The Homeodomain Iroquois Proteins Control Cell Cycle Progression and Regulate the Size of Developmental Fields.

During development, proper differentiation and final organ size rely on the control of territorial specification and cell proliferation. Although many regulators of these processes have been identified, how both are coordinated remains largely unknown. The homeodomain Iroquois/Irx proteins play a key, evolutionarily conserved, role in territorial specification. Here we show that in the imaginal discs, reduced function of Iroquois genes promotes cell proliferation by accelerating the G1 to S transition. Conversely, their increased expression causes cell-cycle arrest, down-regulating the activity of the Cyclin E/Cdk2 complex. We demonstrate that physical interaction of the Iroquois protein Caupolican with Cyclin E-containing protein complexes, through its IRO box and Cyclin-binding domains, underlies its activity in cell-cycle control. Thus, Drosophila Iroquois proteins are able to regulate cell-autonomously the growth of the territories they specify. Moreover, our results provide a molecular mechanism for a role of Iroquois/Irx genes as tumour suppressors.

EGFR-dependent downregulation of Capicua and the establishment of Drosophila dorsoventral polarity.

Dorsoventral (DV) axis formation in Drosophila begins during oogenesis through the graded activation of the EGF receptor (EGFR)-Ras-MAPK signaling pathway in the follicle cell layer of the egg chamber. EGFR signaling, which is higher in dorsal follicle cells, represses expression of the sulfotransferase-encoding gene pipe, thereby delimiting a ventral domain of Pipe activity that is critical for the subsequent induction of ventral embryonic fates. We have characterized the transcriptional circuit that links EGFR signaling to pipe repression: in dorsal follicle cells, the homeodomain transcription factor Mirror (Mirr), which is induced by EGFR signaling, directly represses pipe transcription, whereas in ventral follicle cells, the HMG-box protein Capicua (Cic) supports pipe expression by repressing mirr. Although Cic is under negative post-transcriptional regulation by Ras-MAPK signaling in different contexts, the relevance of this mechanism for the interpretation of the EGFR signal during DV pattern formation remains unclear. Here, we consider a model where EGFR-mediated downregulation of Cic modulates the spatial distribution of Mirr protein in lateral follicle cells, thereby contributing to define the position at which the pipe expression border is formed.

Mirror represses pipe expression in follicle cells to initiate dorsoventral axis formation in Drosophila.

Dorsoventral (DV) axis formation in Drosophila begins with selective activation of EGFR, a receptor tyrosine kinase (RTK), in dorsal-anterior (DA) ovarian follicle cells. A critical event regulated by EGFR signaling is the repression of the sulfotransferase-encoding gene pipe in dorsal follicle cells, but how this occurs remains unclear. Here we show that Mirror (Mirr), a homeodomain transcription factor induced by EGFR signaling in DA follicle cells, directly represses pipe expression by binding to a conserved element in the pipe regulatory region. In addition, we find that the HMG-box protein Capicua (Cic) supports pipe expression in ventral follicle cells by repressing Mirr in this region. Interestingly, this role of Cic resembles its function in regulating anteroposterior (AP) body patterning, where Cic supports gap gene expression in central regions of the embryo by repressing Tailless, a repressor induced by RTK signaling at the embryonic poles. Thus, related RTK-Cic repressor circuits regulate the early stages of Drosophila DV and AP body axis formation.

An ancient genomic regulatory block conserved across bilaterians and its dismantling in tetrapods by retrogene replacement.

Developmental genes are regulated by complex, distantly located cis-regulatory modules (CRMs), often forming genomic regulatory blocks (GRBs) that are conserved among vertebrates and among insects. We have investigated GRBs associated with Iroquois homeobox genes in 39 metazoans. Despite 600 million years of independent evolution, Iroquois genes are linked to ankyrin-repeat-containing Sowah genes in nearly all studied bilaterians. We show that Iroquois-specific CRMs populate the Sowah locus, suggesting that regulatory constraints underlie the maintenance of the Iroquois-Sowah syntenic block. Surprisingly, tetrapod Sowah orthologs are intronless and not associated with Iroquois; however, teleost and elephant shark data demonstrate that this is a derived feature, and that many Iroquois-CRMs were ancestrally located within Sowah introns. Retroposition, gene, and genome duplication have allowed selective elimination of Sowah exons from the Iroquois regulatory landscape while keeping associated CRMs, resulting in large associated gene deserts. These results highlight the importance of CRMs in imposing constraints to genome architecture, even across large phylogenetic distances, and of gene duplication-mediated genetic redundancy to disentangle these constraints, increasing genomic plasticity.

Drosophila araucan and caupolican integrate intrinsic and signalling inputs for the acquisition by muscle progenitors of the lateral transverse fate.

A central issue of myogenesis is the acquisition of identity by individual muscles. In Drosophila, at the time muscle progenitors are singled out, they already express unique combinations of muscle identity genes. This muscle code results from the integration of positional and temporal signalling inputs. Here we identify, by means of loss-of-function and ectopic expression approaches, the Iroquois Complex homeobox genes araucan and caupolican as novel muscle identity genes that confer lateral transverse muscle identity. The acquisition of this fate requires that Araucan/Caupolican repress other muscle identity genes such as slouch and vestigial. In addition, we show that Caupolican-dependent slouch expression depends on the activation state of the Ras/Mitogen Activated Protein Kinase cascade. This provides a comprehensive insight into the way Iroquois genes integrate in muscle progenitors, signalling inputs that modulate gene expression and protein activity.

Apposition of iroquois expressing and non-expressing cells leads to cell sorting and fold formation in the Drosophila imaginal wing disc.

BACKGROUND: The organization of the different tissues of an animal requires mechanisms that regulate cell-cell adhesion to promote and maintain the physical separation of adjacent cell populations. In the Drosophila imaginal wing disc the iroquois homeobox genes are expressed in the notum anlage and contribute to the specification of notum identity. These genes are not expressed in the adjacent wing hinge territory. These territories are separated by an approximately straight boundary that in the mature disc is associated with an epithelial fold. The mechanism by which these two cell populations are kept separate is unclear. RESULTS: Here we show that the Iro-C genes participate in keeping the notum and wing cell populations separate. Indeed, within the notum anlage, cells not expressing Iro-C tend to join together and sort out from their Iro-C expressing neighbours. We also show that apposition of Iro-C expressing and non-expressing cells induces invagination and apico-basal shortening of the Iro-C- cells. This effect probably underlies formation of the fold that separates the notum and wing hinge territories. In addition, cells overexpressing a member of the Iro-C contact one another and become organized in a network of thin strings that surrounds and isolates large groups of non-overexpressing cells. The strings appear to exert a pulling force along their longitudinal axis. CONCLUSION: Apposition of cells expressing and non-expressing the Iro-C, as it occurs in the notum-wing hinge border of the Drosophila wing disc, influences cell behaviour. It leads to cell sorting, and cellular invagination and apical-basal shortening. These effects probably account for keeping the prospective notum and wing hinge cell populations separate and underlie epithelial fold formation. Cells that overexpress a member of the Iro-C and that confront non-expressing cells establish contacts between themselves and become organized in a network of thin strings. This is a complex and unique phenotype that might be important for the generation of a straight notum-wing hinge border.