Visualization of binding patterns for five Leucine-rich repeat proteins in the Drosophila embryo

Leucine-rich repeat (LRR) domain-containing proteins play central roles in organizing neural connectivity. The LRR is a protein-recognition motif and proteins with extracellular LRR (eLRR) domains mediate intercellular communication and cell adhesion, which in turn regulate neuronal processes such as axon guidance, target selection, synapse formation and stabilization of connections (de Wit et al. 2011). The LRR-domain containing Slits and their Robo receptors are one of the best characterized examples of ligand-receptor pairs that regulate midline crossing and axon guidance in both Drosophila and vertebrates (Brose et al. 1999; Dickson and Gilestro 2006). There are 66 eLRR proteins in Drosophila, many of which are expressed in the nervous system and exhibit strikingly specific expression patterns, often labeling distinct subpopulations of neurons (Lauren et al. 2003; Dolan et al. 2007). The binding partners and functions of many of these eLRR proteins remain unknown. We have previously described a novel method to identify ligands and/or binding partners for extracellular proteins (Fox and Zinn 2005; Lee et al. 2013; Ozkan et al. 2013). This method involves using fusion proteins containing the extracellular domain (ECD) of a protein fused to a pentamerization domain (COMP), followed by human placental alkaline phosphatase (AP). These AP fusion proteins are used to stain live-dissected stage 16 Drosophila embryos. The resulting staining patterns can be used as a template to identify expression patterns of the binding partners of the AP fusion protein. Using this technique, we have identified ligands for the receptor tyrosine phosphatases Ptp10D, Lar and Ptp69D (Bali et al. 2019; Fox and Zinn 2005; Lee et al. 2013). Here, we describe novel binding patterns for 5 eLRR proteins using their respective AP fusion proteins. Tartan (trn) and Capricious (caps) are two closely-related eLRR proteins with known functions in embryonic motor axon guidance and the innervation of antennal lobe glomeruli by olfactory sensory axons (Kurusu et al. 2008; Hong et al. 2009). Studies of trn and caps single and double mutants suggest that the two genetically interact and may function through a common binding partner (Milan et al. 2005; Kurusu et al. 2008). Tartan may be a substrate for the receptor tyrosine phosphatase Ptp52F (Bugga et al. 2009). We stained wild-type live-dissected stage 16 Drosophila embryos with trn-AP and caps-AP fusion proteins separately, and found distinct as well as overlapping staining patterns for both fusion proteins. Both trn-AP and caps-AP bind to longitudinal axons in the ventral nerve cord (VNC), with stronger binding seen in one particular axon bundle close to the midline (arrows, a1’ and b2’). Both also show binding to muscles (arrows, a2’ and b3’), indicating that they interact with a binding partner expressed on the surface of muscles. trn-AP shows binding to a subset of sensory neurons (arrow, a3’), which caps-AP does not. In addition, caps-AP binds to the transverse nerve, which emanates from the midline and is located on the dorsal side of the VNC (arrow, b1’). Fish-lips (Fili) is an eLRR with roles in the regulation of apoptosis (Adachi-Yamada et al. 2005) and olfactory receptor neuron (ORN) targeting in the antennal lobe (Xie et al. 2019). It is expressed at moderately-high levels during embryonic stages 12 – 17 and during 24 – 48 hours after puparium formation (modENCODE Temporal Expression Data, FlyBase). These developmental stages correspond to peak synaptogenesis times, implying a developmental role of Fili in regulating synaptogenesis. Thus, identification of binding partners of Fili is crucial to understand its roles in CNS development. Staining of wild-type stage 16 embryos with Fili-AP fusion protein shows a restricted binding pattern in the CNS, indicating a similar restricted expression pattern of its binding partners. It binds to a set of dorsal midline neurons (arrow, c1’) and a subset of longitudinal axons in the VNC (arrow, c2’). A subset of midline cells, putatively glial cells are also labeled with Fili-AP. Strong binding is seen to the transverse nerve in the VNC (c1) and in the periphery (arrow, c3’), while no labeling is seen to the SNa in the same focal plane (arrow, c3). Reduced ocelli (rdo) is a gene that regulates ocelli development (Caldwell et al. 2007) and encodes an eLRR protein of unknown function. Caldwell et al. 2007 showed a broad expression pattern of the encoded protein in the adult nervous system. We performed staining of wild-type stage 16 embryos with rdo-AP fusion protein and found a very strong binding signal in the longitudinal and commissural axons of the VNC (arrow, d1’). This binding was limited to the VNC, and no binding was observed to the muscles (data not shown), indicating that the eLRR encoded by rdo interacts with neuronal-specific ligands. We also observed binding in a subset of midline glial cells in the VNC (arrow, d2’). 2mit is another gene encoding a putative eLRR and is expressed in the developing nervous system. It has a putative role in regulating short-term memory (Baggio et al. 2013). No other information is known about this eLRR. We stained wild-type stage 16 embryos with 2mit-AP fusion protein and saw a wide pattern of binding by this fusion protein, unlike the other restricted patterns observed above. Both longitudinal, commissural as well as exiting motor axons in the VNC are labeled by 2mit-AP (arrows, e1’). Moreover, we observed a pan-cellular pattern of labeling in the periphery as well as in the VNC, where 2-mit-AP binding signal is seen on the surface of cells, resulting in a cell-membrane staining pattern (arrow, e2’). This implies that the eLRR encoded by 2mit is capable of interacting with ligands expressed on neuronal as well as non-neuronal cell types. These binding patterns provide clues to the expression patterns of proteins that these eLRRs might interact with to regulate various developmental processes

Visualization of binding patterns for five Leucine-rich repeat proteins in the Drosophila embryo

Leucine-rich repeat (LRR) domain-containing proteins play central roles in organizing neural connectivity. The LRR is a protein-recognition motif and proteins with extracellular LRR (eLRR) domains mediate intercellular communication and cell adhesion, which in turn regulate neuronal processes such as axon guidance, target selection, synapse formation and stabilization of connections (de Wit et al. 2011). The LRR-domain containing Slits and their Robo receptors are one of the best characterized examples of ligand-receptor pairs that regulate midline crossing and axon guidance in both Drosophila and vertebrates (Brose et al. 1999; Dickson and Gilestro 2006). There are 66 eLRR proteins in Drosophila, many of which are expressed in the nervous system and exhibit strikingly specific expression patterns, often labeling distinct subpopulations of neurons (Lauren et al. 2003; Dolan et al. 2007). The binding partners and functions of many of these eLRR proteins remain unknown. We have previously described a novel method to identify ligands and/or binding partners for extracellular proteins (Fox and Zinn 2005; Lee et al. 2013; Ozkan et al. 2013). This method involves using fusion proteins containing the extracellular domain (ECD) of a protein fused to a pentamerization domain (COMP), followed by human placental alkaline phosphatase (AP). These AP fusion proteins are used to stain live-dissected stage 16 Drosophila embryos. The resulting staining patterns can be used as a template to identify expression patterns of the binding partners of the AP fusion protein. Using this technique, we have identified ligands for the receptor tyrosine phosphatases Ptp10D, Lar and Ptp69D (Bali et al. 2019; Fox and Zinn 2005; Lee et al. 2013). Here, we describe novel binding patterns for 5 eLRR proteins using their respective AP fusion proteins. Tartan (trn) and Capricious (caps) are two closely-related eLRR proteins with known functions in embryonic motor axon guidance and the innervation of antennal lobe glomeruli by olfactory sensory axons (Kurusu et al. 2008; Hong et al. 2009). Studies of trn and caps single and double mutants suggest that the two genetically interact and may function through a common binding partner (Milan et al. 2005; Kurusu et al. 2008). Tartan may be a substrate for the receptor tyrosine phosphatase Ptp52F (Bugga et al. 2009). We stained wild-type live-dissected stage 16 Drosophila embryos with trn-AP and caps-AP fusion proteins separately, and found distinct as well as overlapping staining patterns for both fusion proteins. Both trn-AP and caps-AP bind to longitudinal axons in the ventral nerve cord (VNC), with stronger binding seen in one particular axon bundle close to the midline (arrows, a1’ and b2’). Both also show binding to muscles (arrows, a2’ and b3’), indicating that they interact with a binding partner expressed on the surface of muscles. trn-AP shows binding to a subset of sensory neurons (arrow, a3’), which caps-AP does not. In addition, caps-AP binds to the transverse nerve, which emanates from the midline and is located on the dorsal side of the VNC (arrow, b1’). Fish-lips (Fili) is an eLRR with roles in the regulation of apoptosis (Adachi-Yamada et al. 2005) and olfactory receptor neuron (ORN) targeting in the antennal lobe (Xie et al. 2019). It is expressed at moderately-high levels during embryonic stages 12 – 17 and during 24 – 48 hours after puparium formation (modENCODE Temporal Expression Data, FlyBase). These developmental stages correspond to peak synaptogenesis times, implying a developmental role of Fili in regulating synaptogenesis. Thus, identification of binding partners of Fili is crucial to understand its roles in CNS development. Staining of wild-type stage 16 embryos with Fili-AP fusion protein shows a restricted binding pattern in the CNS, indicating a similar restricted expression pattern of its binding partners. It binds to a set of dorsal midline neurons (arrow, c1’) and a subset of longitudinal axons in the VNC (arrow, c2’). A subset of midline cells, putatively glial cells are also labeled with Fili-AP. Strong binding is seen to the transverse nerve in the VNC (c1) and in the periphery (arrow, c3’), while no labeling is seen to the SNa in the same focal plane (arrow, c3). Reduced ocelli (rdo) is a gene that regulates ocelli development (Caldwell et al. 2007) and encodes an eLRR protein of unknown function. Caldwell et al. 2007 showed a broad expression pattern of the encoded protein in the adult nervous system. We performed staining of wild-type stage 16 embryos with rdo-AP fusion protein and found a very strong binding signal in the longitudinal and commissural axons of the VNC (arrow, d1’). This binding was limited to the VNC, and no binding was observed to the muscles (data not shown), indicating that the eLRR encoded by rdo interacts with neuronal-specific ligands. We also observed binding in a subset of midline glial cells in the VNC (arrow, d2’). 2mit is another gene encoding a putative eLRR and is expressed in the developing nervous system. It has a putative role in regulating short-term memory (Baggio et al. 2013). No other information is known about this eLRR. We stained wild-type stage 16 embryos with 2mit-AP fusion protein and saw a wide pattern of binding by this fusion protein, unlike the other restricted patterns observed above. Both longitudinal, commissural as well as exiting motor axons in the VNC are labeled by 2mit-AP (arrows, e1’). Moreover, we observed a pan-cellular pattern of labeling in the periphery as well as in the VNC, where 2-mit-AP binding signal is seen on the surface of cells, resulting in a cell-membrane staining pattern (arrow, e2’). This implies that the eLRR encoded by 2mit is capable of interacting with ligands expressed on neuronal as well as non-neuronal cell types. These binding patterns provide clues to the expression patterns of proteins that these eLRRs might interact with to regulate various developmental processes

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

Cornell-SEWA-WIEGO 2008 Dialogue - Ahmedabad and Delhi Compendium of Personal and Technical Notes

Food Security and Poverty, International Development, International Relations/Trade,

INFORMAL SECTOR AND SOCIAL POLICY Compendium of Personal and Technical Reflections Cornell-SEWA-WIEGO Exposure and Dialogue Program Oaxaca, Mexico

Food Security and Poverty, International Development, International Relations/Trade,

Differential Responses of Progesterone Receptor Membrane Component-1 (Pgrmc1) and the Classical Progesterone Receptor (Pgr) to 17β-Estradiol and Progesterone in Hippocampal Subregions that Support Synaptic Remodeling and Neurogenesis

Progesterone (P4) and estradiol (E2) modulate neurogenesis and synaptic remodeling in the hippocampus during the rat estrous cycle and in response to deafferenting lesions, but little is known about the steroidal regulation of hippocampal progesterone receptors associated with these processes. We examined the neuronal expression of progesterone receptor membrane component-1 (Pgrmc1) and the classical progesterone receptor (Pgr), by in situ hybridization and immunohistochemistry. Pgr, a transcription factor, has been associated with synaptic remodeling and other major actions of P4, whereas Pgrmc1 is implicated in P4-dependent proliferation of adult neuroprogenitor cells and with rapid P4 effects on membranes. Ovariectomized adult rats were given E2, P4, or E2+P4 on two schedules: a 4-d model of the rodent estrous cycle and a 30-d model of postmenopausal hormone therapy. Pgr was hormonally responsive only in CA1 pyramidal neurons, and the induction of Pgr by E2 was partly antagonized by P4 only on the 30-d schedule. In CA3 pyramidal and dentate gyrus (DG) neurons, Pgr was largely unresponsive to all hormone treatments. In contrast to Pgr, Pgrmc1 was generally induced by E2 and/or P4 throughout the hippocampus in CA1, CA3, and DG neurons. In neuroprogenitor cells of the DG (immunopositive for bromodeoxyuridine and doublecortin), both Pgrmc1 and Pgr were detected. The differential regulation of hippocampal Pgrmc1 and Pgr by E2 and P4 may guide drug development in hormonal therapy for support of neurogenesis and synaptic regeneration.This work was supported by National Institute on Aging Grants 1PO1 AG026572 (to R.D.B.); Project 4 (to C.E.F. and T.E.M.), Animal Core A (to T.E.M.), and Analytic Core C (to L.Z.)

Cornell-SEWA-WIEGO 2008 Dialogue - Ahmedabad and Delhi Compendium of Personal and Technical Notes

WP 2008-15 August 200

THE CORNELL-SEWA-WIEGO Exposure and Dialogue Programme: An Overview of the Process and Main Outcomes

WP 2012-13 July 201