jumeau: Biological Overview | Developmental Biology | Evolutionary Homologs | References
Gene name - jumeau
Synonyms - CG4029, Domina, Jumeaux
Cytological map position - 86A2--4
Function - Transcription factor
Symbol - jumu/Dom
FlyBase ID: FBgn0015396
Genetic map position -
Classification - forkhead/winged-helix class
Cellular location - nuclear
|Recent literature||Zhang, G., Hao, Y. and Jin, L. H. (2016). Overexpression of jumu induces melanotic nodules by activating Toll signaling in Drosophila. Insect Biochem Mol Biol 77: 31-38. PubMed ID: 27507244
Melanotic nodules are commonly assumed to be caused by an abnormal immune response. Several hematopoietic mutants and signaling pathways, including the Toll, JAK/STAT, Ras and JNK pathways, can cause melanotic nodules to develop when specifically activated in hemocytes. This study used the UAS-Gal4 system to overexpress jumeau (jumu) in the fly immune response system. jumeau (Jumu) is a new member of the winged-helix/forkhead (WH/FKH) gene family of transcription factors, which plays an important role in the growth and morphogenesis of Drosophila and participates in the proliferation and differentiation of hemocytes. Overexpressing jumu in both hemocytes and the fat body generated many melanotic nodules in larvae and adult flies. The nodules observed in the fat body were surrounded by large numbers of blood cells through a process that appeared similar to foreign body encapsulation. This phenomenon is caused by Toll pathway activation and leads to blood cells deposited in the fat body. In addition, the dissociation of fat cells and the abnormal proliferation and differentiation of blood cells are also reported. These results suggest a Jumu-mediated crosstalk between hematopoiesis and the fat body, especially during the Toll-dependent formation of melanotic nodules.
|Hao, Y. and Jin, L. H. (2017). Dual role for Jumu in the control of hematopoietic progenitors in the Drosophila lymph gland. Elife 6. PubMed ID: 28350299
The Drosophila lymph gland is a hematopoietic organ in which the maintenance of hematopoietic progenitor cell fate relies on intrinsic factors and extensive interaction with cells within a microenvironment. The posterior signaling center (PSC) is required for maintaining the balance between progenitors and their differentiation into mature hemocytes. Moreover, some factors from the progenitors cell-autonomously control blood cell differentiation. This study shos that Jumeau (Jumu), a member of the forkhead (Fkh) transcription factor family, controls hemocyte differentiation of lymph gland through multiple regulatory mechanisms. Jumu maintains the proper differentiation of prohemocytes by cell-autonomously regulating the expression of Col in medullary zone and by non-cell-autonomously preventing the generation of expanded PSC cells. Jumu can also cell-autonomously control the proliferation of PSC cells through positive regulation of dMyc expression. Deficiency of jumu throughout the lymph gland can induce the differentiation of lamellocytes via activating Toll signaling.
|Hao, Y., Yu, S., Luo, F. and Jin, L. H. (2018). Jumu is required for circulating hemocyte differentiation and phagocytosis in Drosophila. Cell Commun Signal 16(1): 95. PubMed ID: 30518379
The regulatory mechanisms of hematopoiesis and cellular immunity show a high degree of similarity between insects and mammals, and Drosophila has become a good model for investigating cellular immune responses. Jumeau (Jumu) is a member of the winged-helix/forkhead (FKH) transcription factor family and is required for Drosophila development. Adult junu mutant flies show defective hemocyte phagocytosis and a weaker defense capability against pathogen infection. This study investigated the role of junu in the regulation of larval hemocyte development and phagocytosis. In vivo phagocytosis assays, immunohistochemistry, Real-time quantitative PCR and immunoblotting were performed to investigate the effect of junu on hemocyte phagocytosis. 5-Bromo-2-deoxyUridine (BrdU) labeling, phospho-histone H3 (PH3) and TdT-mediated dUTP Nick-End Labeling (TUNEL) staining were performed to analyze the proliferation and apoptosis of hemocyte; immunohistochemistry and Mosaic analysis with a repressible cell marker (MARCM) clone analysis were performed to investigate the role of Jumu in the activation of Toll pathway. Jumu was found to indirectly control hemocyte phagocytosis by regulating the expression of NimC1 and cytoskeleton reorganization. The loss of junu also causes abnormal proliferation and differentiation in circulating hemocytes. Thesee results suggest that a severe deficiency of junu leads to the generation of enlarged multinucleate hemocytes by affecting the normal cell mitosis process and induces numerous lamellocytes by activating the Toll pathway. It is concluded that Jumu regulates circulating hemocyte differentiation and phagocytosis in Drosophila. These findings provide new insight into the mechanistic roles of cytoskeleton regulatory proteins in phagocytosis and establish a basis for further analyses of the regulatory mechanism of the mammalian ortholog of Jumu in mammalian innate immunity.
|Wang, X. C., Liu, Z. and Jin, L. H. (2019). Drosophila jumu modulates apoptosis via a JNK-dependent pathway and is required for other processes in wing development. Apoptosis. PubMed ID: 30796611
Previous studies in several model organisms have revealed that members of the Forkhead (Fkh) transcription factor family have multiple functions. Drosophila Jumeau (Jumu), a member of this family, participates in cardiogenesis, hematopoiesis and immune system homeostasis. This study shows that loss of jumu function positively regulates or triggers apoptosis via a JNK-dependent pathway in wing development. jumu mutants showed reduced wing size and increased apoptosis. Moreover, a loss was observed of the anterior cross vein (ACV) phenotype that was similar to that observed in wings in which JNK signaling has been ectopically activated. The JNK signaling markers puckered (puc) and p-JNK were also significantly increased in the wing discs of jumu mutants. In addition, apoptosis induced by the loss of jumu was rescued by knocking down JNK, indicating a role for JNK in reducing jumu-induced apoptosis. Jumu could also control wing margin development via the positive regulation of cut expression, and the observed wing margin defect did not result from a loss of jumu-induced apoptosis. Further, jumu deficiency in the pupal wing could induce multiple wing hairs via a Rho1-mediated planar cell polarity pathway, but abnormal Rho1 expression was not why jumu loss induced apoptosis via a JNK-dependent pathway in wing discs.
|He, S., Zhang, G., Wang, J., Gao, Y., Sun, R., Cao, Z., Chen, Z., Zheng, X., Yuan, J., Luo, Y., Wang, X., Zhang, W., Zhang, P., Zhao, Y., He, C., Tao, Y., Sun, Q. and Chen, D. (2019). 6mA-DNA-binding factor Jumu controls maternal-to-zygotic transition upstream of Zelda. Nat Commun 10(1): 2219. PubMed ID: 31101825
A long-standing question in the field of embryogenesis is how the zygotic genome is precisely activated by maternal factors, allowing normal early embryonic development. N6-methyladenine (6mA) DNA modification has been shown to be highly dynamic in early Drosophila embryos and forms an epigenetic mark. However, little is known about how 6mA-formed epigenetic information is decoded. This study reports that the Fox-family protein Jumu binds 6mA-marked DNA and acts as a maternal factor to regulate the maternal-to-zygotic transition. zelda encoding the pioneer factor Zelda was found to be marked by 6mA. Genetic assays suggest that Jumu controls the proper zygotic genome activation (ZGA) in early embryos, at least in part, by regulating zelda expression. Thus, these findings not only support that the 6mA-formed epigenetic marks can be read by specific transcription factors, but also uncover a mechanism by which the Jumu regulates ZGA partially through Zelda in early embryos.
The gene Domina (Dom) has been identifed as a Drosophila member of the Forkhead/winged-helix (FKH/WH) gene family; it is a suppressor of position effect variegation (PEV), and affects and regulates eye and bristle development (Strodicke, 1996 and 1999). Domina [termed jumeau (Jumu) by Cheah, 2000] is required for generating asymmetric sibling neuronal cell fates. The Dom/Jumu protein is expressed in the developing embryonic CNS, including the neuroblast GMC4-2a. Dom/Jumu appears to play a role in fate determination during CNS lineage development. The proteins Inscuteable, Partner of Inscuteable and Bazooka form a complex that is localized to the apical cortex of neural progenitors. They function to both coordinate and mediate several aspects of neural progenitor asymmetric cell divisions required for the resolution of distinct fates for the sibling neurons derived from (at least some) GMC divisions. It is thought that this complex of proteins, localized to the apical domain of neural progenitors, acts to provide positional information necessary to coordinate and mediate processes that ensure the correct execution of asymmetric cell divisions. Dom/Jumu is dispensable for Inscuteable apical localization but necessary for the basal localization of Partner of numb and Numb. These results suggest that in addition to the correct formation of an apical complex, transcription mediated by molecules like Dom/Jumu is also required to facilitate the correct asymmetric localization and segregation of cell fate determinants like Numb (Cheah, 2000).
jumeau was identified in a mutant screen of lethal P-element insertions that affect the number of RP2 motorneurons. The anti-Even-skipped antibody (anti-Eve) stains the nuclei of approximately 20 neurons in each of the hemineuromeres of the CNS: the EL cluster, the aCC and pCC neurons, the RP2 neuron and its sibling cell and the CQ neurons. Using anti-Eve and anti-beta-gal, 500 lethal single P-element enhancer trap lines were screened, focusing on mutations that affected RP2 cell number. P1683 was identified as an insertion at cytological position 86B that expresses beta-gal in NB subsets and that exhibits an occasional duplication of the Eve positive RP2 neuron. In P1683 homozygous embryos, the expressivity of the RP2 duplication phenotype is low. Although P1683 is lethal, the P element is inserted in the large first intron of the transcription unit and represents a weak hypomorphic allele. The P element was mobilized in order to ascertain that the phenotype was due to the P-element insertion and also to obtain stronger alleles. Twelve alleles show an increase in the expressivity of the RP2 duplication phenotype, as compared to the P1683 homozygote. jumu L40 and jumu L70, which show the strongest phenotypes, were further analysed. jumu L40, a strong hypomorph which deletes sequences from the transcribed region of jumu, increases the expressivity of the RP2 duplication phenotype 4 fold; jumu L70, that shows a seven-fold increase in expressivity appears to be a total loss-of-function allele. The analyses of the jumu mutant phenotype was carried out with either jumu L40/Df(3)B22-5 [Df(3)B22-5 is a deficiency for the jumu region] or jumu L70 homozygous embryos that show high penetrance (>95%) for the RP2 duplication phenotype (Cheah, 2000).
In stage 14 or older wild-type embryos stained with anti-Eve, the nucleus of one Eve + RP2 neuron can be seen at its characteristic position in each hemisegment. In anti-Eve stained jumu mutant embryos of a similar age, many hemisegments exhibit two Eve + nuclei, of unequal size, at the characteristic RP2 position. On the basis of anti-Eve, anti-Zfh1 and monoclonal antibody 22C10 stainings, the duplicated cells both express markers consistent with a RP2 identity. If jumu is exerting its effect on sibling cell fate choice at the level of the postmitotic neurons one would not expect to observe an alteration in the GMC4-2a cell identity in jumu mutant embryos. To see whether the GMC4-2a undergoes a cell fate transformation, the identity of mutant GMC4-2a cells was assessed using a variety of markers that label the wild-type GMC4- 2a, including anti-Eve, anti-Ftz, anti-Pros and anti-Pdm1. The expression of these markers in GMC4-2a is unaffected in the mutant. These results suggest that in jumu mutant embryos the mutant GMC4-2a appears to retain a wild-type GMC4-2a identity (Cheah, 2000).
In order to understand the origin of the extra RP2-like cell in jumu mutant embryos, the appearance of Eve positive cells from the NB4-2 lineage was followed. In the wild-type temporal series, the first born GMC, GMC4-2a, buds off from the dorsal/lateral cortex at late stage 10 and by mid-stage 11 becomes Eve positive; GMC4-2a divides to produce the postmitotic RP2 and RP2sib, both of which express Eve at stage 11; RP2 retains Eve expression until the end of embryogenesis but its sibling cell extinguishes Eve expression such that by stage 15, the sibling cell can no longer be detected by anti-Eve staining and only the Eve positive RP2 neuron can be seen. In a temporal series of jumu mutant embryos, the birth of the Eve + GMC4-2a and its division to produce two Eve + postmitotic neurons appear to parallel those of wild-type animals; however, in contrast to the wild-type situation, Eve expression does not become extinguished in the mutant RP2sib; both cells remain Eve + at stage 13 and stage 15, and until stage 17. These results indicate that the extra RP2-like cell found in jumu embryos is derived from the RP2 sibling cell (Cheah, 2000).
What might be the underlying mechanism responsible for the RP2 duplication phenotype associated with the jumu loss-of-function mutants? Several observations suggest that Jumu may be acting at the level of the GMC4-2a cell division. Jumu is not expressed in NB4-2 prior to its first divisions, making it unlikley that it would be acting at the level of the NB4-2 cell divisions. Moreover, it is only transiently detected in the postmitotic RP2 and RP2sib and it is not asymmetrically segregated to one of these cells. Therefore it seems unlikely that jumu would be acting at the level of the postmitotic neurons. Since nuclear Jumu can be detected in GMC4-2a, the possibility was examined that jumu may be required for the asymmetric localization of the cell fate determinant Numb during the GMC4-2a cell division. Since Numb always colocalizes with Pon, which acts to facilitate its localization (Lu, 1998), an anti-Pon antibody was used to illustrate the localization of Numb. Examination of late prophase to metaphase GMC4-2a cells triple labelled with anti-Eve, DNA stain and anti-Pon indicates that Pon localization is defective in dividing jumu GMC4-2a cells. In essentially all of the dividing wild-type GMC4-2a cells, Pon and Numb always form basal cortical crescents; it has been suggested that the more basal progeny, which preferentially inherits Numb, becomes the RP2 neuron. However, in jumu mutant embryos, many dividing GMC4-2a cells fail to localize Pon as a basal crescent: about 36% show either cortical Pon distribution or misplaced crescents. The frequency of the Pon mislocalization roughly coincides with the frequency of hemisegments showing the RP2 duplication phenotype (29%). Similar conclusions can be drawn using anti-Numb. These data are therefore consistent with the notion that the duplication of RP2 neurons in jumu embryos arises as a result of the symmetric segregation of Numb to both the postmitotic RP2 and RP2sib leading to a RP2sib to RP2 cell fate transformation. In contrast to Pon/Numb, Insc localization does not appear to be affected in jumu embryos. Essentially all of the dividing GMC4-2a cells in both wild-type and jumu embryos localize Insc as an apical cortical crescent. Hence, the loss of jumu function does not exert a general effect on the protein localization machinery per se but appears to specifically affect the localization of Pon/Numb. Loss of jumu also does not alter the localization of any of the asymmetrically localized proteins, i.e. Miranda, Pros, Insc, during NB divisions (Cheah, 2000).
These data indicate that the failure to localize Numb is the primary defect responsible for the failure to resolve distinct RP2/RP2sib cell fates. The localization of known asymmetric components is not affected in mutant NBs. Moreover, the localization of Insc in GMC4-2a (and other GMCs) remains apical in mutant embryos. Therefore the effect of loss of jumu does not affect protein localization in a general way but rather appears to be specific to Pon/Numb. Moreover, other aspects of the GMC4-2a division appear to occur normally in jumu mutants. The RP2 and RP2sib nuclei are distinct in size; furthermore, in most mutants that cause an RP2sib to RP2 cell fate transformation, including jumu, the duplicated RP2 neurons exhibit distinct nuclear size differences. The only example in which the nuclei of the sibling neurons adopt equivalent size are in insc and pins embryos. Hence the generation of different sized sibling nuclei, which requires insc function, is not affected by the loss of jumu. Similarly, the orientation of the GMC4-2a cell division is also not affected in jumu mutants. These results indicate that jumu acts downstream of Insc, or in a parallel pathway, to mediate Pon/Numb localization but is not required for other aspects of the GMC4-2a division (Cheah, 2000).
jumu mutant embryos also exhibit an additional unique phenotype. In wild-type embryos, RP2 and RP2sib clearly separate from one another. In all of the known mutants that fail to resolve distinct sibling cell fates and cause RP2 duplication, e.g. insc, sanpodo, N, mastermind, the two RP2 neurons separate from one another. In jumu embryos, although there is clearly cell membrane between the nuclei of the duplicated RP2 neurons, these cells invariably fail to separate following cytokinesis. The phenotype is reminiscent of a number of mutations in yeast that show similar defects in cell separation. The gene associated with one of these mutations, sep1, encodes a putative winged-helix transcription factor like jumu, suggesting possible parallel function(s) in these related proteins. sep1 is not essential and its deletion leads to hyphal growth due to the failure of the daughter cells to separate. The fact that both sep1 and jumu encode transcription factors suggest that the separation of daughter cells may require the expression of genes late in the cell cycle (Cheah, 2000).
It is speculated that the jumu neuronal cell fate phenotype and the cell separation phenotype may be related by a common mechanism. The occurrence of the two defects appears to show a complete correlation; in jumu mutant embryos the RP2 and RP2sib neurons in the hemisegments that undergo normal sibling cell fate resolution always undergo separation; whereas the duplicated RP2 neurons in the hemisegments that fail to resolve distinct sibling cell fates, also fail to separate. Little is known about the separation of sibling cells. However, it seems likely that cytoskeletal and membrane components must play a role in the separation of postmitotic sibling neurons. Similarly, the localization of components of asymmetric cell division is likely to be dependent on components of cell cortex. Therefore it is possible that a transcription regulator like Jumu might mediate the expression of cortical/membrane components necessary for both processes (Cheah, 2000).
A common theme in developmental biology is the repeated use of the same gene in diverse spatial and temporal domains, a process that generally involves transcriptional regulation mediated by multiple separate enhancers, each with its own arrangement of transcription factor (TF)-binding sites and associated activities. By contrast, the expression of the Drosophila Nidogen (Ndg) gene at different embryonic stages and in four mesodermal cell types is governed by the binding of multiple cell-specific Forkhead (Fkh) TFs [including Biniou (Bin), Checkpoint suppressor homologue (CHES-1-like) and Jumeau (Jumu)] to three functionally distinguishable Fkh-binding sites in the same enhancer. Whereas Bin activates the Ndg enhancer in the late visceral musculature, CHES-1-like cooperates with Jumu to repress this enhancer in the heart. CHES-1-like also represses the Ndg enhancer in a subset of somatic myoblasts prior to their fusion to form multinucleated myotubes. Moreover, different combinations of Fkh sites, corresponding to two different sequence specificities, mediate the particular functions of each TF. A genome-wide scan for the occurrence of both classes of Fkh domain recognition sites in association with binding sites for known cardiac TFs showed an enrichment of combinations containing the two Fkh motifs in putative enhancers found within the noncoding regions of genes having heart expression. Collectively, these results establish that different cell-specific members of a TF family regulate the activity of a single enhancer in distinct spatiotemporal domains, and demonstrate how individual binding motifs for a TF class can differentially influence gene expression (Zhu, 2012).
To drive expression in the visceral mesoderm (VM), the Fkh1 site in the Ndg enhancer is required in concert with either the Fkh2 or Fkh3 site. The trans-acting factor responsible for this activity of the Ndg enhancer is likely to be Bin because: (1) Bin binds to all three Fkh sites in vitro; (2) among the three candidate Fkh genes with appropriate VM expression, eliminating the function of only bin resulted in a significant reduction of Ndg expression in this tissue; and (3) Bin overexpression in the mesoderm is associated with Ndg enhancer activity in additional mesodermal cells. There are multiple precedents for Bin activating the expression of other VM genes. Moreover, chromatin immunoprecipitation assays show that Bin binds in vivo to the Ndg enhancer throughout embryonic stages 14 to 15, precisely when it would be expected to regulate this element in the visceral musculature (Zhu, 2012).
Somatic muscles in Drosophila are formed by the sequential fusion of individual muscle founder cells (FCs) with multiple fusion-competent myoblasts (FCMs). Both the endogenous Ndg gene and the reporter driven by the minimal enhancer used in this study are expressed in a subset of FCs, but not in any FCMs. Mutating all three Fkh-binding sites had no effect on Ndg expression in FCs, suggesting that Fkh factors do not play a role either in activating Ndg reporter expression in certain FCs, or in repressing it in other FCs. By contrast, binding of the FCM-expressed Fkh TF CHES-1-like to the Fkh2 or Fkh3 sites mediated repression of Ndg expression in FCMs. The design of the experiment prevented unambiguous determination of whether this repression also required CHES-1-like binding to the Fkh1 site. These results reveal a mechanism for regulating somatic myoblast gene expression that has not been previously recognized (Zhu, 2012).
Prior studies have focused on the contributions of signal-activated, tissue-specific and FC identity TFs in specifying the unique genetic programs of this class of myoblast. Similarly, TFs such as Lmd are known to be responsible for activating FCM-specific genes. However, this study has uncovered a novel mode of regulation in which FC genes are excluded from FCMs by an FCM-restricted repressor, in this case in the form of a Fkh domain protein. CHES-1-like is unlikely to be the only repressor playing such a role, as the de-repression in CHES-1-like mutants is limited to only a subset of the FCMs and is weaker than that seen for the Ndg enhancer with mutated Fkh sites. Although not verified functionally, it is possible that Lmd could play a similar repressive role in FCMs as a chromatin immunoprecipitation study found that this TF is bound extensively to FC genes (Cunha, 2010). However, given the widespread expression of CHES-1-like in FCMs, it is anticipated that many other FC genes will also be repressed by this Fkh domain TF (Zhu, 2012).
Finally, in the heart, it was shown that CHES-1-like and Jumu repress Ndg expression in Odd-PCs and in all Cardial cells (CCs) other than Tin- Lb-CCs. Repression in these cardiac cell types is mediated by binding of CHES-1-like to all three of the Fkh sites in the Ndg enhancer, and of Jumu to at least the Fkh2 site (Zhu, 2012).
A common occurrence in development is the repeated function of the same gene in multiple biological contexts and regulatory processes, requiring that the gene be expressed in distinct spatial and temporal domains. Such expression patterns are often generated by differential transcription mediated by multiple enhancers, each with its own arrangement of TF-binding sites and associated activities (Davidson, 2006). A notable exception is the case of genes regulated by Hox TFs, where different family members exhibit similar binding sequence specificity but exert differential effects on the same target genes (Zhu, 2012).
The results of the present study identify another class of TFs, the Fkh proteins, which exhibit a similar role in the Drosophila embryonic mesoderm. Specifically, it was shown that various cell-specific members of the Fkh TF family associate with the same binding sites within a single enhancer, thereby regulating the different spatiotemporal expression patterns of the associated target gene. Furthermore, it was shown that the distinct tissue-specific gene expression responses to these Fkh TFs are mediated by the TFs binding to different combinations of Fkh primary and secondary motifs that are represented by these sites. Thus, it was interesting to see that in several Drosophila species, where the Fkh1 site (which is the only site corresponding to the Fkh primary motif in D. melanogaster) is absent, its role may be compensated for by the overlapping Fkh2 and Fkh3 sites (which match only the secondary motif in D. melanogaster), which correspond to both Fkh primary and secondary motifs in these species. Similar evolutionary shuffling of motifs has been described previously (Zhu, 2012).
Finally, this study used a computational approach to attempt to generalize the potential involvement of the two classes of Fkh sites in cardiac gene regulation. Specifically, within putative enhancers in the noncoding regions of heart-expressed genes, a statistically significant overrepresentation of combinations of binding sites for known cardiogenic TFs was observed along with primary and secondary Fkh motifs. These observations are in agreement with previous studies that have documented an inability of a single consensus binding site to explain all aspects of in vivo TF binding. In addition, it has recently been shown that the regulatory specificity of a myoblast homeodomain TF is mediated by sequences preferentially bound by that particular homeodomain and not by other related family members (Busser et al., 2012b). In light of these findings, it will be interesting to determine whether other Fkh TFs and members of other TF families mediate differential gene expression responses by acting through distinct sequence motifs (Zhu, 2012).
Domina/jumeau is expressed in neural progenitors including NB4-2 and GMC4-2a. Antiserum was raised against a Jumu fusion protein. This polyclonal antibody (anti-Jumu) specifically recognizes the Jumu protein as evidenced by the fact that it fails to stain mutant embryos (e.g. jumu L70 homozygotes); in addition, the general embryonic staining pattern of anti-Jumu appears to parallel the expression pattern of the lacZ reporter gene in P1683. Consistent with its homology to the winged-helix family of transcription factors, Jumu protein shows nuclear localisation. Protein expression is first detected in the nuclei of syncitial embryos, indicating maternal expression, and appears to be present in all nuclei during cellular blastoderm. During germ band extension, and in the germ band extended embryo, nuclear expression is seen throughout the ectoderm and in the CNS primordia. During germ band retraction the ectodermal expression fades and Jumu expression is seen predominantly in the brain lobes, in the segmented CNS and elements of the PNS. The CNS expression persists into late embryogenesis. The NB expression pattern of Jumu is highly dynamic. To elucidate how jumu might act to generate the duplicated RP2 neurons, the expression of Jumu protein within the NB4-2 lineage was assessed. Pros is expressed in the nuclei of many GMCs in the developing CNS, including GMC4-2a, and can be used as a general GMC marker. At stage 10 (following SIII NB segregation), anti-Pros staining shows that there is a Pros + cell dorsal to NB4-2, indicating that the first round of NB4-2 cell division is complete and GMC4-2a is formed at this stage; the NB seen at the NB4-2 position at this time is NB4-2a; anti-Jumu and anti-Pros double-labellings indicate that NB4-2a is Jumu + at a time when GMC4-2a is not yet expressing Jumu protein; however, no Jumu + NB4-2 are seen prior to the formation of the Pros + GMC4-2a; therefore, Jumu is expressed in NB4-2 only after its first division. Later, during mid-stage 11, GMC4-2a expresses Eve just prior to its division (note that unlike Pros which is present in GMC4-2a when it is born, Eve expression commences only late in the GMC4-2a cell cycle); double labelling experiments with anti-Eve and anti-Jumu demonstrate that GMC4-2a also expresses Jumu at this time. Late in stage 11, GMC4-2a divides to produce the Eve + postmitotic RP2 and RP2 sibling cell; double labelling experiments indicate that both of these cells are also positive for Jumu shortly after their births. However, Jumu protein does not persist for long in the postmitotic neurons and can no longer be detected by the beginning of stage 12. These data indicate that within the NB4-2 lineage, Jumu accumulates only following the first NB cell division, in the nuclei of both NB4-2a and GMC4-2a. Jumu expression in NB4-2a precedes its expression in GMC4-2a (since a Jumu + GMC4-2a is never seen in conjunction with a Jumu - NB4-2a). Furthermore, Jumu protein is present in the nuclei of the postmitotic RP2 and its sibling (Cheah, 2000).
Hybridization of DIG-labeled probes shows ubiquitous distribution of Dom RNA in embryos of stages 1-4. In stages 5-6 Dom RNA amount is slightly reduced between 10% and 30% and is absent in pole cells. From stage 7, transcripts show very distinct concentration in cells of neurogenic regions, in cardial or pericardial cells, and possibly in gonad precursor cells -- this reflects the beta-galactosidase staining pattern of the enhancer trap strain. At the end of embryogenesis, Dom transcripts are found in the CNS, in maxillary cells, in gonad and imaginal disc precursor cells. In larvae, DIG-labeled Dom antisense RNA-probes and anti-Dom antibodies give strong signals in imaginal discs especially in neurogenic cells. In salivary glands, the strongest labeling is found in the ducts, in eye-antenna discs behind the morphogenic furrow and in developing ommatidia. The reduced fertility of homozygous Dom females and the high abundance of Dom RNA in early embryos as well as the lacZ expression in the enhancer trap line indicates maternal Dom expression. This is corroborated by the in situ hybridization of DIG-labeled RNA probes in ovaries. Dom RNA signals appear and increase in germ line cells of egg chambers from stages 1 to 9. Dom RNA is produced and stored in nurse cells until stage 10 when the RNA starts to be completely transferred to the oocyte (Strodicke, 2000).
Homozygous mutants show rough eyes, irregular arrangement of bristles, extended wings, defective posterior wing margins, and a severely diminished vitality and fertility. Heterozygous Dom flies are morphologically wild type but show suppression of position-effect variegation. Consistently with this chromatin effect Dom protein is accumulated in the chromocenter and, as expected from a transcription factor, is found at specific euchromatic loci. Besides the suppression of PEV, the mutant morphological Dom phenotype, caused by all P-element alleles with exception of Doml(3)06142, involves aberrant structures of eyes and wings and a mutant arrangement of bristles. Rough eyes with only a few mechanosensory bristles is the most obvious feature of this phenotype. The eyes of homozygous mutant Dom fiies are smaller than wild-type eyes; adjacent ommatidia are often fused and the eyes are composed of ommatidia of variable size and shape This is probably due to the incompletely formed mesh of pigment cells that usually shapes ommatidia into a regular hexagonal structure. In histological tangential sections of wild-type eyes, seven round rhabdomeres are observed in a regular array whereas the outer irregular facet array observed in homozygous Dom mutants corresponds to an inaccurate underlying cell pattern. Sections from homozygous DomD631 eyes reveal a variable number of rhabdomeres with distorted shapes in unusual positions in the ommatidia The mutant bristle phenotype suggests that Dom is involved in adult PNS development. In most cases homozygous mutant Dom flies show loss or doubling of macrochaetae, however, bristles appear at correct positions. This indicates that extra bristles arise from the normal complement of proneural clusters. They have sockets and shafts and, therefore, obviously represent complete sensory organs (Strodicke, 2000).
The wings are weakly affected by Dom mutations. Wing size is reduced, hairs are irregularly arranged, posterior wing margins are notched and L5 is sometimes shortened. In some cases wings are extended. The mutant Dom phenotype is caused by the P[lArB] integration D631 while the wild-type phenotype is restored in excision lines after remobilization of the P[lArB] transposon. In accordance with the mutant phenotype, lacZ expression of the P[lArB] transposon was found in all affected tissues. In late embryos and in first and second instar larvae, the beta-galactosidase staining suggests Dom expression mainly in the CNS. In larvae, the CNS, imaginal discs, gonadal anlagen, and salivary glands are stained. Strong lacZ expression is observed in eye discs behind the morphological furrow and in sensory and bristle precursor cells of wing and leg discs. In adults, lacZ is expressed in ovaries and in testes (Strodicke, 2000).
Mutations at the nude locus of mice and rats disrupt normal hair growth and thymus development, causing nude mice and rats to be immune-deficient. The mouse nude locus has been localized on chromosome 11 within a region of less than 1 megabase. One of the genes from this critical region, designated whn (winged-helix nude), encodes a new member of the winged-helix domain family of transcription factors, and it is disrupted on mouse nu and rat rnuN alleles. Mutant transcripts do not encode the characteristic DNA-binding domain, strongly suggesting that the whn gene is the nude gene. Mutations in winged-helix domain genes cause homeotic transformations in Drosophila and distort cell-fate decisions during vulval development in Caenorhabditis elegans. The whn gene is thus the first member of this class of genes to be implicated in a specific developmental defect in vertebrates (Nehls, 1994).
The development of the thymus depends initially on epithelial-mesenchymal interactions, and subsequently on reciprocal lympho-stromal interactions. The genetic steps governing development and differentiation of the thymic microenvironment are unknown. By a targeted disruption of the whn gene, which recapitulates the phenotype of the athymic nude mouse, the Whn transcription factor has been shown to be the product of the nude locus. Formation of the thymic epithelial primordium before the entry of lymphocyte progenitors does not require the activity of Whn. However, subsequent differentiation of primitive precursor cells into subcapsular, cortical, and medullary epithelial cells of the postnatal thymus depends on activity of the whn gene. These results define the first genetically separable steps during thymic epithelial differentiation (Nehls, 1996).
Mutations in the winged-helix nude (whn) gene are associated with the phenotype of congenital athymia and hairlessness in mouse and rat. The whn gene encodes a presumptive transcription factor with a DNA binding domain of the forkhead/winged-helix class. Two previously described null alleles encode truncated whn proteins lacking the characteristic DNA binding domain. In the rat rnu allele described here, a nonsense mutation in exon 8 of the whn gene was identified. The truncated whnrnu protein contains the DNA binding domain but lacks the 175 C-terminal amino acids of the wild-type protein. To facilitate the identification of functionally important regions in this region, a whn homolog from the pufferfish Fugu rubripes was isolated. Comparison of derived protein sequences with the mouse whn gene reveals the presence of a conserved acidic protein domain in the C terminus, in addition to the highly conserved DNA binding domain. Using fusions with a heterologous DNA binding domain, a strong transcriptional activation domain was localized to the C-terminal cluster of acidic amino acids. Since the whnrnu mutant protein lacks this domain, these results indicate that a transactivation function is essential for the activity of the whn transcription factor (Schuddekopf, 1996).
Mutations in the winged-helix nude (whn) gene result in the nude mouse and rat phenotypes. The pleiotropic nude phenotype, which affects the hair, skin, and thymus, suggests that whn plays a pivotal role in the development and/or maintenance of these organs. However, little is known about whn function in these organs. In skin whn is specifically expressed in epithelial cells and not the mesenchymal cells: using a hair reconstitution assay, it has been demonstrated that the abnormal nude mouse hair development is attributable to a functional defect of the epithelial cells. Examination of nude mouse primary keratinocytes in culture reveals that these cells have an increased propensity to differentiate in an abnormal fashion, even under conditions that promote proliferation. Furthermore, nude mouse keratinocytes display a 100-fold increased sensitivity to the growth-inhibitory/differentiation effects of the phorbol ester TPA. In parallel with these findings, it has been directly shown that Whn functions as a transcription factor that can specifically suppress expression of differentiation/TPA-responsive genes. The region of Whn responsible for these effects was mapped to the carboxy-terminal transactivating domain. These results establish whn as a key regulatory factor involved in maintaining the balance between keratinocyte growth and differentiation. The general implications of these findings for an epithelial self-renewal model are discussed (Brissette, 1996).
In the mouse, the product of the nude locus, Whn, is required for the keratinization of the hair shaft and the differentiation of epithelial progenitor cells in the thymus. A bacterially expressed peptide representing the presumptive DNA binding domain of the mouse whn gene in vitro specifically binds to an 11-bp consensus sequence containing the invariant tetranucleotide 5'-ACGC. In transient transfection assays, such binding sites stimulate reporter gene expression about 30- to 40-fold, when positioned upstream of a minimal promotor. Whn homologs from humans, bony fish (Danio rerio), cartilaginous fish (Scyliorhinus caniculus), agnathans (Lampetra planeri), and cephalochordates (Branchiostoma lanceolatum) share at least 80% of amino acids in the DNA binding domain. In agreement with this remarkable structural conservation, the DNA binding domains from zebrafish, which possesses a thymus but no hair, and amphioxus, which possesses neither thymus nor hair, recognize the same target sequence as the mouse DNA binding domain in vitro and in vivo. The genomes of vertebrates and cephalochordates contain only a single whn-like gene, suggesting that the primordial whn gene was not subject to gene-duplication events. Although the role of whn in cephalochordates and agnathans is unknown, its requirement in the development of the thymus gland and the differentiation of skin appendages in the mouse suggests that changes in the transcriptional control regions of whn genes accompanied their functional reassignments during evolution (Schlake, 1997).
Nude mice are characterized by the absence of visible hair, epidermal defects, and the failure to develop a thymus. This phenotype results from loss-of-function mutations in Whn (Hfh11), a winged-helix transcription factor. In murine epidermis and hair follicles, endogenous whn expression is induced as epithelial cells initiate terminal differentiation. Using the promoter for the differentiation marker involucrin, transgenic mice that ectopically express whn in stratified squamous epithelia, hair follicles, and the transitional epithelium of the urinary tract were generated. Transgenic epidermis and hair follicles display impaired terminal differentiation and a subset of hair defects, such as delayed growth, a waved coat, and curly whiskers, correlated with decreased transforming growth factor (TGF)-alpha expression. The exogenous Whn protein also stimulates epithelial cell multiplication. In the epidermis, basal keratinocytes exhibits hyperproliferation, though transgene expression is restricted to suprabasal, postmitotic cells. Hair follicles fail to enter telogen (a resting period) and remain continuously in an abnormal anagen (the growth phase of the hair cycle). Ureter epithelium develop severe hyperplasia, leading to the obstruction of urine outflow and death from hydronephrosis. Though an immune infiltrate is present occasionally in transgenic skin, the infiltrate is not the primary cause of the epithelial hyperproliferation, because the immune reaction is not observed in all affected transgenics, and the transgene induces identical skin and urinary tract abnormalities in immunodeficient Rag1-null mice. Given the effects of the transgene on cell proliferation and TGFalpha expression, the results suggest that Whn modulates growth factor production by differentiating epithelial cells, thereby regulating the balance between proliferative and postmitotic populations in self-renewing epithelia (Prowse, 1999).
Loss-of-function mutations in Whn (wing-helix nude; Hfh 11), a winged-helix/forkhead transcription factor, result in the nude mouse phenotype. To determine the whn expression pattern during development, mice were used in which a beta-galactosidase reporter gene was placed under the control of the wild-type whn promoter by homologous recombination. Sites of reporter expression were confirmed by immunohistochemical staining for Whn protein or by in situ hybridization for whn mRNA. At all developmental stages, whn expression is restricted to epithelial cells. In addition to the skin and thymus, whn is expressed in the developing nails, nasal passages, tongue, palate, and teeth. In embryonic epidermis, suprabasal cells induce whn expression at the same time that terminal differentiation markers first appear. As the epidermis matures, whn promoter activity is found primarily in the first suprabasal layer, which contains keratinocytes in the early stages of terminal differentiation. In developing and mature anagen hair follicles, whn is expressed at high levels in the postmitotic precursor cells of the hair shaft and inner root sheath. Though principally associated with terminal differentiation, whn expression is also detected in progenitor cell compartments: in the hair bulb matrix and basal epidermal layer, a small subclass of cells expresses whn, while in the outer root sheath, whn promoter activity is induced as the follicle completes its elongation. Within these compartments, rare cells exhibit both whn expression and the nuclear proliferation marker Ki-67. The results suggest that whn expression encompasses the transition from a proliferative to a postmitotic state and that whn regulates the initiation of terminal differentiation. During thymus development, whn expression first appears in epithelial cells of the thymic primordium, and in the mature thymus, whn expressing epithelial cells are present throughout the medulla, cortex, and subcapsular region. Given the sites of whn expression in the mouse, as well as the presence of homologs in lower vertebrates and cephalochordates, the whn gene may influence either fundamental or common features of epithelial cell differentiation (Lee, 1999).
The nude locus encodes Whn, a transcription factor of the forkhead/winged-helix class. Mutations in Whn cause failure of differentiation of thymic epithelium with a corresponding lack of intrathymic T-cell development; in the skin, differentiation of follicular keratinocytes is disturbed resulting, in the formation of fragile hair shafts. A novel nude allele, nu(StL), has been identified and characterized. nu(StL) encodes a truncated Whn transcription factor protein, designated Whn(StL), lacking the activation domain but retaining the characteristic DNA binding domain. In contrast, the previously described Whn(nu) mutant protein lacks both domains. nu(StL)/nu(StL) mice show an alymphoid thymic rudiment and lack of peripheral T cells, similar to nu/nu mice. In the skin, impaired expression of hair keratin genes mHa1, mHa2, mHa3 and mHa4, mHb3, mHb4, mHb5, and mHb6 is observed in a pattern that parallels that of nu/nu mice: both mutant alleles behave as hypomorphs with respect to the expression of these hair keratin genes. However, a significant difference between these two alleles exists for mHa5 expression, which is reduced in nu(StL)/nu(StL) but not in nu/nu mice. The mutant Whn protein in nu/nu mice cannot enter the nucleus, whereas the mutant Whn protein in nu(StL)/nu(StL) mice is present in the nucleus. The antimorphic characteristic of the activation-deficient Whn(StL) protein with respect to mHa5 expression is therefore most likely caused by its non-productive interaction with other proteins at cis-regulatory regions of the mHa5 gene. These results indicate that the molecular consequences of mutations of the Whn gene can be different and demonstrate an unexpected complexity of transcriptional control mechanisms of hair keratin genes (Schorpp, 2000).
The molecular characteristics of the nude phenotype (alopecia and thymic aplasia) in humans and rodents are unknown. The nude locus encodes Whn, a transcription factor of the forkhead/winged-helix class. Expression of Whn in HeLa cells induces expression of human hair keratin genes Ha3-II and Hb5. Correspondingly, in nude mice, which are homozygous for a loss-of-function mutation of Whn, expression of mouse mHa3 and mHb5 hair keratin genes is severely reduced. Characterization of a previously identified nude allele, nu(Y), reveals a mis-sense mutation (R320C) in the DNA binding domain of Whn. This mutant protein is unable to activate hair keratin gene expression in HeLa cells. When the Whn transcription factor is expressed in two parts, one containing the N-terminal DNA binding domain and the other the C-terminal activation domain, no activation of hair keratin genes in HeLa cells is observed. However, when these two proteins are noncovalently linked by means of synthetic dimerizers, hair keratin gene expression is induced. This finding suggests that target gene activation by Whn depends on the structural integrity and physical proximity of DNA binding and activation domains, providing a molecular framework to explain the loss-of-function phenotypes of all previously characterized nude mutations. These results implicate Whn as a transcriptional regulator of hair keratin genes and reveal the nude phenotype as the first example of an inherited skin disorder that is caused by loss of expression rather than mutation of keratin genes (Schlake, 2000).
During vertebrate retinogenesis, seven classes of cells are specified from multipotent progenitors. To date, the mechanisms underlying multipotent cell fate determination by retinal progenitors remain poorly understood. The Foxn4 winged helix/forkhead transcription factor is shown to be expressed in a subset of mitotic progenitors during mouse retinogenesis. Targeted disruption of Foxn4 largely eliminates amacrine neurons and completely abolishes horizontal cells, while overexpression of Foxn4 strongly promotes an amacrine cell fate. These results indicate that Foxn4 is both necessary and sufficient for commitment to the amacrine cell fate and is nonredundantly required for the genesis of horizontal cells. Furthermore, evidence is provided that Foxn4 controls the formation of amacrine and horizontal cells by activating the expression of the retinogenic factors Math3, NeuroD1, and the Prospero-like transcription factor Prox1. These data suggest a model in which Foxn4 cooperates with other key retinogenic factors to mediate the multipotent differentiation of retinal progenitors (Li, 2004).
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Brissette, J. L., et al. (1996). The product of the mouse nude locus, Whn, regulates the balance between epithelial cell growth and differentiation. Genes Dev. 10(17): 2212-21. PubMed Citation: 8804315
Cheah, P. Y., Chia, W. and Yang, X. Jumeaux, a novel Drosophila winged-helix family protein, is required for generating asymmetric sibling neuronal cell fates. Development 127(15): 3325-3335. PubMed Citation: 10887088
Cunha, P. M., et al. (2010). Combinatorial binding leads to diverse regulatory responses: Lmd is a tissue-specific modulator of Mef2 activity. PLoS Genet. 6(7): e1001014. PubMed Citation: 20617173
Lee, D., Prowse, D. M. and Brissette, J. L. (1999). Association between mouse nude Gene expression and the initiation of epithelial terminal differentiation. Dev. Biol. 208(2): 362-374. PubMed Citation: 10191051
Li, S., Mo, Z., Yang, X., Price, S. M., Shen, M. M. and Xiang, M. (2004). Foxn4 controls the genesis of amacrine and horizontal cells by retinal progenitors. Neuron 43(6): 795-807. 15363391
Lu, B., Rothenberg, M., Jan, L. Y. and Jan, Y. N. (1998). Partner of numb colocalizes with numb during mitosis and directs Numb asymmetric localization in Drosophila neural and muscle progenitors. Cell 95: 225-35. PubMed Citation: 9790529
Nehls, M., et al. (1994). New member of the winged-helix protein family disrupted in mouse and rat nude mutations. Nature 372(6501): 103-7. PubMed Citation: 7969402
Nehls, M., et al. (1996). Two genetically separable steps in the differentiation of thymic epithelium. Science 272(5263): 886-9. PubMed Citation: 8629026
Prowse, D. M., et al. (1999). Ectopic expression of the nude gene induces hyperproliferation and defects in differentiation: implications for the self-renewal of cutaneous epithelia. Dev Biol 212: 54-67. PubMed Citation: 10419685
Schlake, T., et al. (1997). The nude gene encodes a sequence-specific DNA binding protein with homologs in organisms that lack an anticipatory immune system. Proc. Natl. Acad. Sci. 94(8): 3842-7. PubMed Citation: 9108066
Schlake, T., et al. (2000). Forkhead/winged-helix transcription factor whn regulates hair keratin gene expression: molecular analysis of the nude skin phenotype. Dev. Dyn. 217(4): 368-76. PubMed Citation: 10767081
Schorpp, M., et al. (2000). Genetically separable determinants of hair keratin gene expression. Dev. Dyn. 218: 537-543. PubMed Citation: 10767081
Schuddekopf, K., Schorpp, M. and Boehm, T. (1996). The whn transcription factor encoded by the nude locus contains an evolutionarily conserved and functionally indispensable activation domain. Proc. Natl. Acad. Sci. 93(18): 9661-4. PubMed Citation: 8790387
Strodicke, M., Karberg, S., Korge, G. (1996). Domina is a female-specific suppressor of position effect variegation and regulates eye and bristle development. Annual Dros. Res. Conf. 37: 163
Strodicke, M., Karberg, S., Korge, G. (1999). The forkhead box gene Domina (Dom) is a suppressor of position effect variegation (PEV) and affects the morphogenesis of the eye and the PNS in Drosophila melanogaster. Annual Dros. Res. Conf. 40: 822B
Strodicke, M., Karberg, S. and Korge, G. (2000). Domina (Dom), a new Drosophila member of the FKH/WH gene family, affects morphogenesis and is a suppressor of position-effect variegation. Mech. Dev. 96: 67-78. PubMed Citation: 10940625
Zhu, X., et al. (2012). Differential regulation of mesodermal gene expression by Drosophila cell type-specific Forkhead transcription factors. Development 139(8): 1457-66. PubMed Citation: 22378636
date revised: 20 July 2012
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