Identification of four Drosophila Toll-related proteins as ligands for the PTP69D receptor tyrosine phosphatase

The nine Toll-related receptors in Drosophila (Toll-1 – Toll-9) (Valanne et al. 2011) mediate a range of functions, ranging from embryonic development and immunity (Valanne et al. 2011) to larval locomotion, motor axon targeting and neuronal survival (McIlroy et al. 2013). Some members of the Toll family in Drosophila have been shown to bind to members of the Spaetzle family (Valanne et al. 2011; McIlroy et al. 2013; Ballard et al. 2014). Toll-6 and Toll-7 bind to Spz2 and Spz5 in a promiscuous manner (McIlroy et al. 2013). Toll-8 (also known as Tollo) has been shown to bind to Spz3 (Ballard et al. 2014). Our group has conducted several screens to identify ligands for Drosophila receptor-like protein tyrosine phosphatases (RPTPs). A deficiency screen identified Syndecan as a ligand for Lar (Fox and Zinn 2005), and a gain-of-function screen identified Stranded at second (Sas) as a ligand for PTP10D (Lee et al. 2013). Here we show that members of the Toll family are ligands for PTP69D, an RPTP expressed exclusively on CNS axons in the embryo. Extracellular domains (ECD) of cell surface and secreted proteins can be used to stain live embryos, and the observed binding patterns may represent the expression patterns of ligand(s) for these ECDs (Fox and Zinn 2005; Lee et al. 2013; Ozkan et al. 2013). Here, we used ECDs of Toll proteins fused to pentameric Alkaline Phosphatase (AP) to create AP fusion proteins (Ozkan et al. 2013). Live-dissected late stage 16 Drosophila embryos were incubated with these AP fusion proteins, using methods described in (Bali et al. 2016), to reveal in vivo binding patterns of Toll proteins in the Drosophila CNS. Wild-type embryos were incubated with Toll-2 (also known as 18w) AP, Toll-6 AP, Toll-7 AP and Toll-8 AP separately and immunostained for AP and FasII. Surprisingly, we observed a similar binding pattern for the four Toll proteins, suggesting common binding partners. All four Toll proteins showed binding to longitudinal CNS axons (A), and maximum staining intensity was observed at the junctions between the longitudinal and the commissural tracts (A, ai’ – aiv’, yellow arrowheads). These regions are where many synaptic connections between neurons projecting in the longitudinal and commissural tracts will later form. Binding of Toll proteins was also seen to a bundle of axons in the posterior commissure (A, ai’ – aiv’, white arrows). The anterior commissure was weakly labeled. Weak binding was also seen to motor axons as they leave the CNS. No binding was seen to muscles for any of the Toll proteins examined (data not shown). We had identified Toll-8 as a putative ligand for PTP69D in the gain-of-function embryo binding screen. In this screen, RPTP-AP proteins were used to stain embryos from crosses of a collection of ~300 lines with UAS-containing P elements upstream of cell surface protein genes to a pancellular GAL4 driver line (Lee et al. 2013). Since Toll-2, Toll-6 and Toll-7 showed a similar binding pattern to Toll-8 in wild-type embryos, we sought to examine whether they also bind to PTP69D. PTP69D was ectopically expressed in embryos by crossing tubulin-GAL4 to a line with an insertion of a UAS-containing P element in the 5’ end of the PTP69D gene. This conferred overexpression of PTP69D, especially in the CNS. Both wild-type embryos and embryos with ectopic PTP69D expression were incubated with Toll2-AP, Toll6-AP, Toll7-AP and Toll8-AP in separate experiments. In each case, we saw significantly increased binding of individual AP fusion proteins to the ectopically expressed PTP69D (B, compare bi to bi’, bii to bii’, biii to biii’ and biv to biv’). This shows that all four Toll proteins examined are able to bind to ectopically expressed PTP69D. Interestingly, although PTP69D expression was driven using a pancellular driver, we observed increased staining only on CNS axons. This suggests that the Toll proteins might be able to bind to PTP69D only when a cofactor expressed in the CNS is present. Alternatively (or in addition), PTP69D might only be able to localize to the cell surface on CNS axons. Here we also show Toll-8 binding to PTP69D in the reverse orientation, as in the gain-of-function screen. We ectopically expressed Toll-8 using tubulin-GAL4 and a line with a UAS-containing P element insertion upstream of the Toll-8 gene. We incubated both wild-type embryos and embryos with ectopic expression of Toll-8 with PTP69D-AP fusion protein and saw greatly increased binding of 69D-AP fusion protein to ectopically expressed Toll-8, both in the CNS and in the periphery. In the CNS, staining is observed only on axons and not on cell bodies, suggesting that Toll-8 localizes to axons, as does PTP69D. Thus Toll-8 and PTP69D bind to each other in vivo when either is over-expressed. Taken together, our results show that we have identified four Toll proteins that are likely to be novel ligands for PTP69D, either individually or as part of a larger complex

Identification of four Drosophila Toll-related proteins as ligands for the PTP69D receptor tyrosine phosphatase

The nine Toll-related receptors in Drosophila (Toll-1 – Toll-9) (Valanne et al. 2011) mediate a range of functions, ranging from embryonic development and immunity (Valanne et al. 2011) to larval locomotion, motor axon targeting and neuronal survival (McIlroy et al. 2013). Some members of the Toll family in Drosophila have been shown to bind to members of the Spaetzle family (Valanne et al. 2011; McIlroy et al. 2013; Ballard et al. 2014). Toll-6 and Toll-7 bind to Spz2 and Spz5 in a promiscuous manner (McIlroy et al. 2013). Toll-8 (also known as Tollo) has been shown to bind to Spz3 (Ballard et al. 2014). Our group has conducted several screens to identify ligands for Drosophila receptor-like protein tyrosine phosphatases (RPTPs). A deficiency screen identified Syndecan as a ligand for Lar (Fox and Zinn 2005), and a gain-of-function screen identified Stranded at second (Sas) as a ligand for PTP10D (Lee et al. 2013). Here we show that members of the Toll family are ligands for PTP69D, an RPTP expressed exclusively on CNS axons in the embryo. Extracellular domains (ECD) of cell surface and secreted proteins can be used to stain live embryos, and the observed binding patterns may represent the expression patterns of ligand(s) for these ECDs (Fox and Zinn 2005; Lee et al. 2013; Ozkan et al. 2013). Here, we used ECDs of Toll proteins fused to pentameric Alkaline Phosphatase (AP) to create AP fusion proteins (Ozkan et al. 2013). Live-dissected late stage 16 Drosophila embryos were incubated with these AP fusion proteins, using methods described in (Bali et al. 2016), to reveal in vivo binding patterns of Toll proteins in the Drosophila CNS. Wild-type embryos were incubated with Toll-2 (also known as 18w) AP, Toll-6 AP, Toll-7 AP and Toll-8 AP separately and immunostained for AP and FasII. Surprisingly, we observed a similar binding pattern for the four Toll proteins, suggesting common binding partners. All four Toll proteins showed binding to longitudinal CNS axons (A), and maximum staining intensity was observed at the junctions between the longitudinal and the commissural tracts (A, ai’ – aiv’, yellow arrowheads). These regions are where many synaptic connections between neurons projecting in the longitudinal and commissural tracts will later form. Binding of Toll proteins was also seen to a bundle of axons in the posterior commissure (A, ai’ – aiv’, white arrows). The anterior commissure was weakly labeled. Weak binding was also seen to motor axons as they leave the CNS. No binding was seen to muscles for any of the Toll proteins examined (data not shown). We had identified Toll-8 as a putative ligand for PTP69D in the gain-of-function embryo binding screen. In this screen, RPTP-AP proteins were used to stain embryos from crosses of a collection of ~300 lines with UAS-containing P elements upstream of cell surface protein genes to a pancellular GAL4 driver line (Lee et al. 2013). Since Toll-2, Toll-6 and Toll-7 showed a similar binding pattern to Toll-8 in wild-type embryos, we sought to examine whether they also bind to PTP69D. PTP69D was ectopically expressed in embryos by crossing tubulin-GAL4 to a line with an insertion of a UAS-containing P element in the 5’ end of the PTP69D gene. This conferred overexpression of PTP69D, especially in the CNS. Both wild-type embryos and embryos with ectopic PTP69D expression were incubated with Toll2-AP, Toll6-AP, Toll7-AP and Toll8-AP in separate experiments. In each case, we saw significantly increased binding of individual AP fusion proteins to the ectopically expressed PTP69D (B, compare bi to bi’, bii to bii’, biii to biii’ and biv to biv’). This shows that all four Toll proteins examined are able to bind to ectopically expressed PTP69D. Interestingly, although PTP69D expression was driven using a pancellular driver, we observed increased staining only on CNS axons. This suggests that the Toll proteins might be able to bind to PTP69D only when a cofactor expressed in the CNS is present. Alternatively (or in addition), PTP69D might only be able to localize to the cell surface on CNS axons. Here we also show Toll-8 binding to PTP69D in the reverse orientation, as in the gain-of-function screen. We ectopically expressed Toll-8 using tubulin-GAL4 and a line with a UAS-containing P element insertion upstream of the Toll-8 gene. We incubated both wild-type embryos and embryos with ectopic expression of Toll-8 with PTP69D-AP fusion protein and saw greatly increased binding of 69D-AP fusion protein to ectopically expressed Toll-8, both in the CNS and in the periphery. In the CNS, staining is observed only on axons and not on cell bodies, suggesting that Toll-8 localizes to axons, as does PTP69D. Thus Toll-8 and PTP69D bind to each other in vivo when either is over-expressed. Taken together, our results show that we have identified four Toll proteins that are likely to be novel ligands for PTP69D, either individually or as part of a larger complex

Sticks and Stones, a conserved cell surface ligand for the Type IIa RPTP Lar, regulates neural circuit wiring in Drosophila

Control of tyrosine phosphorylation is an essential element of many cellular processes, including proliferation, differentiation neurite outgrowth, and synaptogenesis. Receptor-like protein-tyrosine phosphatases (RPTPs) have cytoplasmic phosphatase domains and cell adhesion molecule (CAM)-like extracellular domains that interact with cell-surface ligands and/or co-receptors. We identified a new ligand for the Drosophila Lar RPTP, the immunoglobulin superfamily CAM Sticks and Stones (Sns). Lar is orthologous to the three Type IIa mammalian RPTPs, PTPRF (LAR), PTPRD (PTPδ), and PTPRS (PTPσ). Lar and Sns bind to each other in embryos and in vitro. The human Sns ortholog, Nephrin, binds to PTPRD and PTPRF. Genetic interaction studies show that Sns is essential to Lar′s functions in several developmental contexts in the larval and adult nervous systems. In the larval neuromuscular system, Lar and sns transheterozygotes (Lar/sns transhets) have synaptic defects like those seen in Lar mutants and Sns knockdown animals. Lar and Sns reporters are both expressed in motor neurons and not in muscles, so Lar and Sns likely act in cis (in the same neurons). Lar mutants and Lar/sns transhets have identical axon guidance defects in the larval mushroom body in which Kenyon cell axons fail to stop at the midline and do not branch. Pupal Kenyon cell axon guidance is similarly affected, resulting in adult mushroom body defects. Lar is expressed in larval and pupal Kenyon cells, but Sns is not, so Lar-Sns interactions in this system must be in trans (between neurons). Lastly, R7 photoreceptor axons in Lar mutants and Lar/sns transhets fail to innervate the correct M6 layer of the medulla in the optic lobe. Lar acts cell-autonomously in R7s, while Sns is only in lamina and medulla neurons that arborize near the R7 target layer. Therefore, the Lar-Sns interactions that control R7 targeting also occur in trans

Live Dissection of Drosophila Embryos: Streamlined Methods for Screening Mutant Collections by Antibody Staining

Drosophila embryos between stages 14 and 17 of embryonic development can be readily dissected to generate "fillet" preparations. In these preparations, the central nervous system runs down the middle, and is flanked by the body walls. Many different phenotypes have been examined using such preparations. In most cases, the fillets were generated by dissection of antibody-stained fixed whole-mount embryos. These "fixed dissections" have some disadvantages, however. They are time-consuming to execute, and it is difficult to sort mutant (GFP-negative) embryos from stocks in which mutations are maintained over GFP balancer chromosomes. Since 2002, our group has been conducting deficiency and ectopic expression screens to identify ligands for orphan receptors. In order to do this, we developed streamlined protocols for live embryo dissection and antibody staining of collections containing hundreds of balanced lines. We have concluded that it is considerably more efficient to examine phenotypes in large collections of stocks by live dissection than by fixed dissection. Using the protocol described here, a single trained individual can screen up to 10 lines per day for phenotypes, examining 4-7 mutant embryos from each line under a compound microscope. This allows the identification of mutations conferring subtle, low-penetrance phenotypes, since up to 70 hemisegments per line are scored at high magnification with a 40X water-immersion lens

Interactions between a Receptor Tyrosine Phosphatase and a Cell Surface Ligand Regulate Axon Guidance and Glial-Neuronal Communication

We developed a screening method for orphan receptor ligands, in which cell-surface proteins are expressed in Drosophila embryos from GAL4-dependent insertion lines and ligand candidates identified by the presence of ectopic staining with receptor fusion proteins. Stranded at second (Sas) binds to the receptor tyrosine phosphatase Ptp10D in embryos and in vitro. Sas and Ptp10D can interact in trans when expressed in cultured cells. Interactions between Sas and Ptp10D on longitudinal axons are required to prevent them from abnormally crossing the midline. Sas is expressed on both neurons and glia, whereas Ptp10D is restricted to CNS axons. We conducted epistasis experiments by overexpressing Sas in glia and examining how the resulting phenotypes are changed by removal of Ptp10D from neurons. We find that neuronal Ptp10D restrains signaling by overexpressed glial Sas, which would otherwise produce strong glial and axonal phenotypes