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


cDNA length - 4.3 kb

Bases in 5' UTR - 2033

Bases in 3' UTR - 1669


Amino Acids - 653

Structural Domains

The hdc cDNA sequence has the potential to encode more than one polypeptide. There is a 3241 nucleotide long ORF starting with an AUG flanked by a conserved Drosophila translational initiation site. The sequence of this putative ORF is interrupted by an internal UAA stop codon at position 2981 and continues in frame until it is interrupted again by another UAA stop codon, followed by a polyadenylation signal. This intriguing structure suggests that HDC mRNA has the potential to encode at least two proteins: One with a predicted molecular mass of 70 kD generated by termination at the first UAA codon, and one with a molecular mass of 125 kD, generated by suppression of translational termination. The shorter protein proves to be four times more abundant than the longer product. Thus hdc codes for two overlapping protein products requiring unusual suppression of the translational termination mechanism (Weaver, 1995 and Steneberg,1998).

Database searches reveal no sequence similarity or conserved protein motifs within either the upstream or downstream ORF. However, Hdc is cysteine-rich and has a predicted pI of 9.6. Since the Hdc protein is concentrated in the cytoplasm, the extreme basicity may reflect an affinity for protein-protein or protein-RNA interactions (Weaver, 1995).


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).

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

date revised: 18 May 98

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.