The gene associated with the regulatory sequences responsible for driving E8-2-46 expression is potentially a target of both cut and ttk. To approach this question, reporter gene expression of E8-2-46 was first examined in homozygous embryos for ctc145 and ctdb7, which belong to the lethal II class, are the strongest cut mutants, and are likely to be null. In these backgrounds, the expression of the E8-2-46 reporter gene in the PNS is severely reduced. Normally, the reporter gene is expressed in 26 es support cells in each abdominal hemisegment. In cut mutants, es organs are often transformed into ch organs, albeit this phenotype is not completely penetrant. Accordingly, the number of cells expressing the E8-2-46 reporter in ctdb7 is reduced to 15 cells, and the level of expression is significantly lower. In the complex es organs of the thoracic segments, the humidity receptive Keilin organs, the number of E8-2-46-expressing cells is not decreased significantly, only the level of expression, consistent with the observation that these organs are less affected in cut mutant embryos than simple es organs (Hardiman, 2002).
E8-2-46 reporter gene expression was examined in mutations of the lineage genes numb and ttk. In numb1 mutants, the number of cells expressing the reporter gene increases as would be expected if the neural pIIb secondary precursors are transformed into pIIa, the support cell precursors. Similarly, Keilin organ cells are also increased. In ttk mutants the opposite phenotype is expected, since ttk is required for support cell development. Indeed, in ttk702/7 mutants E8-2-46 expression per hemisegment is reduced, similar in extent to the reduction observed in cut mutants (Hardiman, 2002).
Whether cut function activates or modulates bft transcription in the PNS was investigated by examining cutdb7 null mutant embryos. In the absence of cut function, E8-2-46 reporter gene expression is reduced in es support cells. In wild-type embryos, bft is also expressed in the developing posterior spiracles, which is severely reduced or absent in cut mutants (Hardiman, 2002).
Does cut suffice to activate bft transcription? The UAS-Gal4 system or a heat shock promoter was used to drive cut ectopically in embryos and to examine the resulting pattern of bft transcription. When hairy-Gal4 is used to drive cut expression in the odd-numbered segments, bft is expressed ectopically, most noticeably near the dorsal abdominal PNS clusters, which are the embryonic origins of the lateral chordotonal organs. Ectopic expression of cut causes es-specific gene expression in these chordotonal organs and prevents their lateral migration. Thus, the cell fate changes in the PNS induced by cut result in ectopic bft expression. Furthermore, cells that normally never express cut, in particular ectodermal cells overlying the central nervous system, are induced to express bft when cut is ectopically expressed. Since cut can induce ectopic bft expression outside the PNS, it may participate directly in the regulation of bft. Consistent with this hypothesis, consensus Cut binding sites were identified immediately upstream of the bft 5' RACE products (Hardiman, 2002).
The course of bft expression during sensory organ development was visualized in whole mount wild-type embryos either by itself or in combination with Cut protein. bft transcripts in the PNS coincide with the onset of Cut protein expression in some PNS precursor cells, suggesting bft is already turned on in neural progenitor cells, just as is cut. bft transcripts appear to be punctate and perinuclear in the vicinity of nuclear Cut staining. After es organ precursors have begun dividing, bft expression levels are sometimes higher in cells next to strongly Cut-positive nuclei, consistent with the observation that reporter gene expression in the E8-2-46 enhancer trap line is higher in forming trichogen than tormogen cells (the opposite is the case for Cut) (Hardiman, 2002).
In addition to the PNS, bft is highly expressed in the head and terminal regions. bft expression first appears at stage 6 in the cephalic region of the future posterior transverse furrow and of the acron primordia; this expression persists until after the clypeolabrum has formed. At stage 8/9, bft-expressing cells appear ventrally in the head, at the anterior lip of the cephalic furrow; these then appear to invaginate during head involution. At early stage 11, bft RNA is present in two stripes of cells corresponding to the anlagen of the pharyngeal ridges. Later during stage 11, the expression expands to include strong staining in the maxillary and labial lobes and weaker staining in the mandibular lobe. Most of this staining in the gnathal segments persists throughout embryonic development and probably corresponds to PNS precursors (such as the antenno-maxillary organ), which also express Cut. As the hypopharyngeal lobes form, they also express bft. In the terminal, proctodeal region of stage 10 embryos, bft transcripts appear in endodermal cells corresponding to the anlagen of the posterior spiracles, which also express Cut. The primordia of anterior spiracles begin to express bft only at late stage 11 (Hardiman, 2002).
Since the E8-2-46 flies are viable and exhibit no visible phenotype, attempts were made to generate mutations in the gene responsible for the bft expression pattern by excising the P element. Often, these excisions are imprecise, resulting in the loss of flanking sequences. A total of 244 fly strains were generated in which the P element had excised or had excised and reinserted. Candidate alleles were detected by identifying those strains in which the DNA was disrupted (PCR screening), or they were detected by examining es organ structures for defects. Twenty-one mutant strains were recovered that contained small deletions or that exhibited defective sensory organs or both. The 7 strains that were chosen for further study have reduced viability, form a single complementation group, and exhibit a similar bristle phenotype. In bft6 and bft24, genomic lesions have been identified that eliminate the first (and longest) putative ORF and the transcript start. bft6 contains a deletion of 1.6 kb that removes sequences distal to the site of the P-element insertion, eliminating bft's first exon and 0.75 kb of intron 1. bft24 lacks 2.8 kb of sequence, extending not only distal but also proximal to the insert, removing both bft's first exon and 1.5 kb of bft's first intron. Furthermore, in bft24 (and in bft225), the 7-kb transcript is missing. Thus, the 7-kb bft transcript is disrupted in the mutant alleles examined (Hardiman, 2002).
In the bft mutants, the majority of the interommatidial bristles (IOBs) are missing. bft24 and bft225 are the strongest alleles, in that each fly lacks 50%-90% of the normal complement of IOBs. In most cases, severely defective structures are found where the IOBs normally form. The most severe defect is the complete absence of shaft and socket morphogenesis, resulting in a slight bump or cap in a shallow pit, without any other distinguishing characteristics. Other structures found in bft mutants were a relatively normal socket and a round, spherical shape protruding from it, reminiscent of mechanosensitive campaniform sensilla, found in other regions of the fly. Another phenotype consists of discontinuous sockets, seemingly composed of two halves, without any remnant of a shaft, as if the shaft were transformed into another socket. To determine if the precursors of the IOBs form in these flies, pupal eye discs were stained with Cut antibodies to visualize the precursor cells and their progeny. In wild-type flies, all four IOB sensillum cells express Cut; in bft mutants, these cells express Cut normally, suggesting that bft is not required to produce the normal number of Cut-expressing progeny. Thus, bft must act at a later step in IOB differentiation. Interestingly, as is observed in the embryo, the presumptive trichogen cells within the forming IOBs express the E8-2-46 reporter most strongly. This prevalent expression in the shaft-forming cell may reflect the possibility that one of bft's crucial functions is in bristle morphogenesis (Hardiman, 2002).
The cells comprising the interommatidial bristles (IOB) do form in bft mutants, but the cuticular structures they secrete are severely defective. These observations indicate that bft may be required to direct the secretion of the cuticular shaft (and socket) structures. The shaft is formed from a cytoplasmic extension of the trichogen cell, and its structure is provided by a core of microtubules surrounded by actin fiber bundles. A number of different genes encoding actin-associated proteins have been shown to affect bristle morphology; among them is sanpodo, a tropomodulin homolog, which also acts downstream of numb, as does bft (Hardiman, 2002).
bft mutants were examined for defects in mechanosensory bristles of the head, thorax, abdomen, and legs. While wild-type flies occasionally lack vertical bristles, postvertical or humeral bristles were never missing. bft homozygous mutants lack bristle shafts on the head and thorax at a significantly higher incidence than wild type. The vertical, postvertical, and humeral bristles were most often missing in bft mutants (up to 80% of the flies lack one or more of these bristles). Notably, the sockets of the missing bristle shafts in bft mutants are still present and normal in appearance, even when examined with scanning electron microscopy. These results again suggest that primarily bristle shaft formation is affected in bft mutants and that the observed high levels of bft activity in trichogen cells may be required autonomously for this process (Hardiman, 2002).
Although no es organ defects can be detected in bft mutants during embryonic stages, a requirement for bft in bristle morphogenesis might manifest itself during larval stages. Indeed, third instar bft larvae often exhibit abnormal trichoid sensilla in which the shaft is missing, similar to the adult phenotype. In some cases, the sensory structure resembles that of a campaniform sensillum similar to what was observed with IOBs. The sensory organs established in the embryo further differentiate during larval stages. Thus, it was reasoned that sensory organ defects associated with bft mutant alleles might be detected during larval stages. First instar larvae were examined, but no defects were found in their sensory organ structure. However, third instar bft larvae often exhibit abnormal trichoid sensilla in which the shaft is missing, similar to the adult phenotype. In some cases, the sensory structure resembles that of a campaniform sensillum. A function reminiscent of bft has been found for the paired homeobox gene pox neuro, which is expressed in one of the es support cells during larval stages. Interestingly, in pox neuro as in bft mutants, not only do the trichoid sensilla show bristle shaft abnormalities, but these defects do not manifest themselves earlier than in second instar larvae (Hardiman, 2002).
Although some of the E8-2-46 P-element excision alleles generated do have molecular lesions at the bft locus, it is conceivable that the observed bft bristle phenotype of these alleles is caused by a background mutation in the E8-2-46 enhancer trap stock. To determine whether the observed defects in bristle morphogenesis are indeed associated with the bft locus, cytological deficiencies at chromosome position 33B were crossed to bft excision alleles. Df(2L)escP3-0, Df(2L)esc10, and Df(2L) prd1.7 fail to complement the reduced viability of bft mutants. The reported cytology of these deficiencies suggests that they overlap only in the 33B region. Flies trans-heterozygous for bft alleles and any of these three deficiencies exhibit a loss of IOBs, as is typically observed in bft homozygous flies. Thus, the observed bristle phenotype is unlikely due to a background mutation, but rather due to a lesion at the bft locus. Df(2L)esc10 and Df(2L)prd1.7 both affect the bft locus but their deficiencies extend in opposite chromosomal directions. Therefore, it was reasoned that the deficiency overlap may be small enough to yield some viable trans-heterozygotes that lack the bft locus. Indeed, survivors of the genotype Df(2L)esc10/Df(2L)prd1.7 do display the bft eye phenotype and show an almost complete absence of IOBs. The complementation pattern of the lethal allele bft97 with the large deficiencies of the bft locus suggests it contains a larger deletion than bft6 and bft24. As assessed by PCR, bft97 lacks genomic DNA centromere-distal to the site of the E8-2-46 insertion corresponding to the location of the bft locus. Furthermore, bft97 but none of the other bft alleles fails to complement the lethality of Df(2L) escP2-0. These data provide strong evidence that the bft phenotype results from a loss of gene activity in the 33B region. Moreover, the fact that bft6, bft24, and bft97 contain molecular lesions of the bft locus, removing the first exon or likely the entire locus, and that they do exhibit the same phenotype in trans-heterozygous combination with deficiency flies, leaves little doubt that a defect in the bft locus is responsible for the observed phenotype in bristle morphogenesis (Hardiman, 2002).
The relationship between bft, cut, and ttk was examined in genetic interaction experiments. For this purpose, flies of the genotype ctc145/FM6;bft6/CyO were generated. These strains never yielded bft6 homozygous females that are also heterozygous for cut (e.g., ctc145/FM6;bft6/bft6), although bft6 homozygotes are semiviable. Thus, mutating one copy of cut eliminates the viability of bft6. In an attempt to generate viable bft flies that lack some but not all cut function, bft6 was crossed to the viable ctk allele, which by itself exhibits bristle and wing margin defects. Similarly, a stock of ctk/FM6;bft6/CyO flies never produces any bft6 homozygous females. In contrast, some ctk/ctk;bft6/CyO females do survive and exhibit a typical ctk phenotype. To address the possibility that the interaction between bft and cut may be attributable to a background mutation in bft6, ctk/ctk;bft6/CyO females were crossed to bft24/bft24 homozygotes. Indeed, female ctk/+;bft6/bft24 survivors are observed, and they exhibit the bft eye phenotype. However, male hemizygotes of this cross that are also trans-heterozygous for these two bft alleles, ctk/Y;bft6/bft24, are never observed. Similar experiments were carried out with another bft allele, bft225. When bft225 is crossed into a ctk mutant background, neither doubly homozygous females (ctk/ctk;bft225/bft225) nor males hemizygous for ctk and homozygous for bft225 (ctk/Y;bft225/bft225) are observed (Hardiman, 2002).
To determine if removing ttk function augments the bft phenotype, bft6 was crossed into a ttk702/7/+ background. Indeed, survivors of the genotype, bft6/bft6;ttk702/7/+, were never observed. Thus, losing one copy of ttk is completely fatal for bft6 flies. Taken together, these findings demonstrate that cut and ttk exhibit genetic interactions with bft, consistent with the idea they affect some of the same developmental pathways (Hardiman, 2002).
Hardiman, K. E., Brewster, R., Khan, S. M., Deo, M. and Bodmer, R. (2002). The bereft gene, a potential target of the neural selector gene cut, contributes to bristle morphogenesis. Genetics 161: 231-247. 12019237
date revised: 10 August 2002
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