BIOLOGICAL OVERVIEW

The neural selector gene cut, a homeobox transcription factor, is required for the specification of the correct identity of external (bristle-type) sensory organs. Targets of cut function, however, have not been described. bereft (bft) mutants exhibit loss or malformation of a majority of the interommatidial bristles of the eye and cause defects in other external sensory organs. These mutants were generated by excising a P element located at chromosomal location 33AB, the enhancer trap line E8-2-46, indicating that a gene near the insertion site is responsible for this phenotype. Similar to the transcripts of the gene nearest to the insertion, reporter gene expression of E8-2-46 coincides with Cut in the support cells of external sensory organs, that secrete the bristle shaft and socket. Although bft transcripts do not obviously code for a protein product, bereft's expression is abolished in bft deletion mutants, and the integrity of the bft locus is required for (interommatidial) bristle morphogenesis. This suggests that disruption of the bft gene is the cause of the observed bristle phenotype. Attempts were made to determine what factors regulate the expression of bft and the enhancer trap line. The correct specification of individual external sensory organ cells involves not only cut, but also the lineage genes numb and tramtrack. Mutations of these three genes affect the expression levels at the bft locus. Furthermore, cut overexpression is sufficient to induce ectopic bft expression in the PNS and in nonneuronal epidermis. On the basis of these results, it is proposed that bft acts downstream of cut and tramtrack to implement correct bristle morphogenesis (Hardiman, 2002).

Bft's longest transcript is 7 kb, but surprisingly the longest open reading frame (potential protein-coding sequence) is only 465 base pairs contained within the first exon. While other genes encoding small proteins have been reported, for instance reaper, the size of this transcript makes it difficult to identify which ORF is likely translated into a protein product. In addition, none of the sequences, at the nucleotide or amino acid level, exhibit any homology to other genes. Considering the lack of an obvious ORF, the 7-kb bft transcript may not code for a protein product, but perhaps acts as an RNA. The mechanisms of action by noncoding, nonribosomal RNAs are poorly understood (see RNAi and posttranscriptional gene silencing). A few apparently noncoding mRNAs have been proposed to act by hybridizing to the mRNAs of other genes, thereby preventing their translation. Further experiments are needed to decide if bft acts primarily as a protein or as a noncoding RNA and what is its mechanism of action (Hardiman, 2002).

The PNS sensory organs are categorized on the basis of sensillum structure and sensory neuron morphology: type I sense organs are innervated by monopolar, cilium-containing dendrites, whereas the type II sense neurons extend multiple dendrites and are thought to be touch receptors. Type I organs are further classified as external sensory (es) organs, which secrete cuticular structures from the larval epidermis, and as internal chordotonal (ch) organs, which form internal attachments to the larval cuticle. es organs serve as mechano- and chemo-receptors, whereas ch organs function as proprioceptors. The genetic distinction between es and ch organs is under the control of the homeobox gene cut. cut is expressed in the es sensory organ precursors and their progeny and is required to correctly specify their identity. cut acts similarly to homeotic selector genes: when cut function is removed, es organs are directed to assume the ch fate, whereas ectopic expression of cut in ch organ lineages causes transformation of ch organs into es organs. Although from this perspective cut behaves as an 'activator' of es organ fate, evidence from in vitro experiments using mammalian cut homologs suggests that Cut may act to transcriptionally repress target genes. Recent genetic experiments in flies also support the idea that cut suppresses non-es organ-derived cell fates. While these data suggest cut may act as a transcriptional repressor, understanding of the mechanism by which cut specifies cell fates remains limited. Thus, the identification of cut targets would aid in elucidating how it regulates sensory organ cell fates (Hardiman, 2002).

In the Drosophila embryo, a simple bristle-type es organ is composed of a neuron, a glial-like cell (thecogen), and two external support cells, the shaft-forming trichogen cell and the socket-forming tormogen cell. These cells are generated from a single ectodermal precursor through asymmetric divisions, involving the segregation of Numb, a membrane-associated protein, to one daughter cell of the dividing precursor, but not the other. The daughter cell that receives Numb protein, the pIIb cell, ultimately produces the neuron and thecogen cell, whereas the pIIa cell is the precursor to the external support cells. The asymmetric inheritance of Numb within the sensory organ lineages [and those of the central nervous system (CNS)] is necessary and sufficient to distinguish between alternative daughter cell fates. Numb exerts its function by inhibiting signal transduction of the transmembrane protein encoded by Notch. ttk, a lineage gene encoding a zinc-finger protein, appears to act downstream of numb to implement sensory organ cell fates. ttk mutant embryos exhibit a phenotype opposite that of numb, in that pIIa is transformed into pIIb, resulting in excess neurons and glia. Furthermore, overexpression of ttk results in a phenotype similar to that observed in numb mutants, namely the sensilla lack neurons and glia, consisting entirely of support cells. ttk acts epistatically to numb, since embryos doubly mutant for both genes exhibit a ttk phenotype. Consistent with this result, Ttk protein, which normally is excluded from the neurons, exhibits ectopic neural expression in numb mutants, whereas the distribution of Numb protein appears unaffected in ttk mutants. Thus, ttk is likely to promote cell-type-specific gene expression in the daughter cells produced from asymmetric divisions of sensory organ precursors (Hardiman, 2002).

Lineage genes and selector genes clearly must regulate different aspects of sensory organ formation: the lineage genes direct the asymmetric divisions of the sensory organ precursors, but they do not appear to take part in specifying the identity of the sensory organ itself. The lineage genes are required and expressed in es as well as ch organs to distinguish the daughter cells from each other. By contrast, the selector gene cut is expressed only in those sensory organs it specifies. For appropriate organogenesis of the sensillum structures to take place, organ identity and lineage information must ultimately be integrated within individual cells of a sensory organ. Thus, a cell needs to acquire at least two pieces of information: for example, (a) support cell information (provided by lineage genes) and (b) es organ-type information (provided by selector genes) (Hardiman, 2002).

In an effort to identify and characterize genes that might integrate information from cut and ttk, bereft was cloned. bft is expressed in es, but not in ch support cells. Analysis of cDNA, reverse transcribed, and genomic sequence of the bft locus does not suggest an obvious protein-coding region. Thus, bft either encodes a very small protein or may act as an RNA. Analysis of flies with deletions of the bft locus, together with the es support cell-specific expression pattern, suggest that bft function is required for correct morphogenesis of the cuticular structure forming support cells, in particular those of the interommatidial bristles of the eye. Moreover, bft expression in es organs is reduced in cut and ttk mutants, and cut and ttk interact genetically with bft. These data are consistent with the idea that bft is a target for cut and ttk in the implementation of es organ-specific structures (Hardiman, 2002).

Targets of both cut and ttk were sought on the basis of the expression pattern of candidate genes within the PNS. cut is expressed in all the cells of es organs (at higher levels in support cells), whereas ttk is found in three es and two ch support cells, but not the neurons. Thus, the support cells of es organs express both cut and ttk, suggesting that genes responsive to these two pathways (i.e., the pathways leading to organ identity specification and lineage decisions, respectively) should also be expressed in these cells. An enhancer trap line, E8-2-46, was identified in which the lacZ reporter gene is expressed primarily in the support cells of es organs within the PNS, on the basis of position, morphology, and cut expression. Although E8-2-46 is expressed in both es support cells (as identified by high levels of cut expression), the level of expression is lower in one of them. To determine which of the two cell support cells express the reporter gene more strongly, the dorsal-most abdominal es organ (desD) were examined. DesD are aligned in a stereotyped linear fashion: tormogen, trichogen, thecogen, and neuron (from dorsal to ventral). Strong reporter activity is observed in the bristle shaft-forming trichogen cell, whereas cut expression predominates in the shaft-forming tormogen cell (Hardiman, 2002).

Three lines of evidence indicate that the bristle phenotype observed in bft mutants results from a mutation in the bft gene. (1) The tissues and cells in which bft transcripts are expressed are affected in bft mutant flies. bft is expressed in the precursor cells that secrete the sensory structures, consistent with bft being required for appropriate differentiation of these cells. (2) The alleles bft⁶, bft²⁴, and bft⁹⁷ contain molecularly characterized deletions of the bft coding region: bft⁶ and bft²⁴ contain deletions of 1.6 and 2.8 kb, respectively, that remove the first exon harboring the largest open reading frame, and bft⁹⁷ contains a larger deletion, probably removing the entire bft locus. (3) The 7-kb bft transcript is absent in bft⁶ and bft²²⁵ homozygotes. Taken together, this evidence strongly indicates that the bft phenotype results from a disruption of the bft locus and that it is likely that the absence of or a defect in the 7-kb bft transcript is the cause of the observed bristle phenotype. A further consideration is that bft alleles in trans to cytological deficiencies of the 33A-B genomic region do not noticeably increase the observed phenotypes, suggesting strong bft alleles have been isolated. However, without having corrected the phenotype using a bft transgene, the possibility cannot be completely rule out that the molecular lesions of bft⁶ and bft²⁴ (also) affect a regulatory region of a distant gene. Centromere distal to bft are (or are predicted to be) odorant receptor 33C (Or33c; 7 kb 3' to the 7-kb bft transcript), Drosocrystallin (also known as Cry), and CG16964 (novel). Centromere proximal are alpha-alpha trehalase (similar to an enzyme involved in stress response in Saccharomyces cerevisiae, 10 kb 5' to the 7-kb bft transcript); CG6686 (predicted to be a cytoskeleton-associated protein with homologies to human and rodent tumor-rejection antigen SART-1), and CG12314 (novel). None of these genes, however, are predicted by the Drosophila genome sequence project to span the bft locus (Hardiman, 2002).

Considering the lack of an obvious ORF, the 7-kb bft transcript may not code for a protein product, but perhaps acts as an RNA. The mechanisms of action by noncoding, nonribosomal RNAs are poorly understood. A few apparently noncoding mRNAs have been proposed to act by hybridizing to the mRNAs of other genes, thereby preventing their translation. For instance, the lin-4 gene of C. elegans encodes small, noncoding transcripts that are thought to post-transcriptionally regulate the lin-14 gene. In mammals, the Xist gene, involved in X chromosome inactivation, appears to lack a coding region, and its transcript, like bft's, is quite large: 17 kb in humans and 15 kb in mice. In Drosophila, the rox1 and rox2 genes also appear to encode only RNAs and not proteins, and they have a redundant but essential function in dosage compensation (Hardiman, 2002 and references therein).

By examining both Cut protein and bft transcripts in the same embryo, it has been found that the es precursors express bft transcripts almost coincident with the onset of Cut expression. At later stages, bft transcripts are restricted to the support cells of es organs. Furthermore, bft transcripts are expressed in nonneural tissues that also express Cut, such as in the cephalic segments, and the precursors of both the anterior and posterior spiracles. In the absence of Cut activity, bft expression is reduced or absent. Conversely, the ectopic expression of Cut drives ectopic bft transcription. Moreover, consensus Cux/Cut-binding sites have been identified upstream of the bft transcript: ATC GATTA is found 600 and 660 bp upstream of the transcript start site, and a CCAAT motif, recognized by Cut repeat II, is also found near one of these sites. This, together with the overexpression data, suggests that Cut may activate bft transcription directly. However, Cut is unlikely to be the only factor regulating bft transcription, since in cut null mutants, bft expression is not completely absent (Hardiman, 2002).

The data suggest that bft may be responsive to both organ identity (cut) and lineage (ttk) information. Other candidate genes active in the Drosophila PNS that may respond to both the lineage and selector gene pathways include BarHI and BarHII. These genes are also expressed specifically in es organs, as is bft, but in contrast to bft, they are present in the neurons and glia. The evidence presented here suggests bft is one of a group of genes that must be activated in es support cells to ensure their proper differentiation (Hardiman, 2002).

GENE STRUCTURE

A transcript of 7 kb is detected in Northern blots. Northern analysis and sequencing of PCR products suggests the existence of two transcripts. The longest, 7-kb transcript is detected using three different probes. It is most abundant in 6- to 8-hr embryos (stage 11), which corresponds to the time when the PNS precursors divide and show expression by in situ hybridization. The 7-kb transcript is also expressed at pupal stages, again correlating with the development of the adult sensory organs. RT-PCR products also suggest a potential transcript of 3.5 kb, which was, however, not reliably detected using Northern hybridization, suggesting that it may be less abundant than the 7-kb transcript. Analysis of bft's exon/intron structure reveals that the 3.5-kb transcript contains three exons, whereas the 7-kb transcript contains only two. 5' RACE experiments using two separate sets of primers indicate the 5' end of the transcript is located 37–41 bp upstream of the first exon's potential ORF and 39–43 bp distal to the P-element insertion. Using the neural network promoter prediction program, a putative transcription initiation site is predicted within 4 bp of the 5' end obtained with 5' RACE, providing additional support for having identified bft's transcript start site (Hardiman, 2002).

Bidirectional sequencing of the RT-PCR products corresponding to the 7-kb and the 3.5-kb transcripts has revealed four short ORFs. The first exon (common to both transcripts) contains a 465-bp ORF, but no start ATG. Two putative ORFs are found in the second exon of the 7-kb transcript that do not occur in the 3.5-kb transcript (242 and 305 bp), and the fourth (280 bp) is again common to both transcripts. All sequences and conceptual translations were blasted against the GenBank database but no homologies or motifs were found. Thus, bft either encodes a short, novel protein or perhaps may function as an RNA (Hardiman, 2002).

Part of bft's first intron matches the 5' and 3' ends of a 4.5-kb expressed sequence tag (EST; LP 06727) isolated by the Berkeley Drosophila Genome Project from a directionally cloned library. In this EST, the longest ORF (317 bp) also has no ATG, and no transcripts are detected on Northern blots. However, anti-sense riboprobes from this EST by whole mount in situ hybridization do show a bft-like expression, but in a punctate pattern that is typical of nascent transcripts. These results do not rule out the possibility that this EST may encode a short protein or a noncoding RNA as well. The genome project has not identified any protein-coding regions within the bft locus in the direction of bft transcription. In the opposite direction, the genome project predicts an ORF that falls within the LP06727 sequence, but corresponding riboprobes do not detect any expression in whole mount in situ hybridization. The next closest known protein-coding regions are ~7 kb (distal, odorant receptor 33C) and 10 kb (proximal, alpha-alpha trehalase) from the bft locus (Hardiman, 2002).

PROTEIN STRUCTURE

Structural Domains

bft either encodes a short, novel protein or perhaps may function as an RNA (Hardiman, 2002).

date revised: 10 August 2002

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