InteractiveFly: GeneBrief

headcase : Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

Gene name - headcase

Synonyms -

Cytological map position - 99F1--99F11

Function - biochemical function unknown

Keywords - trachea, neuroblasts, imaginal discs, head

Symbol - hdc

FlyBase ID:FBgn0010113

Genetic map position - 3-[101]

Classification - novel basic protein

Cellular location - cytoplasmic

NCBI links: Precomputed BLAST | Entrez Gene
Recent literature
Resende, L.P., Truong, M.E., Gomez, A. and Jones, D.L. (2017). Intestinal stem cell ablation reveals differential requirements for survival in response to chemical challenge. Dev Biol [Epub ahead of print]. PubMed ID: 28104389
The Drosophila intestine is maintained by multipotent intestinal stem cells (ISCs). Although increased intestinal stem cell (ISC) proliferation has been correlated with a decrease in longevity, there is some discrepancy regarding whether a decrease or block in proliferation also has negative consequences. This study identifies headcase (hdc) as a novel marker of ISCs and enteroblasts (EBs) and demonstrates that Hdc function is required to prevent ISC/EB loss through apoptosis. Hdc depletion was used as a strategy to ablate ISCs and EBs in order to test the ability of flies to survive without ISC function. While flies lacking ISCs show no major decrease in survival under unchallenged conditions, flies depleted of ISCs and EBs exhibit decreased survival rates in response to damage to mature enterocytes (EC) that line the intestinal lumen. These findings indicate that constant renewal of the intestinal epithelium is not absolutely necessary under normal laboratory conditions, but it is important in the context of widespread chemical-induced damage when significant repair is necessary.

headcase (hdc) is the first gene to be described that is specifically expressed in all imaginal cells; this has allowed the identification of many imaginal primordia in the embryo and the opportunity to follow their morphogenesis throughout embryonic and larval development. headcase was initially characterized as a random P element insertion into a gene that turned out to be expressed in all imaginal tissues. Hdc protein is an extremely basic cytoplasmic protein with no obvious sequence similarities or conserved motifs. Interestingly, the spatial-temporal pattern of hdc expression prefigures imaginal cell re-entry into the mitotic cell cycle and persists until the final cell divisions (Weaver, 1995). Headcase has a second identity with a developmental role that has very little overlap with its potential role in regulating re-entry into the mitotic cycle. Headcase suppresses tracheal cell branching in a cell non-autonomous fashion (Steneberg,1998). These two activities -- expression in imaginal discs and suppression of tracheal branching -- are reviewed below in detail, but no attempt will be made here to find a uniform explanation for the biological effects of Headcase.

Although hdc is expressed in all imaginal lineages, it initiates in the different imaginal cell groups according to a developmental sequence. It is clear from the embryonic expression of hdc in imaginal disc primordia that hdc activation in these cells occurs at least 24 hours before their re-entry into the mitotic cell cycle, since mitosis in these cells is not observed until late first/early second instar. Similarly, for the adepithelial cells, headcase expression precedes entry into a mitotic cycle. Adepithelial cells, of mesodermal origin, constitute the primordia of adult somatic musculature and are found closely associated with the imaginal discs to which they ultimately attach; this is an association that can be traced back into embryonic stages using twist as a marker for adepithelial cells and hdc as a marker for the disc primordia. In the embyonic stage of adepithelial development, approximately half of the adepithelial cells express hdc; a complete overlap of twist- and hdc-expressing cells is apparent only at larval stages. Since the muscle precursors are mitotically quiescent until second larval instar (as judged by susceptibility to a DNA synthesis inhibitor, it can be concluded that hdc expression prefigures the activation of imaginal somatic mesoderm cell proliferation. Similarly, for the imaginal histoblast lineage, hdc-positive histoblast cells are not detected in the embryo, or in 1st, 2nd or early 3rd instar larvae, even though histoblast cells are present and can be visualized by several histoblast-specific markers. Instead, hdc expression is first detected in wandering third instar, corresponding to 12-24 hours before the various histoblast nests begin division during pupal stages. The most prominent hdc defects involve head development and can result in complete deletion of the head capsule, or duplication of head cuticle or antennae (Weaver, 1995).

A correlation also exists between hdc repression and the end of imaginal proliferation and the onset of differentiation. This is best illustrated by imaginal neuroblast proliferation, which has been studied extensively with bromodeoxyuridine incorporation. Double-labelling experiments using Grainyhead protein as an imaginal neuroblast marker (Bray, 1991) demonstrate that during the intense proliferative stages of the second and early third instars, hdc is expressed in imaginal neuroblasts, as well as their progeny. This relationship changes when the larval CNS prepares for pupariation in late third instar. At this point, the levels of hdc expression are severely reduced in the imaginal neuroblast, but not their progeny, presumably because these cells are competent to continue cell proliferation. The timing of this change precedes final neuroblast division, which, for these neuroblasts located in the thoracic region of the ventral nerve cord, occurs sometime within the first 12 hours following pupariation. This observation is broadly supported by the pupal expression of hdc during disc morphogenesis. Although expression is uniformly strong throughout the proliferative stages, this fades during the first 24 hours of pupal development; after the initial 24 hours, cell division has ended. It has been concluded that the activation of hdc expression correlates with the onset of imaginal proliferation and hdc inactivation correlates with its cessation (Weaver, 1995). Although hdc expression correlates with imaginal tissue proliferation, a specific defect in cell proliferation in hdc mutants has not been documented.

Tracheal branching and fusion involves a complex succession of cellular events accompanied by expression of different classes of genes involved in sprouting of new branches, terminal branching and branch fusion. Here attempts will be made to describe the course of events in this process and consider the role of Headcase. Mutations in a gene expressed in trachea, Fus-6 (from a series of markers termed fusion markers expressed during tracheal fusion), increases the number of unicellular sprouts emanating from dorsal primary branches. Cloning of Fus-6 shows it to be identical to headcase. Understanding the nature of the headcase branching phenotype required the examination of tracheal genes known to be involved in branching. In flies, the number of cells in each branch that undergo terminal sprouting is regulated by the breathless (btl) gene encoding an FGF receptor homolog and branchless (bnl), a fly member of the FGF growth factor family. The sprouting process is associated with the activation of a set of marker genes (pantip markers) in the leading cells of each primary branch in response to FGF signaling (Samakovlis,1996a and Sutherland, 1996). The expression pattern of these markers is dynamic; pantip markers gradually become restricted to the cells that form unicellular branches. Depending on the primary branch from which these unicellular sprouts originate, they either retain the expression of pantip markers and further differentiate into terminal sprouts, ramifying in response to respiratory demand or they migrate and fuse to similar tubular extensions from adjacent tracheal metameres to interconnect the network. The processes of terminal branching and branch fusion, controlled as they are by different genes, are accompanied by the expression of separate classes of marker genes (Samakovlis, 1996a). Mutants in blistered, which codes for the Drosophila Serum response factor, abolish terminal branching (Guillemin, 1996). Blistered is termed Terminal-1 marker and is a terminal marker. The Fusion-1 (Fus-1) gene, one of a series of so-called fusion markers encodes the Escargot transcription factor, specifically affects branch fusion (Samakovlis,1996b and Tanaka-Matakatsu,1996). Esg is an activator of the fusion program as well as a repressor of terminal branching that can drive ectopic tracheal fusion events and repress terminal branching when misexpressed in all tracheal cells (Samakovlis, 1996b). Both terminal and fusion genes are under the control of the pantip gene pointed (pnt), which encodes an Ets domain transcription factor. pnt is required for the transcriptional activation of blistered in the terminal cells and the repression of esg in the cells of the pantip group that do not acquire the fusion cell fate (Samakovlis, 1996a).

What is the sequence of gene expression at the tip of sprouting branches? At embryonic stage 12, 3-4 cells at the tip of the dorsal branch express the group of markers termed pantip markers. In the dorsal and lateral trunk branches of each tracheal metameric unit in which hdc is expressed, the expression of this marker gene becomes restricted in two steps. In the first step, at stage 14, strong pantip marker expression is maintained in the two cells of the initial group that is selected to sprout; in the second step, at stage 15, marker expression ultimately becomes refined to one of the sprouting cells that will generate terminal branches. The first step in the restriction of the domain of pantip marker expression is preceded by the activation of separate classes of marker genes in a subset of the cells expressing pantip markers at stage 13. These cells either express terminal markers and undergo terminal branching or fusion markers and connect to similar fusion sprouts deriving from neighboring metameres. The remaining cells of the group, those expressing neither fusion nor terminal markers, stop migrating, lose the expression of pantip markers, and acquire their position at the stalk of the dorsal branch. The extra branching cells in the Fus-6 (headcase) mutant express the two pantip and the two terminal marker genes tested (including blistered). During larval life, these cells go on to form extensive networks of fine tracheoles. Thus, the extra branching cells arise from the group of cells expressing both pantip markers and display the molecular and morphological features of terminal cells. These additional branching cells can first be detected in the mutant embryos at stage 14 with the appearance of an extra cell expressing pantip and terminal markers. The initiation of expression of the earliest terminal marker, both in wild-type and mutant embryos, occurs in a single cell of the dorsal branch at stage 13, which argues that the initial selection of a terminal cell from the pantip group is not affected in the mutants (Steneberg,1998).

What then is the identity of the extra sprouting cells observed in mutant trachea and when do they arise? The additional terminal sprouts in the mutant could arise by a change in the cell fate specification program of the fusion cell. This is not the case, because the expression of the three fusion markers tested is unchanged in the Fus-6 mutant. Furthermore, the formation of the dorsal anastomosis is not affected in the mutants. Two opposing fusion cells are able to migrate toward one another and are connected by intercellular junctions. Asymmetric cell division of the fusion cell to generate a terminal and a fusion sprout in the mutant is also excluded as a possible mechanism for the generation of new terminal branches because no cell divisions were detected in the trachea of mutant embryos. What then is the cellular mechanism behind the increased branching in the mutant? The dorsal primary branch is consistently decreased by one cell in all of the mutant metameres examined, as compared with wild-type metameres, which do not exhibit the extra sprouting phenotype. This suggests that the extra branching cells arise by a cell fate transformation of a nonbranching cell into a sprouting cell, and that the Fus-6 gene product (Headcase) acts nonautonomously to prevent neighboring tracheal cells from branching (Steneberg, 1998).

Since headcase mutants possess extra branches, it is reasonable to expect that ectopic headcase expression would suppress branching. This is indeed true, and ectopic headcase expression also suppresses the expression of the terminal cell marker blistered. An unusual feature of the HDC mRNA is that it contains an internal translational termination codon. headcase thus encodes two overlapping protein products, the longer of the two resulting from an unusual suppression of translational termination mechanism. Translational readthrough is necessary for hdc function because rescue of the tracheal mutant phenotype requires the full-length HDC mRNA. In ectopic expression experiments with full-length and truncated hdc gene constructs, only the full-length cDNA encoding both proteins can inhibit terminal branching. Generalized misexpression of the truncated form of hdc, a form that is normally expressed in developing flies, is found to have a marginal effect on terminal branching; this argues for the requirement of the longer ORF for full hdc function. No biological role for the shorter Hdc protein has been established, but because it represents the majority of Hdc protein in cells, a role for the short form of Hdc in fly development might in fact exist (Steneberg,1998).


Transcriptional Regulation

headcase expression in the trachea is regulated by Escargot. Branch fusion in the Drosophila trachea is a complex process involving two specialized cells at the tip of each fusing branch; they undergo a series of morphological changes to generate a bicellular anastomosis and connect the two tracheal branches. In esg mutants, the fusion cells of the dorsal branches fail to undergo the fusion process and express later fusion markers; instead, they express terminal markers and ramify into tracheoles during larval life. Ectopic expression of esg in all tracheal cells is sufficient to induce ectopic branch fusions and suppress terminal branching and expression of terminal genes. hdc expression is absent in the fusion cells of esg mutants. esg was misexpressed in all tracheal cells using the UAS-GAL4 system. This was found to be sufficient to induce hdc expression in one to two additional tracheal cells at the tips of the dorsal and lateral trunk branches. Thus, esg is not only necessary for hdc expression in the fusion cells, but it is also sufficient to induce hdc expression in the cells of the pantip group of the dorsal and lateral branches. Because hdc acts nonautonomously as a branching inhibitor, the above results predict that in esg mutants not only the fusion cell, but also one of the stalk cells may acquire the terminal cell fate. The extra terminal branching phenotype of esg strong loss-of-function mutants was analyzed. An additional cell extends a unicellular branch and expresses the terminal marker blistered (coding for the Drosophila Serum response factor) in ~16% of the 176 dorsal branches analyzed. These results show that esg can suppress terminal cell fate in the fusion cells by repressing terminal genes like blistered, and in neighboring cells by the activation of hdc and perhaps other fusion genes that act non-cell autonomously as branching inhibitors (Steneberg, 1998).

buttonhead and Sp1 are activators of the headcase and Distal-less in ventral imaginal discs

The related genes buttonhead (btd) and Drosophila Sp1 (the Drosophila homolog of the human SP1 gene) encode zinc-finger transcription factors known to play a developmental role in the formation of the Drosophila head segments and the mechanosensory larval organs. A novel function of btd and Sp1 is reported: they induce the formation and are required for the growth of the ventral imaginal discs. They act as activators of the headcase (hdc) and Distal-less (Dll) genes, which allocate the cells of the disc primordia. The requirement for btd and Sp1 persists during the development of ventral discs: inactivation by RNA interference results in a strong reduction of the size of legs and antennae. Ectopic expression of btd in the dorsal imaginal discs (eyes, wings and halteres) results in the formation of the corresponding ventral structures (antennae and legs). However, these structures are not patterned by the morphogenetic signals present in the dorsal discs; the cells expressing btd generate their own signalling system, including the establishment of a sharp boundary of engrailed expression, and the local activation of the wingless and decapentaplegic genes. Thus, the Btd product has the capacity to induce the activity of the entire genetic network necessary for ventral imaginal discs development. It is proposed that this property is a reflection of the initial function of the btd/Sp1 genes that consists of establishing the fate of the ventral disc primordia and determining their pattern and growth (Estella, 2003).

In a search for genes with restricted expression in the adult cuticle, the MD808 Gal4 line was found to direct expression in the ventral derivatives of the adult body; proboscis, antennae, legs and genitalia. In the abdomen and analia no clear expression was discerned. It was also noticed that the insertion was located in the first chromosome and associated with a lethal mutation. The mutant larvae showed a head phenotype resembling that described for mutants at the btd gene: loss of antennal organ and the ventral arms of the cephalopharyngeal skeleton, and complementation analysis indicated that the chromosome carrying the insert contained a mutation at btd. The expression pattern found in MD808/UAS-lacZ embryos was also similar to that reported for btd, suggesting that the Gal4 insertion was located at this gene. In addition, the imaginal expression of MD808 and of btd was largely coincident (Estella, 2003).

Further to the genetic analysis and the expression data, DNA fragments at the insertion site were cloned to map the position of the P-element on the genome. It is located 753 bp 5' of the btd gene. The related gene Sp1 is immediately adjacent. It is likely that btd and Sp1 have originated by a tandem duplication of a primordial btd-like gene (Estella, 2003).

In early embryos btd is expressed in the head region, but by the extended germ band stage the expression domain has expanded to the ventral region of cephalic, thoracic and abdominal segments. During germ band retraction most of the abdominal and thoracic expression is lost, except in derivatives of the peripheral nervous system and the primordia of the imaginal discs. Sp1 is not expressed in early embryos, but from stage 11 onwards it shows the same pattern as btd (Estella, 2003).

Special attention was paid to the btd/Sp1 expression domain in the thoracic imaginal discs primordia, as it may suggest a novel function related to imaginal development. Double labelling with Dll and btd probes indicates that btd precedes Dll expression, but by stage 12 the two genes are co-expressed in a group of thoracic cells. However, the Dll domain is smaller and is included within the btd/Sp1 domain: there are cells expressing btd that do not show Dll activity, although all the cells expressing Dll express btd (Estella, 2003).

The ventral disc primordia include not only cells expressing Dll but also other cells containing expression of escargot (esg) and hdc, markers of the diploid cells that form the imaginal primordia. In late embryonic stages, esg is expressed in a ring domain surrounding the Dll-expressing cells and hdc is expressed in a similar pattern. Double label experiments were carried out with btd, hdc and esg probes; the expression of the two latter genes overlaps with that of btd (and with Sp1) in the thoracic disc primordia (Estella, 2003).

The overlap of the btd and of esg domains indicates that btd is also expressed in the hth domain, which is coincident with that of esg. As the hth/esg domain marks the precursor cells of the proximal region of the adult leg the embryonic expression data indicate that btd and Sp1 are active in the entire primordia of the ventral adult structures, including the distal and the proximal parts (Estella, 2003).

In the mature antennal disc, btd expression is restricted mostly to the region corresponding to the second antennal segment, where it co-localizes with both Dll and hth. In the leg disc btd also overlaps in part with Dll and with hth. The latter result is significant, for the expression of Dll and hth define two major genetic domains, which are kept apart by antagonistic interactions. The fact that btd is expressed in the two domains suggests that its regulation and function is independent from the interactions between the two domains. This observation is consistent with the results obtained in embryos and suggests that the btd domain includes the precursors of the whole ventral thoracic region from the beginning of development (Estella, 2003).

This work demonstrates a novel and also redundant function of btd and Sp1: they are responsible for the formation of the ventral imaginal discs by activating the genetic network necessary for their development. Furthermore, Btd protein retains the capacity of inducing the entire ventral genetic network during the larval period. It is proposed that the activation of btd/Sp1 is the crucial event in the determination of the ventral structures of the adult fly (Estella, 2003).

This argument is based on the finding that btd and Sp1 appear to mediate all events connected with the formation of the ventral discs. The discussion deals with the leg disc, but there is evidence that antennal primordium also requires btd. Moreover, the genital primordium is lacking in Df(1)C52 embryos, suggesting that this disc is also under the same control. Most of the experiments concern the function of btd but given the expression and functional similarities between the two genes, it is assumed that Sp1 fulfils the same or a very similar role. Therefore, btd/Sp1 will be considered to carry out a single function (Estella, 2003).

One crucial feature is that btd is an upstream activator of Dll and hdc, which are considered developmental markers of disc primordia: (1) btd expression precedes that of Dll and of hdc; (2) the btd expression domain includes those of Dll and hdc; (3) in btd mutants, Dll and hdc activity is much reduced, and completely absent in Df(1)C52 embryos; (4) ectopic btd function induces ectopic activation of Dll and hdc (Estella, 2003).

The role of btd in embryogenesis can be illustrated in the light of the models of Dll regulation. Dll is activated by wg and its expression modulated by the EGF spitz and by dpp, whereas it is repressed in the abdominal segments by the BX-C genes. The current experiments suggest that Dll regulation is mediated by btd: in wg mutants there is no btd expression and hence neither Dll nor hdc activity. In dpp mutant embryos, btd expands to the dorsal region resembling the effect on Dll. In Ubx- embryos there is an additional group of cells in the first abdominal segment expressing btd; the same cells that also express Dll in those embryos. The interpretation of the role of btd mediating Dll regulation by Ubx is complicated by previous observations showing direct repression of Dll by the Ubx protein. It is possible that Ubx regulates Dll both directly and by controlling btd activity (Estella, 2003).

It is proposed that the localization of btd/Sp1 activity to a group of ventral cells is a major event in the specification of adult structures. btd and Sp1 are activated in response to spatial cues from Wg, Dpp, EGF and BX-C, and in turn their function induces the activity of the genes necessary for ventral imaginal development (Estella, 2003).

This hypothesis is strongly supported by the results obtained inducing ectopic btd activity in the dorsal discs; just the presence of the Btd product alone is sufficient to bring about ventral disc development. In the wing and the haltere discs, Btd induces a transformation into leg, whereas in the eye it induces antennal development. This indicates that it specifies ventral disc identity jointly with other factors, e.g., the Hox genes, possibly through the activation of subsidiary genes such as Dll, known to contribute to ventral appendage identity in combination with Hox genes (Estella, 2003).

The requirement for btd and Sp1 activity appears to be restricted only to the ventral discs, even during the early phases of the thoracic disc primordia. In this context it is worth considering the observation that in Df(1)C52 embryos there is esg expression in the wing and haltere disc primordia, even though it is absent in the leg discs. Thus, the wing and haltere discs are formed in the absence of btd and Sp1. Because in these embryos there is an almost complete lack of Dll expression, this observation raises the question of the origin of the dorsal thoracic discs, which are currently considered to derive from the original ventral primordium, formed by cells expressing Dll. Although some of the original group of ventral cells may contribute to the dorsal disc primordia, the data suggest that there may be cells recruited to form the dorsal discs that do not originate in the initial ventral primordium. Accordingly, it is worth considering that in the absence of Dll activity the leg and wing discs are formed, although the leg only differentiates proximal disc derivatives. Thus, the activity of Dll cannot be considered a reliable marker for imaginal discs (Estella, 2003).

Tramtrack is genetically upstream of genes controlling tracheal tube size in Drosophila

The Drosophila transcription factor Tramtrack (Ttk) is involved in a wide range of developmental decisions, ranging from early embryonic patterning to differentiation processes in organogenesis. Given the wide spectrum of functions and pleiotropic effects that hinder a comprehensive characterisation, many of the tissue specific functions of this transcription factor are only poorly understood. Multiple roles of Ttk have been discovered in the development of the tracheal system on the morphogenetic level. This study sought to identify some of the underlying genetic components that are responsible for the tracheal phenotypes of Ttk mutants. Gene expression changes were profiled after Ttk loss- and gain-of-function in whole embryos and cell populations enriched for tracheal cells. The analysis of the transcriptomes revealed widespread changes in gene expression. Interestingly, one of the most prominent gene classes that showed significant opposing responses to loss- and gain-of-function was annotated with functions in chitin metabolism, along with additional genes that are linked to cellular responses, which are impaired in ttk mutants. The expression changes of these genes were validated by quantitative real-time PCR and further functional analysis of these candidate genes and other genes also expected to control tracheal tube size revealed at least a partial explanation of Ttk's role in tube size regulation. The computational analysis of tissue-specific gene expression data highlighted the sensitivity of the approach and revealed an interesting set of novel putatively tracheal genes (Rotstein, 2011).

The microarray results confirm previous observations and provide new data for the different Ttk tracheal requirements. For instance, the transcription factor Esg, which plays a pivotal role in fusion cell identity specification is lost when Ttk is over-expressed, but still present in Ttk loss-of-function conditions. The microarray data confirm this regulation, and in addition identifies other genes already shown to directly or indirectly modulate fusion fate as Ttk targets, like hdc, CG15252, or pnt. Similarly, polychaetoid (pyd), which has been identified as a Ttk target in in situ hybridisation analysis, is differentially expressed in the microarray conditions (it should be noted however that pyd is not formally a candidate due to inconsistencies between microarray replicates; in fact only splice variant pyd-RE shows a response), explaining in part the requirement of Ttk in tracheal cell intercalation. In addition, it is tempting to speculate about other candidate targets to mediate this function of Ttk in intercalation, like canoe for instance, which has been recently shown to act with pyd during embryogenesis (Rotstein, 2011).

The microarray analysis pointed to a regulation of the Notch signalling pathway or its activity by Ttk, likely acting as a negative regulator. In contrast, it has previously been observed that Ttk acts as a downstream effector of N activity in the specification of different tracheal identitites. Indeed, it was shown that Ttk levels depend on N activity in such a way that when N is active, Ttk levels are high, whereas when N is not active, Ttk levels are low. Thus, lower levels of Ttk were observed in tracheal fusion cells due to the inactivity of N there. Therefore, Ttk acts as a target of N in fusion cell determination. Now, the results of the microarray add an extra level of complexity to the Ttk-N interaction. The observation that in turn Ttk also transcriptionally regulates several N pathway components suggests that Ttk is involved in a feedback mechanism that could play a pivotal role in biasing or amplifying N signalling outcome (Rotstein, 2011).

Interactions between Ttk and N have been observed in different developmental contexts, emphasising the importance of such regulations. Several examples illustrate the regulation, either positive or negative, of Ttk expression by N activity. In addition, a recent report provides evidence of a regulation of N activity by Ttk and proposes a mutually repressive relationship between N and Ttk which would also involve Ecdysone signalling. The results are consistent with many of these observations, indicating that they could represent general molecular mechanisms of morphogenesis. Thus, tracheal cell specification could serve as an ideal scenario to investigate the intricate, and often contradictory, interactions between N and Ttk and the complexity of N signaling (Rotstein, 2011).

Protein Interactions

Unkempt is negatively regulated by mTOR and uncouples neuronal differentiation from growth control

Neuronal differentiation is exquisitely controlled both spatially and temporally during nervous system development. Defects in the spatiotemporal control of neurogenesis cause incorrect formation of neural networks and lead to neurological disorders such as epilepsy and autism. The mTOR kinase integrates signals from mitogens, nutrients and energy levels to regulate growth, autophagy and metabolism. The insulin receptor (InR)/mTOR pathway has been identified as a critical regulator of the timing of neuronal differentiation in the Drosophila melanogaster eye. This pathway has also been shown to play a conserved role in regulating neurogenesis in vertebrates. However, the factors that mediate the neurogenic role of this pathway are completely unknown. To identify downstream effectors of the InR/mTOR pathway transcriptional targets of mTOR were screened for neuronal differentiation phenotypes in photoreceptor neurons. The conserved gene unkempt (unk), which encodes a zinc finger/RING domain containing protein, as a negative regulator of the timing of photoreceptor differentiation. Loss of unk phenocopies InR/mTOR pathway activation and unk acts downstream of this pathway to regulate neurogenesis. In contrast to InR/mTOR signalling, unk does not regulate growth. unk therefore uncouples the role of the InR/mTOR pathway in neurogenesis from its role in growth control. The gene headcase (hdc) was identified a second downstream regulator of the InR/mTOR pathway controlling the timing of neurogenesis. Unk forms a complex with Hdc, and Hdc expression is regulated by unk and InR/mTOR signalling. Co-overexpression of unk and hdc completely suppresses the precocious neuronal differentiation phenotype caused by loss of Tsc1. Thus, Unk and Hdc are the first neurogenic components of the InR/mTOR pathway to be identified. Finally, Unkempt-like is expressed in the developing mouse retina and in neural stem/progenitor cells, suggesting that the role of Unk in neurogenesis may be conserved in mammals (Avet-Rochex, 2014).

Neural progenitors in the developing human brain generate up to 250,000 neurons per minute. After differentiating from these neural progenitors, neurons migrate and are then integrated into neural circuits. Temporal control of neurogenesis is therefore critical to produce a complete and fully functional nervous system. Loss of the precise temporal control of neuronal cell fate can lead to defects in cognitive development and to neurodevelopmental disorders such as epilepsy and autism (Avet-Rochex, 2014).

Mechanistic target of rapamycin (mTOR) signalling has recently emerged as a key regulator of neurogenesis. mTOR is a large serine/threonine kinase that forms two complexes, known as mTORC1 and mTORC2. mTORC1 is rapamycin sensitive and is regulated upstream by mitogen signalling, such as the insulin receptor (InR)/insulin like growth factor (IGF) pathway, amino acids, hypoxia, cellular stress and energy levels. mTORC1 positively regulates a large number of cellular processes including growth, autophagy, mitochondrial biogenesis and lipid biosynthesis and activation of mTOR has been linked to cancer. Hyperactivation of mTOR signalling in neurological disease is best understood in the dominant genetic disorder tuberous sclerosis complex (TSC), which causes epilepsy and autism. mTOR signalling has also been shown to be activated in animal models of epilepsy and in human cortical dysplasia (Avet-Rochex, 2014).

The control of neurogenesis by the InR/mTOR pathway was first discovered in the developing Drosophila melanogaster retina, where activation of the pathway caused precocious differentiation of photoreceptor neurons and inhibition caused delayed differentiation. Subsequent in vitro studies demonstrated that insulin induces neurogenesis of neonatal telencephalonic neural precursor cells in an mTOR dependent manner and that Pten negatively regulates neuronal differentiation of embryonic olfactory bulb precursor cells. More recently, in vivo studies have shown that inhibition of mTOR suppresses neuronal differentiation in the developing neural tube. Furthermore, knock-down of the mTOR pathway negative regulator RTP801/REDD1 causes precocious differentiation of neural progenitors in the mouse embryonic subventricular zone (SVZ), while overexpression of RTP801/REDD1 delays neuronal differentiation. Loss of Pten, Tsc1, or overexpression of an activated form of Rheb, also cause premature differentiation of neurons in the SVZ. These studies have demonstrated that InR/mTOR signalling plays a conserved role in regulating neurogenesis in several different neural tissues. However, the downstream effectors of InR/mTOR signalling in neurogenesis are completely unknown (Avet-Rochex, 2014 and references therein).

To identify neurogenic downstream regulators of InR/mTOR signalling, genes were screened that were previously shown to be transcriptionally regulated by mTOR in tissue culture cells, for in vivo neurogenic phenotypes in the developing Drosophila retina. From this screen the zinc finger/RING domain protein Unkempt (Unk) was identified as a negative regulator of photoreceptor differentiation. Loss of unk phenocopies the differentiation phenotype of InR/mTOR pathway activation and Unk expression is negatively regulated by InR/mTOR signalling. Importantly, unk does not regulate cell proliferation or cell size and so uncouples the function of InR/mTOR signalling in growth from its role in neurogenesis. The evolutionarily conserved basic protein Headcase (Hdc) was identified as a physical interactor of Unk, and it was shown that loss of hdc causes precocious differentiation of photoreceptors. Hdc expression is regulated by the InR/mTOR pathway and by unk, demonstrating that Hdc and Unk work together downstream of InR/mTOR signalling in neurogenesis. Unk also regulates the expression of and interacts with D-Pax2 (Shaven/Sparkling), suggesting a model for the regulation of neurogenesis by the InR/mTOR pathway. It was also shown that one of the mammalian homologs of Unk, Unkempt-like, is expressed in the developing mouse retina and in the early postnatal brain. This study has thus identified the Unk/Hdc complex as the first component of the InR/mTOR pathway that regulates the timing of neuronal differentiation (Avet-Rochex, 2014).

Several lines of evidence together demonstrate that unk and hdc act downstream of InR/mTOR signalling to negatively regulate the timing of photoreceptor cell fate. First, loss of either unk or hdc causes precocious differentiation of the same cells and to the same degree as activation of InR/mTOR signalling. Second, the expression of both Unk and Hdc are regulated by InR/mTOR signalling. Third, loss of unk suppresses the strong delay in photoreceptor differentiation caused by inhibition of the InR/mTOR pathway and combined overexpression of unk and hdc suppresses the precocious photoreceptor differentiation caused by loss of Tsc1. Fourth, although Unk has been shown to physically interact with mTOR, neither unk nor hdc regulate cell or tissue growth. Taken together these data show that unk and hdc are novel downstream components of the InR/mTOR pathway that regulate the timing of neuronal differentiation (Avet-Rochex, 2014).

InR/mTOR signalling is a major regulator of cell growth. In Drosophila activation of InR/mTOR signalling by loss of either Tsc1, Tsc2, Pten, or overexpression of Rheb causes increased cell size and proliferation. In the genetic disease TSC, which is caused by mutations in Tsc1 or Tsc2, patients develop benign tumours in multiple organs including the brain. The previously identified components of the InR/mTOR pathway regulate both growth and neurogenesis in Drosophila and vertebrate model. unk and hdc therefore represent a branchpoint in the pathway where its function in neurogenesis bifurcates from that in growth control. Moreover, analysis of unk and hdc demonstrates that regulation of cell growth can be uncoupled from and is not required for the function of InR/mTOR signalling in the temporal control of neuronal differentiation (Avet-Rochex, 2014).

At the protein level this study shows that Unk and Hdc physically interact in S2 cells. Although this interaction remains to be demonstrated in vivo, the additional observations that they both regulate each other's expression and act synergistically in vivo strongly support the model that they physically interact (see A model for the regulation of the timing of neuronal differentiation by the Unk/Hdc complex acting downstream of InR/mTOR signalling). Moreover, Unk and Hdc have also previously been shown to physically interact by yeast-2-hybrid and co-immunoprecipitation. Unk and Hdc are both expressed in all developing photoreceptors and so it is hypothesised that they control the timing of differentiation through the regulation of neurogenic factors whose expression is restricted to R1/6/7 and cone cells. Loss of unk causes increased expression of D-Pax2, the main regulator of cone cell differentiation. hdc and Tsc1 mutant clones also cause a similar increase in D-Pax2 expression. Overexpression of D-Pax2 alone is insufficient to induce cone cell differentiation, which requires overexpression of both D-Pax2 and Tramtrack88 (TTK88). Thus, regulation of D-Pax2 expression by mTOR signalling may contribute to the rate of cone cell differentiation, while overall control would require the regulation of additional factors such as TTK88. Pax8, part of the Pax2/Pax5/Pax8 paired domain transcription factor subgroup that is homologous to D-Pax2, has been shown to physically interact with one of the two human homologs of Unkempt. This study found that Drosophila Unk physically interacts with D-Pax2, demonstrating that the physical interaction between Unk and this group of transcription factors is conserved. It is suggested that D-Pax2 may be one of several neurogenic factors regulated by InR/mTOR signalling, through a physical interaction with the Unk/Hdc complex, to control the timing of R1/6/7 and cone cell fate (Avet-Rochex, 2014).

Unk has been shown to physically interact with mTOR and the strength of this interaction is regulated by insulin. This suggests the intriguing possibility that the inhibition of Unk activity by InR/mTOR signalling is dependent on the strength of the physical interaction between Unk and the mTORC1 complex. Unk was also identified as part of the mTOR-regulated phosphoproteome in both human and murine cells. Thus, Unk may potentially be regulated by mTOR through phosphorylation. Future studies will fully characterise the mechanism by which mTORC1 regulates Unk activity (Avet-Rochex, 2014).

This study represents the first demonstration of a role for unk in specific developmental processes. By contrast, hdc has previously been shown to regulate dendritic pruning during metamorphosis and to act as a branching inhibitor during tracheal developmen. A screen for genes affecting tracheal tube morphogenesis and branching recently identified Tsc1, suggesting that InR/mTOR also regulates tracheal development. Thus, hdc and unk may act repeatedly as downstream effectors of the InR/mTOR pathway during Drosophila development (Avet-Rochex, 2014).

The one previous study of either of the mammalian Unk homologs showed that Unkl binds specifically to an activated form of the Rac1 GTPase. If this function is conserved in Drosophila then the defects in photoreceptor apical membrane morphogenesis caused by activation of mTOR signalling or loss of unk/hdc may be mediated through Rac1 (Avet-Rochex, 2014).

The function of the two unk homologs, unk and unkl, in mammalian development is not known, but unk has been shown to be expressed in the mouse early postnatal mouse retina. This study found that Unkl is also expressed in the developing mouse retina, suggesting that Unk may play a conserved role in eye development in both flies and mammals. InR/mTOR signalling acts as a pro-survival pathway preventing retinal degeneration, but its role in mammalian eye development has not been characterised. By contrast InR/mTOR signalling has a well characterised role in NSC self-renewal and differentiation in the mouse SVZ. Loss of Tsc1 or expression of a constitutively active form of Rheb in neural progenitor cells in the postnatal mouse SVZ causes the formation of heterotopias, ectopic neurons and olfactory micronodules. Furthermore, individuals with TSC, which results in activated mTOR signalling, have aberrant cortical neurogenesis and develop benign cortical tumours during foetal development and throughout childhood. mTOR signalling has been shown to be active in proliferative NSCs and TAPs in the neonatal SVZ and inhibition of mTOR signalling prevents NSC differentiation. This study found that Unkl is expressed in both NSCs and TAPs in the early postnatal SVZ. Thus, Unkl may regulate NSC differentiation downstream of mTOR signalling in the mammalian brain. Unkempt may therefore play a conserved role in regulating the timing of neural cell fate downstream of mTOR signalling in both flies and mammals (Avet-Rochex, 2014).



The rudiments of the strong B5-lacZ staining in the thoracic segments of the CNS, directed by the headcase promoter, are discernible by stage 13 of embryogenesis, and mature as the ventral nerve cord condenses. This pattern prefigures the larval pattern of postembryonic imaginal neuroblasts (IN), which is unusual, since other IN markers (such as Grainyhead protein) do not show segmental modulation until the very last stages of embryogenesis. As in the CNS, the pattern of B5-lacZ expression in the epidermis differs significantly between thoracic and abdominal segments. The reason for this is the presence of the imaginal disc primordia in thoracic segments. The ventral discs and the dorsal prothoracic disc are the first to be detected following germ band retraction, early in stage 13. The wing (w) and haltere (h) primordia appear later during head involution and dorsal closure [Images], as cells appear to migrate dorsally from the ventral discs and form small pockets clinging to tracheal branches. This migration has also been observed for Distalless-lacZ-expressing cells. The dorsal prothoracic disc primordia, initially adjacent to the posterior T1 segment boundary, rounds up and invaginates with the anterior spiracle during head involution. A very different pattern is seen in abdominal segments. Three groups of strong lacZ-staining cells are reiterated in each segment: (1) the imaginal tracheoblasts, which are situated along the same position in the embryonic tracheal network as the elongate clusters described in the mature larva; (2) two epidermal cells of unknown identity and (3) two cells located on the ventral internal oblique muscles, also of unknown identity. B5-lacZ expression is also seen in the location of the genital disc primordium. Expression appears in three cell clusters: two transverse stripes of cells, one on each side of the midline and a more posterior, median cluster. These clusters subsequently associate together, presumably to form the fused genital disc (Weaver, 1995).

One hundred of the ~2000 cells of the tracheal epithelium express a set of fusion cell-specific marker genes and undergo a complex program of sprouting unicellular branches that fuse to each other and connect the independent metameric units of the trachea. headcase, identified by the expression of the marker Fus-6, is expressed in a subset of the fusion cells of each tracheal metamere from stage 14 until the end of embryogenesis. In Fus-6 embryos, the marker is selectively expressed in the fusion cells of the dorsal anastomoses in metameres 2-9, and in the fusion cells of the lateral trunk and ventral anastomoses. In these branches, the fusion cells are in contact with terminal cells that sprout off to generate tracheoles. The fusion cells of the dorsal trunk and dorsal branches in metameres 1 and 10 that do not contact terminal cells, do not express lacZ (Weaver, 1995).


In the enhancer trap line B5, lacZ expression driven by the hdc promoter is occurs in imaginal cell populations of the mature larva. At this stage, the imaginal cells can be distinguished from their larval counterparts by a characteristic small cell size, tissue morphology and positioning within the larval body plan. In addition to the classic imaginal discs, the B5-lacZ pattern also highlights the imaginal precursors of gut, genitalia, CNS, respiratory system and epidermis. The B5-lacZ pattern in the ventral nerve cord corresponds to the distribution of imaginal neuroblasts, as well as their postembryonic lineages, with many more expressing cells in the thoracic than in the abdominal neuromeres. The posterior tip of the ventral nerve cord also has many more lacZ-expressing cells than in abdominal neuromeres. Interestingly, it is this region where sexually dimorphic neuroblasts are proliferating and most likely represent the precursors of neurons destined to innervate the genitalia. The larval pattern diminishes during the early stages of pupal development, and is replaced by strong expression in the developing central brain and optic centers (Weaver, 1995).

In the larval respiratory system, B5-lacZ expression is observed in imaginal tracheoblasts, which are easily identified by their small size compared to neighboring larval tracheal cells and close association with the larval tracheal network. Two types of clusters are seen, one round and one elongate. Round clusters are found in thoracic segments only, and are situated next to the dorsal longitudinal trunk. Elongate clusters are stretched along the visceral and spiracular branches, which emanate from the transverse connectives and are segmentally repeated down the length of the larval trunk. The other respiratory-associated cell types represented in the B5- lacZ pattern include the dorsal prothoracic discs, associated with the anterior spiracles and the spiracular histoblast nest found in the epidermis of abdominal segments A1-A7. Larval glands that express B5-lacZ include the imaginal ring of the salivary gland, the ring gland, the lymph gland and pericardial cells flanking the length of the aorta. Alimentary expression includes the foregut and hindgut imaginal rings, and midgut imaginal islands. B5-lacZ is also expressed in the larval gonads. In the male, B5-lacZ is found in apically associated cells of the testis. These cells correspond to the mitotically dividing gonial cells and remain positive in the adult male, where expression is concentrated at the anterior tip of the testis. The terminal cells at the posterior tip are of unknown identity; however, they continue expressing B5-lacZ during pupation as this group of cells grow out toward the developing genital disc, presumably forming the seminal duct epithelium. In the female, B5-lacZ is expressed uniformly throughout the ovary (Weaver, 1995).

Drosophila metamorphosis is characterized by diverse developmental phenomena, including cellular proliferation, tissue remodeling, cell migration, and programmed cell death. Cells undergo one or more of these processes in response to the hormone 20-hydroxyecdysone (ecdysone), which initiates metamorphosis at the end of the third larval instar and before puparium formation (PF) via a transcriptional hierarchy. Additional pulses of ecdysone further coordinate these processes during the prepupal and pupal phases of metamorphosis. Larval tissues such as the gut, salivary glands, and larval-specific muscles undergo programmed cell death and subsequent histolysis. The imaginal discs undergo physical restructuring and differentiation to form rudimentary adult appendages such as wings, legs, eyes, and antennae. Ecdysone also triggers neuronal remodeling in the central nervous system (White, 1999).

Wild-type patterns of gene expression in D. melanogaster during early metamorphosis were examined by assaying whole animals at stages that span two pulses of ecdysone. Microarrays were constructed containing 6240 elements that included more than 4500 unique cDNA expressed sequence tag (EST) clones along with a number of ecdysone-regulated control genes having predictable expression patterns. These ESTs represent approximately 30% to 40% of the total estimated number of genes in the Drosophila genome. In order to gauge expression levels, microarrays were hybridized with fluorescent probes derived from polyA+ RNA isolated from developmentally staged animals. The time points examined are relative to PF, which last approximately 15 to 30 min, during which time the larvae cease to move and evert their anterior spiracles. Nineteen arrays were examined representing six time points relative to PF: one time point before the late larval ecdysone pulse; one time point just after the initiation of this pulse (4 hours BPF), and time points at 3, 6, 9, and 12 hours after PF (APF). The prepupal pulse of ecdysone occurs 9 to 12 hours APF (White, 1999).

In order to manage, analyze, and disseminate the large amount of data, a searchable database was constructed that includes the average expression differential at each time point. The analysis set consists of all elements that reproducibly fluctuate in expression threefold or more at any time point relative to PF, leaving 534 elements containing sequences represented by 465 ESTs and control genes. More than 10% of the genes represented by the ESTs display threefold or more differential expression during early metamorphosis. This may be a conservative estimate of the percentage of Drosophila genes that change in expression level during early metamorphosis, because of the stringent criteria used for their selection (White, 1999).

To interpret these data, genes were grouped according to similarity of expression patterns by two methods. The first relied on pairwise correlation statistics, and the second relied on the use of self-organizing maps (SOMs). Differentially expressed genes fall into two main categories. The first category contains genes that are expressed at >18 hours BFP (before the late larval ecdysone pulse) but then fall to low or undetectable levels during this pulse. These genes are potentially repressed by ecdysone and make up 44% of the 465 ESTs identified in this set. The second category consists of genes expressed at low or undetectable levels before the late larval ecdysone pulse but then are induced during this pulse. These genes are potentially induced by ecdysone and make up 31% of the 465 ESTs. Consequently, 75% of genes that changed in expression by threefold or more do so during the late larval ecdysone pulse that marks the initial transition from larva to prepupa. This result is consistent with the extreme morphological changes that are about to occur in these animals. There are clearly discrete subdivisions of gene expression within these categories (White, 1999).

The expression levels of genes involved in cellular differentiation also dynamically change during metamorphosis. The gene headcase is expressed in all proliferating imaginal cell lineages. This gene is induced during the prepupal ecdysone pulse but does not substantially change expression levels during the late larval ecdysone pulse. Imaginal tissues in headcase null mutants appear normal in size and shape but fail to differentiate normally. These mutants are invariably pupal lethal and show pleiotropic effects in adult tissues. The predominant headcase loss of function phenotype is defective head development. Mutants can display deletion of the head capsule, leaving only a protruding proboscis. Another gene expressed in this manner with a known role in ecdysone-mediated differentiation of imaginal discs is IMP-L2, an essential secreted immunoglobulin family member implicated in neural and ectodermal development in Drosophila. These data demonstrate that factors required for cellular differentiation during metamorphosis are present in the data set. There are 29 other EST sequences encoding novel genes that display a greater than threefold induction from PF to 12 hours APF but do not display a threefold or greater change in expression level during the late larval ecdysone pulse. Perhaps some of these genes, such as headcase and IMP-L2, are involved in differentiation of adult-specific tissues (White, 1999).


In order to assess the possible role of the headcase gene in imaginal tissue development, deletions removing part or all of the mapped hdc transcription unit were generated by imprecise P-element excision. All deletions cause pupal lethality when homozygous and heterallelic combinations show similar mutant phenotypes, suggesting that all alleles are functionally null. Although hdc mutants die as pupae, all larval imaginal discs are present, and their size and shape appear normal. Therefore the loss of hdc does not affect cell growth in any obvious way. The penetrance of pupal lethality is 100%; however, the developmental stage at which the pupae die is variable, ranging from brown pupae with no obviously differentiated tissue, to dead pharate adults. The most prominent hdc defects involve head development and can result in complete deletion of the head capsule, or duplication of head cuticle or antennae. Although the majority of pharate adults exhibit normal external head morphology, they possess massive defects in internal head structures, including the CNS, muscular and tracheal tissues. Other tissues are also affected (including wings, halteres, legs and epidermis) but these effects vary among mutant individuals. Such pleiotropy is consistent with the broad range of imaginal cell-type specificity of the hdc gene expression; however, the reason for variable expressivity is unclear (Weaver, 1995).

Mutation of headcase causes ectopic branching from terminal tip cells of developing trachea. Five new early pupal lethal alleles in the same complementation group as the original Fus-6 (headcase) insert have been isolated and as well as several revertants of the lethal and tracheal phenotypes, suggesting that these phenotypes are associated with the transposon insertion. The Fus-650 and Fus-620 alleles showed the strongest tracheal phenotypes, and Fus-650 was selected for further analysis. All of the mutant embryos have additional fine branches emanating from the dorsal branches. These new branches sprout at the tips of the dorsal branches in a position where the fusion and terminal sprouts are found in the wild-type flies. The effect of the mutation is sporadic in each embryo, on average 21% of the tracheal metameres have additional sprouts, as compared with 1% seen in the wild type. Extra sprouting is also detected in the lateral trunk of the mutants, but here the frequency of increased branching is much lower (Steneberg, 1998).

Headcase promotes cell survival and niche maintenance in the Drosophila testis

At the apical tip of the Drosophila testis, germline and somatic stem cells surround a cluster of somatic cells called the hub. Hub cells produce a self-renewal factor, Unpaired (Upd), that activates the JAK-STAT pathway in adjacent stem cells to regulate stem cell behavior. Therefore, apical hub cells are a critical component of the stem cell niche in the testis. headcase (hdc) was identified in the course of a screen to identify factors involved in regulating hub maintenance. Hub cells depleted for hdc undergo programmed cell death, suggesting that anti-apoptotic pathways play an important role in maintenance of the niche. Using hdc as paradigm, this study describes the first comprehensive analysis on the effects of a progressive niche reduction on the testis stem cell pool. Surprisingly, single hub cells remain capable of supporting numerous stem cells, indicating that although the size and number of niche support cells influence stem cell maintenance, the testis stem cell niche appears to be remarkably robust in the its ability to support stem cells after severe damage (Resende, 2013; free access article).


One of the C. elegans homologs identified by BLAST database searches is homologous to the amino acids 440-574 shared by the two headcase translation products, whereas the other is homologous to an 84-amino acid amino-terminal region common to both products and a region of 24 amino acids at the carboxy-terminal end, unique to the longer product. The presence of two C. elegans genes with homologies to distinct domains of the two hdc products suggests that both products may be functional. Attempts to detect the proteins in more distant Drosophila species and C. elegans have failed, probably because the epitope recognized by the monoclonal antibody used is not conserved (Steneberg, 1998).


Search PubMed for articles about Drosophila headcase

Avet-Rochex, A., Carvajal, N., Christoforou, C. P., Yeung, K., Maierbrugger, K. T., Hobbs, C., Lalli, G., Cagin, U., Plachot, C., McNeill, H., Bateman, J. M. (2014). Unkempt is negatively regulated by mTOR and uncouples neuronal differentiation from growth control. PLoS Genet 10: e1004624. PubMed ID: 25210733

Bray, S. J. and Kafatos, F. C. (1991). Developmental function of Elf-1: an essential transcription factor during embryogenesis in Drosophila. Genes Dev. 5: 1672-1683. PubMed ID: 1909284

Estella, C., et al. (2003). The role of buttonhead and Sp1 in the development of the ventral imaginal discs of Drosophila. Development 130: 5929-5941. 14561634

Guillemin, K., et al. (1996). The pruned gene encodes the Drosophila serum response factor and regulates cytoplasmic outgrowth during terminal branching of the tracheal system. Development 122: 1353-1362

Resende, L. P., Boyle, M., Tran, D., Fellner, T. and Jones, D. L. (2013). Headcase promotes cell survival and niche maintenance in the Drosophila testis. PLoS One 8: e68026. PubMed ID: 23874487

Rotstein, B., Molnar, D., Adryan, B. and Llimargas, M. (2011). Tramtrack is genetically upstream of genes controlling tracheal tube size in Drosophila. PLoS One 6(12): e28985. PubMed Citation: 22216153

Samakovlis, C., et al. (1996a). Branching morphogenesis of the Drosophila tracheal system occurs by a series of morphologically distinct but genetically coupled branching events. Development 122: 1395-1407. PubMed ID: 8625828

Samakovlis, C., et al. (1996b). Genetic control of epithelial tube fusion during Drosophila tracheal development. Development 122: 3531-3536. PubMed ID: 8951068

Steneberg, P., et al. (1998). Translational readthrough in the hdc mRNA generates a novel branching inhibitor in the Drosophila trachea. Genes & Dev. 12: 956-967. PubMed ID: 9531534

Sutherland, D., C. Samakovlis, and M.A. Krasnow. 1996. branchless encodes a Drosophila FGF homolog that controls tracheal cell migration and the pattern of branching. Cell 87: 1091-1101

Tanaka-Matakatsu, M., et al (1996). Cadherin-mediated cell adhesion and cell motility in Drosophila trachea regulated by the transcription factor escargot. Development 122: 3697-3705

Weaver, T.A. and White, R.A. (1995). headcase, an imaginal specific gene required for adult morphogenesis in Drosophila melanogaster. Development 121: 4149--4160. PubMed ID: 8575315

White, K., et al. (1999). Microarray analysis of Drosophila development during metamorphosis. Science 286: 2179-2184

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date revised: 10 December 2014

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