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Molecular Endocrinology 20 (1): 194-203
Copyright © 2006 by The Endocrine Society

Genetic Analysis of Early Endocrine Pancreas Formation in Zebrafish

Hyon J. Kim1, Saulius Sumanas1, Sharina Palencia-Desai, Yuan Dong, Jau-Nien Chen and Shuo Lin

Department of Molecular, Cellular, and Developmental Biology, University of California, Los Angeles, California 90095

Address all correspondence and requests for reprints to: Shuo Lin, Department of Molecular Cellular and Developmental Biology University of California, Los Angeles, 4–317 Life Science, 621 Charles E. Young Drive South, Los Angeles, California 90095. E-mail: shuolin{at}ucla.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Endocrine pancreas of zebrafish consist of at least four different cell types that function similarly to mammalian pancreatic islet. No mutants specifically affecting formation of the endocrine pancreas have been identified during the previous large-scale mutagenesis screens in zebrafish due to invisibility of a pancreatic islet. We combined in situ hybridization method to visualize pancreatic islet with an ethyl-nitroso-urea mutagenesis screen to identify novel genes involved in pancreatic islet formation in zebrafish. We screened 900 genomes and identified 11 mutations belonging to nine different complementation groups. These mutants fall into three major phenotypic classes displaying severely reduced insulin expression, reduced insulin expression with abnormal islet morphology, or abnormal islet morphology with relatively normal number of insulin expressing cells. Seven of these mutants do not have any other visible phenotypes associated. These mutations affect different processes in pancreatic islet development. Additional analysis on glucagon and somatostatin cell specification revealed that somatostatin cells are specified at a separate domain from insulin cells whereas glucagon cells are specified adjacent to insulin cells. Furthermore, glucagon cells and somatostatin cells are always associated with insulin cells in mutants that have scattered insulin expression. These data indicate that there are separate mechanisms regulating endocrine cell migration, proliferation, and differentiation. Further study on these mutants will reveal important information on novel genes involved in pancreatic islet cell specification and morphogenesis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ENDOCRINE PANCREAS, known as Islets of Langerhans, secrets hormones that regulate body metabolism and glucose use in all vertebrates. At least four different cell types secreting unique hormones are organized in a characteristic structure that consists of the core insulin secreting ß-cells surrounded by glucagon-secreting {alpha}-cells, somatostatin-secreting {delta}-cells, and pancreatic polypeptide-secreting PP-cells at the periphery.

Previous studies in mammalian model systems showed that a highly complex and coordinated gene regulation and cell-cell signaling is required for the proper pancreatic islet development. First, early pancreatic endoderm is specified within the early primitive endoderm via signaling from the different neighboring tissues. For example, specification of dorsal pancreatic bud requires signaling from the notochord between six and 10 somite stage in both mouse and rat (1). Later signaling from the aortic endothelial cells to the endoderm is required to turn on an important early pancreatic transcription factor, pdx-1 (2).

All pancreatic lineage cells are thought to arise from the pdx-1-positive cells in mammals (3). Pdx-1-positive cells further differentiate by complex regulation of transcription factors. As hormone-positive cells are specified within the pancreatic duct epithelial layer (4), they migrate out to surrounding mesenchyme to form an islet (5) and organize into a characteristic structure of insulin expressing ß-cell core surrounded by cells secreting other hormones (6). Despite extensive studies that have been done on pancreatic ß-cell development and specification in mammals, however, our knowledge of molecular mechanisms regulating endocrine cell differentiation, migration, and morphogenesis is very limited (7).

In zebrafish, endocrine pancreas regulates metabolism and blood glucose level similar to mammals (4, 8). Recent studies showed that the zebrafish pdx-1 is expressed in bilateral rows of cells from the 14-somite stage onwards (9). However, the process of pancreatic endoderm specification and subsequent pdx-1 expression during pancreatic development is not so well understood in zebrafish. For example, sonic hedgehog was shown to be a negative regulator of endoderm to adopt a pancreatic fate in the anterior endoderm in mammalian model systems (10). In zebrafish, however, ectopic expression of sonic hedgehog induces pancreatic fate rather than repress it differently from mammalian models (11, 12). Also, cloche mutants, which lack dorsal aorta, were shown to have normal pancreas arguing against a role of aortic endothelial cells in pancreas development of zebrafish (13). Therefore, it is not so clear how pancreatic endoderm is specified in zebrafish.

At the 16-somite stage, insulin expression becomes apparent (9). The insulin-positive cells actively migrate posteriorly and converge medially (25). By the 24 h post fertilization (hpf) stage, all insulin-positive cells have coalesced into a single islet in the midline. Some of the important genes involved in mammalian pancreatic islet formation such as pdx-1, islet-1, and nkx 2.6 were shown to have similar function in zebrafish, indicating conserved signaling pathways and mechanisms of pancreatic islet formation between zebrafish and other vertebrates (14). Therefore, zebrafish provides the simplest model pancreatic islet with conserved physiological complexity.

We performed ethyl-nitroso-urea (ENU)-mediated mutagenesis screen in zebrafish to identify novel genes required for the specification of ß-cells and islet morphogenesis. Previous large-scale mutagenesis screens showed that zebrafish can be used successfully to study early developmental processes (15, 16). However, the studies failed to identify endocrine pancreas-specific mutants because the pancreatic islet is not visible in living embryos. To overcome this problem, we combined in situ hybridization using insulin as an endocrine pancreas marker to screen F3 embryos with defects in pancreatic ß-cell specification and morphogenesis at the 24 hpf. By this stage, all insulin-positive cells have finished the early morphogenesis and formed a compact islet with insulin cells at the core intermingled with somatostatin cells and glucagon cells at the periphery (9).

In total, we have screened 900 genomes and isolated 11 mutants representing nine genes required for different processes in pancreatic islet development. Here we report for the first time three different classes of zebrafish mutants with defects in early pancreatic islet formation. In addition, we provide genetic evidence that two distinctively separate mechanisms are required for islet precursor cell migration and formation of islet architecture. These mutants will allow us to identify novel regulators of early pancreas development and to understand better mechanisms of pancreatic islet formation and morphogenesis.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Three Classes of Mutants Reveal Different Processes Involved in Early Zebrafish Pancreatic Development
We performed a diploid F3 ENU mutagenesis screen to identify zygotic recessive mutations resulting in abnormal insulin expression. F3 embryos were fixed at the 24 hpf stage and processed for in situ hybridization to detect insulin mRNA expression. We screened 2051 pairs to cover 900 genomes and isolated 11 mutants. Subsequent complementation crosses placed these mutants into nine different groups (genes). These mutants can be divided into three different classes. Class I mutants show severely reduced number of insulin-positive cells. This group was further subdivided according to the presence of other visible morphological defects at 24 hpf. Class IA mutants have visible morphological abnormality in addition to severely reduced pancreatic islet phenotype. Class IB mutants have no other visible phenotype associated with the severely reduced pancreatic islet at 24 hpf. Class II mutant shows moderately reduced number of insulin-positive cells with abnormal islet morphology and no other visible morphological abnormality. Class III mutants show abnormally split and scattered islet morphology but have relatively normal number of insulin-positive cells and no other visible morphological abnormality (Table 1Go).


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  		Table 1. Summary of Pancreas Mutants

 
Mutations with Severely Reduced Insulin Expression
We isolated four mutants that have the severely reduced insulin expression. We subdivided this group into two different categories. Class IA mutants have abnormal morphology at 24hpf in addition to the reduced insulin expression. Further analysis showed that allele 620 in Class 1A is an allele of an already published mutant, floating head (flh). This mutant shows a truncated tail without any visible notochord similar to flh mutants (17). Flh mutants previously also have been reported to have a severely reduced insulin expression (9). Sea dragon (sdr) shows severely reduced insulin expression associated with thinner and ventrally curved tail at 24 hpf (Fig. 1BGo). Insulin-positive cell coalescing at the midline into a single islet is not affected at 24 hpf stage. Number of glucagon and somatostatin cells is also reduced (Fig. 1Go, D and F). Interestingly, pdx-1 expression is expanded at 24 hpf in sdr mutants compared with wild-type siblings (Fig. 1HGo). At 36 hpf, pdx-1 expression still remain diffused and slightly enlarged than in wild-type embryos (Fig. 1JGo). At the 3 d post fertilization (dpf) stage, pancreatic islet is embedded within the exocrine pancreas and positioned on the right side of the body, illustrating asymmetry of internal organs in wild-type embryos (Fig. 1KGo). In sdr mutant embryos, however, insulin-positive cells remain close to the midline. The reduced islet is split bilaterally along the midline at this stage (Fig. 1LGo). Analysis of carboxypeptidase A and ceruloplasmin revealed duplicated exocrine pancreas and liver respectively in sdr mutants (Fig. 1Go, N and P). FoxA2 staining at the same stage confirmed duplicated liver and pancreas, whereas intestine is not duplicated (Fig. 1RGo).



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  		Fig. 1. Analysis of Endodermal Defects in the sdr Mutant (Class 1A)

A, C, E, G, I, K, M, O, and Q, Wild type; B, D, F, H, J, L, N, P, and R, sdr mutant; A–H, 24 hpf; A and B, insulin (notice reduced insulin expression in sdr mutant). C and D, Glucagon; E and F, somatostatin; G and H, pdx-1 (pdx-1 expression is expanded and diffused in sdr mutant). I and J, pdx-1, 36 hpf; K–R, 3 dpf; K and L, insulin; M and N, carboxypeptidase A (notice duplicated exocrine pancreas). O and P, Ceruloplasmin, small and duplicated liver is observed in sdr mutant. Q and R, foxA2. Arrow, Exocrine pancreas; black arrowhead, liver; white arrowhead, intestine.

 
Class IB mutants also have severely reduced insulin expression but do not show any visible morphological defects at the 24 hpf stage in contrast to class 1A mutants. We isolated two complementing mutants in this class, mislet (msl) and angelina (agl). Although msl mutants do not have any visible morphological defects at 24 hpf, they do not respond to touch stimuli at 2 dpf stage (see wild-type and mislet movies). We performed a more detailed molecular analysis on msl embryos. Analysis of insulin expression showed severely reduced number of insulin-positive cells located bilaterally along the midline (Fig. 2BGo). Expression of other endocrine pancreas markers, glucagon and somatostatin, is also severely reduced (Fig. 2Go, D and F). Expression of pdx-1 is also severely reduced at 24 hpf (Fig. 2HGo). Expression of the exocrine pancreas marker, carboxypeptidase A, is significantly reduced at 3dpf in msl mutants. A liver marker, ceruloplasmin, is also reduced in the mutants, whereas intestine is not affected as evident from foxA2 expression (Fig. 2Go, N and P). These data suggest that msl is an early gene involved in the whole pancreas and liver development.



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  		Fig. 2. Analysis of Endodermal Defects in the msl Mutant (Class 1B)

A, C, E, G, I, K, M, and O, Wild type (WT); B, D, F, H, J, L, N, and P, msl mutant; A–H, 24 hpf stage; I–P, 3 dpf stage. A and B, Insulin (ins) (insulin expression is severely reduced in msl mutant). C and D, Glucagon (glu); E and F, somatostatin (sst); G and H, pdx-1; I and J, insulin (ins); K and L, carboxypeptidase A (carA) (exocrine pancreas is absent in msl mutant). M and N, Ceruloplasmin (cp), liver is also absent in msl mutant; O and P, foxA2. Arrow, Exocrine pancreas, black arrowhead, liver; white arrowhead, intestine.

 
Mutation Affecting the Size and Morphology of Pancreatic Islet
We isolated one mutant that shows a reduced size and abnormal morphology of pancreatic islet. In minime (mnm) mutant, the number of insulin cells is significantly reduced, but not as much as in class I mutants (Table 1Go). Glucagon- and somatostatin-positive cells are also reduced in number and have abnormal morphology (Fig. 3Go, D and F). Interestingly, the morphology of pdx-1 expression is abnormal, whereas the area of expression is not significantly affected compared with the wild-type embryos at 24 hpf stage (Fig. 3Go, G and H). Analysis of insulin expression shows that the insulin-positive cells remain split and do not coalesce into a single islet at 3 dpf stage (Fig. 3JGo). In addition, the location of insulin-expressing cells at 3 dpf remains close to the midline instead of moving toward the right side as observed in wild-type embryos (Fig. 3Go, I and J). Carboxypeptidase A expression at 3 dpf is reduced even though pdx-1 expression appears not affected (Fig. 3LGo). On the other hand, liver marker ceruloplasmin expression is normal in mnm embryos, indicating that mnm is likely to function specifically in pancreas development. Further analysis of foxA2 expression showed that the liver and intestine is normal, whereas the entire pancreas is slightly reduced in size compared with wild-type embryos (Fig. 3Go, O and P). These data suggest that mnm is required for the formation of both endocrine and exocrine pancreas.



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  		Fig. 3. Analysis of Endodermal Defects in mnm Mutant (Class II)

A, C, E, G, I, K, M, and O, Wild type (WT); B, D, F, H, J, L, N, and P, mnm mutant; A–H, 24 hpf stage; I–P, 3 dpf stage. A and B, Insulin (ins); C and D, glucagon (glu); E and F, somatostatin (sst); G and H, pdx-1; I and J, insulin (ins); K and L, carboxypeptidase A (carA); M and N, ceruloplasmin (cp); O and P, foxA2. Arrow, Exocrine pancreas; black arrowhead, liver; white arrowhead, intestine.

 
Mutations Affecting the Morphology of Pancreatic Islet
We found five mutants representing four different genes in this group. These mutants have no other visible morphological abnormalities other than scattered pancreas showed by in situ hybridization for insulin at 24 hpf. All of the mutants we examined in this class are normal until 3 dpf stage (data not shown). Whereas wild-type embryos have a single islet in the midline at 24 hpf stage (Fig. 4AGo), different patterns of scattered and split insulin-positive cells were observed in mutants. Cheetah (che) mutants have two to three patches of scattered insulin-positive cells along the anterior-posterior axis that appear different or equal in size (Fig. 4BGo). Scarlet (sle) mutants have three patches of insulin expression that can vary in size (Fig. 4CGo). Dalmatian (dal) mutants have a single large patch of insulin cells, and two to three smaller insulin-positive cells scattered anterior to the main patch (Fig. 4DGo). Peppershaker (ppp) mutants have multiple patches (3, 4, 5, 6, 7, 8) split laterally and along the anterior-posterior axis (Fig. 4EGo). At the 3 dpf stage, insulin expression in these mutants remains scattered. In addition, cells expressing insulin stay close to the midline instead of moving to the right side (Fig. 4Go, G–J). The insulin expression remains split and does not recover to make a single islet, although insulin cells are surrounded by the exocrine pancreas in sle mutants at 5 dpf (Fig. 4LGo). Progeny from all the mutant carriers remain alive and are phenotypically indistinguishable from the wild-type siblings at 5dpf. However, we failed to recover any adult homozygous mutant pairs so far, suggesting that the homozygous mutant embryos are not viable up to the adult stage.



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  		Fig. 4. Analysis of Insulin Expression in Class III Mutants

A–E, 24 hpf; F–J, 3 dpf; K–L, 5 dpf; A, F, and K, wild type (WT); B and G, che mutants; C and H, dal mutants; D, I, and L, sle mutants; and E and J, ppp mutants.

 
We performed a detailed molecular analysis of che mutants. Pdx-1 expression is scattered but was not reduced compared with wild type (Fig. 5Go, A and B). Glucagon and somatostatin expression is also scattered similar to insulin expression but the number of cells appear normal (Fig. 5Go, C–F). A normal exocrine pancreas in wild-type embryos at 3dpf has a head domain containing the main pancreatic islet and a tail domain extending to posterior as analyzed by expression of an exocrine marker, carboxypeptidase A (Fig. 5GGo). In che embryos, the head and tail domains of exocrine pancreas are not distinguished (Fig. 5HGo). Analysis of ceruloplasmin showed that liver formation is not affected (Fig. 5Go, I and J). Analysis of foxA2 showed that the liver and intestine are normal, but pancreas is disorganized (Fig. 5Go, K and L). These data suggest that genes in this class affect pancreatic islet morphogenesis.



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  		Fig. 5. Analysis of Endodermal Defects in che Mutant (Class III)

A, C, E, G, I, and K, Wild type; B, D, F, H, J, and L, che mutant; A–F, 24 hpf; G-L, 3 dpf. A and B, pdx-1; C and D, glucagon; E and F, somatostatin; G and H, carboxypeptidase A (notice the disorganized exocrine pancreas in che mutant); I and J, ceruloplasmin; K and L, foxA2. Arrow, Exocrine pancreas; black arrowhead, liver; white arrowhead, intestine.

 
Formation of Pancreatic Islet Architecture in Zebrafish Is Independent from Migration of Insulin Cells
In all Class III pancreatic islet mutants we examined, we noticed that endocrine pancreatic markers glucagon and somatostatin are also scattered. In wild-type embryos, glucagon and somatostatin cells are always closely associated with insulin-positive cells at 24 hpf. We asked whether scattered somatostatin and glucagon expressing cells are still associated with insulin cells despite the abnormal morphology, or positioned separately from insulin-positive cells in class III mutants.

In mammals, insulin and glucagon cells are specified within the primitive pancreatic epithelium before they migrate out to the surrounding mesenchyme to form an islet (4). In wild-type zebrafish embryos, glucagon cells first appear at 24 somatostatin stage and positioned at the periphery of insulin core after insulin cells have already finished migration (9). However, the position of glucagon cells relative to insulin cells during the pancreatic islet morphogenesis has not been studied before in zebrafish. We performed double in situ hybridization of insulin and glucagon in wild-type embryos at different developmental stages. At the 22 hpf stage, low level of glucagon expression is intermingled with insulin expression (Fig. 6AGo). At 24 hpf stage, glucagon expression becomes stronger and positioned at the periphery of insulin cells (Fig. 6BGo). By the 28 hpf stage, the number of glucagon cells has increased and the cells become positioned at the periphery of insulin cells in wild-type embryos (Fig. 6CGo). We also performed double in situ hybridization of insulin and glucagon different class III mutants. Interestingly, in che and sle mutant embryos, scattered glucagon cells are always associated with scattered insulin-positive cells (Fig. 6Go, D and E).



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  		Fig. 6. Analysis of Insulin/Glucagon, and Insulin/Somatostatin Expression Pattern in Wild-Type and Class III Mutant Embryos

A–E, Double in situ hybridization of insulin and glucagon. Insulin, red; glucagon, purple. A–C, Wild-type (WT) glucagon cells (purple) are specified adjacent to insulin cells (A and B) and are located at the periphery of insulin cells (red) (C). B, che mutant; C, sle mutant (insulin cells and glucagon cells are still closely associated in mutant embryos). F–L, Double in situ hybridization of insulin and somatostatin—insulin, red; somatostatin, purple; F, 16 som, somatostatin cells (purple) are located separate from insulin cells (red). G, 18 som; H, 20 som (somatostatin cells are already coalesced into an islet, whereas insulin cells are still dispersed). I, 22 hpf; J, 24 hpf (insulin and somatostatin cells are coalesced into a single islet at this stage). K, che mutant; L, dal mutant (somatostatin cells and insulin cells are associated closely in mutant embryos).

 
Somatostatin cells in zebrafish are intermingled with insulin cells differently from mammals in which somatostatin cells are located at the periphery of pancreatic islets (9). Somatostatin expression starts around the 16 somite stage after insulin cells are specified in zebrafish (9). However, the position of somatostatin cells relative to insulin-positive cells also has not been studied before. As analyzed by the double in situ hybridization with insulin and somatostatin, somatostatin cells are first positioned posteriorly to the insulin expressing cells (Fig. 6FGo). These cells proliferate and coalesce into an islet between the 20–22 somite stages when cells expressing insulin are still in the process of migration (Fig. 6Go, G–I). At the 24 hpf stage, insulin cells finish migration and together with somatostatin cells form a single islet (Fig. 6JGo). Double in situ hybridization of insulin and somatostatin in che and dal mutants shows that even though the morphology of islet is abnormal in these mutants, insulin and somatostatin cells are still associated together (Fig. 6Go, K and L). These data argue that class III mutations affect only migration of islet precursors, but not the formation of characteristic islet architecture.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Early Pancreatic Development in Zebrafish
In all vertebrates, the development of a mature pancreatic islet involves at least three distinctively different processes. First, endocrine cells producing different hormones are specified within the pancreatic endoderm. In both mammals and zebrafish, this event is initially marked by the expression of pdx-1 followed by further differentiation of the islet cells expressing different hormones such as insulin, glucagon, or somatostatin (4, 9). Second, specified cells migrate and proliferate to form an islet. In mammals, differentiated cells migrate out to surrounding mesenchyme to form an islet (5). In zebrafish, insulin-positive cells migrate posteriorly to form a single pancreatic islet medially (25). Third, characteristic mature pancreatic islet architecture is formed with different hormone-secreting cells positioned at a particular location within a pancreatic islet. In mammals, insulin cells are positioned at the core of the islet, whereas somatostatin and glucagon cells are positioned at the periphery of the islet (4). In zebrafish, the position of insulin and glucagon cells is the same as in mammals, but somatostatin cells are dispersed and intermingled with insulin cells differently from mammals (9). This shows that some fundamental processes in pancreatic development are conserved despite other major differences between mammals and zebrafish.

Mutants described in this paper represent genes required for early pancreatic endoderm specification, insulin cell specification and/or proliferation, and pancreatic islet morphogenesis (Fig. 7Go). Because our screening concentrated on isolating mutants without any other associated morphological phenotypes, genes with multiple roles in different tissues at different time points were not likely to be identified during this screen. In addition, mutants that have a normal number of ß-cells with reduced or increased insulin transcript level were possibly missed in the screening because it is difficult to quantify transcript level with in situ hybridization. Mutants that specifically affect formation of glucagon or somatostatin cells also would not be identified in this screen.



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  		Fig. 7. Diagram Illustrating Putative Roles of Genes Isolated from the Screening

Process of early pancreas development involves three different steps. First, pancreatic endoderm is specified within the endoderm. Second, endocrine cells are specified within the pancreatic endoderm. Third, specified endocrine cells migrate to form a mature islet. Based on our molecular analysis, sdr, msl, agl genes are required for pancreatic endoderm specification, whereas mnm is involved in endocrine cell specification. Sle, che, dal, and ppp mutation are involved in the islet morphogenesis.

 
Despite these limitations, however, we isolated mutants that are deficient in pancreatic endoderm specification, insulin cell specification, and islet morphogenesis. By the 24 hpf stage, exocrine and liver tissues are not observed by histological examination and exocrine and liver markers, such as trypsin or ceruloplasmin, respectively, are not detected by in situ hybridization. Thus, any defect in pancreatic islet formation at this stage is likely not secondary defects in exocrine pancreas or liver formation. Further study of these mutants and the subsequent isolation of affected genes will help us to advance our knowledge of different processes of pancreatic development.

The Class III mutants, in particular, represent a new class of genes required for islet morphogenesis, a process that is still not understood very well, but likely to be perturbed in different pancreatic diseases (7, 18). In mammals, it has been difficult to study islet morphogenesis because of the in utero development of mammalian embryos. Positional cloning of these genes is currently in the process and with the development of transgenic technology and the advantages of zebrafish model system, we hope to gain further insight into the process of pancreatic islet morphogenesis.

Regulation of pdx-1 and Pancreatic Endoderm Specification
Two of the identified mutants may have a direct or indirect role in regulating pdx-1 expression. Sdr mutants show expanded pdx-1 expression at 24 hpf stage. This suggests that the down-regulation of pdx-1 expression is required at certain developmental stages to maintain proper pancreatic specification and proliferation. This is consistent with the observation that pdx-1 expression is progressively reduced in the exocrine pancreas but remain high level of expression in the islet and parts of the intestine after 2 d of development in mouse and zebrafish (9, 19). Alternatively, sdr could have two separate functions affecting pdx-1 expression during the early stages of pancreatic development and required for the insulin cell specification and/or proliferation at the later stage. However, because sdr mutants display other phenotypes such as ventral tail curvature, increased pdx-1 expression may be an indirect consequence of other defects. In this case, studying this mutant will help us to identify signals required for proper pancreatic endoderm specification.

In contrast to sdr mutants, msl mutants show reduced pdx-1 expression at 24 hpf stage, suggesting that msl is required for the induction of pancreatic fate. At this stage, no other visible abnormalities were apparent. Further study of these two mutants will determine their direct or indirect roles in regulating pdx-1 expression and pancreatic endoderm specification.

Endocrine Cell Subtype Specification and Pancreatic Architecture Formation
Pancreatic {alpha}- and {delta}-cell specification process is poorly understood in all vertebrates in contrast to ß-cell specification. We show here that glucagon cells are specified adjacent to insulin cells in zebrafish and that glucagon cells associate with insulin cells despite abnormal islet morphology in class III mutants (Fig. 6Go, A–E). We also report for the first time that somatostatin cells are specified separately from insulin cells in zebrafish (Fig. 6FGo). Specified somatostatin cells coalesce into an islet before insulin cells finish migration (Fig. 6Go, G–I). Insulin cells later catch up with somatostatin cells and form an islet (Fig. 6JGo). Insulin and somatostatin cells associate together in the class III mutants despite split insulin expression (Fig. 6Go, K and L).

Taken together, our data show that, regardless of islet morphology, insulin, glucagon, and somatostatin cells associate together. This argues for two distinctively separate mechanisms governing the formation of islet morphology and the association of different endocrine cell to form a mature pancreatic islet. One of the simplest ways for different endocrine cells to achieve this association would be to specify cells adjacent to insulin cells by lateral inhibition mechanism. This may be the case for the specification of glucagon cells, but it cannot be the mechanism for somatostatin cells because somatostatin cells are specified separately from insulin cells. Further study will be required to understand the mechanism of endocrine cell subtype specification and association into a mature islet.

Islet Morphogenesis in Zebrafish
Our data argue for a specific role of class III genes in pancreatic islet morphogenesis. First, class III mutants did not have any visible phenotypes associated until at least 3 dpf. Second, despite abnormal islet morphology, insulin cells were still associated with glucagon and somatostatin cells, indicating that only the process of islet morphogenesis was perturbed in these mutants. Therefore, studying these genes and mutant phenotypes in greater detail will help us to understand better the process of islet morphogenesis.

In summary, this paper reports novel genes involved in early endocrine pancreatic development by analyzing mutant embryos generated by ENU mutagenesis using zebrafish as a model system. We have isolated 11 mutants affecting different aspects of pancreatic development including pancreatic endoderm specification, insulin cell specification, and islet morphogenesis. We also show a genetic evidence that the process of islet morphogenesis and endocrine cell association into an islet is a separate process involving different molecular mechanisms. This study will allow us to understand further the early processes of vertebrate endocrine pancreas development and help us to gain insight into the molecular mechanisms of pancreas development and diseases.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Mutagenesis and Fish Husbandry
Wild-type AB strain 6-month-old male zebrafish was treated with ENU as described (15, 16). Briefly, F1 generation was obtained by mating ENU mutagenized male AB strain adult zebrafish with wild-type AB adult female. F2 families were obtained by raising progenies from a cross between an individual F1 fish to a wild-type fish. We screened up to 10 pairs of each F2 family. Mutants were scored by analyzing insulin expression pattern at 24 hpf. Embryo clutches with approximately 25% of abnormal phenotype from at least three independent mating were scored positive. Mutagenesis rate was calculated by mating to a carrier of golden mutation. Flh mutation carrier was obtained from International Zebrafish Stock Center.

In Situ Hybridization
Single and double in situ hybridization was performed as previously described (20). The following probes were used: pdx-1, insulin (21), glucagon, somatostatin (22), carboxypeptidase A (11), ceruloplasmin (23), and foxA2 (24).

Imaging
Images were captured by a digital camera (Axiocam) attached to a microscope (AxioPlan II; Zeiss, Thornwood, NY) using Openlab program (Improvision, Lexington, MA).


   ACKNOWLEDGMENTS
 
We thank H. D. Pan for excellent support from the fish facility.


   FOOTNOTES
 
This work was supported by grants from the National Institutes of Health (R01HD041367 to J.C. and S.L., R21ES012990 to S.L., and T32-GM008244 to H.K.).

First Published Online August 11, 2005

1 H.J.K. and S.S. contributed equally for the project. Back

Abbreviations: agl, Angelina; che, cheetah; dal, dalmatian; dpf, days post fertilization; ENU, ethyl-nitroso-urea; flh, floating head; hpf, hours post fertilization; mnm, minime; msl, mislet; ppp, perppershaker; sdr, sea dragon; sle, scarlet.

Received for publication May 11, 2005. Accepted for publication August 2, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Kim SK, Hebrok M, Melton DA 1997 Notochord to endoderm signaling is required for pancreas development. Development 124:4243–4252[Abstract]
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