Integration of an abdominal Hox complex with Pax2 yields cell-specific EGF secretion from Drosophila sensory precursor cells

Abstract

SUMMARYCis-regulatory modules (CRMs) ensure specific developmental outcomes by mediating both proper spatiotemporal gene expression patterns and appropriate transcriptional levels. In Drosophila, the precise transcriptional control of the serine protease rhomboid regulates EGF signaling to specify distinct cell types. Recently, we identified a CRM that activates rhomboid expression and thereby EGF secretion from a subset of abdominal sensory organ precursor cells (SOPs) to induce an appropriate number of lipid-processing cells called oenocytes. Here, we use scanning mutagenesis coupled with reporter assays, biochemistry and genetics to dissect the transcriptional mechanisms regulating SOP-specific rhomboid activation. Our results show that proper spatial activity of the rhomboid CRM is dependent upon direct integration of the abdomen-specific Hox factor Abdominal-A and the SOP-restricted Pax2 factor. In addition, we show that the Extradenticle and Homothorax Hox co-factors are differentially integrated on the rhomboid CRM by abdominal versus thoracic Hox proteins in the presence of Pax2. Last, we show that Abdominal-A uses both Pax2-dependent and Pax2-independent mechanisms to stimulate rhomboid CRM activity to induce proper oenocyte numbers. Thus, these data demonstrate how a CRM integrates Hox and neural transcriptional inputs to regulate the appropriate spatial pattern and levels of EGF secretion to specify an essential cell fate.

Thecis-regulatory logic underlying abdominal Hox-mediated repression versus activation of regulatory elements inDrosophila

“…Consistent with this idea, previous studies demonstrated that Abd-A and Pax2 could be coimmunoprecipitated in cell culture, whereas a thoracic Hox factor (Antennapedia, Antp) that fails to activate RhoA also failed to form such complexes with Pax2 [26]. Moreover, the vertebrate Hox11 proteins were also found to form protein complexes with Pax2 to regulate target gene expression in the mammalian kidney [46].…”

“…To better understand the mechanisms underlying abdominal Hox outputs, we next compared the ability of Ubx and Abd-A to regulate the RhoBAD cis-regulatory element. Unlike Dll and the DCRE, which are repressed by abdominal Hox factors, rhomboid (rho) is activated by Abd-A in a subset of abdominal sensory organ precursor cells (SOPs) via a highly conserved Exd/Hth/Hox binding site within RhoBAD [25,26]. rho encodes a serine protease that triggers the release of an EGF ligand and neighboring cells that receive the EGF signal are specified to form larval oenocytes [30,31].…”

“…In contrast, the RhoA element contains a single contiguous set of Exd/Hth/Hox sites that mediate activation in conjunction with a nearby Pax2 site (Fig 3A). To determine if the Hox/Hox co-factor sites from the DCRE can similarly mediate RhoA activation in abdominal SOP cells, we used a previously established transgenic reporter assay based on three copies of the RhoA element (RhoAAA-lacZ) which is sufficient to mediate activation in abdominal SOP cells [26,28] (Fig 6B). To do so, we made RhoAAA-DF (forward) and RhoAAA-DR (reverse) constructs in which the Pax2 site was maintained but the RhoA Exd/Hth/Hox sites were replaced with the DCRE Hox/Exd-Hth/Hox sites in the "forward" and "reverse" orientations ( Fig 6A).…”

“…Moreover, studies on additional cis-regulatory modules (CRMs) revealed that the Abd-A Hox factor can either activate or repress target gene expression when in complex with the Exd and Hth proteins. The RhoBAD regulatory element contains a highly conserved sequence (RhoA) encoding an adjacent set of Exd/Hth/Hox sites that recruits an Abd-A complex to mediate rhomboid (rho) activation in a subset of sensory organ precursor (SOP) cells [25][26][27][28]. The activation of rho, which encodes a serine protease that triggers the release of an EGF ligand, results in the induction of neighboring cells to form an essential set of hepatocyte-like cells known as oenocytes [29][30][31][32].…”

“…Current models suggest that the RhoA and DCRE CRMs integrate additional transcription factor inputs that dictate the sign of transcription. For example, RhoA requires a nearby Pax2 transcription factor binding site to mediate activation whereas the DCRE contains a nearby FoxG binding site (Sloppy-paired 1 (Slp1) and Slp2 are largely redundant Drosophila FoxG (FoxG) proteins) to mediate repression [26,34]. How these factors are integrated with the specific Hox transcription factor complexes remain relatively unknown.…”