Gene name - klumpfuss
Cytological map position - 68A1--68A9
Function - transcription factor
Symbol - klu
Genetic map position - 3-
Classification - zinc finger, egr family
Cellular location - nuclear
Klumpfuss is a zinc finger protein related to mammalian early growth response genes. The zinc fingers of Klu are similar to those of two other Drosophila proteins, Stripe and Huckebein. The name Klumpfuss means club-foot and refers to leg defects found in mutants. The distal regions of the leg segments are preferentially affected. For all three leg pairs in homozygotes for hypomorphic alleles, tarsal segments 3-5 are fused together, as well as the fusion of trochanter and femur. For more severe alleles, the defects are stronger (Klein, 1997).
klumpfuss mutants show loss of bristles at certain positions. In mutant flies a number of macrochaetae are missing in head and thorax, particularly from the anterior margin of the wing, the wing veins, antenna and legs of homozygotes. Non-innervated bristles at the margin of the alula are also affected. Observations suggest that in some positions, klu is require in order for epidermal cells to develop as sensory organ precursors and, in other positions, for proper differentiation of the progeny cells (Klein, 1997).
Klumpfuss is also involved in determination of the identities of secondary precursor cells within neuroblast lineages in the central nervous system. The approximately 300 distinct neurons comprising each hemineuromere of the fly's embryonic central nervous system are derived from a segmentally reiterated array of approximately 30 progenitor cells known as neuroblasts (NBs). Each NB has a unique identity and undergoes repeated cell divisions to produce several smaller secondary precursor cells, termed ganglion mother cells (GMCs); each GMC divides once to produce two neurons and/or glia, thereby generating a specific lineage of neurons/glia. Since the NB4-2 lineage is the best characterized of all NB lineages in the embryonic CNS, this lineage was studied in detail to determine the role of Klu in cell fate determination.
Klu is expressed in the NB4-2 lineage only after two rounds of NB cell division, that is, Klu is expressed in those GMCs born second, indicated as GMC4-2b. Klu fails to be expressed in the first progeny of NB4-2 (GMC4-2a). The two progeny of GMC4-2a are the RP2 motoneuron and a sibling cell. In loss-of-function klu mutant embryos, the first born GMC (GMC4-2a) as well as its progeny RP2 neurons are duplicated; this abnormal duplication of the GMC4-2a sublineage arises because GMC4-2b, in a homeotic shift, adopts the identity of GMC4-2a and divides to produce the GMC4-2a progeny. The GMC4-2a lineage is characterized by the expression of Even-skipped, Fushi tarazu, PDM1 and PDM2 and Zinc finger homeodomain 1. Each of these markers is expressed in duplicated progeny, consistent with their RP2 identity (Yang, 1997).
When Klu is ectopically expressed in GMC4-2a, it fails to acquire its normal identity and fails to produce correctly specified progeny. The extent to which EVE+ RP2 neurons are lost depends on the developmental stage at which Klu is expressed. Expression at early stage 10 shows the maximum inhibition of Eve+ RP2 formation, while little or no inhibition is observed when Klu is expressed later. The window of time when Klu expression prohibits formation of the mature RP2 neuron coincides with the time frame of the birth of GMC4-2a. It is possible that ectopically expressed Klu in GMC42a forces it to adopt a GMC4-2b identity; however, because of the lack of information on the nature of the progeny of GMC4-2b, this cannot be directly tested (Yang, 1997).
It is concluded that klu acts to specify the identity of GMC4-2b and to make it distinct from GMC4-2a. These findings suggest that the determination of GMC cell fate occurs in two steps; the initial GMC identity is the consequence of inheritance from the maternal NB. The gene wingless and its targets patched and gooseberry are involved in the specification of NB4-2. The subsequent stabilization of this identity requires functions like klu in the GMC. GMC4-2a appears to retain a default program that will confer a GMC4-2a cell identity if the program initiated by Klu (which is necessary to establish a GMC4-2b cell identity) is not implemented. It is likely that a balance between alternative cell fates is held under control of genes like klu, and transformation from one fate to another depends on the pattern of expression of this and similar genes (Yang, 1997).
The mechanisms that generate neuronal diversity within the Drosophila central nervous system (CNS), and in particular in the development of a single identified motoneuron called RP2, are of great interest. Expression of the homeodomain transcription factor Even-skipped (Eve) is required for RP2 to establish proper connectivity with its muscle target. The mechanisms by which eve is specifically expressed within the RP2 motoneuron lineage have been examined. Within the NB4-2 lineage, expression of eve first occurs in the precursor of RP2, called GMC4-2a. A small 500 base pair eve enhancer has been identified that mediates eve expression in GMC4-2a. Four different transcription factors (Prospero, Huckebein, Fushi tarazu, and Pdm1) are all expressed in GMC4-2a, and are required to activate eve via this minimal enhancer; one transcription factor (Klumpfuss) represses eve expression via this element. All four positively acting transcription factors act independently, regulating eve but not each other. Thus, the eve enhancer integrates multiple positive and negative transcription factor inputs to restrict eve expression to a single precursor cell (GMC4- 2a) and its RP2 motoneuron progeny (McDonald, 2003).
GMC4-2a forms at stage 9, becomes Eve+ at stage 11, and generates the Eve+ RP2/sib neurons at late stage 11. The second-born Eve-negative GMC4-2b forms at stage 10, and generates an unknown pair of neurons. The first transcription factors detected in GMC4-2a are Pros and Hkb, due to inheritance of the proteins from the neuroblast. The next transcription factors detected in GMC4-2a are Ftz and Pdm1. Ftz is first detected at stage 10, and Pdm1 is first detected at stage 11. The de novo expression of Pdm1 is distinct from its inheritance in GMCs produced by Pdm+ neuroblasts during the assignment of temporal identity. The last protein to be detected is Eve, which appears only at late stage 11. Pros, Hkb, Ftz, and Pdm1 are each expressed transiently in the RP2/sib neurons at stage 12, but by stage 16 none of these proteins is detectable in the mature RP2 neuron. It is concluded that there is a temporal sequence of transcription factor expression in GMC4-2a: first Pros and Hkb, then Ftz, then Pdm1, and that Eve is detected only after all of these proteins are present (McDonald, 2003).
GMC4-2b forms at late stage 10, never expresses Eve, and generates two unknown Eve-negative neurons. Three transcription factors that positively regulate eve expression are detected in GMC4-2b: Pros, Ftz, and Hkb. The pattern of Pdm1 expression is too complex to score at the time GMC4-2b is born. The negative regulator Klu is detected in GMC4-2b but not GMC4-2a. It is concluded that GMC4-2b expresses at least three of the four positively acting transcription factors that are required to activate eve (Pros, Ftz, Hkb), and at least one negative regulator of eve expression (Klu). The absence of eve expression is likely due to the presence of Klu, rather than the absence of a positive regulator, because klu mutants can activate eve transcription in GMC4-2b (McDonald, 2003).
The sequential expression of Pros, Hkb, Ftz, Pdm1, and Eve in GMC4-2a raises the possibility that these four transcription factors act in a linear pathway to regulate eve expression. If so, then a mutant in an early-acting gene should lead to loss of expression of all later-acting genes in the pathway. Alternatively, the four transcription factors could all act directly to activate eve transcription, with expression of eve occurring only after all transcription factors are present. In this case, mutants in one gene should have no effect on any other gene except eve. To distinguish between these two models, pros, hkb, ftz, and pdm1 mutants were examined for expression of all four transcription factors and eve. Pdm1 is detected in GMC4-2a in all mutant genotypes: Ftz is detected in GMC4-2a in all mutant genotypes: pros, hkb, and pdm1, and Hkb is detected in GMC4-2a in all mutant genotypes. Finally, Pros is observed in GMC4-2a in all mutant genotypes, as expected because Pros is transcribed and translated in neuroblasts and is asymmetrically partitioned into each GMC. Taken together, these data support the model that all four transcription factors act directly to activate eve transcription, with expression of eve occurring only after all transcription factors are present (McDonald, 2003).
To test the model that Pros, Hkb, Ftz, and Pdm1 transcription factors directly regulate eve expression, the eve cis-regulatory DNA that confers regulated expression in the NB4-2 lineage was identified. Eve is expressed in a subset of neurons in the embryonic CNS, including the aCC/pCC neurons derived from NB1-1, the U1-5 neurons derived from NB7-1, the EL neurons derived from NB 3-3, and the RP2/sib neurons derived from NB4-2. An eve cis-regulatory element [R79R92; from ~7.9 and ~9.2 kilobase pair (kb) on the eve genomic map] has been defined that accurately directs lacZ expression to the Eve+ cells within two NB lineages: GMC4-2a and its RP2 progeny and GMC1-1a and its aCC/pCC progeny. The properties of this element are examined in this study in detail. When the R79R92 eve element was truncated to ~7.9 to ~8.6 kb (R79N86), lacZ expression in RP2 and aCC was normal, whereas expression in the pCC neuron was reduced. Truncation of the eve element to ~7.9 to ~8.4 kb (R79S84) almost completely abolished expression of lacZ in pCC, although occasionally expression in pCC was observed at low levels, whereas expression in RP2 and aCC remained high. Further truncation of the left end point to ~8.0 kb (S80S84) resulted in a reduction of expression in both aCC and RP2. Addition of the region ~8.4 to ~8.6 kb to this fragment (S80N86) increased the level of expression. However, because the region ~8.4 to ~9.2 kb (S84R92) did not show any ability to activate lacZ, the region ~8.4 to ~8.6 kb is apparently insufficient on its own to direct expression, and thus serves an auxiliary function. The removal of ~8.2 to ~8.4 kb from P80N86 abolished expression (SNdeltaSC). Together with the fact that each of the fragments ~7.9 to ~8.2 kb (S79C82) and ~8.2 to ~9.2 kb (C82R92) failed to activate lacZ, this indicates that both of the regions ~7.9 to ~8.2 kb and ~8.2 to ~8.4 kb are necessary to direct expression, and that neither alone is sufficient. Consistent with this, two tandem copies of ~8.2 to ~8.4 kb failed to activate lacZ (C82S84x2), suggesting that the two regions may provide qualitatively different activities. In summary, the critical eve cis-regulatory element for the GMC4-2a and RP2 lies in a 0.5 kb fragment of genomic DNA between ~7.9 and ~8.4 kb (McDonald, 2003).
Do the genes that activate or repress eve expression in the NB4-2 lineage work through the minimal 500 bp RP2/aCC eve enhancer? Expression of R79S84-lacZ was assayed in pros, ftz, hkb, pdm1, and klu mutant embryos, and whether it was regulated identically to the endogenous eve gene was tested. ftz, pdm1, and hkb mutant embryos show loss of R79S84-lacZ in the RP2 neuron but not the aCC neuron, identical to the pattern of endogenous eve expression in these mutants. pros mutants show loss of eve-lacZ in both RP2 and aCC, identical to the pattern of endogenous eve expression in pros mutants. In embryos lacking klu, R79S84-lacZ is expressed in two cells at the RP2 position, whereas expression in aCC is normal; this matches the pattern of endogenous eve expression in klu mutant embryos. It is concluded that the R79S84 minimal eve cis-regulatory element precisely reproduces the pattern of endogenous eve expression within the NB4-2 lineage, and that transcription factors regulating eve in GMC4-2a can act through this enhancer to activate or repress eve expression (McDonald, 2003).
Expression of eve is not detected in GMC4-2b in wild-type embryos, but mutations in the klu gene result in ectopic expression of eve in GMC4-2b. Klu contains four predicted zinc fingers, one of which is highly homologous to the WT1 zinc finger domain. The consensus binding site for the WT1 zinc finger transcription factor is a ten nucleotide sequence, 5'-(C/G/T)CGTGGG( A/T)(G/T)(T/G)-3', with variable nucleotides shown in parentheses. It was reasoned that if Klu directly binds to the eve enhancer to repress expression in GMC4-2b, one or more WT1 consensus binding sites should be found in the minimal eve enhancer R79S84. Three conserved putative Klu-binding sites were found in the R79S84 sequence: site 1, GGGTGGGGAG at nucleotides ~8066 to ~8075; site 2, GCGTGGGTGA at nucleotides ~8090 to ~8099; and site 3, TCGCCCACCA at ~8262 to ~8271. Based on the fact that altering the C2, G3, G5, G6, and G7 to T or T4 to A in the WT1-consensus binding site abolished WT1 binding, nucleotide substitutions were made in the three putative Klu-binding sites. In sites 1 and 2, As were substituted for T4, G6, and G7. In site 3, which is a reversed binding site, Ts were substituted for C4, C6, and A7. These substitutions were made at all three sites; transgenic lines were constructed expressing the mutant enhancer driving lacZ (eveK123-lacZ), and the pattern of lacZ expression was examined in the CNS of wild-type embryos and embryos misexpressing Klu protein in the NB4-2 lineage (McDonald, 2003).
In wild-type embryos, the eveK123-lacZ transgene is expressed in the aCC and RP2 neurons, similar to the wild-type (R79S84) eve-lacZ transgene. However, in one or two hemisegments per embryo, an extra cell expressing eveK123-lacZ adjacent to the RP2 neuron was observed. This phenotype is very similar to wild-type (R79S84) eve-lacZ expression in klu mutant embryos, although slightly less penetrant. It is concluded that the eveK123-lacZ transgene mimics the klu mutant phenotype, and it is proposed that Klu represses eve expression via direct binding to one or more of these sites (McDonald, 2003).
To further test this hypothesis, gain of function experiments were used to test whether ectopic Klu in GMC4-2a can repress eve-lacZ expression via these sites. Expression of a wild-type (R79S84) eve-lacZ transgene was compared with a transgene containing three mutated Klu consensus binding sites (eveK123-lacZ) in embryos where Scabrous-Gal4 (Sca-Gal4) drives ectopic expression of UAS-klu in all neuroblast lineages. The wild-type (R79S84) eve-lacZ expression is partially repressed by ectopic Klu expression, but the eveK123-lacZ transgene with mutated Klu sites is repressed to a lesser extent. This difference in repression is only observed when the levels of transgene expression are lowered by raising the embryos at 18°C; when the transgenes are more strongly expressed (by raising the embryos at 23°C) no detectable repression was observed. Taken together, Klu loss of function and misexpression studies indicate that Klu acts partly, but not completely, through three predicted Klu-binding sites to repress eve expression in the NB4-2 lineage (McDonald, 2003).
In summary, hkb, ftz, pdm1, and pros are independently required to activate eve expression in GMC4-2a. This suggests that the eve enhancer is capable of integrating the input of all four of these transcription factors to activate transcription. Hb and Ind are also necessary for eve expression in GMC4-2a, but it is not known if they act directly on the eve element or via one of the four transcription factors described in this study. Putative binding sites were found for each of the positively acting transcription factors within the minimal eve element, but mutation of these sites had no effect on expression of the eve-lacZ transgene in embryos (M. Fujioka, J.A. McDonald, and C.Q. Doe, unpublished results reported in McDonald, 2003). It remains to be determined whether Pros, Hkb, Ftz, or Pdm1 activate eve transcription via direct binding to the minimal eve element, or indirectly by activating or facilitating the binding of other transcriptional activators (McDonald, 2003).
Based on functional dissection of the RP2/aCC/pCC eve element, it seems to be composed of three parts. The regions ~7.9 to ~8.2 kb and ~8.2 to ~8.4 kb are each necessary to direct the expression pattern (together they comprise the minimal element for expression in RP2 and aCC), while the region ~8.4 to ~8.6 kb enhances the level of expression. Expression in the pCC neuron is further enhanced by the region extending to ~9.2 kb. The two regions within the minimal element seem to be regulated by different factors, because two copies of ~8.2 to ~8.4 kb (increasing the number of activator binding sites within this region by twofold) could not substitute for the function of the region ~7.9 to ~8.2 kb. This is consistent with the fact that at least four factors are independently required to activate eve in RP2 neurons. How does Klu repress eve expression in GMC4-2b? Negative regulation of eve expression by Klu is due to direct binding to the eve minimal element. (1) It is shown that klu mutants exhibit similar derepression of the eve minimal element transgene and the endogenous eve gene in the NB4-2 lineage; (2) three consensus binding sites are detected for Klu in the eve minimal element (comparison of Drosophila virilis and Drosophila melanogaster shows that the three identified sites are highly conserved); (3) mutation of these sites results in ectopic expression of eve-lacZ in the NB4-2 lineage in wild-type, and (4) mutation of these sites impairs repression of eve-lacZ by ectopic Klu in the NB4-2 lineage. The predicted Klu binding sites (K123) are probably only a subset of relevant Klu binding sites, however, because mutation of the sites gives only partially penetrant phenotypes (McDonald, 2003).
Surprisingly, it was not possible to separate the GMC4-2a/ RP2 element from the GMC1-1a/aCC/pCC element. In both NB 1-1 and NB 4-2 lineages, eve is expressed in the first-born GMC and its neuronal progeny. Both first-born GMCs share expression of several transcription factors, including Pros and Ftz. However, many other transcription factors are differentially expressed, such as the GMC1-1a specific expression of Vnd and Odd-skipped, and the GMC4-2a specific expression of Hkb, Pdm1, and Ind. It is possible that one or more commonly expressed transcription factors are required for expression of eve in both GMC1-1a and GMC4-2a, such as Pros, and this is why the elements cannot be subdivided (McDonald, 2003).
The Klumpfuss sequence is characterized by four zinc-finger domains of the C2H2 class; part of the N-terminus is negatively charged; the C-terminus, including the zinc-fingers, is positively charged. The N-terminal region contains glutamine-, histidine- and proline-rich stretches, features found in transcriptional activation and repression domains. There are three poly-alanine stretches, two in the N- and one in the C-terminal region. Such stretches are implicated in transcriptional repression. Three putative nuclear localization sites are found in the protein. Klu has a high degree of similarity to the zinc-finger domains of the members of the EGR family. As in WT-1, Klu contains an additional zinc finger (the first) that is only distantly related to those of the EGR proteins. Besides WT-1, Klu is the only other member of the family with four zinc fingers. There is complete conservation of the amino acids that contact the DNA-binding consensus sequence. So far only two other proteins are described as EGR-like in invertebrates, the Drosophila genes stripe and huckebein. Whereas the zinc fingers of Stripe have the characteristic amino acids for the binding of the DNA consensus sequence, in the zinc fingers of Huckebein, four out of the six amino acids are not conserved. However, the overall homology of the Huckebein finger region shows no greater homology to the EGR sequences than does the finger region of Klumpfuss. In comparison, the Stripe sequence shows a much more marked homology to the vertebrate EGR proteins than do the sequences of either Huckebein or Klu (Klein, 1997).
Gene expression analyses have led to a better understanding of growth control of prostate cancer cells. Several zinc finger transcription factors has been identified in the neoplastic prostate, suggesting a potential role for these genes in the regulation of the prostate cancer transcriptome. One of the transcription factors (TFs) identified in the prostate cancer epithelial cells was the Wilms tumor gene (WT1). To rapidly identify coordinately expressed prostate cancer growth control genes that may be regulated by WT1, an in silico approach was used. Evolutionary conserved transcription factor binding sites (TFBS) recognized by WT1, EGR1, SP1, SP2, AP2 and GATA1 were identified in the promoters of 24 differentially expressed prostate cancer genes from eight mammalian species. To test the relationship between sequence conservation and function, chromatin of LNCaP prostate cancer and kidney 293 cells were tested for TF binding using chromatin immunoprecipitation (ChIP). Multiple putative TFBS in gene promoters of placental mammals were found to be shared with those in human gene promoters and some were conserved between genomes that diverged about 170 million years ago (i.e., primates and marsupials), therefore implicating these sites as candidate binding sites. Among those genes coordinately expressed with WT1 was the kallikrein-related peptidase 3 (KLK3) gene commonly known as the prostate specific antigen (PSA) gene. This analysis located several potential WT1 TFBS in the PSA gene promoter and led to the rapid identification of a novel putative binding site confirmed in vivo by ChIP. Conversely for two prostate growth control genes, androgen receptor (AR) and vascular endothelial growth factor (VEGF), known to be transcriptionally regulated by WT1, regulatory sequence conservation was observed and TF binding in vivo was confirmed by ChIP. Overall, this targeted approach rapidly identified important candidate WT1-binding elements in genes coordinately expressed with WT1 in prostate cancer cells, thus enabling a more focused functional analysis of the most likely target genes in prostate cancer progression. Identifying these genes will help to better understand how gene regulation is altered in these tumor cells (Eisermann, 2008).
Store-operated calcium entry (SOCE) is a key evolutionarily conserved process whereby decreases in endoplasmic reticulum Ca(2+) content lead to the influx of Ca(2+) across the plasma membrane. How this process is regulated in specific tumor cell types is poorly understood. In an effort to address this concern, primary Wilms tumor cells were obtained and tested, since no detectable SOCE was found in this cell type. Analysis of the expression levels of STIM1 and ORAI1 (the molecular mediators of SOC) revealed poor STIM1 expression. Analysis of the STIM1 promoter using the TESS search system revealed four putative response elements to the zinc-finger proteins WT1 (Wilms tumor suppressor 1) and EGR1 (early growth response 1). Either overexpression of WT1 or knockdown of EGR1 resulted in loss of STIM1 expression and a resultant decrease in SOCE. Furthermore, examination of Egr1 knock-out animals revealed loss of STIM1 expression in multiple tissues. Finally, using chromatin immunoprecipitation, direct binding was detected of both WT1 and EGR1 to putative response elements located within 500 bp of the transcriptional start site of STIM1. Considering that WT1 and EGR1 are well described oncogenes and tumor suppressors, these observations may reveal new mechanisms responsible for distinct Ca(2+) signals in cancer cells (Ritchie, 2010).
Wilms' tumor gene 1 (WT1) is a transcription factor involved in developmental processes. In adult hematopoiesis, only a small portion of early progenitor cells express WT1, whereas most leukemias show persistently high levels, suggesting an oncogenic role. Previous studies have characterized oncogenic BCR/ABL1 tyrosine kinase signaling pathways for increased WT1 expression. This study showed that overexpression of BCR/ABL1 in CD34+ progenitor cells leads to reduced expression of interferon regulatory factor 8 (IRF8), in addition to increased WT1 expression. Interestingly, IRF8 is known as a tumor suppressor in some leukemias and whether WT1 might repress IRF8 expression was investigated. When analyzed in four leukemia mRNA expression data sets, WT1 and IRF8 were anticorrelated. Upon overexpression in CD34+ progenitors, as well as in U937 cells, WT1 strongly downregulated IRF8 expression. All four major WT1 splice variants induced repression, but not the zinc-finger-deleted WT1 mutant, indicating dependence on DNA binding. A reporter construct with the IRF8 promoter was repressed by WT1, dependent on a putative WT1-response element. Binding of WT1 to the IRF8 promoter was demonstrated by chromatin immunoprecipitation. These results identify IRF8 as a direct target gene for WT1 and provide a possible mechanism for oncogenic effects of WT1 in leukemia (Vidovic, 2010).
Mammalian kidney development requires the functions of the Wilms tumor gene WT1 and the WNT/beta-catenin signaling pathway. Recent studies have shown that WT1 negatively regulates WNT/beta-catenin signaling, but the molecular mechanisms by which WT1 inhibits WNT/beta-catenin signaling are not completely understood. This study identified a gene, CXXC5, which was renamed WID (WT1-induced Inhibitor of Dishevelled), as a novel WT1 transcriptional target that negatively regulates WNT/beta-catenin signaling. WT1 activates WID transcription through the upstream enhancer region. In the developing kidney, Wid and Wt1 are coexpressed in podocytes of maturing nephrons. Structure-function analysis demonstrated that WID interacts with Dishevelled via its C-terminal CXXC zinc finger and Dishevelled binding domains and potently inhibits WNT/beta-catenin signaling in vitro and in vivo. WID is evolutionarily conserved, and ablation of wid in zebrafish embryos with antisense morpholino oligonucleotides perturbs embryonic kidney development. Taken together, these results demonstrate that the WT1 negatively regulates WNT/beta-catenin pathway via its target gene WID and further suggest a role for WID in nephrogenesis (Kim, 2010).
Wt1 regulates the epithelial-mesenchymal transition (EMT) in the epicardium and the reverse process (MET) in kidney mesenchyme. The mechanisms underlying these reciprocal functions are unknown. This study shows in both embryos and cultured cells that Wt1 regulates Wnt4 expression dichotomously. In kidney cells, Wt1 recruits Cbp and p300 as coactivators; in epicardial cells it enlists Basp1 as a corepressor. Surprisingly, in both tissues, Wt1 loss reciprocally switches the chromatin architecture of the entire Ctcf-bounded Wnt4 locus, but not the flanking regions; this mode of action was termed 'chromatin flip-flop.' Ctcf and cohesin are dispensable for Wt1-mediated chromatin flip-flop but essential for maintaining the insulating boundaries. This work demonstrates that a developmental regulator coordinates chromatin boundaries with the transcriptional competence of the flanked region. These findings also have implications for hierarchical transcriptional regulation in development and disease (Essafi, 2011).
The Wilms' tumor suppressor 1 (WT1) gene encodes a DNA- and RNA-binding protein that plays an essential role in nephron progenitor differentiation during renal development. To identify WT1 target genes that might regulate nephron progenitor differentiation in vivo, chromatin immunoprecipitation (ChIP) was performed, coupled to mouse promoter microarray (ChIP-chip), using chromatin prepared from embryonic mouse kidney tissue. 1663 genes bound by WT1 were identified, 86% of which contain a previously identified, conserved, high-affinity WT1 binding site. To investigate functional interactions between WT1 and candidate target genes in nephron progenitors, a novel, modified WT1 morpholino loss-of-function model in embryonic mouse kidney explants was used to knock down WT1 expression in nephron progenitors ex vivo. Low doses of WT1 morpholino resulted in reduced WT1 target gene expression specifically in nephron progenitors, whereas high doses of WT1 morpholino arrested kidney explant development and were associated with increased nephron progenitor cell apoptosis, reminiscent of the phenotype observed in Wt1(-/-) embryos. Collectively, these results provide a comprehensive description of endogenous WT1 target genes in nephron progenitor cells in vivo, as well as insights into the transcriptional signaling networks controlled by WT1 that might direct nephron progenitor fate during renal development.
Despite the importance of taste in determining nutrient intake, understanding of the processes that control the development of the peripheral taste system is lacking. Several early regulators of taste development have been identified, including sonic hedgehog, bone morphogenetic protein 4 and multiple members of the Wnt/β-catenin signaling pathway. However, the regulation of these factors, including their induction, remains poorly understood. This study identified a crucial role for the Wilms' tumor 1 protein (WT1) in circumvallate (CV) papillae development. WT1 is a transcription factor that is important in the normal development of multiple tissues, including both the olfactory and visual systems. In mice, WT1 expression is detectable by E12.5, when the CV taste placode begins to form. In mice lacking WT1, the CV fails to develop normally and markers of early taste development are dysregulated compared with wild type. Expression of the WT1 target genes Lef1, Ptch1 and Bmp4 is significantly reduced in developing tongue tissue derived from Wt1 knockout mice, and, in normal tongue, WT1 was shown to be bound to the promoter regions of these genes. Moreover, siRNA knockdown of WT1 in cultured taste cells leads to a reduction in the expression of Lef1 and Ptch1. These data identify WT1 as a crucial transcription factor in the development of the CV through the regulation of multiple signaling pathways that have established roles in the formation and patterning of taste placodes (Gao, 2014).
date revised: 5 Sept. 97
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