Lateral asymmetry in multicellular organisms.
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Animals↗
Biomedical subjects
C V Wright
Publications and source records attributed to C V Wright.
An endocrine-exocrine switch in the activity of the pancreatic homeodomain protein PDX1 through formation of a trimeric complex with PBX1b and MRG1 (MEIS2).
HOX proteins and some orphan homeodomain proteins form complexes with either PBX or MEIS subclasses of homeodomain proteins. This interaction can increase the binding specificity and transcriptional effectiveness of the HOX partner. Here we show that specific members of both PBX and MEIS subclasses form a multimeric complex with the pancreatic homeodomain protein PDX1 and switch the nature of its transcriptional activity. The two activities of PDX1 are exhibited through the 10-bp B element of the transcriptional enhancer of the pancreatic elastase I gene (ELA1). In pancreatic acinar cells the activity of the B element requires other elements of the ELA1 enhancer; in beta-cells the B element can activate a promoter in the absence of other enhancer elements. In acinar cell lines the activity is mediated by a complex comprising PDX1, PBX1b, and MRG1 (MEIS2). In contrast, beta-cell lines are devoid of PBX1b and MRG1, so that a trimeric complex does not form, and the beta-cell-type activity is mediated by PDX1 without PBX1b and MRG1. The presence of specific nuclear isoforms of PBX and MEIS is precisely regulated in a cell-type-specific manner. The beta-cell-type activity can be detected in acinar cells if the B element is altered to retain binding of PDX1 but prevent binding of the PDX1-PBX1b-MRG1 complex. These observations suggest that association with PBX and MEIS partners controls the nature of the transcriptional activity of the organ-specific PDX1 transcription factor in exocrine versus endocrine cells.
Animals↗
XBMPRII, a novel Xenopus type II receptor mediating BMP signaling in embryonic tissues.
Bone Morphogenetic Proteins (BMPs) are potent regulators of embryonic cell fate that are presumed to initiate signal transduction in recipient cells through multimeric, transmembrane, serine/threonine kinase complexes made up of type I and type II receptors. BMPRII was identified previously in mammals as the only type II receptor that binds BMPs, but not activin or TGFbeta, in vitro. We report the cloning and functional analysis in vivo of its Xenopus homolog, XBMPRII. XBMPRII is expressed maternally and zygotically in an initially unrestricted manner. Strikingly, XBMPRII transcripts then become restricted to the mesodermal precursors during gastrulation. Expression is lower in the dorsal organizer region, potentially providing a mechanism to suppress the actions of BMP4 on dorsally fated tissues. Similar to the results seen for a truncated type I BMP receptor (tBR), a dominant-negative form of XBMPRII (tBRII) can dorsalize ventral mesoderm, induce extensive secondary body axes, block mesoderm induction by BMP4 and directly neuralize ectoderm, strongly suggesting that XBMPRII mediates BMP signals in vivo. However, although both tBRII and tBR can induce partial secondary axes, marker analysis shows that tBRII-induced axes are more anteriorly extended. Additionally, coinjection of tBRII and tBR synergistically increases the incidence of secondary axis formation. A truncated activin type II receptor (deltaXAR1) is known to block both activin and BMP signaling in vivo. Here we show that such crossreactivity does not occur for tBRII, in that it does not affect activin signaling. Furthermore, our studies indicate that the full-length activin type II receptor (XAR1) overcomes a block in BMP4 signaling imposed by tBRII, implicating XAR1 as a common component of BMP and activin signaling pathways in vivo. These data implicate XBMPRII as a type II receptor with high selectivity for BMP signaling, and therefore as a critical mediator of the effects of BMPs as mesodermal patterning agents and suppressors of neural fate during embryogenesis.
Activin Receptors↗
Hepatocyte nuclear factor 3beta is involved in pancreatic beta-cell-specific transcription of the pdx-1 gene.
The mammalian homeobox gene pdx-1 is expressed in pluripotent precursor cells in the dorsal and ventral pancreatic bud and duodenal endoderm, which will produce the pancreas and the rostral duodenum. In the adult, pdr-1 is expressed principally within insulin-secreting pancreatic islet beta cells and cells of the duodenal epithelium. Our objective in this study was to localize sequences within the mouse pdx-1 gene mediating selective expression within the islet. Studies of transgenic mice in which a genomic fragment of the mouse pdx-1 gene from kb -4.5 to +8.2 was used to drive a beta-galactosidase reporter showed that the control sequences sufficient for appropriate developmental and adult specific expression were contained within this region. Three nuclease-hypersensitive sites, located between bp -2560 and -1880 (site 1), bp -1330 and -800 (site 2), and bp -260 and +180 (site 3), were identified within the 5'-flanking region of the endogenous pdx-1 gene. Pancreatic beta-cell-specific expression was shown to be controlled by sequences within site 1 from an analysis of the expression pattern of various pdr-1-herpes simplex virus thymidine kinase promoter expression constructs in transfected beta-cell and non-beta-cell lines. Furthermore, we also established that this region was important in vivo by demonstrating that expression from a site 1-driven beta-galactosidase reporter construct was directed to islet beta-cells in transgenic mice. The activity of the site 1-driven constructs was reduced substantially in beta-cell lines by mutating a hepatocyte nuclear factor 3 (HNF3)-like site located between nucleotides -2007 and -1996. Gel shift analysis indicated that HNF3beta present in islet beta cells binds to this element. Immunohistochemical studies revealed that HNF3beta was present within the nuclei of almost all islet beta cells and subsets of pancreatic acinar cells. Together, these results suggest that HNF3beta, a key regulator of endodermal cell lineage development, plays an essential role in the cell-type-specific transcription of the pdx-1 gene in the pancreas.
Animals↗
Functional characterization of the transactivation properties of the PDX-1 homeodomain protein.
Pancreas formation is prevented in mice carrying a null mutation in the PDX-1 homeoprotein, demonstrating a key role for this factor in development. PDX-1 can also bind to and activate transcription from cis-acting regulatory sequences in the insulin and somatostatin genes, which are expressed in pancreatic islet beta and delta cells, respectively. In this study, we compared the functional properties of PDX-1 with those of the closely related Xenopus homeoprotein XIHbox8. Analysis of chimeras between PDX-1, XIHbox8, and the DNA-binding domain of the Saccharomyces cerevisiae transcription factor GAL4 revealed that their transactivation domain was contained within the N-terminal region (amino acids 1 to 79). Detailed mutagenesis of this region indicated that transactivation is mediated by three highly conserved sequences, spanning amino acids 13 to 22 (subdomain A), 32 to 38 (subdomain B), and 60 to 73 (subdomain C). These sequences were also required by PDX-1 to synergistically activate insulin enhancer-mediated transcription with another key insulin gene activator, the E2A-encoded basic helix-loop-helix E2-5 and E47 proteins. These results indicated that N-terminal sequences conserved between the mammalian PDX-1 and Xenopus XIHbox8 proteins are important in transcriptional activation. Stable expression of the PDX-1 deltaABC mutant in the insulin- and PDX-1-expressing betaTC3 cell line resulted in a threefold reduction in the rate of endogenous insulin gene transcription. Strikingly, the level of the endogenous PDX-1 protein was reduced to very low levels in these cells. These results suggest that PDX-1 is not absolutely essential for insulin gene expression in betaTC3 cells. We discuss the possible significance of these findings for insulin gene transcription in islet beta cells.
Amino Acid Sequence↗
Differentiation of new insulin-producing cells is induced by injury in adult pancreatic islets.
The ability of the adult pancreas to generate new insulin (beta) cells has been controversial because of difficulties in unequivocally identifying the precursor population. We recently determined that beta cells were generated during development from precursors that expressed the homeodomain-containing transcription factor pancreas duodenum homeobox gene-1 (PDX-1). To investigate whether PDX-1+ stem cells are present in adult pancreas, we examined two animal models of diabetes. One model was produced by injecting adult mice with streptozotocin (SZ), a toxin that produces hyperglycemia due to rapid and massive beta cell death. After SZ-mediated elimination of existing IN+/PDX-1+ cells, a population of somatostatin (SOM)+/PDX-1+ cells, a cell type thought to represent an embryonic islet precursor cell, appeared in islets. The appearance of SOM+/PDX-1+ cells was followed in time by the differentiation to SOM+/IN+/PDX-1+ cells. SOM+/PDX-1+ cells also appeared in islets of nonobese diabetic mice, a strain of mice in which beta cell destruction is immune-mediated. Our findings establish the existence of PDX-1+ beta cell precursors in the adult pancreas and indicate that their differentiation is induced by islet injury.
Animals↗
Functional differences among Xenopus nodal-related genes in left-right axis determination.
An association has been noted previously in chick, mouse and frog embryos between asymmetric nodal-related gene expression and embryonic situs, implying an evolutionarily conserved role in left-right specification. Of the four Xenopus nodal-related genes expressed during gastrulation, only Xnr-1 is re-expressed unilaterally in the left lateral plate mesoderm at neurula/tailbud stages. Here, we show that the asymmetric expression of Xnr-1 can be made bilaterally symmetric by right-sided microinjection of RNA encoding active Xenopus hedgehog proteins. Moreover, we provide the first evidence that Xnr-1 expression per se is a causal factor in left-right axis determination. When plasmids expressing Xnr-1 were delivered unilaterally to the right side of Xenopus embryos, a reversed laterality of both the heart and gut (homotaxic reversal) was induced in 40% of surviving embryos, while an additional 10-20% showed reversal of the heart or gut alone (heterotaxia). This effect on laterality was specific to Xnr-1, since neither Xnr-2 nor Xnr-3 plasmids had this activity. In addition, we find that Xnr-1 and Xnr-2, which have both been defined as mesoderm inducers from overexpression studies, show quantitative differences in their ability to induce dorsal mesoderm. Together, these findings suggest that the various Xnrs perform substantially different functions during Xenopus embryogenesis. Moreover, they strongly support the hypothesis that left lateral plate expression of nodal-related genes is a causative factor in the determination of asymmetry in vertebrate embryos.
Animals↗
Conserved left-right asymmetry of nodal expression and alterations in murine situs inversus.
Vertebrates have characteristic and conserved left-right (L-R) visceral asymmetries, for example the left-sided heart. In humans, alterations of L-R development can have serious clinical implications, including cardiac defects. Although little is known about how the embryonic L-R axis is established, a recent study in the chick embryo revealed L-R asymmetric expression of several previously cloned genes, including Cnr-1 (for chicken nodal-related-1), and indicated how this L-R molecular asymmetry might be important for subsequent visceral morphogenesis. Here we show that nodal is asymmetrically expressed in mice at similar stages, as is Xnr-1 (for Xenopus nodal related-1) in frogs. We also examine nodal expression in two mouse mutations that perturb L-R development, namely situs inversus viscerum (iv), in which assignment of L-R asymmetry is apparently random and individuals develop either normally or are mirror-image-reversed (situs inversus), and inversion of embryonic turning (inv), in which all individuals develop with situs inversus. In both, nodal expression is strikingly affected, being reversed or converted to symmetry. These results further support a key role for nodal and nodal-related genes in interpreting and relaying L-R patterning information in vertebrates. To our knowledge, our results provide the first direct evidence that iv and inv normally function well before the appearance of morphological L-R asymmetry.
Animals↗
PDX-1 is required for pancreatic outgrowth and differentiation of the rostral duodenum.
It has been proposed that the Xenopus homeobox gene, XlHbox8, is involved in endodermal differentiation during pancreatic and duodenal development (Wright, C.V.E., Schnegelsberg, P. and De Robertis, E.M. (1988). Development 105, 787-794). To test this hypothesis directly, gene targeting was used to make two different null mutations in the mouse XlHbox8 homolog, pdx-1. In the first, the second pdx-1 exon, including the homeobox, was replaced by a neomycin resistance cassette. In the second, a lacZ reporter was fused in-frame with the N terminus of PDX-1, replacing most of the homeodomain. Neonatal pdx-1 -/- mice are apancreatic, in confirmation of previous reports (Jonsson, J., Carlsson, L., Edlund, T. and Edlund, H. (1994). Nature 371, 606-609). However, the pancreatic buds do form in homozygous mutants, and the dorsal bud undergoes limited proliferation and outgrowth to form a small, irregularly branched, ductular tree. This outgrowth does not contain insulin or amylase-positive cells, but glucagon-expressing cells are found. The rostral duodenum shows a local absence of the normal columnar epithelial lining, villi, and Brunner's glands, which are replaced by a GLUT2-positive cuboidal epithelium resembling the bile duct lining. Just distal of the abnormal epithelium, the numbers of enteroendocrine cells in the villi are greatly reduced. The PDX-1/beta-galactosidase fusion allele is expressed in pancreatic and duodenal cells in the absence of functional PDX-1, with expression continuing into perinatal stages with similar boundaries and expression levels. These results offer additional insight into the role of pdx-1 in the determination and differentiation of the posterior foregut, particularly regarding the proliferation and differentiation of the pancreatic progenitors.
Animals↗
Bone morphogenetic protein-4 (BMP-4) acts during gastrula stages to cause ventralization of Xenopus embryos.
Injection of RNA encoding BMP-4 into the early Xenopus embryo suppresses formation of dorsal and anterior cell types. To understand this phenomenon, it is necessary to know the stage at which BMP-4 acts. In this paper, we present three lines of evidence showing that BMP-4 misexpression has no effect on the initial steps of mesoderm induction, either dorsal or ventral, but instead causes ventralization during gastrulation. Firstly, activation of organizer-specific genes such as goosecoid, Xnot, pintallavis and noggin occurs normally in embryos injected with BMP-4 RNA, but transcript levels are then rapidly down-regulated as gastrulation proceeds. Similarly, BMP-4 does not affect the initial activation of goosecoid by activin in animal caps, but expression then declines precipitously. Secondly, embryos made ventral by injection with BMP-4 RNA cannot be rescued by grafts of Spemann's organizer at gastrula stages. Such embryos therefore differ from those made ventral by UV-irradiation, where the defect occurs early and rescue can be effected by the organizer. Finally, the dorsalizing effects of the organizer, and of the candidate dorsalizing signal noggin, both of which exert their effects during gastrulation, can be counteracted by BMP-4. Together, these experiments demonstrate that BMP-4 can act during gastrulation both to promote ventral mesoderm differentiation and to attenuate dorsalizing signals derived from the organizer.
Animals↗
Differential localization of Mox-1 and Mox-2 proteins indicates distinct roles during development.
Transcript localizations for Mox genes have implicated this homeobox gene subfamily in the early steps of mesoderm formation. We have extended these studies by determining the protein expression profile of Mox-1 and Mox-2 during mouse development. The time of onset of Mox protein expression has been accurately obtained to provide clues as to their roles during gastrulation. Expression of Mox-1 protein is first detected in the newly formed mesoderm of primitive streak stage mouse embryos (7.5 days post-coitum, d.p.c.). In contrast, Mox-2 protein is first detected at 9.0 d.p.c. in thr already formed somites. Additionally, immunostaining reveals new and distinct areas of Mox expression in the branchial arches and limbs that were not reported in our previous mRNA localization analysis. Mouse Mox-2 antibodies cross-react specifically in similar embryonic tissues in chick indicating the conservation of function of Mox genes in vertebrates. These expression data suggest that the Mox genes function transiently in the formation of mesodermal and mesenchymal derivatives, after their initial specification, but before their overt differentiation. Furthermore, while there appears to be some overlap in protein expression between Mox-1 and Mox-2 during somitogenesis, unique areas of expression indicate several distinct roles for the Mox genes during development.
Alcohol Oxidoreductases↗
Reduction of insulin gene transcription in HIT-T15 beta cells chronically exposed to a supraphysiologic glucose concentration is associated with loss of STF-1 transcription factor expression.
Chronic exposure of HIT-T15 beta cells to elevated glucose concentrations leads to decreased insulin gene transcription. The reduction in expression is accompanied by diminished binding of a glucose-sensitive transcription factor (termed GSTF) that interacts with two (A+T)-rich elements within the 5' flanking control region of the insulin gene. In this study we examined whether GSTF corresponds to the recently cloned insulin gene transcription factor STF-1, a homeodomain protein whose expression is restricted to the nucleus of endodermal cells of the duodenum and pancreas. We found that an affinity-purified antibody recognizing STF-1 supershifted the GSTF activator complex formed from HIT-T15 extracts. In addition, we demonstrated a reduction in STF-1 mRNA and protein levels that closely correlated with the change in GSTF binding in HIT-T15 cells chronically cultured under supraphysiologic glucose concentrations. The reduction in STF-1 expression in these cells could be accounted for by a change in the rate of STF-1 gene transcription, suggesting a posttranscriptional control mechanism. In support of this hypothesis, no STF-1 mRNA accumulated in HIT-T15 cells passaged in 11.1 mM glucose. The only RNA species detected was a 6.4-kb STF-1 RNA species that hybridized with 5' and 3' STF-1-specific cDNA probes. We suggest that the 6.4-kb RNA represents an STF-1 mRNA precursor and that splicing of this RNA is defective in these cells. Overall, this study suggests that reduced expression of a key transcriptional regulatory factor, STF-1, contributes to the decrease in insulin gene transcription in HIT-T15 cells chronically cultured in supraphysiologic glucose concentration.
3T3 Cells↗
Cloning of a Xenopus laevis cDNA encoding focal adhesion kinase (FAK) and expression during early development.
Focal adhesion kinase (FAK) is a widely produced nonreceptor protein-tyrosine kinase thought to participate in signalling pathways activated in response to cell interaction with the extracellular matrix. Fibronectin-dependent cell adhesion mediated by integrin receptors plays a critical role in mesodermal cell migration during amphibian gastrulation in early development. As a first step toward understanding the role of FAK in Xenopus laevis (Xl) early development, we isolated cDNAs encoding Xl FAK and deduced the entire amino acid (aa) sequence. Xl FAK has 89-91% overall identity to the homologs previously described from mouse, human and chicken sources. Within the catalytic domain, the aa identity is about 97%. Northern blot analysis revealed that abundant maternal FAK transcript is present in Xl eggs, with levels decreasing slightly through cleavage and early blastula stages. At early gastrulation, the FAK mRNA level becomes modestly elevated, followed by a steady decline through late gastrulation. The mRNA level undergoes a further drop at the neurula stage, then begins a steady increase through the tailbud and tadpole stages. These data indicate that the steady-state level of FAK mRNA is regulated during Xl early development, and are consistent with a proposed role for FAK in the process of gastrulation.
Amino Acid Sequence↗
Localization of human homeodomain transcription factor insulin promoter factor 1 (IPF1) to chromosome band 13q12.1.
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Base Sequence↗
Autonomous endodermal determination in Xenopus: regulation of expression of the pancreatic gene XlHbox 8.
In neural plate stage Xenopus embryos, XlHbox 8 expression marks anterior endodermal cells fated to develop into pancreas/duodenum, and expression continues in adult pancreas in exocrine duct, acinar, and islet cells. Here, XlHbox 8 is used as a marker in experiments addressing the mechanisms of early endodermal patterning, particularly with respect to the role of specific polypeptide growth factors. When mesoderm-free vegetal explants (VEs) from early blastula stage embryos are cultured in isolation, XlHbox 8 expression develops autonomously in the dorsal region, strongly suggesting that endodermal region-specific determination occurs before MBT. Data from microinjection experiments using RNA encoding the activin and FGF dominant negative receptors and growth factor treatments of isolated VEs suggest that activin positively regulates XlHbox 8 expression, whereas bFGF is a potent negative regulator. Moreover, bFGF induces mesodermal marker expression in VEs. This suggests that the early endodermal determination state is plastic and that elevated levels of bFGF may convert vegetal (endodermal) cells into mesoderm. We propose a model for XlHbox 8 regulation in which an early signal from the Nieuwkoop center (whose eventual fate is endoderm) predisposes dorsovegetal cells for autonomous XlHbox 8 expression, in an area of high local activin (or activin-like) ligand concentration, and low relative concentrations of bFGF.
Activins↗
Effects of the TWis mutation on notochord formation and mesodermal patterning.
The mouse T (Brachyury) gene is required for early mesodermal patterning. Mice homozygous for mutations in T die at midgestation and display defects in mesodermal tissues such as the notochord, the allantois and the somitic mesoderm. To examine the role of T in patterning of somitic and posterior mesoderm along the anterior-posterior axis, we have examined the expression of a panel of molecular markers normally localized to the sub-set of cell types affected in TWis mutant mice. Through the use of whole-mount antibody double labelling techniques, we have analysed the spatial relationships of distinct mesodermal populations relative to cells expressing the T protein. We have also examined the consequences of the TWis mutation on mesodermal populations recognised by these markers. We demonstrate that TWis homozygous mutants retain the ability to form notochordal precursor cells, as identified both by the T antibody and the expression of sonic hedgehog/vertebrate homolog of hedgehog 1 (Shh/vhh-1) and goosecoid, however, these cells fail to proliferate or differentiate. These early notochordal defects appear to result in aberrant somitic differentiation as revealed by the distribution of mox-1 protein and twist RNA expression. Moreover, twist expression in paraxial mesoderm appears to be dependent on normal T activity, while Shh/vhh-1, goosecoid, mox-1 and cdx-4 are not T dependent. We propose that T is required for the maintenance of notochordal tissue and subsequent signals required for somite differentiation.
Alleles↗
The expression pattern of Xenopus Mox-2 implies a role in initial mesodermal differentiation.
We have isolated a Xenopus homolog of the murine Mox-2 gene. As is the case for the mouse homolog, mesoderm specific expression of Xenopus Mox-2 (X. Mox-2) expression begins during gastrulation. Using whole mount in situ hybridization, we show that X. Mox-2 is expressed in undifferentiated dorsal, lateral and ventral mesoderm in the posterior of neurula/tailbud embryos, with expression more anteriorly detected in the dermatomes. In the tailbud tadpole, X. Mox-2 is expressed in tissues of the tailbud itself that represent a site of continued gastrulation-like processes resulting in mesoderm formation. X. Mox-2 is not expressed in the marginal zone of blastula, nor in the dorsal lip of gastrula, nor midline tissues (i.e. prospective notochord). Treatments that affect mesodermal patterning during embryonic development, including LiCl and ultraviolet light, and injection of mRNAs encoding BMP-4, or dominant negative activin and FGF receptors, produce changes in X. Mox-2 expression consistent with the types of tissues affected by these manipulations. X. Mox-2 expression is induced more in animal caps treated with FGF than those treated with activin. Together with the fact that X. Mox-2 activation in animal caps requires protein synthesis, our data suggest that X. Mox-2 is involved in initial mesodermal differentiation, downstream of molecules affecting mesoderm induction and determination such as Brachyury and goosecoid, and upstream of factors controlling terminal differentiation such as MyoD and myf5. X. Mox-2, therefore, is another useful marker for understanding the formation of mesoderm in amphibian development.
Amino Acid Sequence↗
Managing the passive-aggressive resident.
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Adaptation, Psychological↗