BIOLOGICAL OVERVIEW

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 Jumeaux (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).

jumeaux 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).

GENE STRUCTURE

Domina is a novel member of the FKH/WH transcription factor gene family of Drosophila. Two alternatively polyadenylated Dom transcripts of 2.9 and 3.9 kb encode a 719-amino-acid protein with a FKH/WH domain and a putative acidic transactivation domain. On the basis of several Dom-cDNA clones, the two alternative Dom transcripts have been shown to differ only in the 3' untranslated region (UTR). The Dom antisense RNA probes RP1-RP3 corresponding to the first and second exon and the 5' end of the third exon, indeed detect the two postulated transcripts of 2.9 and 3.9 kb in RNA from wild-type larvae. The antisense probe RP4 corresponding to the 3' end of the third exon detects only the 3.9 kb transcript. These results coincide with the assumption that both transcripts differ only in the length of their 3' UTR because of the usage of alternative polyadenylation signals. The 3.9 kb transcript contains a 3' UTR of 1385 bp and the 2.9 kb transcript a 3' UTR of 324bp. This means that the Dom gene encodes two transcripts but only one protein. The 3' UTR of the 3.9 kb Dom transcript contains ten copies of the mRNA degradation motifs AUUUA, AUUUUA and AUUUUUA. Due to the shortened 3' UTR, only three mRNA degradation motifs remain in the 2.9 kb Dom transcript. Near the 3' end of the 3.9 kb transcript a K-box is located that has been shown to be important for the post-transcriptional regulation of E(spl)-C genes. Domina consists of three exons. The first 403 bp exon and the second 1800 bp exon are separated by a 7.4 kb large intron; the second and third exons are separated by a 60 bp intron. The transcription start site was determined by 5'-RACE. Three primers corresponding to the second intron were used for the amplification of 5' ends. For both transcripts only a single transcription start site could be detected. The 5' UTR is 343 bp long; the putative translation start site was found at the 3' end of the first exon and the translation stop in the third exon (Strodicke, 2000).

Bases in 5' UTR - 344

Bases in 3' UTR - 1381

PROTEIN STRUCTURE

Amino Acids - 720

Structural Domains

The Dom/Jumu protein contains two regions with features reminiscent of trancriptional activation domains: a high proportion of serine and proline residues in its amino terminal half and a highly acidic region [aa 536-592 (31/57 residues are aspartic or glutamic acids)]. Protein homology searches revealed that residues 409-526 from the deduced Jumu protein contain a high degree of homology to the DNA-binding domains found in the winged-helix family of transcription factors which include the Drosophila homeotic protein Fork-Head, the C. elegans lin-31 gene product, and in mammals the Hepatocyte Nuclear Factor3 family and the Winged-Helix-Nude protein. Alignment of the winged-helix domains show that the highest homology (68% identity; 85% similarity) is with the Winged-Helix-Nude protein (Nehls, 1994), which is associated with the gene that is mutated in nude mice. These results suggest that Jumu effects asymmetric sibling cell fate choice through its actions as a transcription regulator (Cheah, 2000).

Sequence comparison suggests that Dom of Drosophila is homologous to the chordate WHN proteins. The chromatin modifying capability of Dom is probably based on the FKH/WH domain, which shows a remarkable structural similarity to the winged-helix structures of H1 and the central globular domain of H5. The DNA-binding fork head/winged helix (FKH/WH) domain starts at amino acid 409 and extends to amino acid 525. It is followed by two acidic regions from amino acids 526-592 and 616-659, respectively, which represent a putative transactivating domain. The FKH/WH domain of DOM shows the highest degree of sequence identity to the FKH/WH domain of Winged Helix Nude (WHN) proteins. A region of a lower degree of sequence identity between DOM and WHN proteins from pufferfish, zebrafish and human is recognizable in the C-terminal acid region of the DOM protein. The FKH/WH domain and a putative transactivating domain characterize DOM as a member of the FKH/WH transcription factor family (Strodicke, 2000).

date revised: 25 July 2000

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