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Correspondence to: Kevin Pang, Ontogeny, Inc., 45 Moulten Street, Cambridge, MA 021381118. Email: kpang@ontogeny.com
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Summary |
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We report the isolation and characterization of a serine/threonine kinase expressed during rat pancreas development. This kinase was cloned as part of a general screen using degenerate oligonucleotides to map expression of kinases and receptors during the course of pancreatic development. Sequence analysis showed it to be a member of the ste20-like serine/threonine kinase family. Northern blotting analysis against both fetal and adult tissues showed two transcripts, one of 2 kb and the other of 4 kb. The ratio of transcript expression varied with the tissue. In situ hybridization analysis showed that this gene is expressed in the early gut and pancreatic epithelium. By embryonic Day 15, the transcript is localized to cells that will eventually become exocrine in nature. In situ hybridization analysis also demonstrated high levels of expression in the choroid plexus, the developing myocardium, kidney, CNS, dorsal root ganglia, and testes. In addition, a search of the EST database revealed a related human kinase not previously described. (J Histochem Cytochem 48:13911400, 2000)
Key Words: serine/threonine kinase, pancreas, development, in situ hybridization, CNS
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Introduction |
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Pancreatic development occurs through a series of signaling events between the mesenchyme and epithelium (
A number of soluble growth factors can influence the survival, growth, and differentiation of pancreatic epithelium grown in explant culture ( in transgenic mice has been shown to result in duct hyperplasia (
To better understand the process of pancreatic development and how choices might be made with regard to endocrine vs exocrine differentiation, we have begun mapping the expression patterns of signaling molecules and receptors during development. As part of this approach, we have used degenerate PCR to detect and map the expression of members of the receptor tyrosine kinase (RTK) and serine/threonine kinase (STK) signaling molecule families. RNA from discrete stages of pancreatic development was prepared and subjected to analysis. In our first series of experiments, a number of known signaling molecules previously unmapped to the pancreas were detected.
We describe here the identification of an STK that is expressed during pancreatic development. This STK is identical to the gene PASK identified by
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Materials and Methods |
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Cloning and Sequencing of cDNAs
Timed-pregnant SpragueDawley rats were obtained from Taconic Labs (Germantown, NY). Appropriately staged embryos were dissected and the pancreases collected on dry ice for RNA and either 2% or 4% paraformaldehyde for histology. Total RNA was obtained by Trizol (Gibco BRL; Rockville, MD) extraction. Each RNA sample was quantitated and stored as an EtOH precipitate at -80C until further use. Reverse transcription was performed using SuperScript II (Gibco). The resulting single-stranded cDNA was then amplified using degenerate primers to the conserved IHRDL and WMAPE kinase motifs (
Northern Blotting Analysis of PASK mRNA Expression
RNA was isolated from e18 fetal and adult pancreas using the guanidinium isothiocyanate method (
Localization of PSTK1 mRNA by Whole-mount and Section In Situ Hybridization Analysis
In situ probes were made using a digoxigenin RNA synthesis kit (Boehringer). Digoxigenin-based whole-mount in situ hybridization was performed as described previously (
Immunohistochemistry
Guinea pig anti-insulin antibodies were purchased from Linco Research (St Charles, MO). The antibodies were used at a final concentration of 1:2000 on paraffin sections in a PBSTriton (0.3%) buffer using 1% BSA and 5% normal donkey serum as blocking agent. Secondary antibody was a biotinylated donkey anti-guinea pig antibody purchased from Jackson Immunoresearch (West Grove, PA) and used at a final concentration of 1:500, followed by avidin DHRP (1:200) from Vector Labs (Burlingame, CA). Antibody binding was visualized using a diaminobenzidine substrate kit (Zymed; South San Francisco, CA).
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Results |
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Isolation and Nucleotide Sequence Analysis of PASK cDNAs
Kinase members were identified by RT-PCR amplification of total RNA isolated from four different stages of rat embryonic pancreatic development spanning e12e18. Degenerate primers were chosen from conserved nucleotide (nt) sequences between Domains VIII and IX of receptor tyrosine kinases (RTK) (
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Twelve different kinases were detected in our study, and the frequency of their detection varied with development (Table 1). However, it is noteworthy that the sample size of the analyzed populations was small and therefore the detected differences in clone frequency observed might be as much due to relative stage-specific cloning efficiencies as reflective of relative expression levels. Nevertheless, we include Table 1 as an example of signaling molecules detected by this methodology and heretofore unmapped to pancreatic development. The stages chosen for analysis span both the early expansion phase of pancreatic development (e12e14) and the time period between e15 and e18, a time in which the pancreas is undergoing differentiation (
Interestingly, PDGFR was detected most often in the e12e14 period, whereas c-abl was found more often in the later differentiative phase. Molecules such as Flk1 showed some differences in detected levels and a few, such as the IGF-1 receptor and JAK1, showed low detectable levels.
One of the cloned RT-PCR products detected in our analysis of e18 cDNA encoded what appeared to be a novel kinase on database comparison using BLAST (
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On the basis of the expression pattern, we screened both a pancreas and a testis cDNA library and isolated five independent clones, apparently derived from the same mRNA species because no alternative splice forms were found. The longest cDNA clone was 1.86 kb and was designated pstk1, for pancreas serine/threonine kinase one. It contains a predicted 181-bp 5'-untranslated region followed by a 1659-bp open reading frame encoding a protein of 563 amino acids plus a stop codon, leaving a predicted 3'-untranslated region of 104 bp. The polyadenylation site was not found in any of the clones examined. The predicted amino acid sequence of the protein encoded by this clone is shown in Fig 2. Comparison of this sequence to the human EST database revealed the presence of a homologue that is 97.6% identical to the rat protein. Our analysis also discovered a similar but not identical kinase, which we termed human PSTK-2, which is 77.6% identical to the rat PASK/PSTK1 protein. We believe that these two genes might be part of a separate kinase subgroup. Sequence alignments and cladistic analysis (not shown) revealed pask/pstk-1 to be most similar to the Ste20-like family of kinases (
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Expression in the e14 Rat Embryo
Fig 3 shows a parasagittal section of an e14 rat embryo hybridized with antisense digoxigenin-labeled pask probe. Hybridization was visualized using an alkaline phosphatase-labeled anti-digoxigenin antibody and BM purple (Boehringer). The sections were then counterstained with eosin. Hybridization with sense control showed no staining expression (data not shown). There was a high level of pstk1 expression in the choroid plexus, myocardium, mesonephros, and in the dorsal root ganglia. The choroid plexus, myocardium, and mesonephros, respectively, are shown under higher magnification from top to bottom in the accompanying inset. Expression in the choroid plexus was observed throughout the anterior CNS from telencephalon to myelencephalon. Whole-mount staining also showed strong expression throughout the developing adrenal gland (data not shown).
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Expression in the e15 Embryo
Fig 4 is a more medial parasagittal section of an e15 rat embryo. There was continued strong expression of pask in the choroid plexus. In addition, a number of large ganglia in the CNS now showed strong pask expression. Froriep's ganglion, which is a vestigial DRG whose components later break down to give rise to the motor neuron elements of the hypoglossal (XII) ganglion (
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Pancreatic Expression
Whole-mount in situ hybridization of e12 rat gut tubes showed distinct expression of pstk1 in the early pancreatic epithelium (Fig 5A) and in the epithelium of the developing gut tube (Fig 5B, arrows). The surrounding mesenchyme was negative for pstk1 expression and at this stage the entire pancreatic epithelium appeared to be pask-positive. Fig 5C shows an e12 gut tube hybridized with sense control. Little expression was found in the stomach anlage and no detectable expression was found in the lung. Expression in the gut epithelium could be detected as early as e11 and increased throughout the gut tube at e13, but subsequently decreased with age in an anterior to posterior fashion (data not shown).
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Fig 5A shows whole-mount in situ hybridizations of the foregut region encompassing the stomach and pancreas at e15 and e18 of development. The age-matched sense controls are placed above and the antisense hybridized tissue below. Note that the stomach and the attached spleen are devoid of signal at all stages examined. Expression in the e15 pancreas is most intense along the distal edge of the developing pancreas, where acinus formation begins. As the pancreas develops, there is increased expression and signal strength of pask along the more distal aspects. The proximal portion of the pancreas, in which the larger ducts reside closest to the duodenal origin of the pancreas, is negative for pask expression. The signal for pancreatic expression of pask1 increased significantly by e18, accompanied by the explosion in formation of exocrine epithelium.
As early as e15, exocrine structures could be seen in the developing pancreas. Fig 5B shows a 5-µm paraffin section of an e15 foregut hybridized with pask. The stomach was devoid of pstk1 signal but the nascent pancreatic acini had strong expression. Pask was not seen in the mesenchyme. Note that the duct bed that gives rise to the branching acini (arrows) has little pask signal. The inset shows a higher-magnification photo detailing the strong acinar expression. Fig 5C is a 5-µm paraffin section of an e17 pancreas hybridized for pask (purple) and immunohistochemically stained for insulin (brown). The exocrine acini were again strongly positive for pask hybridization. The duct bed had very low levels of pask expression and was giving rise to strongly insulin-immunoreactive cells. The expression of PSTK1 mRNA and insulin protein appeared mutually exclusive. Further in situ studies in the adult pancreas showed continued extensive exocrine acinar expression (data not shown).
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Discussion |
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Our screen for kinases expressed in the developing pancreas uncovered a number of molecules whose expression levels varied with stage of development. Of particular interest are the PDGF- and Flk-1 receptors, which bind PDGF and VEGF (
During the course of our mapping studies, expression of pask was discovered and studied. The expression pattern of this gene suggests that it may be important in the early formation of a variety of tissues, including the pancreas. The amino acid sequence of pask indicates that it is most closely related to the OPK Group III family of protein kinases as defined by
Rat pask transcripts were detectable in the embryo by in situ hybridization as early as e9 (data not shown). Expression was strong in the heart, telencephalic neuroepithelium, and the first two aortic arches, and was weak in the region of the developing gut endoderm. This pattern of expression was observed at all stages studied, with spreading of signal observed in tissues and organs such as the pancreas, adrenal medulla, kidney, dorsal root ganglia, heart, and choroid plexus. CNS expression appeared primarily in the cochlear ganglion at e15, and was not detectable in any other neural structures. Expression of pask increased with time in most positive tissues studied, an exception being the early gut epithelium in which signal progressively decreased in an anterior to posterior fashion, thus indicating that pask is a marker of early gut epithelium but is subsequently lost with differentiation.
In contrast, the pancreas exhibits early expression in the entire bud epithelium, which then progressively localizes to the exocrine acini. In the e12 rat pancreas, expression of pask is found in the early epithelial bud, but by e14/15 expression is predominantly found in the distal portions of the pancreas, where the bulk of the acini are budding off the main epithelial trunk. By e17, there is clear exclusion of pask expression from the main branch of epithelium that gives rise to the insulin-positive ß-cells and clear localization to the branching exocrine acini. Studies of the adult pancreas show continued high expression of pask in the exocrine tissue (data not shown).
The role of PASK in vertebrate development is not yet known. However, our spatial expression studies in the embryo might provide insight as to its role in early tissue formation. The expression of pask in early organogenesis of a number of tissues indicates a possible early role. It is interesting to speculate that many of the tissues that express pstk1 also express, and in some cases are known to be regulated by, members of the bone morphogenetic protein (BMP) gene family, a branch of the TGF-ß superfamily, whose signal is mediated by activating serine/threonine receptor kinases (
The recent work of
The approach of using degenerate PCR to detect the expression of specific gene families as a function of time in development, followed by spatial localization either by in situ hybridization or immunohistochemistry, is useful in studying the ontogeny of that tissue. For example, the expression of many of the kinases and receptors detected during this study had not been previously mapped to the pancreas. The expression of key regulatory molecules can be rapidly detected and mapped to particular cell types at key physiological transitions during development in tissue targets of interest, e.g., the gain of exocrine or endocrine function in the case of the pancreas through the use of these methods. This mapping also provides markers by which specific cell types can be identified and perhaps isolated and, in the process, information regarding possible regulation of either growth or differentiation can be obtained. In addition, as demonstrated here, this approach also provides the possibility of discovering new and interesting gene family members.
In conclusion, the approach that we have taken in terms of mapping the temporal and spatial expression of specific gene family members as a function of tissue formation and development is a useful one. This process combines precise dissection of stage-specific tissue with degenerate PCR, sequencing, and high-throughput in situ hybridization analysis. This approach can be applied to any target tissue or organ of choice. We are now exploring subtractive array hybridization technologies to increase the throughput of discovering genes within given families that are differentially expressed either as a function of time in development and/or spatial expression within a given tissue. The knowledge gained through these efforts will help to develop novel markers by which specific cell types can be identified in subsequent lineage studies, map the expression of important signal transduction genes as demonstrated herein and, finally, provide insights into mechanisms of growth and differentiation.
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Acknowledgments |
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We would like to thank David Kagan for help with molecular biology, David Bumcrot for help with in situ hybridization, Nagesh Mahanthappa and Irina Karavanova for help with in situ analysis, and Mina Peshavaria for critical analysis of the manuscript.
Received for publication November 4, 2000; accepted April 13, 2000.
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