InteractiveFly: GeneBrief

bereft: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References


Gene name - bereft

Synonyms - mir-263a, nuclear untranslated RNA gene bft

Cytological map position - 33AB

Function - potential role in post-transcriptional gene silencing

Keywords - peripheral nervous system, interommatidial bristles, post-transcriptional gene silencing

Symbol - bft

FlyBase ID: FBgn0041606

Genetic map position -

Classification - potential non-coding RNA

Cellular location - cytoplasmic



NCBI link: Entrez Gene Precomputed BLAST
Recent literature
Aw, S. S., Lim, I. K. H., Tang, M. X. M. and Cohen, S. M. (2017). A glio-protective role of mir-263a by tuning sensitivity to Glutamate. Cell Rep 19(9): 1783-1793. PubMed ID: 28564598
Summary:
Glutamate is a ubiquitous neurotransmitter, mediating information flow between neurons. Defects in the regulation of glutamatergic transmission can result in glutamate toxicity, which is associated with neurodegeneration. Interestingly, glutamate receptors are expressed in glia, but little is known about their function, and the effects of their misregulation, in these non-neuronal cells. This study reports a glio-protective role for Drosophila mir-263a mediated by its regulation of glutamate receptor levels in glia. mir-263a mutants exhibit a pronounced movement defect due to aberrant overexpression of glutamate receptor CG5621/Grik, Nmdar1, and Nmdar2. mir-263a mutants exhibit excitotoxic death of a subset of astrocyte-like and ensheathing glia in the CNS. Glial-specific normalization of glutamate receptor levels restores cell numbers and suppresses the movement defect. Therefore, microRNA-mediated regulation of glutamate receptor levels protects glia from excitotoxicity, ensuring CNS health. Chronic low-level glutamate receptor overexpression due to mutations affecting microRNA (miRNA) regulation might contribute to glial dysfunction and CNS impairment.
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).

miR-71 and miR-263 jointly regulate target genes Chitin synthase and Chitinase to control locust molting

Chitin synthase (see Drosophila Chitin synthase)  and chitinase (see Drosophila chitinase) play crucial roles in chitin biosynthesis and degradation during insect molting (see Drosophila molting). Silencing of Dicer-1 (see Drosophila Dicer-1) results in reduced levels of mature miRNAs and severely blocks molting in the migratory locust. However, the regulatory mechanism of miRNAs in the molting process of locusts has remained elusive. This study found that in chitin metabolism, two crucial enzymes, chitin synthase (CHS) and chitinase (CHT) are regulated by miR-71 and miR-263 (see Drosophila bereft) during nymph molting. The coding sequence of CHS1 and the 3'-untranslated region of CHT10 contain functional binding sites for miR-71 and miR-263, respectively. miR-71/miR-263 display cellular co-localization with their target genes in epidermal cells and directly interact with CHS1 and CHT10 in the locust integument, respectively. Injections of miR-71 and miR-263 agomirs suppresses the expression of CHS1 and CHT10, which consequently alters chitin production of new and old cuticles and results in a molting-defective phenotype in locusts. Unexpectedly, reduced expression of miR-71 and miR-263 increases CHS1 and CHT10 mRNA expression and leads to molting defects similar to those induced by miRNA delivery. This study reveals a novel function and balancing modulation pattern of two miRNAs in chitin biosynthesis and degradation, and it provides insight into the underlying molecular mechanisms of the molting process in locusts (Yang, 2016). 

miR-263a regulates ENaC to maintain osmotic and intestinal stem cell homeostasis in Drosophila

Proper regulation of osmotic balance and response to tissue damage is crucial in maintaining intestinal stem cell (ISC) homeostasis. The Drosophila genome encodes an exceptionally large number of DEG/ENaC subunits termed Pickpocket (Ppk) 1-31. This study found that Drosophila miR-263a downregulates the expression of epithelial sodium channel (ENaC) subunits in enterocytes (ECs) to maintain osmotic and ISC homeostasis. In the absence of miR-263a, the intraluminal surface of the intestine displays dehydration-like phenotypes, Na+ levels are increased in ECs, stress pathways are activated in ECs, and ISCs overproliferate. Furthermore, miR-263a mutants have increased bacterial load and expression of antimicrobial peptides. Strikingly, these phenotypes are reminiscent of the pathophysiology of cystic fibrosis (CF) in which loss-of-function mutations in the chloride channel CF transmembrane conductance regulator can elevate the activity of ENaC, suggesting that Drosophila could be used as a model for CF. Evidence is provided that overexpression of miR-183, the human ortholog of miR-263a, can also directly target the expressions of all three subunits of human ENaC (Kim, 2016).

The Drosophila intestinal system is an attractive model for studying signaling events that control stem cell homeostasis given its anatomical and functional similarities to human epithelial systems, including the intestine. The adult midgut is continuously damaged during feeding as well as by chemicals and pathogens they encounter in the food, and thus needs to be constantly renewed. The renewal process requires tight regulation of the activities of multiple conserved signaling pathways in response to various types of intestinal epithelial injuries. These responses promote both intestinal stem cell (ISC) proliferation and enteroblast (EB) differentiation, expediting the rapid generation of new midgut epithelial cells to replace damaged (Kim, 2016).

MicroRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally regulate gene expression. In the past few years, miRNAs have been shown to play an important role in stem cell homeostasis by regulating differentiation and self-renewal. This study found that a well-conserved miRNA, miR-263a, is necessary for maintaining ISC homeostasis. Deletion of miR-263a in the adult midgut enterocytes (ECs) activates a stress response that, in turn, activates signaling pathways required for ISC proliferation, resulting in midgut hyperplasia. Well-conserved subunits of the epithelial sodium channel (ENaC) were found to be biologically important targets of miR-263a,and regulation of these subunits by miR-263a was found to be critical for maintaining proper osmotic homeostasis in the midgut epithelium. Remarkably, many of the phenotypes of miR-263a mutants are reminiscent of the pathophysiology of cystic fibrosis (CF), an autosomal recessive disorder caused by mutations in the gene encoding the chloride channel CF transmembrane conductance regulator (CFTR). In CF patients, loss-of-function mutations in the CFTR can elevate the activity of ENaC through a mechanism that is not fully understood. ENaC is present at the apical plasma membrane in many epithelial tissues throughout the body to regulate sodium reabsorption, and control total body salt and water homeostasis. The most common symptoms of CF are potential lethal blockages of distal small intestines, airway mucus obstruction, and chronic airway inflammation, which are consistent with the model that upregulation in ENaC activity increases sodium and water reabsorption, ultimately leading to dehydration of the intraluminal surface and reduction in mucus transport. Interestingly, this study provides evidence that overexpression of miR-183, the human ortholog of miR-263a, can also directly target all three subunits of human ENaC to regulate its activity. Altogether, these findings describe the role of a miRNA in regulating ENaC levels and suggest that the Drosophila intestine could be used as a model for CF (Kim, 2016).

In CF, two different models have been proposed regarding the role of hydration and salt concentration in normal airway defense. The hydration model proposes that increased absorption of fluid by the epithelium leads to dehydrated mucus and impaired mucociliary clearance that contributes to the establishment of an environment promoting colonization of the lungs by bacteria. In contrast, the salt model proposes that the salt content of airway fluid in CF is too high and thus prevents salt-sensitive defensin molecules in the airway surface liquid from killing bacteria, leading to increased susceptibility to lung infections. In Drosophila, the phenotypes associated with perturbation of ENaC are consistent with the hydration model, as misregulation of ENaC subunits in miR-263a mutants result in increased sodium reabsorption across the midgut epithelium. Furthermore, a dehydration-like phenotype of the PM, which is analogous to mucous secretions in the vertebrate digestive tract, was observed. Consistent with the PM providing protection against abrasive food particles and pathogens, miR-263a mutants appear more susceptible to bacterial infections as they succumb to P. aeruginosa infection more rapidly than the controls. In addition, increased bacterial load and antimicrobial peptide levels, and disruption of the intestinal pH were observed in miR-263a mutants. Interestingly, ECs in miR-263a mutants appear swollen, which is likely due to increased water reabsorption through osmosis. Finally, an activation was observed of stressed pathways characteristic of damaged ECs, which correlates with increased proliferation of ISCs (Kim, 2016).

Consistent with previous reports that cell swelling can activate the JNK pathway, JNK signaling is activated in miR-263a mutants that have large ECs. In addition, the JAK/STAT and EGFR pathways that regulate ISC proliferation are hyperactivated. Similarly, in CF airway and small intestine epithelia, cells in the airway epithelium and submucosal glands are more proliferative than cells in non-CF airways. In addition, in all CF mouse models in which CFTR has been deleted, goblet cell hyperplasia was observed in the small intestine (Kim, 2016).

Although the existence of Drosophila CFTR is yet to be determined, given its phenotypic similarities to the pathophysiology of CF, miR-263a mutants may provide a cost-effective and high-throughput animal model for identifying potential therapeutics that can specifically target ENaC in vivo, as the Drosophila gut is amenable to large-scale small-molecule screens. In addition, miR-183 might itself be a potential therapeutic agent for regulating ENaC activity in CF, based on the data that overexpression of miR-183 can directly target the expression of all three ENaC subunits in CFBE41o cells. Thus, possibly a combinational therapy for CF using the CFTR potentiator, Ivacaftor (also known as Kalydeco, which improves the transport of chloride through the mutated CFTR, together with overexpression of miR-183, could be imagined (Kim, 2016).


REGULATION

Transcriptional Regulation

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).


DEVELOPMENTAL BIOLOGY

Embryonic

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).


EFFECTS OF MUTATION

Effects of Mutation or Deletion

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).


REFERENCES

Search PubMed for articles about Drosophila bereft

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

Kim, K., Hung, R. J. and Perrimon, N. (2016). miR-263a regulates ENaC to maintain osmotic and intestinal stem cell homeostasis in Drosophila. Dev Cell 40(1):23-36. PubMed ID: 28017617

Yang, M., Wang, Y., Jiang, F., Song, T., Wang, H., Liu, Q., Zhang, J., Zhang, J. and Kang, L. (2016). miR-71 and miR-263 jointly regulate target genes Chitin synthase and Chitinase to control locust molting. PLoS Genet 12: e1006257. PubMed ID: 27532544


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date revised: 30 April 2017

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