The Drosophila Perlecan gene trol regulates multiple signaling pathways in different developmental contexts

<p>Abstract</p> <p>Background</p> <p>Heparan sulfate proteoglycans modulate signaling by a variety of growth factors. The mammalian proteoglycan Perlecan binds and regulates signaling by Sonic Hedgehog, Fibroblast Growth Factors (FGFs), Vascular Endothelial Growth Factor (VEGF) and Platelet Derived Growth Factor (PDGF), among others, in contexts ranging from angiogenesis and cardiovascular development to cancer progression. The <it>Drosophila </it>Perlecan homolog <it>trol </it>has been shown to regulate the activity of Hedgehog and Branchless (an FGF homolog) to control the onset of stem cell proliferation in the developing brain during first instar. Here we extend analysis of <it>trol </it>mutant phenotypes to show that <it>trol </it>is required for a variety of developmental events and modulates signaling by multiple growth factors in different situations.</p> <p>Results</p> <p>Different mutations in <it>trol </it>allow developmental progression to varying extents, suggesting that <it>trol </it>is involved in multiple cell-fate and patterning decisions. Analysis of the initiation of neuroblast proliferation at second instar demonstrated that <it>trol </it>regulates this event by modulating signaling by Hedgehog and Branchless, as it does during first instar. Trol protein is distributed over the surface of the larval brain, near the regulated neuroblasts that reside on the cortical surface. Mutations in <it>trol </it>also decrease the number of circulating plasmatocytes. This is likely to be due to decreased expression of <it>pointed</it>, the response gene for VEGF/PDGF signaling that is required for plasmatocyte proliferation. Trol is found on plasmatocytes, where it could regulate VEGF/PDGF signaling. Finally, we show that in second instar brains but not third instar brain lobes and eye discs, mutations in <it>trol </it>affect signaling by Decapentaplegic (a Transforming Growth Factor family member), Wingless (a Wnt growth factor) and Hedgehog.</p> <p>Conclusion</p> <p>These studies extend the known functions of the <it>Drosophila </it>Perlecan homolog <it>trol </it>in both developmental and signaling contexts. These studies also highlight the fact that Trol function is not dedicated to a single molecular mechanism, but is capable of regulating different growth factor pathways depending on the cell-type and event underway.</p

The Perlecan gene regulates multiple signaling pathways in different developmental contexts-6

<p><b>Copyright information:</b></p><p>Taken from "The Perlecan gene regulates multiple signaling pathways in different developmental contexts"</p><p>http://www.biomedcentral.com/1471-213X/7/121</p><p>BMC Developmental Biology 2007;7():121-121.</p><p>Published online 2 Nov 2007</p><p>PMCID:PMC2174950.</p><p></p> BrdU labeled neuroblasts falling within the control range, some data originally published in Park et al, 2003a. B) Lethal phase phenotype presented as the percentage of mutant animals capable of survival and development to third instar (grey bars) or to pupal formation (black bars) compared to sibling controls. Error bars indicate s.e.m. C) Cartoon of second instar larval brain with dividing TNBs in ventral ganglion. Boxed area indicates portion of brain shown in panels D-G below. A = anterior, P = posterior. D-G) Examples of the five classes of BrdU incorporation into TNbs are shown. In all panels anterior is to the left, posterior is to the right. Scale bar in panel D indicates 25 um. D) None (class 1). E) Few (class 2). F) Segmentally repeated lines with few extra neuroblasts (class 3). G) Segmentally repeated lines with several scattered neuroblasts (class 4). H) Heavily populated segmental pattern (class 5)

The Perlecan gene regulates multiple signaling pathways in different developmental contexts-0

<p><b>Copyright information:</b></p><p>Taken from "The Perlecan gene regulates multiple signaling pathways in different developmental contexts"</p><p>http://www.biomedcentral.com/1471-213X/7/121</p><p>BMC Developmental Biology 2007;7():121-121.</p><p>Published online 2 Nov 2007</p><p>PMCID:PMC2174950.</p><p></p> BrdU labeled neuroblasts falling within the control range, some data originally published in Park et al, 2003a. B) Lethal phase phenotype presented as the percentage of mutant animals capable of survival and development to third instar (grey bars) or to pupal formation (black bars) compared to sibling controls. Error bars indicate s.e.m. C) Cartoon of second instar larval brain with dividing TNBs in ventral ganglion. Boxed area indicates portion of brain shown in panels D-G below. A = anterior, P = posterior. D-G) Examples of the five classes of BrdU incorporation into TNbs are shown. In all panels anterior is to the left, posterior is to the right. Scale bar in panel D indicates 25 um. D) None (class 1). E) Few (class 2). F) Segmentally repeated lines with few extra neuroblasts (class 3). G) Segmentally repeated lines with several scattered neuroblasts (class 4). H) Heavily populated segmental pattern (class 5)

The Perlecan gene regulates multiple signaling pathways in different developmental contexts-2

<p><b>Copyright information:</b></p><p>Taken from "The Perlecan gene regulates multiple signaling pathways in different developmental contexts"</p><p>http://www.biomedcentral.com/1471-213X/7/121</p><p>BMC Developmental Biology 2007;7():121-121.</p><p>Published online 2 Nov 2007</p><p>PMCID:PMC2174950.</p><p></p>Bnl response gene (grey bars) in the ventral ganglia of mutant normalized to controls at one hour post molt to second instar. βwas used to as an internal control to normalize message levels. All analyses were done in triplicate and three different concentrations to ensure samples were within linear range of amplification. Error bars indicate standard deviation

The Perlecan gene regulates multiple signaling pathways in different developmental contexts-1

<p><b>Copyright information:</b></p><p>Taken from "The Perlecan gene regulates multiple signaling pathways in different developmental contexts"</p><p>http://www.biomedcentral.com/1471-213X/7/121</p><p>BMC Developmental Biology 2007;7():121-121.</p><p>Published online 2 Nov 2007</p><p>PMCID:PMC2174950.</p><p></p>region shown in panels B-E. One hour BrdU incorporation in TNbs in B) a ; /+ brain from 2–3 hours pm; C) a sibling control +/, ; +/or /+ ; brain 2–3 hours pm (see text); D) a ; /+ brain from 2–3 hours pm and a E) sibling control +/, ; +/or /+ ; brain from 2–3 hours pm (see text). Scale bar in panel A indicates 10 um

The Perlecan gene regulates multiple signaling pathways in different developmental contexts-4

<p><b>Copyright information:</b></p><p>Taken from "The Perlecan gene regulates multiple signaling pathways in different developmental contexts"</p><p>http://www.biomedcentral.com/1471-213X/7/121</p><p>BMC Developmental Biology 2007;7():121-121.</p><p>Published online 2 Nov 2007</p><p>PMCID:PMC2174950.</p><p></p>ple consisted of hemolymph pooled from three third instar larvae. Five squares were counted for each sample. Each genotype was analyzed in triplicate. B) Brightfield image of plasmatocytes from Trol-GFP stock. C) Fluorescence image of plasmatocytes from Trol-GFP stock demonstrating presence of Trol on plasmatocytes. D) Expression of the VEGF/PDGF response gene in and mutant hemocytes compared to sibling controls by qRT-PCR. Samples of hemolymph from three third instar larvae of each genotype were pooled, RNA extracted, amplified and analyzed. All reactions were carried out in triplicate at three different template concentrations to ensure amplification was in the linear range. βwas used as an internal normalization control

The Perlecan gene regulates multiple signaling pathways in different developmental contexts-3

<p><b>Copyright information:</b></p><p>Taken from "The Perlecan gene regulates multiple signaling pathways in different developmental contexts"</p><p>http://www.biomedcentral.com/1471-213X/7/121</p><p>BMC Developmental Biology 2007;7():121-121.</p><p>Published online 2 Nov 2007</p><p>PMCID:PMC2174950.</p><p></p> optic lobe neuroblasts, CNbs = central brain neuroblasts, TNbs = thoracic neuroblasts. In panels B-F, Anterior is to the left, Posterior is to the right. B) Optical section of Trol-GFP brains at first instar, brain surface, scale bar indicates 10 um for both panels B and C. C) Trol-GFP localization in first instar internal section. D) Trol-GFP localization in second instar internal section, scale bar indicates 15 um. E) Trol-GFP localization in third instar internal section, scale bar indicates 25 um. F) First instar brain stained with anti-Trol antibody, showing staining over the entire surface of the brain. Scale bar indicates 10 um. G) Trol-GFP localization in internal section of third instar salivary gland. Scale bar indicates 25 um

The Perlecan gene regulates multiple signaling pathways in different developmental contexts-5

<p><b>Copyright information:</b></p><p>Taken from "The Perlecan gene regulates multiple signaling pathways in different developmental contexts"</p><p>http://www.biomedcentral.com/1471-213X/7/121</p><p>BMC Developmental Biology 2007;7():121-121.</p><p>Published online 2 Nov 2007</p><p>PMCID:PMC2174950.</p><p></p>in the ventral ganglia of second instar larvae. Data for the Hh response gene is shown in Fig. 3. B) Expression levels of , its response gene , and its response gene , and its response gene in mutant third instar brain lobes/eye discs. C) Expression of and in second instar ventral ganglia and controls. D) Expression of and in second instar ventral ganglia and controls. In all panels, error bars indicate standard error. All reactions were carried out in triplicate at three different template concentrations to ensure amplification was in the linear range. βwas used as an internal normalization control