Size compensation in Drosophila after generalised cell death.

Regeneration is a response mechanism aimed to restore tissues that have been damaged. We are studying in the wing disc of Drosophila the regenerative response to a dose of Ionizing Radiation that kills over 35% of the cells distributed all over the disc. After such treatment the discs are able to restore normal size, indicating there is a mechanism that repairs generalised damage. We have tested the role of the JNK, JAK/STAT and Wg pathways, known to be required for regeneration after localised damage in the disc. We find that after irradiation there is size compensation in the absence of function of these pathways, indicating that they are not necessary for the compensation. Furthermore, we also find that generalised damage does not cause an increase in the proliferation rate of surviving cells. We propose that irradiated discs suffer a developmental delay and resume growth at normal rate until they reach the final stereotyped size. The delay appears to be associated with a developmental reversion, because discs undergo rejuvenation towards an earlier developmental stage. We argue that the response to generalized damage is fundamentally different from that to localized damage, which requires activity of JNK and Wg.

Chromatin remodelling and retrotransposons activities during regeneration in Drosophila.

Regeneration is a response mechanism aiming to reconstruct lost or damaged structures. To achieve this, the cells repopulating the lost tissue often have to change their original identity, a process that involves chromatin remodelling.We have analysed the issue of chromatin remodelling during regeneration in the wing disc of Drosophila . In this disc the ablation of the central region (the pouch) induces the regenerative response of the cells from the lateral region (the hinge), which reconstitute the wing pouch. We have examined euchromatin and heterochromatin histone marks during the process and find that heterochromatin marks disappear but are recovered when regeneration is complete. Euchromatin marks are not modified. We also describe the transcription of two retrotransposons, Roo and F-element in the regenerating cells. We have established a temporal correlation between the alterations of heterochromatin marks and the levels of transcription of two retrotransposons, Roo and F-element, both during embryonic development and in the regeneration process.

The Pax protein Eyegone (Eyg) interacts with the pi-RNA component Aubergine (Aub) and controls egg chamber development in Drosophila.

The eyegone (eyg) gene encodes Eyg, a transcription factor of the Pax family with multiple roles during Drosophila development. Eyg has been shown to be nuclear in the cells where it functions. In this report we describe a new functional cytoplasmic distribution of Eyg during egg chamber development in the female ovarioles. The protein is present from the germarium until stage 10 of cyst development. The majority of egg chambers that develop in the absence of Eyg arrest their development before stage 10, show augmented levels of the telomeric retro-transposon TART-A and low levels of heterochromatin marks in the oocyte nucleus. During the maternal to zygotic transition (MTZ) Eyg seems to play a role in destabilizing germ cell less (gcl) and oo 16 RNA binding protein (orb) mRNAs. We were able to show that Eyg interacts with Aubergine (Aub), a component of the pi-RNA pathway during egg chamber development. This interaction could be essential for Eyg to be retained in the cytoplasm and fulfill its functions there.

The TALE transcription factor homothorax functions to assemble heterochromatin during Drosophila embryogenesis.

We have previously identified Homothorax (Hth) as an important factor for the correct assembly of the pericentromeric heterochromatin during the first fast syncytial divisions of the Drosophila embryo. Here we have extended our studies to later stages of embryonic development. We were able to show that hth mutants exhibit a drastic overall reduction in the tri-methylation of H3 in Lys9, with no reduction of the previous di-methylation. One phenotypic outcome of such a reduction is a genome instability visualized by the many DNA breaks observed in the mutant nuclei. Moreover, loss of Hth leads to the opening of closed heterochromatic regions, including the rDNA genomic region. Our data show that the satellite repeats get transcribed in wild type embryos and that this transcription depends on the presence of Hth, which binds to them as well as to the rDNA region. This work indicates that there is an important role of transcription of non-coding RNAs for constitutive heterochromatin assembly in the Drosophila embryo, and suggests that Hth plays an important role in this process.

Functional dissection of the splice variants of the Drosophila gene homothorax (hth).

Homothorax belongs to the TALE-homeodomain family of transcription factors, together with its vertebrate counterparts, the Meis family of proto-oncogenes. It fulfills many important different functions during embryonic and larval developments in Drosophila, which encompass from subdivision and specification of body parts to assembly of heterochromatin structures. Hth interacts with Extradenticle, another member of the TALE-homeodomain family of conserved transcription factors, to facilitate its entrance to the nucleus. The many different functions described for Hth rely on the complexity of the locus, from which six different isoforms arise. The isoforms can be grouped into full-length and short versions, which contain either one or the two conserved domains of the protein (homeodomain and Exd-interacting domain). We have used molecular and genetic tools to analyze the levels of expression, the distribution and the function of the isoforms during embryonic development. Our results clearly show that the isoforms display distinct levels of expression and are differentially distributed in the embryo. This detailed study also shows that during normal embryonic development not all the Hth isoforms translocate Exd into the nucleus, suggesting that both the proteins can also function separately. We have demonstrated that the full-length Hth protein activates transcription of exd, augmenting the levels of exd mRNA in the cell. The higher levels of Exd protein in those cells facilitate its entrance to the nucleus. Our work demonstrates that hth is a complex gene that should not be considered as a functional unit. The roles of the different isoforms probably rely on their distinct protein domains and conformations and, at the end, on interactions with particular partners.

Functional association between eyegone and HP1a mediates wingless transcriptional repression during development.

The eyegone (eyg) gene encodes Eyg, a transcription factor of the Pax family with multiple roles during Drosophila development. Although Eyg has been shown to act as a repressor, nothing is known about the mechanism by which it represses its target genes. Here, we show that Eyg forms a protein complex with heterochromatin protein 1a (HP1a). Both proteins bind to the same chromatin regions on polytene chromosomes and act cooperatively to suppress variegation and mediate gene silencing. In addition, Eyg binds to a wingless (wg) enhancer region, recruiting HP1a to assemble a closed, heterochromatin-like conformation that represses transcription of the wg gene. We describe here the evidence that suggests that Eyg, encoded by eyegone (eyg), represses wingless (wg) during eye development by association with HP1a. We show that Eyg forms a protein complex with HP1a and both proteins colocalize on salivary gland polytene chromosomes. Using position effect variegation (PEV) experiments, we demonstrated that eyg has a dose-dependent effect on heterochromatin gene silencing and identified a genetic interaction with HP1a in this process. We further demonstrated that HP1a binds to the same wg enhancer element as Eyg. DNase I sensitivity assays indicated that this enhancer region has a closed heterochromatin-like conformation, which becomes open in eyg mutants. In these mutants, much less HP1a binds to the wg enhancer region, as shown by ChIP experiments. Furthermore, as previously described for Eyg, a reduction in the amount of HP1a in the eye imaginal disc derepresses wg. Together, our results suggest a model in which Eyg specifically binds to the wg enhancer region, recruiting HP1a to that site. The recruitment of HP1a prevents transcription by favoring a closed, heterochromatin-like structure. Thus, for the first time, we show that HP1a plays a direct role in the repression of a developmentally regulated gene, wg, during Drosophila eye development.

A new role for hth in the early pre-blastodermic divisions in drosophila.

In drosophila, the preblastodermic syncytial nuclear divisions occur very fast. In this short period of time chromosomes must condense, segregate and decondense, in conditions governed by maternally provided RNAs and proteins. In this report, we show that the Homothorax (Hth) transcription factor is maternally provided and that its function is necessary for the proper assembly of the centric/centromeric heterochromatin during preblastodermic divisions. Embryos lacking the hth maternally-derived transcript, show abnormal localisation of the centromeric CID protein, and aberrant chromosomal segregation. In this syncytial context, Hth presumably acts together with its partner Extradenticle (Exd) and the RNA PolII, to facilitate transcription of satellite repeats. The transcripts derived from these sequences are needed for the correct assembly of the centric heterochromatin.

Patterning function of homothorax/extradenticle in the thorax of Drosophila.

In Drosophila, the morphological diversity is generated by the activation of different sets of active developmental regulatory genes in the different body subdomains. Here, we have investigated the role of the homothorax/extradenticle (hth/exd) gene pair in the elaboration of the pattern of the anterior mesothorax (notum). These two genes are active in the same regions and behave as a single functional unit. We find that their original uniform expression in the notum is downregulated during development and becomes restricted to two distinct, alpha and betasubdomains. This modulation appears to be important for the formation of distinct patterns in the two subdomains. The regulation of hth/exd expression is achieved by the combined repressing functions of the Pax gene eyegone (eyg) and of the Dpp pathway. hth/exd is repressed in the body regions where eyg is active and that also contain high levels of Dpp activity. We also present evidence for a molecular interaction between the Hth and the Eyg proteins that may be important for the patterning of the alpha subdomain.

The Pax-homeobox gene eyegone is involved in the subdivision of the thorax of Drosophila.

The eyegone (eyg) gene is known to be involved in the development of the eye structures of Drosophila. We show that eyg and its related gene, twin of eyegone (toe), are also expressed in part of the anterior compartment of the adult mesothorax (notum). We report experiments concerning the role of these genes in the notum. In the absence of eyg function the anterior-central region does not develop, whereas ectopic activity of either eyg or toe induces the formation of the anterior-central pattern in the posterior or lateral region of the notum. These results demonstrate that eyg and toe play a role in the genetic subdivision of the notum, although the experiments indicate that eyg exerts the principal function. However, by itself the Eyg product cannot induce the formation of notum patterns; its thoracic function requires co-expression with the Iroquois (Iro) genes. We show that the restriction of eyg activity to the anterior-central region of the wing disc is achieved by the antagonistic regulatory activities of the Iro and pnr genes, which promote eyg expression, and those of the Hh and Dpp pathways, which act as repressors. We argue that eyg is a subordinate gene of the Iro genes, and that pnr mediates their thoracic patterning function. The activity of eyg gives rise to a new notum subdivision that acts upon the pre-extant one generated by the Iro genes and pnr. As a result the notum becomes subdivided into four distinct genetic domains.

Distinct functions of homothorax in leg development in Drosophila.

The Drosophila leg is subdivided into two mutually antagonistic proximal and distal domains. The proximal domain is defined by the activity of the homeobox genes homothorax and extradenticle and the distal one by the Dpp/Wg targets Distal-less (Dll) and dachshund (dac). It is known that hth/exd function prevents the activity of Dpp and Wg response genes and that cells deficient for exd activity in the proximal domain differentiate pattern elements corresponding to more distal leg regions. We report new results on the role of hth/exd antagonising the Dpp pathway. In cells expressing hth in the distal leg, there is a debilitation of the Dpp pathway which is reflected in lower levels of Mad phosphorylation and in increased levels of the receptor thick veins. Ectopic hth expression in the distal leg results in JNK-mediated apoptosis, decreased growth and pattern abnormalities. It also causes a general proximalisation of the appendage, which can be explained by interference with the Dpp and Wg pathways. We also report that the repression by hth/exd of the Dpp and Wg target Distal-less is not achieved at the level of transcription but preventing the activation of Dll target genes. We propose that hth/exd function contributes to the normal identity of proximal cells both by limiting the influence of the Dpp and Wg pathways and by activating proximal genes like teashirt (tsh) and aristaless (al).