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Howard Hughes Medical Institute, Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois 60637
Address all correspondence and requests for reprints to: W. M. Milewski, Howard Hughes Medical Institute, University of Chicago, 5841 South Maryland Avenue, MC 1028, Chicago, Illinois 60637. E-mail: wmilewsk{at}haven.uchicago.edu
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To learn more about the mechanisms that regulate the process of
pancreas development, recent studies have focused on the function of
islet-specific transcription factors. The homeodomain protein PDX-1 has
been shown to be required for full proliferation and differentiation of
the pancreatic anlagen into the mature pancreas. Several studies showed
that PDX-1 expression during embryonic development is restricted to
dorsal gut endoderm and later to dorsal and ventral pancreatic anlaga
and duodenal epithelium (2, 3, 4). In adulthood, its expression is mainly
restricted to the insulin producing ß and somatostatin producing 
cells of the pancreas, and epithelial cells of the duodenum (4, 5, 6, 7).
PDX-1 has been shown to activate both the insulin and somatostatin
promoters by binding to specific DNA sequences and interacting with
other DNA binding proteins (8, 9, 10, 11, 12, 13, 14). The importance of PDX-1 in the
development of the pancreas was recently illustrated in PDX-1 deficient
mice. Pdx-1-/- mice, generated by targeted gene disruption,
are born without a pancreas and die shortly after birth (15, 16).
Although much has been learned from studies on mice, the emergence of the zebrafish as a model for vertebrate development (17) suggests that investigation into pancreatic development of this organism might be informative. We therefore decided to search for a PDX-1 homolog in zebrafish, determine its pattern of expression during development and in adults, and to evaluate its ability to activate the insulin promoter.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Animals
Adult zebrafish (Brachydanio rerio) were obtained
from local commercial sources and the embryos were purchased from
Scientific Hatcheries (Huntington Beach, CA).
Preparation of zebrafish DNA, RNA, and complementary DNA (cDNA)
Genomic DNA and total RNA were isolated from adult zebrafish
using standard procedures (18). Poly(A+) messenger RNA
(mRNA) was purified from total RNA by oligo(dT) cellulose
chromatography (Stratagene, La Jolla, CA) and used as a template for
MMLV reverse transcriptase (Life Technologies, Grand Island, NY) to
synthesize cDNA.
cDNA library construction
cDNA with cohesive EcoRI ends was synthesized from
Poly(A+) mRNA using a cDNA synthesis kit (Pharmacia,
Piscataway, NJ), then ligated to EcoRI digested lambda Zap
II Express vector (
Zap II, Stratagene) and packaged into phage
(Stratagene).
Nucleotide sequencing
PCR products were screened by Southern blotting and positive PCR
products were subcloned into the EcoRV site of pBluescript
II SK+ (pBSK+, Stratagene) and sequenced using
oligonucleotide primers and the Sequenase 2.0 DNA sequencing kit
(United States Biochemical Corp., Cleveland, OH).
Isolation of the cDNA encoding zebrafish PDX-1
A 110-bp homeodomain sequence from zebrafish cDNA was obtained
by PCR with degenerate oligonucleotides corresponding to the conserved
region of known homeoboxes. Primer 1 (5'GAPCTNGAPA-APGAPTTQCAQTT-3')
and primer 2 (5'-TTQTGPAACCA(G/A/T)ATQTT(G/A/T)AT-3'),
(P = A or G, Q = C or T and N = A, T, G,
or C) were designed to encode, respectively, the sense strand
of amino acids ELEKEFHF and the antisense strand of amino acids IKIWFQN
(Fig. 1
). The PCR profile consisted of 35
cycles at 94 C for 60 sec, 50 C for 90 sec, 72 C for 90 sec. In
addition, a partial sequence was also obtained by PCR of a cDNA
library. Additional 5' sequence of the isolated zebrafish homeodomain
was obtained with antisense primer 3 (5'-TTTCTCGAGCTCGAGCAGCTG-3')
specific to the identified nucleotide sequence and degenerate
oligonucleotide primer 4 (5'-CCNGCNTGQCTNTAQATGGG-3') (Fig. 1)
corresponding to the conserved amino acids (PACLYMG) in the
amino-terminal region of known proteins from the PDX-family. PCR
amplification of zebrafish cDNA was performed for 40 cycles at 94 C for
60 sec, 50 C for 90 sec, 72 C for 90 sec. To isolate the remaining cDNA
sequence, specific primers were used for the rapid amplification of
cDNA ends (RACE, 19). Briefly: in the first strand synthesis, the poly
A+ RNA was primed with gene specific antisense primer 5
(5'-TCATCCACGGAAATGGGAGAT-3') (Fig. 1). Following cDNA synthesis,
terminal deoxynucleotidyl transferase (TdT) was used to attach a
homopolymeric tail to the 5' end of the cDNA. Tailed cDNA was then
amplified by PCR using a nested antisense primer 6
(5'-ATCTGTTCC-GATCCCCGCAGA-3') (Fig. 1) and a dT17 adapter.
This allowed amplification of the unknown sequence between primer 6 and
5' end of the cDNA. In the 3' RACE, cDNA was synthesized with the
oligo-dT adapter and then amplified by PCR using dT17 adapter and a
sense primer 7 (5'-AACAAATACATCTCGCGCCC-3') specific for the coding
region of the isolated homeodomain sequence (Fig. 1). This allowed
amplification of unknown sequences between primer 7 and 3' end of the
cDNA.
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), were used in PCR amplification of
zebrafish cDNA. PCR consisted of 35 cycles at 94 C for 60 sec, 53 C for
90 sec, 72 C for 90 sec. The PCR product was subcloned into the
EcoRI/XbaI site of the pCMV-6C to produce plasmid
pCMV-zPDX1.
Isolation of zebrafish preproinsulin
Degenerate oligonucleotide primers ins1
(5'-GGTATQGTNGANCAPTGQ-TG-3') and ins2
(5'-TTAGTTGCAGTAGTTQT(G/C)CAG-3'), corresponding to conserved regions
of A-chain, were designed to encode, respectively, the sense strand of
amino acids GIVEQCC and the antisense strand of amino acids LQNYCN
(Fig. 2). These primers were used in PCR
amplification of zebrafish genomic DNA, with a PCR profile consisting
of 40 cycles at 94 C for 60 sec, 50 C for 90 sec, and 72 C for 90 sec.
To isolate the remaining nucleotide sequence of the zebrafish
preproinsulin cDNA, sense primer ins3 (5'-CAGTGCTGCCA-CAAACCCTGC-3')
and antisense primer ins4 (5'-AGTAGTTCTGCAGCT-CAAAGA-3') (Fig. 2)
specific to the identified nucleotide sequence were synthesized. These
primers were paired with either of two primers (T3 or T7) complementary
to sequences in the Zap II vector, and used to amplify cDNA inserts
from zebrafish Zap II cDNA library by PCR. The PCR profile
consisted of 35 cycles at 94 C for 60 sec, 55 C for 90 sec, and 72 C
for 90 sec.
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),
respectively, and used in PCR amplification of zebrafish cDNA. The PCR
profile consisted of 35 cycles at 94 C for 60 sec, 55 C for 90 sec, and
72 C for 90 sec. The PCR product was cloned into the EcoRV
site of pBluescript II SK+ vector to produce plasmid
pSK-zINS.
Computer analysis
DNA and protein sequence analysis was performed with the DNA
Strider 2.0 sequence analysis software. The National Center for
Biotechnology Information database was screened with the Basic Local
Alignment Sequencing Tool (BLAST) (20). Protein sequence alignments
were performed with the Clustal W (version 1.4) multiple-alignment
program (21).
In situ hybridization
The 44 and 72 hpf (hours postfertilization) zebrafish embryos
and the adult zebrafish internal organs were fixed and sectioned as
described by Njølstad et al. (22), except that the slides
were coated with polylysine and the sections were dried for 4 h
after sectioning and then stored at -80 C before use.
The 35S-labeled antisense and sense riboprobes derived from
the coding regions of zebrafish PDX-1 (nt 478 to 837, Fig. 1) or
preproinsulin (nt 1 to 437, Fig. 2) cDNA, were prepared using riboprobe
in vitro transcription kit (Promega, Madison, WI) and
5'[
35S] UTP (Amersham, Arlington Heights, IL). The
proteinase K pretreatment, hybridization and washing were carried out
according to Simmons et al. (23), except that the proteinase
was used at concentration 0.1 µg/ml (10 min at 37 C for adult
sections and 3 min at room temperature for embryos).
For autoradiography the slides were coated with Kodak NTB2 emulsion (Kodak, New Haven, CT) diluted (1:1) with water, air dried, and exposed at 4 C in light-proof boxes for 2 weeks (adult sections) or 4 weeks (embryo sections). Developing was performed according to manufacturer’s recommendations. For light microscopy slides were stained with hematoxylin, Richardson’s stain, or hematoxylin and eosin.
DNA constructs
The pGL3-Fluc reporter vector containing
the Firefly luciferase (Fluc) gene coding
sequence and the control pRL-SV40-Rluc reporter vector
containing Renilla luciferase (Rluc) gene coding
sequence were purchased from Promega. The p524INS-Fluc
reporter plasmid was constructed by cloning of a 524 bp
BamHI/HindIII fragment from rat insulin I
promoter sequences (24) into BglII/HindIII
digested pGl3-Fluc. The
p524INSmut-Fluc vector was generated by site-directed
mutagenesis of the A1 element at position -70 to -85 (25), from
TAATGGG to TCCTGGG, using the Stratagene Quik Change system. The mouse
RSV-PDX1 (pRSV-mPDX1) expression vector was a gift from Dr. Helena
Edlund, Sweden (3).
Transfections
Transfection experiments were performed in NIH3T3 cells with
lipofectAmine according to the manufacturer’s protocol (Life
Technologies). The cells were cotransfected with 1 µg of the
pCMV-zPDX1 or pRSV-mPDX1 expression vectors and 1 µg of
p524INS-Fluc or p524INSmut-Fluc reporter vector
plus 50 ng of pRL-SV40-Rluc internal control reporter
vector. Control transfections were performed using the pCMV and
p524INS-Fluc plus pRL-SV40-Rluc. Cells were
harvested 48 h after transfection and the cell extracts were
assayed for Fluc and Rluc activities using
Dual-luciferase Reporter Assay System (Promega). The enzymes activities
were quantitated on a Monolight luminator 2010 (Analytical Luminescence
Laboratory, San Diego, CA) and normalized to the Rluc
activity derived from cotransfection with pRL-SV40-Rluc
internal control reporter vector.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The
expected band of 110 bp was cloned into a pBSK+ vector.
Twenty-five clones were randomly selected and analyzed by sequencing.
Several homeobox sequences were identified but only one contained an
unique histidine residue within the predicted homeobox reading frame
and the encoded amino acid sequence was almost identical to the
corresponding sequence of PDX-1 proteins from other species (3, 5, 7, 8, 26). The nucleotide sequence of the zebrafish PDX-1 cDNA was
extended using PCR based methods and is shown in Fig. 1. It contains 96
nt of 5' untranslated region, a 738 nt open reading frame (ORF) and 108
nt of the 3' untranslated region. The 738-bp ORF encodes a protein of
246 amino acids with a predicted mol wt of 27.3K. The 62-amino acid
homeodomain (HD) is flanked at the amino-terminus by 134 amino acids
and 50 amino acids at the carboxy-terminus.
An alignment of the zebrafish protein with PDXs from other species
showed 97% amino acid similarity (95% identity) within the HD region
(Fig. 3), but the sequences differed in
the regions flanking the homeodomain. Individual amino acid sequence
alignments indicated that the zebrafish protein had a very high and
essentially identical degree of similarity to the frog homeodomain
protein XlHbox8 (7), rat IDX-1 (8), and human IPF-1 (26) (69–70%
similarity and 58–59% identity). However, when the comparison was
made in the C-terminal region flanking the HD, the highest similarity
and identity was found between zebrafish PDX-1 and XlHbox8 (68% and
48%, respectively). It is interesting to note that the zebrafish PDX-1
is much shorter (246 amino acids vs. 283 or 271) than the
other PDX-1 proteins, especially in the C-terminal region. Also, of the
four PDX-homologues isolated to date, zebrafish PDX-1 has three unique
amino acid substitutions within the conserved homeodomain motif. The
differences are found in position 172 where valine is substituted by
leucine, position 173 where methionine is substituted by threonine and
position 175 where asparagine is substituted by serine (Fig. 3).
However, the histidine that is unique to PDX-1 homeodomains is
conserved in zebrafish PDX-1. Outside the homeobox, the sequences
differ, although there are some regions of significant homology. There
is a conserved sequence LPFPWMKSTK at position 107 -116. Also, high
homology can be found in the first 38 amino acids of the N-terminal
region and in the region immediately downstream of the HD (Fig. 3). The
proline- and histidine-rich stretches present in the C-terminal and
N-terminal regions of other PDX-1 proteins are completely absent in the
zebrafish sequence.
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). The remaining sequence was
obtained by PCR amplification of a zebrafish Zap II cDNA
library.
The nucleotide sequence of zebrafish preproinsulin contains 44 nt of 5'
untranslated region, 324 nt of open reading frame encoding 108 amino
acids for zebrafish preproinsulin, and 100 nt of 3' untranslated
region. The putative translation initiation codon is assigned at
nucleotide position 45, the putative polyadenylation signal sequence
(AATAAA) was found at position 450–456 (Fig. 2).
The alignment of the nucleotide sequence of zebrafish preproinsulin
with those from other species (salmon, anglerfish, hagfish, carp,
chicken, human) revealed 95% similarity to carp (Cyprinus
carpio) (27) preproinsulin, and less sequence homology to the
other species (especially in the C-domain coding region). Southern blot
analysis indicates that both preproinsulin and PDX-1 are single copy
genes in zebrafish (Fig. 4)
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PDX-1 expression in adult rats and mice is mostly restricted to insulin
secreting ß cells of the pancreatic islets and duodenal epithelium.
Therefore, the 35S-labeled zebrafish PDX-1 riboprobe was
used in parallel with insulin riboprobe as a ß-cell marker. The adult
or embryonic sections were hybridized alternately with PDX-1 and
insulin sense or antisense riboprobes. The sense riboprobes were used
to confirm the specificity of the hybridization signals in the tissue.
Both control riboprobes gave an uniform, low level of signal intensity
in the adult and embryonic sections as is shown for PDX-1 in Figs. 5D, 6C, and 7C. Some nonspecific labeling was
observed over the eye and trunk region of the 72 hpf embryo with PDX-1
and insulin sense and antisense riboprobes (Fig. 5, F–H).
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, B and C). However, PDX-1
seems to be expressed at a higher level than insulin and the PDX-1
signal extends posteriorly to the insulin positive cells (Fig. 5B). In
contrast, in the 72 hpf embryonic sections, the PDX-1 expression is
much weaker than insulin and is confined only to the region of insulin
positive cells (Fig. 5, F and G). The positive cells are located more
posteriorly and ventrally to the yolk in comparison to the sections
from 44 hpf embryo.
Analysis of serial cross-sections of adult tissue revealed that PDX-1
and insulin were expressed in the same restricted regions, although the
signal intensity was much higher for insulin. In the sections shown in
Fig. 6, A and B, the PDX-1 and insulin transcripts are found in
pancreatic tissue located between the intestine and liver. Under light
microscopy, the positive cells are seen near duct-like structures (Fig. 6D), probably representing pancreatic ducts (28). In the longitudinal
sections of the adult tissue, the PDX-1/insulin positive cells are seen
in several sites consistent with the presence of several islets in the
pancreatic parenchyma (Fig. 7, A and B). Interestingly, additional
PDX-1 expression could be readily detected in the pyloric caeca located
in close proximity to the intestine (Fig. 7B). Insulin expression was
not detected in this tissue.
Zebrafish PDX-1 transactivates the rat insulin I gene promoter
To determine whether zebrafish PDX-1 could activate the rat
insulin I gene promoter, we performed transient transfection assays in
noninsulin producing cells (NIH3T3). As a positive control we used
mouse PDX-1, which is known to bind to and transactivate the rat
insulin I gene promoter (3). The NIH3T3 cells were cotransfected with
the zebrafish PDX-1 (pCMV-zPDX1) or mouse PDX-1 (pRSV-mPDX1) expression
vectors and the p524INS-Fluc reporter vector. The
p524INS-Fluc construct contained nucleotides -480 to +44 of
the rat insulin I gene upstream of the gene encoding Firefly
luciferase. As shown in Fig. 8 the
zebrafish PDX-1 could induce Fluciferase activity about
10-fold, indicating that it was able to transactivate rat insulin I
gene promoter. Comparable results were obtained with mouse PDX-1.
When zPDX-1 or mPDX-1 were cotransfected with the
p524INSmut-Fluc construct, which contains a mutation in one
of the four PDX-1-binding A elements of the rat insulin promoter,
activation was decreased.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, ß, , and PP cell types
(1). Pancreatic development in the zebrafish is less well
characterized, but pancreatic islet and exocrine cell precursors have
been observed in the developing proximal gut of 52 hpf embryo (28). The transcription factor PDX-1 is necessary for pancreatic development in mammals (15, 16, 29). Based on a detailed analysis of gene expression and pancreatic epitheliomesenchymal interactions in normal and pdx1-/- mice, Ahlgren et al. (29) hypothesized that PDX-1 has dual functions in pancreatic development. PDX-1 is first expressed in the patterned embryonic gut epithelium in response to as yet undefined inductive signals. High levels of PDX-1 expression in the dorsal and ventral pancreas diverticulum are believed to result in activation of genes responsible for epithelial growth and differentiation. Early insulin-positive cells appear at this point. As morphogenesis proceeds PDX-1 is down-regulated. This is followed by a secondary transition state that occurs between e11 and e15 in which there is a marked increase in the number of insulin-positive cells that express PDX-1. PDX-1 has been shown to activate the insulin gene promoter (3, 9, 10, 12, 13, 14) and may also play a role in ß-cell differentiation (3, 16, 29).
The expression data presented in this study suggests that the dual function of PDX-1 in pancreas development and ß-cell differentiation has been conserved in the zebrafish. At 44 hpf intense staining for PDX-1 is seen ventral to the notochord in the region of the developing gut. At this point, PDX-1 is expressed at much higher levels than insulin and over a broader area of the cell layer (compare 5B and 5C). By 72 hpf however, PDX-1 expression is barely detectable and is restricted to the insulin positive cells (compare 5F and 5G). In both longitudinal and cross-sections of adult zebrafish PDX-1 is coexpressed with insulin in islet cells. The distribution pattern of insulin expression in zebrafish is typical of that of the Cyprinidae family (30, 31). The pancreatic endocrine cells are not concentrated in one organ. Instead they form one or more principal islets or Brockman bodies, which are located between the intestine and the liver near the pancreatic duct and which consist almost entirely of endocrine cells. It is not unusual to find some additional smaller islets scattered along the intestine. Similar observations on the localization of pancreatic tissue in adult zebrafish were described by Pack et al. (28). Additionally, we have detected insulin producing endocrine cells in the 44 hpf embryo while Pack et al. first detected insulin by immunocytochemistry in the 96 hpf embryo. The discrepancy is likely due to the difference in the sensitivity of in situ hybridization vs. immunostaining. It is not clear when the first insulin transcripts appear, since the earliest stage we studied was the 44 hpf embryo.
In sections of adult tissue, we also observed very high expression levels of PDX-1 in the pyloric caeca. Pyloric caeca are blind pouches of secretory tissue located in the proximal region of the intestine (32). Some fish species possess one or more pyloric caeca that function to increase the gut surface area and may play a role as an additional digestive organ. Pancreatic exocrine cells secreting trypsin, lipase, and amylase have been identified in the pyloric caeca (33). Several endocrine cells producing regulatory peptides such as CCK, IGF-I, IGF-II, glucagon, and somatostatin have also been found in this structure (34, 35, 36). We find that PDX-1 transcripts are widely distributed in the zebrafish caeca. PDX-1 might be expressed in the mucosal epithelium, pancreatic exocrine cells, or both. Because mammalian PDX-1 proteins are expressed in the epithelial cells of rostral duodenum, zebrafish PDX-1 expression is probably restricted to the epithelial cells of the caeca, where it may be required for maintenance of an exocrine cell phenotype. XlHbox8 has been shown to be present in exocrine cell of the adult Xenopus pancreas (2, 6, 7).
Transient transfection experiments in NIH3T3 cells with the rat insulin I promoter-luciferase reporter construct indicate that zebrafish and mouse PDX-1 are equipotent in activating insulin gene expression. When the A1 element was mutated, activation by zebrafish and mouse PDX-1 was decreased, indicating that both transcription factors bind to the same site on the insulin promoter.
This is not surprising given the high degree of conservation of residues that have been shown to be required for DNA binding and/or transcriptional activation by mammalian PDX-1 and the Xenopus homolog XlHbox8. The homeodomain region of zebrafish PDX-1 is 95% identical to the Xenopus and mammalian proteins. Three subdomains of the N-terminal region of PDX-1 and XlHbox8 that are required for synergistic activation of insulin enhancer-mediated transcription with the bHLH proteins E2/5 and E47 have been identified. Subdomain A (residues 13 to 22) and subdomain B (residues 32 to 38) have been well conserved in zebrafish PDX-1, whereas homology is low in subdomain C (residues 60 to 73) (numbers correspond to rat and human PDX-1, Peshavaria et al., 1997). Furthermore, it has recently been demonstrated that mouse PDX-1 can activate the trout insulin gene (37).
Immediately upstream of the homeodomain is the pentapeptide FPWMK that is conserved in all PDX-1 proteins and is required for cooperative binding to the somatostatin promoter with the DNA binding cofactor Pbx (11). Also, the histidine residue that is unique to PDX-1 homeodomains and has been shown to be required for DNA binding and transcriptional activation of the rat somatostatin gene promoter is conserved (13). Conservation of these structures suggests that zebrafish PDX-1 may also regulate the somatostatin gene.
In summary, we have shown that the primary structure of zebrafish PDX-1 is very similar to that of the Xenopus and mammalian homologs. The regions of PDX-1 that are involved in the regulation of insulin and somatostatin gene expression have been conserved in zPDX-1, and the zebrafish transcription factor is equipotent with mouse PDX-1 in activating the rat insulin promoter. Like its mammalian counterparts, zebrafish PDX-1 displays a biphasic expression pattern during development, implying a dual function in growth of the pancreatic buds and in differentiation and insulin expression of ß-cells. In addition, we find very high levels of PDX-1 in the adult pyloric caeca, indicating a potential role in regulation of expression of genes specific to the exocrine cell phenotype.
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Acknowledgments |
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Footnotes |
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Received September 9, 1997.
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G. Weidinger, U. Wolke, M. Koprunner, C. Thisse, B. Thisse, and E. Raz Regulation of zebrafish primordial germ cell migration by attraction towards an intermediate target Development, January 1, 2002; 129(1): 25 - 36. [Abstract] [Full Text] [PDF]
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G. Leibowitz, S. Ferber, A. Apelqvist, H. Edlund, D. J. Gross, E. Cerasi, D. Melloul, and N. Kaiser IPF1/PDX1 Deficiency and {beta}-Cell Dysfunction in Psammomys obesus, an Animal With Type 2 Diabetes Diabetes, August 1, 2001; 50(8): 1799 - 1806. [Abstract] [Full Text] [PDF]
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B. Bisgrove, J. Essner, and H. Yost Multiple pathways in the midline regulate concordant brain, heart and gut left-right asymmetry Development, January 8, 2000; 127(16): 3567 - 3579. [Abstract] [PDF]
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 Y.-T. Yan, K. Gritsman, J. Ding, R. D. Burdine, J. D. Corrales, S. M. Price, W. S. Talbot, A. F. Schier, and M. M. Shen Conserved requirement for EGF-CFC genes in vertebrate left-right axis formation Genes & Dev., October 1, 1999; 13(19): 2527 - 2537. [Abstract] [Full Text]
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