headcase

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).

DEVELOPMENTAL BIOLOGY

Embryonic

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).

Larval

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).

Effects of mutation or deletion

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).

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date revised: 10 February 2010

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