. (E) Genomic DNA sequence of wild-type and akreas a similar function during pancreas development. mutant ptf1a showing the molecular lesion in the akreas ptf1a gene. Like ptf1a mRNA expression, initial Tg(ptf1a:eGFP) (F) Schematic of the Ptf1a bHLH domain and corresponding amino expression in the presumptive ventral pancreas area of acid sequences of wild-type and akreas mutant. (G) Luciferase assay ∼ to evaluate I142N substitution on Ptf1a transcriptional activity. the endoderm occurs at 32 hpf (hours post-fertilization) Wild-type Ptf1a + E47 results in >100-fold induction of the reporter, (data not shown). At this stage, Tg(ptf1a:eGFP)-express- while I142N Ptf1a results in a modest 40-fold induction. (H–K) ing cells are in a ventral endodermal domain, caudal to Retina of 52 hpf Tg(ptf1a:eGFP); akreas mutant (H; green channel the liver bud. The homeodomain transcription factor shown in I) and akreas mutant injected with ptf1aMO1 (J; green Prox1 (Sosa-Pineda et al. 2000; Wang et al. 2005) is ex- channel shown in K) stained for Isl1 (red) showing that Tg(ptf1a:eGFP)- expressing cells are restricted to the horizontal (white arrowheads) pressed in a continuous field of cells that comprises the and amacrine (white arrows) layers in akreas mutants (H,I), but are liver bud and all Tg(ptf1a:eGFP)-expressing cells.
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