ISL1 Research Articles

The LIM Domain Homeobox Gene isl-1 Is a Positive Regulator of Islet Cell-specific Proglucagon Gene Transcription

The LIM domain homeobox gene islet 1 (isl-1) is expressed in the embryonic nervous system and may be an early marker of motor neuron specification. isl-1 is expressed in all 4 islet cell types, but a role for isl-1 in the regulation of insulin gene expression has not been demonstrated, and the genetic targets for isl-1 in the pancreas remain unknown. We show here that the proximal rat proglucagon gene promoter binds an amino-terminally truncated Trp-E-isl-1 fusion protein that lacks the LIM domains. The proglucagon gene promoter also binds full-length in vitro translated isl-1 containing the intact LIM domains. isl-1 antisera detects binding of proglucagon gene sequences to isl-1 present in a slowly-migrating complex in nuclear extracts from InR1-G9 islet cells. The transcriptional properties of the proglucagon gene promoter sequences that bind isl-1 (designated Ga, Gb, and Gc) were assessed after transfection of reporter genes into wild type and isl-1-antisense (isl-1(AS)) InR1-G9 islet cells. The proximal proglucagon gene (Ga) promoter sequence reduced TK-CAT activity by approximately 50%, but no change in the activity of the Ga-TK-CAT plasmid was seen after transfection of isl-1(AS) InR1-G9 cells. In contrast, the Gb/Gc sites activated transcription 2-3 fold in wild type InR1-G9 cells, and the isl-1-dependent activation of gene transcription through the Gb/Gc element was eliminated following transfection of isl-1(AS) InR1-G9 cells. These data demonstrate that the LIM domain homeobox gene isl-1 1) is not constrained from DNA binding by its LIM domains and 2) functions as a positive regulator of proglucagon gene transcription in the endocrine pancreas.

Presence of isl-1-related LIM Domain Homeobox Genes in Teleost and Their Similar Patterns of Expression in Brain and Spinal Cord

Three novel LIM domain homeobox cDNAs encoding proteins structurally related to the Isl-1 protein were isolated from a chinook salmon pituitary cDNA library. Southern blot analysis of genomic DNA indicate that they are derived from three distinct genes, designated as isl-2a, isl-2b, and isl-3 genes. Nucleotide sequence analysis of reverse transcriptase-polymerase chain reaction amplified products reveal that the isl gene family contains two members (a and b) each of both isl-1 and isl-2 genes, and one member of isl-3 gene in the two tetraploid salmonid species, chinook salmon and rainbow trout, and only one member each of isl-1, isl-2, and isl-3 genes in the diploid zebrafish. The expression of the three isl genes in the rainbow trout were studied by reverse transcriptase-polymerase chain reaction analysis of embryonic and adult RNAs, and by in situ hybridization analysis of 8-week-old hatchlings. The transcripts of all three genes could be detected as early as 4 weeks postfertilization (the eye stage) and increased dramatically in 5-week-old embryos. In the adult, the three isl mRNAs appear to be differentially distributed in various tissues. The level of isl-1 mRNA is generally higher than those of isl-2 and isl-3 mRNAs. In situ hybridization analysis indicates that the transcripts of all three genes are localized in subsets of neurons in the brain and spinal cord. In the retina, isl-1 mRNA could be found in both the ganglion and inner nuclear layers while isl-2 and isl-3 mRNAs could only be detected in the ganglion layer. High level of isl-1 mRNA could also be found in mid-gut and interrenal organ where endocrine cells are densely populated. Based on these observations, we speculate that the three structurally related isl genes may play similar roles in cell determination and differentiation in the developing nervous system.

Cell polarity and the mechanism of asymmetric cell division.

During development, one mechanism for generating different cell types is asymmetric cell division, by which a cell divides and contributes different factors to each of its daughter cells. Asymmetric cell division occurs throughout the eukaryotic kingdom, from yeast to humans. Many asymmetric cell divisions occur in a defined orientation. This implies a cellular mechanism for sensing direction, which must ultimately lead to differences in gene expression between two daughter cells. In this review, we describe two classes of molecules: regulatory factors that are differentially expressed upon asymmetric cell division, and components of a signal transduction pathway that may define cell polarity. The lin-11 and mec-3 genes of C. elegans, the Isl-1 gene of mammals and the HO gene of yeast, encode regulatory factors that determine cell type of one daughter after asymmetric cell division. The CDC24 and CDC42 genes of yeast affect both bud positioning and orientation of mating projections, and thus may define a general cellular polarity. We speculate that molecules such as Cdc24 and Cdc42 may regulate expression of genes such as lin-11, mec-3, Isl-1 and HO upon asymmetric cell division.

The LIM domain homeobox gene isl-1: conservation of human, hamster, and rat complementary deoxyribonucleic acid sequences and expression in cell types of nonneuroendocrine lineage.

The LIM domain homeobox gene isl-1 was originally isolated by virtue of its ability to bind DNA sequences from the 5'-flanking region of the rat insulin gene. Initial experiments localized isl-1 to the islet cells of the pancreas, suggesting a possible role for this protein in the regulation of insulin gene expression and/or islet cell development. More recent studies have determined that isl-1 is also expressed in the central and peripheral nervous system. We now report that isl-1 gene expression is not restricted to cells of neuroendocrine lineage. Northern blot analysis of rat and hamster fibroblasts, rat keratinocytes, and human epidermoid carcinoma cells detected messenger RNA transcripts that hybridized, at high stringency, to different isl-1 complementary DNA probes. DNA sequence analysis of human and hamster isl-1 complementary DNAs generated by reverse transcription-polymerase chain reaction from nonneuroendocrine cell lines demonstrated 100% conservation of isl-1 amino acid sequences from rat hamster and human species. Western blot analysis with isl-1 antiserum demonstrated that immunoreactive isl-1 was detectable in nuclear extracts from islet cell lines. In contrast, immunoreactive isl-1 was only detected in cytoplasmic extracts from nonneuroendocrine cell lines. The results of these studies demonstrate striking conservation of mammalian isl-1 coding sequences and widespread expression of the isl-1 gene in heterologous cells. These experiments suggest that the biological importance of isl-1 may not be restricted to neuroendocrine cell lineages and raise the possibility that isl-1 function may be regulated by sequestration in distinct cellular compartments.

The LIM domain homeobox gene isl-1: conservation of human, hamster, and rat complementary deoxyribonucleic acid sequences and expression in cell types of nonneuroendocrine lineage

The LIM domain homeobox gene isl-1: conservation of human, hamster, and rat complementary deoxyribonucleic acid sequences and expression in cell types of nonneuroendocrine lineage

Insulin Gene Can Be Expressed in the Absence of Isl-1

The possible role of Isl-1 in insulin-gene transcription was investigated using Northern blot analysis to determine whether insulin and Isl-1 gene expression are correlated in various somatic cell hybrids. Among several hybrid cell lines obtained by fusing insulin-producing rat insulinoma (RIN) cells and mouse spleen cells, three (RR2, RR5, and RR11) had amounts of insulin transcripts similar to those of the parental RIN cells, although two of them, RR5 and RR11, lacked Isl-1 protein. In contrast, two R1N × mouse L cell hybrids where insulin expression was extinct still expressed Isl-1. RT-PCR analysis showed that Isl-1 transcripts are widely distributed in various mouse tissues, including pancreas, brain, lung, thymus, and ovary. These results indicate that Is-1 is not essential for insulin gene expression.

The LIM domain-containing homeo box gene Xlim-1 is expressed specifically in the organizer region of Xenopus gastrula embryos.

A novel cysteine-rich motif, named LIM, has been identified in the homeo box genes lin-11, Isl-1, and mec-3; the mec-3 and lin-11 genes determine cell lineages in Caenorhabditis elegans. We isolated LIM class homeo box genes from Xenopus laevis that are closely related to lin-11 and mec-3 in the LIM and homeo domains. This paper deals with one of these genes, Xlim-1. Xlim-1 mRNA is found at low abundance in the unfertilized egg, has a major expression phase at the gastrula stage, decreases, and rises again during the tadpole stage. In adult tissues the brain shows the highest abundance, by far, of Xlim-1 mRNA. The maternal and late expression phases of the Xlim-1 gene suggest that it has multiple functions at different stages of the Xenopus life cycle. In the gastrula embryo, Xlim-1 mRNA is localized in the dorsal lip and the dorsal mesoderm, that is, in the region of Spemann's organizer. Explant experiments showed that Xlim-1 mRNA is induced by the mesoderm-inducer activin A and by retinoic acid, which is not a mesoderm inducer but affects patterning during Xenopus embryogenesis; application of activin A and retinoic acid together results in synergistic induction. The structure, inducibility, and localized expression in the organizer of the Xlim-1 gene suggest that it has a role in establishing body pattern during gastrulation.

Islet Cell and Extrapancreatic Expression of the LIM Domain Homeobox Geneisl-1

The phenotypically distinct cell types of the islets of Langerhans express a number of different hormone genes in a regulated and cell-specific manner. Analysis of the molecular factors important for the control of insulin gene transcription has led to the identification of a number of insulin gene-binding proteins. The cDNAs for several of these proteins have been isolated, including isl-1, a LIM domain homeobox protein reported to be preferentially expressed in islet cells. We now report that isl-1 mRNA transcripts are present not only in islet cells, but also in pheochromocytoma (PC-12) cell lines. Isl-1 mRNA transcripts were also detected in several different regions of rat brain as well as in rat kidney. Analysis of the patterns of gene expression in rat islet cell lines and human pancreatic endocrine tumors demonstrated a variable relationship between the hormonal phenotype of the tumor and expression of the isl-1 and insulin genes. Immunocytochemical studies demonstrated both cytoplasmic and nuclear staining for isl-1 in human pancreatic endocrine tumors, in normal pancreatic A, B, D, and PP cells, and in distal tubular cells of the kidney. Isl-1 immunoreactivity was also widely distributed in neurons in both the central and peripheral nervous system, pituitary, and several human pituitary tumors and pheochromocytomas. The results of these studies implicate a potential role for isl-1 outside the endocrine pancreas in the nervous system and kidney.