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. Author manuscript; available in PMC: 2016 Aug 27.
Published in final edited form as: Cell. 2015 Aug 27;162(5):1140–1154. doi: 10.1016/j.cell.2015.08.003

Slit and Receptor Tyrosine Phosphatase 69D Confer Spatial Specificity to Axon Branching via Dscam1

Dan Dascenco 1,2,†, Maria-Luise Erfurth 1,2,3,†, Azadeh Izadifar 1,2, Minmin Song 4, Sonja Sachse 1,2,5, Rachel Bortnick 6, Olivier Urwyler 1,2, Milan Petrovic 1,2, Derya Ayaz 1, Haihuai He 1, Yoshiaki Kise 1,2, Franziska Thomas 6, Thomas Kidd 4, Dietmar Schmucker 1,2,*
PMCID: PMC4699798  NIHMSID: NIHMS724683  PMID: 26317474

SUMMARY

Axonal branching contributes substantially to neuronal circuit complexity. Studies in Drosophila have shown that loss of Dscam1 receptor diversity can fully block axon branching in mechanosensory neurons. Here we report that cell-autonomous loss of the Receptor-Tyrosine-Phosphatase 69D (RPTP69D) and loss of midline-localized Slit inhibit formation of specific axon collaterals through modulation of Dscam1 activity. Genetic and biochemical data support a model in which direct binding of Slit to Dscam1 enhances the interaction of Dscam1 with RPTP69D, stimulating Dscam1 dephosphorylation. Single growth cone imaging reveals that Slit/RPTP69D are not required for general branch initiation, but instead promote the extension of specific axon collaterals. Hence, while regulation of intrinsic Dscam1-Dscam1 isoform interactions is essential for formation of all mechanosensory-axon branches, the local ligand-induced alterations of Dscam1 phosphorylation in distinct growth cone compartments enable the spatial specificity of axon collateral formation.

INTRODUCTION

The Drosophila Dscam1 receptor belongs to the immunoglobulin-superfamily (Ig-SF) of transmembrane proteins and is known to function as a homophilic surface receptor important for neuronal wiring (Hattori et al., 2008; Kise and Schmucker, 2013). Dscam1 is unique due to its extraordinary molecular diversity. Through alternative splicing thousands of structurally diverse receptor isoforms with different binding properties are generated (Schmucker et al., 2000; Wojtowicz et al., 2004), of which up to 18,496 are expressed in vivo (Sun et al., 2013). Loss of Dscam1 disrupts the proper development of many different neurons and in particular affects growth and patterning of neurite arborizations (Chen et al., 2006; Hughes et al., 2007; Hummel et al., 2003; Kim et al., 2013; Matthews et al., 2007; Soba et al., 2007; Wang et al., 2002).

An analysis of single mechanosensory neurons (ms-neurons) has revealed that Dscam1 diversity is essential for growth cones undergoing axonal sprouting as well as for axon growth and branch formation (Chen et al., 2006; He et al., 2014). Dscam1 activity is highly sensitive to the level of Dscam1-Dscam1 isoform interactions in a growth cone. For example, a cell-intrinsic alteration of the number of matching isoforms expressed in ms-neurons can lead to the loss of a subset or even all axon collaterals. The mechanistic basis, however, of how enhanced Dscam1 isoform interactions can block formation of axon collaterals is currently unknown. Moreover, it seems highly likely that axon branching of ms-neurons requires additional instructions by local extracellular signals, which may play an important role in controlling the specific targeting of each axon arbor. What these signals are, or how they influence branching, has not been determined.

In general, the question how branches of the same neuronal compartment (e.g. axon collaterals) can achieve growth towards different targets is still poorly understood and few in vivo studies have addressed this question(Hao et al., 2010; Liu and Halloran, 2005; McLaughlin, 2003). Moreover, some signals controlling branching, such as Semaphorins, Ephrins, or Slit, have been found to be multi-functional and can exert diverse or opposing effects on neurite branching(Liu and Halloran, 2005; Ma and Tessier-Lavigne, 2007; McLaughlin, 2003; Polleux et al., 2000). For example, the guidance cue Slit can function as a potent axon repellent by binding to Robo receptors present on growth cones (Brose et al., 1999a; Chedotal, 2007; Kidd et al., 1999) and can also contribute to axon branching, where it is thought to function as a “positive” factor stimulating axon branching. This was demonstrated by in vitro assays (Wang et al., 1999) where Slit2-N strongly enhanced the number of axonal branchpoints and total neurite length of sensory neurons. However, subsequent genetic studies revealed that the role of Slit in axon branching as more complex and multifunctional (Campbell et al., 2007; Ma and Tessier-Lavigne, 2007; Yeo et al., 2004). For example, loss-of-function of Slit in mice does not lead to less axon branching of sensory neurons of the dorsal root ganglion but results in a disruption of branch guidance consistent with a lack of repulsion. On the other hand, Slit mutants do show a reduction of the sensory field arborization of ophthalmic trigeminal neurons indicating a positive growth promoting activity during neurite arborization (Ma and Tessier-Lavigne, 2007). Taken together, it remains an intriguing question how Slit and other factors contribute to axon branching and exert such diverse roles in different neurons.

Here we present a genetic and biochemical characterization of how Dscam1 activity in growth cones can be regulated by differential phosphorylation through direct cis-interactions with the receptor tyrosine phosphatase RPTP69D. This modulation of Dscam1 signaling activity can be spatially restricted to a subset of axonal branches by the extracellular signal Slit, which functions as a ligand of Dscam1. This function of Slit is independent of Robo1-3 receptors and selectively promotes the consolidation and extension of midline-crossing axon collaterals. Overall, we propose a molecular and cellular model of how compartmentalized regulation of Dscam1 phosphorylation in growth cones contributes to divergent growth decisions underlying the formation of specific axon collateral projections.

RESULTS

RPTP69D promotes formation of specific axon collaterals

The afferent projections of adult ms-neurons such as pSC neurons connect with multiple post-synaptic targets in the CNS through the formation of axon collaterals (Ghysen, 1978; Urwyler et al., 2015). While the main axon shaft and several axonal branches only establish ipsilateral connections, other axon collaterals also project across the midline (Figures 1A-C). Previous work has shown that the neuronal receptor Dscam1 is essential for the formation of the elaborate axon branch pattern of ms-neurons (Chen et al., 2006) (Figure 1D). Importantly, ms-neuron specific increase of Dscam1 expression (Experimental Procedures, Figure S1A and S1B) and reduction of isoform diversity result in a loss of some or all axon collaterals while growth of the main axon shaft is unaffected (Figures 1E and S1C-F). For example, specific Dscam1 alleles where the isoform diversity is decreased show a dominant interference with axon collateral formation, often resulting in a loss of specific axon collaterals (Hattori et al., 2009; He et al., 2014) (Figure 1E). It is important to note that a decrease in isoform diversity concomitantly leads to an increase in the frequency of homotypic isoform interactions and we therefore refer to it as Dscam1 gain-of-function (GOF). Consistent with this notion we find that also ms-neuron specific over-expression of single Dscam1 isoforms in otherwise wild type (WT) ms-neurons leads to a reduction of collateral formation (Figure S1C-F).

Figure 1. RPTP69D and Dscam1 play opposite roles in midline-directed collateral formation.

Figure 1

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(A) Axonal branching of ms-neurons leads to a increase in network complexity by allowing a neuron to connect to different postsynaptic targets. (B) Stereotyped branching pattern of Drosophila ms-neurons. (C-I) Representative pSC neurons dye-filled with carbocyanine dyes. (C) WT pSC ms-neurons displaying the stereotyped branching pattern. (D) Dscam1 LOF phenotypes pSC neurons using MARCM lead to the formation of a “clump” of entangled processes. (E) Dscam1 GOF phenotypes resulting from a loss of isoform diversity. (F, G) RPTP69D LOF partially phenocopies Dscam GOF null clones (G) and RPTP69D strong hypomorph combinations (F). For allele designations and additional quantifications, see Figures S1K, T. (H, H’) RPTP69D1/RPTP69D10 and RPTP69D1/RPTP69D20 mutants display a characteristic absence of the anterior midline-crossing collateral. (I, I’, I”) Heterozygosity of Dscam1 in RPTP69D mutants suppresses RPTP69D branching defects. Dscam121/+; RPTP69D1/RPTP69D10 (I), Dscam121/+; RPTP69D1/RPTP69D20 (I’) and Dscam139/+; RPTP69D1/RPTP69D20 (I”). (J) Quantification of of ms-neurons defects (Chi-square test, **** P ≤ 0.0001). (K) Dscam1 heterozygosity in RPTP69D hypomorphic background also improves adult survival.

For all figures numbers between parentheses represent the numbers of neurons scored. Normal targets area of anterior midline-crossing collaterals are indicated by blue circles. Dotted yellow line indicates the midline. Scale bars are 50µm unless specified otherwise. See also Figures S1 and S2.

In a reverse genetic screen we discovered that selective loss of axon collaterals also occurs in ms-neurons with reduced RPTP69D function. RPTP69D has previously been shown to be important for guidance and targeting of motor as well as sensory neurons (Berger et al., 2008; Garrity et al., 1999; Hofmeyer and Treisman, 2009; Kurusu and Zinn, 2008). We found that RNAi-mediated knock-down (KD) of the receptor tyrosine phosphatase RPTP69D results in a selective loss of a distinct subset of axon collaterals (Figure S1G-J). To confirm this, we further analyzed ms-neuron projections in homozygous hypomorphic adults and in null-mutant clones using Mosaic Analysis with a Repressible Cell Marker (MARCM). In all loss-of-function (LOF) genotypes examined, we found that reduction or complete loss of RPTP69D led to a frequent and specific lack of axon collaterals, which normally grow across the midline (Figures 1F-H’ and S1G-O). The main axon shaft and other axon branches were not aberrant, with the exception of an infrequent (17%) shortening of the anterior ipsilateral branch (Figure S1I). Moreover, by examining MARCM clones or homozygous mutant animals of other receptor tyrosine phosphatases encoded in the fly genome (LAR, RPTP99A, RPTP4E and RPTP10D) (Johnson and Van Vactor, 2002), we found no phenotypic defects of axon collateral formation (Figure S1P-S) suggesting that RPTP69D serves a unique role in the axonal branching of ms-neurons.

We conclude that RPTP69D functions cell-autonomously in ms-neurons and is specifically required for formation of a subset of axon collaterals that cross the CNS midline.

RPTP69D is an inhibitor of Dscam1 function in vivo

Given the similarity of RPTP69D LOF and Dscam1 GOF phenotypes, we set out to determine potentially direct interactions of these receptors. In order to utilize genetic interaction studies we first characterized several hypomorphic non-lethal RPTP69D alleles that were described previously (Figure S1K-O, S1T) (Desai and Purdy, 2003). Combining hypomorphic and null (RPTP69D1) alleles we found that the anterior midline-crossing branch of pSC neurons was absent or truncated in over 70% of mutant flies. (Figures 1F, S1N, S1O, S1T, S1U). As expected, the frequency of phenotypic defects correlated with the strength of hypomorph combinations (Figures S1M, S1N, S1T). Strikingly, removal of only one copy of Dscam1 was sufficient to dominantly suppress these defects in RPTP69D mutants. Specifically, we found that the anterior midline crossing collaterals were intact in the majority of these samples (Figures 1I-J) and that the extent of suppression correlated with the Dscam1 allele strength (Figures 1I-J). As a control, removal of one copy of Robo1, did not affect the penetrance of the RPTP69D hypomorph phenotype (Figure 1J), suggesting that this suppression is specific to Dscam1.

We also tested reciprocal genetic combinations. Although all known hypomorphic allele combinations of Dscam1 already cause severe early axonal growth defects (Figure S2A-A’) (Zhan et al., 2004), we nevertheless found that reducing RPTP69D levels in a Dscam1 mutant background resulted in weak dominant suppression of Dscam1 defects (Figure S2A-E). In addition, the viability of either RPTP69D or Dscam1 homozygous mutant animals is strongly increased upon removal of one copy of Dscam1 or RPTP69D, respectively (Figures 1K and Figure S2F). Furthermore, strong genetic interactions of Dscam1 and RPTP69D are also evident when mushroom body development is examined (Figure S2G-L).

Taken together we show in multiple cellular and functional assays that Dscam1 and RPTP69D genetically interact. The apparent antagonistic functional relations suggest the possibility that RPTP69D is an important negative regulator of Dscam1 function.

RPTP69D phosphatase physically interacts with and directly dephosphorylates Dscam1

We next tested whether RPTP69D directly dephosphorylates Dscam1. We assayed Dscam1 phosphorylation in a cell culture system by engineering a chimeric Dscam1 receptor, “Met-Dscam1”, which can be activated by addition of a non-Drosophila ligand. We fused Dscam1 transmembrane and cytoplasmic tail (CT) (exons 17–24), with the extracellular domain of the mouse Met receptor (Figure 2A). This chimeric receptor can be activated by soluble hepatocyte growth factor (HGF) and provides an on/off trigger for assaying cytoplasmic Dscam1-signaling without interference from endogenous Dscam1.

Figure 2. Dscam1 is an in vitro substrate for RPTP69D.

Figure 2

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(A) Design of the chimeric Met-Dscam1 receptor. (B, C) HGF-mediated activation of Met-Dscam1 leads to an increase of its Y-phosphorylation in S2 or BG3C2 cells. (B) Met-Dscam1 expressed in S2 cells was immunoprecipitated and phosphorylation assessed by semiquantitative Western blot (WB) analysis. All measurements normalized to baseline phosphorylation (Summary of 6 experiments) (C) HGF mediated activation of Met-Dscam1 is stronger and lasts longer in BG3C2 cells than in S2 cells (n=6 for 10 min, n=4 for 20, 60, 90 and 120 min, n=5 for 30 min, n=3 for 40 min). (D) Met-Dscam1 IP from BG3C2 cells before and 30 minutes after HGF addition in the presence or absence RPTP69D shows that RPTP69D KD leads to increased baseline phosphorylation of Met-Dscam1. Top: Representative WB of one experiment. Bottom: Quantification of multiple experiments. (n=6; paired t-test: * P ≤ 0.05.) (E) RNAi tests show that RPTP69D is the only neuronal RPTP affecting Met-Dscam1 baseline phosphorylation (see also Figure S1). Top: Summary of multiple experiments for each dsRNA (n=7 for RPTP69D and Lar, n=2 for RPTP 4E, 10D and 52F). ANOVA/Dunnett: *** P ≤ 0.001. Bottom: Knockdown efficiency assessed by quantitative RT-PCR (n=6 for PTP69D, n=3 for Lar, RPTP10D, 4E and 52F. ANOVA/Dunnett: ** P ≤ 0.01; *** P ≤ 0.001). (F) Endogenous Dscam1 binds to substrate trapping mutants in BG3C2 cells. Top: Mutations introduced in the WPD loop of domain 1 (DA1), or 2 DA2 or both (DA12). Bottom: Co-IP of Dscam1 from S2 cells transiently expressing HA-tagged RPTP69D. Only DA1 and DA2 mutants are able to co-IP Dscam1 (G) Mutational analysis of RPTP69D phosphatase domains. Dscam1 IPs from S2 cells expressing V5-tagged RPTP69D WT and Y>F mutant proteins. Y-phosphorylation status evaluated by semiquantitative WB analysis. Top: WB of one representative experiment. Bottom: Summary of multiple experiments (n=6 for WT, n=4 for DA1 and DA2, n=2 for DA12; ANOVA/Dunnett: ** P ≤ 0.01; *** P ≤ 0.001). Error bars: SEM. See also Figure S3.

We found baseline Met-Dscam1 tyrosine phosphorylation (Y-phosphorylation) in S2 (hemocyte-like) and BG3C2 (neuronal) cell lines in the absence of HGF and a rapid increase of Y-phosphorylation following HGF addition (Figures 2B-D). Activation reached a peak after a few minutes in S2 cells, while the signal increased steadily for more than 30 minutes in BG3C2 cells (Figures 2B, 2C, S3A-A’). Moreover, consistent with previous cell culture studies (Muda et al., 2002; Purohit et al., 2012), we found that the Met-Dscam1 chimeric receptor is Y-phosphorylated by Src-family kinases (SFKs) (Figure S3B-B’) and leads to efficient recruitment of the SH2-domain containing adaptor Dock (Figure S3C-C’).

In order to examine which RPTP contributes to Dscam1 dephosphorylation we combined chimeric receptor expression with RNAi-mediated KD of other RPTPs. Strikingly, we found that of the six known Drosophila RPTPs only RPTP69D reduced Met-Dscam1 baseline phosphorylation (Figure 2E).

Next we examined whether Dscam1 is a direct substrate of RPTP69D. It is known that RPTPs often interact very transiently with their substrates and that enzyme-substrate binding is strongly stabilized by mutating specific residues of the so-called “WPD” loop in the catalytic domains. This approach is referred to as “substrate-trapping” (Blanchetot et al., 2005; Flint et al., 1997). RPTP69D has two phosphatase domains (D1, D2) and we therefore engineered HA-tagged RPTP69D D>A substrate-trap constructs with mutations in either one or both phosphatase domains (DA1, DA2, DA12) (Figures 2F, Figure S3D). Transient expression and immunoprecipitations (IPs) in BG3C2 cells showed that substrate-trapping mutants but not WT RPTP69D bound to endogenous Dscam1 (Figure 2F).

If RPTP69D inhibits Dscam1 signaling through dephosphorylation, then overexpression of RPTP69D should lead to reduction of Y-phosphorylation of Dscam1. We confirmed this by examining Y-phosphorylation of endogenous Dscam1 in BG3C2 cells transiently overexpressing V5-tagged RPTP69D. We found that overexpression of RPTP69D caused a strong reduction of phosphorylation of endogenous Dscam1 (Figure 2G).

These results suggest that RPTP69D can directly dephosphorylate Dscam1 and that endogenous RPTP69D-Dscam1 interactions are normally transient.

Constitutive binding of RPTP69D substrate-trap mutant to Dscam1 phenocopies Dscam1 LOF

For RPTPs bearing two phosphatase domains it is thought that only one domain is catalytically active, whereas the second domain contributes to substrate specificity (Tonks, 2006). Consistent with this, we found that mutations in D1 and D2 of RPTP69D cause strikingly different effects. The RPTP69D-DA1 mutant failed to reduce Dscam1 CT phosphorylation levels suggesting that D1 is the main catalytically active domain (Figure 2G) (Marlo and Desai; 2006). In contrast, the expression of the RPTP69D-DA2 mutant resulted in a pronounced reduction of Dscam1 phosphorylation levels (stronger than WT-RPTP69D) and appears to be toxic for cells. This hyper-dephosphorylation was eliminated when both catalytic domains (RPTP69D-DA12) were mutated (Figure 2G). These results suggest that the RPTP69D-DA2 mutant blocks the dissociation from Dscam1 thereby enhancing dephosphorylation by the catalytically intact D1 domain.

We reasoned that the RPTP69D-DA12 double mutant protein might interfere dominantly with Dscam1 function in vivo. Therefore, we expressed RPTP69D-DA12 in pSC neurons and examined for direct comparison ms-neuron specific KD of Dscam1 expression (Figure 3 C-C’). We indeed found that expression of the RPTP69D-DA12 mutant protein caused strong dominant defects in ms-axon projections almost identical to Dscam1 LOF defects (Figure 3). In contrast, expression of a WT form of RPTP69D had no effect on the branching pattern of ms-neurons (Figure 3D).

Figure 3. Expression of a RPTP69D substrate-trap mutant phenocopies Dscam1 LOF.

Figure 3

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(A-F’) Representative dye-fills of pSC neurons (B) Defects in Dscam39/Dscam47 reveal “clump” formation (arrowheads) and longitudinal axon growth defects (asterisks). (C-C’) RNAi knock-down of Dscam1 results in “clumps” (arrowheads) and longitudinal axon growth defects (asterisks), higher magnification in (C’). (D-F’) Expression of RPTP69DA12 mutant in ms-neurons phenocopies Dscam1 LOF defects. Expression of UAS-RPTP69D-WT (D) and UAS-RPTP69D-DA12 (E-F’) in ms-neurons using pnr-Gal4. Higher magnification (F’).

These results strongly suggest that the D1 domain of RPTP69D is essential for Dscam1 dephosphorylation and that Dscam1 can serve as a direct substrate of RPTP69D, both in vitro and in vivo.

RPTP69D dephosphorylates specific tyrosines of the cytoplasmic domain of Dscam1

We set out to identify which of the 22 tyrosines of the cytoplasmic domain of Dscam1 are RPTP69D substrates (Figures 4A and S4A). We tested tyrosine to phenylalanine mutations (Y>F mutations) in the chimeric Met-Dscam1 receptor assay and found that five tyrosine mutations (Y1707F, Y1857F, Y1890F, Y1911F and Y1981F) significantly reduced Met-Dscam1 baseline phosphorylation (Figure 4B, 4C, S4B) while the Y1857F and Y1890F mutations also showed strong reduction of ligand-induced Met-Dscam1 activation (Figure 4B, 4C). Further sequence motif analysis suggests that Y1857, Y1890, and Y1911, are potential RPTP substrate sites (Figure 4D, S4D) (Ren et al., 2011; Selner et al., 2014; Tonks, 2013).

Figure 4. RPTP69D targets several tyrosine residues of Dscam1 CT.

Figure 4

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(A) Position of 15 conserved tyrosines in Dscam1 IC (in invertebrates). (B-C) Five Y>F mutations lead to significant decrease of baseline Y-phosphorylation. (B) IP of WT and Met-Dscam1 Y>F mutants before and after HGF addition. Semiquantitative WB analysis (C) Summary of multiple experiments. Mean values compared to the baseline phosphorylation of WT Met-Dscam1. Baseline phosphorylation is reduced in all five Y>F mutants, but significant reduction in response to HGF is only observed for mutants in SH2-binding sites (Y1857 and Y1890). (n=5 for YF1707 and YF1911; n=4 for YF1857; n=3 for YF1890 and YF1981; ANOVA/Dunnett to compare the phosphorylation state of Met-Dscam1 in presence and absence of HGF: * or ° P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001). (D) Sequence alignment of the 5 candidate RPTP69D and known PTP tyrosine substrate residues (Selner et al. 2014). Substrate tyrosines (magenta). Acidic or large hydrophobic residues (green). (E, F) RPTP69D interacts with at least two tyrosines of Dscam1 IC. (E) An overexpression-based phosphatase assay to examine RPTP69D-mediated dephosphorylation of Dscam1. Top: Representative WB of the IP of WT Dscam1-HA from S2 cells in the presence and absence of WT RPTP69D-V5. Bottom: Quantification of phosphorylation of mutant Dscam1-HA proteins. Co-expression of RPTP69D significantly reduces the Y-phosphorylation of WT Dscam1-HA as well as Y1707, Y1857 and Y1911 Dscam1-HA mutants. Y-phosphorylation is abolished in YF1890 and YF1981 mutants. Bars represent the mean of several experiments normalized to the baseline phosphorylation of a given construct (in absence of RPTP69D). (n=3 for YF1707; n=11 for YF1857; n=7 for YF1890 and YF1981; n=4 for YF1911; n=6 for WT; paired t-tests: * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001) (F) Representative WBs showing single experiments of the overexpression based phosphatase assay described in (E) Each lane represents data from one gel with lanes from other YF mutations omitted. Error bars: SEM. See also Figure S4.

We also examined Y>F mutations in the context of a full-length HA-tagged Dscam1 isoform (Figure S4C). We reasoned that the dephosphorylation of Dscam1 proteins caused by RPTP69D co-expression should at least be partially diminished in mutant Dscam1 proteins lacking substrate-tyrosines. Co-transfection of RPTP69D with a WT full-length Dscam1 cDNA reduced the Dscam1 Y-phosphorylation levels by approximately 70% (Figures 4E-F, S4C). In contrast, RPTP69D-dependent dephosphorylation of Dscam1 was fully blocked as a result of the Y1890F and Y1981F mutations, and partly blocked for the Y1857F mutation (Figures 4E-F). Taken together, we identified three tyrosines (Y1857, Y1890 and Y1981) in the Dscam1 cytoplasmic domain, which can serve as substrates for RPTP69D-mediated de-phosphorylation. In the following we refer to these sites as “RPTP69D target sites”.

RPTP69D target sites can regulate Dscam1 activity in vivo

We sought to determine whether these potential RPTP69D target sites also play a role for Dscam1 function in vivo. Previous studies have shown that cell-intrinsic UAS-based over-expression of WT Dscam1 can dominantly interfere with branching of ms-neurons (He et. al. 2014). We, therefore, reasoned that these GOF phenotypes might provide a sensitive read out to assay the influence of regulatory mutations on Dscam1 activity in vivo (Figure 5).

Figure 5. The RPTP69D-target sites are important for Dscam1 function in vivo.

Figure 5

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(A-E) Representative confocal images of pDC dye-fills (A, A’) WT pDC neurons have a stereotypical branching pattern similar to pSC neurons. (B-E) Over-expression of WT or mutated Dscam1 isoforms in single pDC neurons (see Figure S1A) results in branching defects. While expression of a WT Dscam1 leads to truncation or shortening of the anterior midline-crossing collateral (B,B’), expression of the Y>F1857 Dscam1 mutant does not (C) or only weakly (C’). Expression of the Y>F1890 Dscam1 mutant (D, D’) results in weak defects(D’). Expression of the Y>F1981 Dscam1 mutant leads to dominant defects that affects all axon collaterals (E,E’). (F) Schematics of the different Dscam1 mutants. (G) Quantification of the phenotypic defects. Statistics: Chi-Square, *** p<0.001.

For each of the three RPTP69D target sites we generated Y>F substitutions in the context of full-length Dscam1 UAS-controlled transgenes and expressed them in ms-neurons (Figure 5F). Expression of WT Dscam1 in ms-neurons neurons leads to frequent aberrant ipsilateral or midline stop of the anterior axon collaterals (Figure 5B-5B’, 5G, for genotypes see SI). The Dscam1 GOF defects were strongly suppressed by the Y1857F mutation (Figures 5C-C’) and weakly by the Y1890F mutation (Figures 5D-5D’, 5G). In contrast, expression of the Y1981F mutant protein resulted in dominant branching defects significantly stronger than expression of the WT Dscam1 protein (Figures 5E-5E’, 5G). We observed complete absence of anterior midline collaterals in approximately 40%, and absence of all branches in over 30% of ms-axons, suggesting a strongly enhanced Dscam1 activity. It is intriguing that the expression of a Dscam1-Y1981F mutant results in a phenotype qualitatively indistinguishable from Dscam1 alleles where isoform diversity is reduced (Figure 1E) (He et al. 2014).

In summary, these results suggest that the Y1857F and less so the Y1890F mutations reduce Dscam1 GOF while the Y1981F mutation strongly enhances Dscam1 GOF activity.

Slit enhances Dscam1-RPTP69D interactions and is important for axon collateral formation

While LOF and GOF alleles of Dscam1 can affect the formation of all branches of ms-neurons (He et al., 2014), the loss of RPTP69D primarily affects the formation of contralateral axon branches. We hypothesized that the apparent spatial specificity of the RPTP69D phenotype is either due to differential subcellular localization of the RPTP69D receptor itself, or a compartmentalized control of RPTP69D-Dscam1 interactions.

However, over-expression of RPTP69D throughout the growth cone and axon did not result in GOF defects (Figure 3D). The localization of an over-expressed HA-tagged RPTP69D was examined by anti-HA staining and was found to be uniformly distributed and present in growth cones as well as all axon branches (data not shown). These findings suggest that the subcellular restriction of RPTP69D protein is unlikely to account for the mechanism of promoting specific axon collateral formation. Alternatively, the branch specific functions could arise from local interactions with extracellular cues and ligands activating or recruiting RPTP69D. A previous genetic analysis has uncovered that the secreted guidance cue Slit influences RPTP69D function in the embryonic nervous system (Sun et al., 2000). Consistent with this, we found that mutations in Slit cause a frequent truncation or lack of the midline crossing axon collateral of both pSC or pDC neurons (Figures 6B, 6C, 6F, and S5A). As Slit null mutations are lethal, we used viable transallelic combinations (Tayler et al., 2004). We found that in Slidui/Slidui and Sli2/Slidui, midline crossing axon branches are absent in 35% to 60% of adults respectively, and closely phenocopy RPTP69D defects (Figures 6B, 6C, 6F, S5A, S5D). Moreover, Slit mutants show dominant genetic interactions with both RPTP69D and Dscam1 in ms-neurons (Figures 6D-F, Figure S5B-D). In addition, increasing and broadening the expression of Slit in the VNC using the pan-glial Gal4-transactivator repo-Gal4 resulted in a GOF phenotype with an increase of ectopic midline-crossing axonal branches (Figure S5G, H).

Figure 6. Slit is required for midline collateral formation and enhances RPTP69D-Dscam complex formation by binding to Dscam1.

Figure 6

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(A-E) Representative confocal images of pSC dye-fills (B, C) Reduction of Slit expression in Slidui/Slidui (B) or Sli2/Slidui (C) mutants results in defects similar to those observed in RPTP69D mutants. Removal of one copy or RPTP69D enhances the penetrance of midline collateral defects in Sli2/Slidui flies (D). Removal of one copy of Dscam1 reduces the defects seen in Slidui/Slidui flies (E). (F) Quantifications of the genetic interactions.(Statistics: Chi-Square test, * P ≤ 0.05; ** P ≤ 0.01, *** P < 0.001). (G) Slit is expressed at the VNC midline during pupal development. Expression is strongly reduced in Slidui/Slidui flies. (H, I) MARCM clones for Robo1 and Robo2 in ms-neurons show that neither Robo1 nor Robo2 LOF affects branching of ms-neurons (for Robo3, see Figure S7F). Dscam1 binding was detected by WB analysis. (J, K) Schematic representation of the protein expression constructs (J). Top: Alkaline phosphatase (AP) was fused to the Slit-N fragment consisting of the N-terminal 4 LRR-repeats (boxes) and the 5 EGF-like domains (circles) and expressed by baculovirus mediated infection of High Five cells (see Experimental Procedures). The optimal multiplicity of infection (MOI, pfu/cell) was found to be 10. Bottom: A Dscam1 EC4 construct consisting of the N-terminal 4 Dscam1-IG domains fused to Fc tag. (K) Representative binding curve for AP-Slit-N and EC4. Five independent experiments with an average dissociation constant (Kd) for Slit-N/Dscam1-EC4 binding of 22.2 ± 2.85 nM. (L) Slit promotes the formation of a Dscam1-RPTP69D complex. Co-IP of Dscam1 and RPTP69D from a stable cell line shows small amounts of Dscam1-RPTP69D complex, which is significantly increased when cells were incubated with Slit-conditioned medium (Experimental Procedures). Complexes were assessed by semiquantitative WB analysis. Top: Representative WB of one experiment. Bottom: Quantification of multiple independent experiments (n=3 experiments; paired t-test: * P ≤ 0.05). (M) Slit incubation leads to Dscam1 dephosphorylation in S2 cells. Endogenous Dscam1 was immunoprecipitated from S2 cells incubated with or without Slit. Phosphorylation levels were evaluated by semiquantitative WB analysis. Top: representative WB from one experiment. Bottom: Quantification of multiple independent experiments (n=5 experiments; paired t-test: * P ≤ 0.05). (N) Slit and the extracellular domain of Dscam1 can physically interact in vitro. IP of Slit and Dscam1 extracellular domain (EC10) from conditioned S2 cell media (Experimental Procedures). Error bars: SEM. See also Figures S5 and S6.

Slit is a well-known ligand for the Robo family of axon guidance receptors (Brose et al., 1999b; Evans and Bashaw, 2010; Kidd et al., 1999; Spitzweck et al., 2010) and is expressed at the pupal VNC midline (Figure 6G, Figure S5E-F). However, clonal analysis of null alleles of Robo1 and Robo2 (Figures 6H, 6I, S6A, S6C), as well as Robo3 (Figure S6F), did not reveal any defects of ms-neuron projections. Moreover, ms-neuron specific KD of each Robo family member or Robo1 and Robo2 together did not reveal defects in ms-neurons (Figure S6B, S6D, S6E and S6G). Furthermore, anti-Robo1 and anti-Robo3 antibody stainings suggest that these receptors are not expressed in ms-neurons (Figure S6H-K). These results confirm a role of Slit for the formation of specific axon collaterals and show that this function of Slit is Robo-independent.

Slit enhances RPTP69D-Dscam1 interactions and can directly bind Dscam1

We next examined whether Slit might be directly modulating RPTP69D-dependent regulation of Dscam1 signaling. We found that Slit-conditioned medium enhanced the complex formation of Dscam1 with RPTP69D (Figure 6L). An enhanced Dscam1-RPTP69D interaction was also observed with purified Slit protein (n=3, Figure S5I). In order to determine if the observed increase of Dscam1-RPTP69D complex formation had an effect on Dscam1 phosphorylation, we incubated S2 cells with Slit-conditioned supernatant and found that this led to a significant decrease in Dscam1 phosphorylation levels (~37% on average, peak value 65% reduction; n=5, Figure 6M).

Importantly, recent studies on Slit processing and receptor binding suggested that an N-terminal fragment of Slit (Slit-N) might directly bind to the N-terminal Ig-domains of Dscam1 (M.S.; T.K., data not shown). We therefore directly measured the binding affinity of a purified N-terminal fragment of Dscam1 and Slit-N by using a standard alkaline phosphatase-based enzymatic assay (Experimental Procedures). We used purified protein of the first four Ig-domains of Dscam1 (Dscam-EC4) fused to the antibody constant region (Fc) domain (Wojtowicz et al., 2004) and Slit-N fused to alkaline phosphatase (AP-Slit-N) (Figures 6J, 6K). From five independent experiments, we determined an average dissociation constant Kd of 22.2 ± 2.85 nM for Slit-N/Dscam-EC4 binding. In addition, co-IP experiments from S2 cell supernatant expressing a secreted form of Dscam1 (DscamEC10; Wojtowicz et al., 2004) revealed that also full-length Slit protein was able to physically interact with the extracellular domain of Dscam1 (Figure 6N).

In summary, the biochemical data suggest that Slit can directly bind Dscam1 and enhance RPTP69D-Dscam1 interactions as well as Dscam1 dephosphorylation.

RPTP69D and Slit are specifically required for axon collateral selection and extension

In order to investigate the developmental mechanism by which RPTP69D and Slit regulate axon collateral formation, we employed a genetic labeling strategy (see SI), (Figure S1A, He et al. 2014) to visualize single pSC growth cones during development (Figure 7). During early pupal development pSC growth cones undergo a highly dynamic sequence of morphological transitions lasting about 12–15 hours. During an early phase (“sprouting phase”), many different processes extend from a highly complex growth cone into all directions (Figure 7A). While some processes are very thin filopodia-like, others are thicker and exhibit their own side branches (e.g. dotted circles in Figure 7A, 7E, 7I). In analogy to what has been described for sprouting sensory neurites in amphibian embryos (Roberts and Taylor, 1983) we refer to these processes as “micropodia”. Comparing WT with RPTP69D or Slit mutant growth cones, we were unable to find any morphological differences between sprouting growth cones, neither in overall shape, extent of sprouting, or presence of filopodia or micropodia (Figures 7A, 7E, 7I).

Figure 7. RPTP69D and Slit are required for specific axon branch consolidation and extension but not branch initiation.

Figure 7

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(A-L) Single Growth cone analysis. Representative confocal images of single pSC axons expressing GFP under a pSC-specific Gal4 driver, at early (first column), intermediate (second and third columns) and later stages (last column). All samples were collected between 23h and 30h after puparium formation (APF). Midline-position indicated by yellow squares. Stochastic ectopic GFP expression in unrelated neurons/axons occurs occasionally and is indicated by asterisks. WT pSC axons (A) as well as RPTP69D (E) and Slit (I) mutant growth cones are highly complex, displaying a great number of filopodia and micropodia processes (circles) during early stages when the growth cones start to expand and sprout. (B, C). In intermediate stages, a separation into a posterior and anterior axon branch compartment is recognizable (B, C, F, G, J, K). Numerous micropodia can be observed, some of which show small globular tips (satellite growth cones) and secondary short filopodia (arrowheads in B,C,F,K). A subset of micropodia are midline-directed (arrows, B, C, D) and form stable axon branches (white arrow C, D). In WT, sometimes two anterior midline-directed collateral branches are present (yellow and white arrows in C,D), but only one is stabilized subsequently. The axons of RPTP69D (E-H) and Slit (I-L) mutants are still able to form many processes early (E, I), but at intermediate stages, midline-directed projections of micropodia with satellite growth cones are shorter and stunted (yellow arrows in G, K). An impairment of the ability to extend towards the midline persists into later stages (H, L, open arrowheads). In most cases mis-directed (H) or stunted (L) micropodia fail to mature into an anterior midline-directed collateral projection. (M) Quantification of the total number of processes (filopodia and micropodia) at different developmental stages in control, RPTP69D and Slit mutants. No significant differences in total process numbers between control and RPTP69D or Slit mutant axons (M). (N, O) Quantifications of midline-directed micropodia with satellite growth cones (intermediate and late stages). Reduced number of midline-directed collateral growth cones in RPTP69D and Slit mutant axons. Maximum length of the main shaft was used assign samples to three stage groups: early: <30µm, intermediate: 30 and 60 µm, and late: >90 µm. (Statistics: One-Way ANOVA followed by Dunnett multiple comparisons, ** P ≤ 0.005; *** P ≤ 0.001 (M, N) Fisher’s exact test, * P ≤ 0.05 (O)). (P) Schematic of the defects observed in RPTP69D and Slit mutant growth cones: while processes are still formed, their midline-directed extension and consolidation is impaired. Scale bars represent 25µm. Error bars: SEM. See also Figure S7.

During an intermediate phase (Figures 7B, 7C, 7F, 7G, 7J, 7K) two primary axon branches become apparent segregating in opposite (anterior-posterior) directions. At this stage some micropodia appear to have small distinct growth cone-like tips, which we refer to as “satellite growth cones” (e.g. arrowheads in Figures 7B, 7C, 7F, 7K). Satellite growth cones are present in WT as well as mutant samples. Importantly, however, in WT micropodia projecting towards the midline can be detected in most samples (e.g. arrows in Figures 7B, 7C). In contrast, in RPTP69D or Slit mutant animals distinct midline directed micropodia are rare and only very short (Figures 7G, 7K). Quantifications of midline-directed micropodia and total number of processes confirm that mutant pSC axons reveal a specific inhibition of midline-directed collaterals (Figures 7M-O).

In later stages, the midline-projecting axon collateral become more prominent and grow contralaterally. During this phase micropodia start disappearing, while filopodia remain concentrated at branching points and distal parts of extending axon branches (Figure 7D). At this stage most mutant axons exhibit only short processes or stubs (Figures 7H, 7L) that are present at axon segments where in WT a prominent commissural axon collateral has formed (Figures 7D).

In summary, the single growth cone analysis suggests that Slit and RPTP69D are unlikely influencing axon sprouting, or branch initiation of ms-neurons, but rather control the selective consolidation and directed extension of midline-crossing axon branches.

DISCUSSION

This study reports on a molecular mechanism regulating Dscam1 activity in growth cones providing insight in the regulation and spatial specificity of axon collateral formation. Biochemical and genetic results are consistent with the molecular model that the specificity of ms-axon branching arises from a spatially restricted change of Dscam1 phosphorylation in growth cones.

Dscam1-Dscam1 interactions can be modulated by extracellular cues

Previous studies on the function of Dscam1 have established the model that isoform specific homophilic Dscam1-Dscam1 interactions trigger repulsion between sister dendrites (Hughes et al., 2007; Matthews et al., 2007; Soba et al., 2007). This controls for regular spacing of sister-dendrites in a process termed neurite self-avoidance. In addition, cell-intrinsic and isoform-specific interactions have also been shown to be important in sensory axons for growth cone sprouting and branching (He et al., 2014). Importantly, for both of these functions it is thought that Dscam1 signaling is primarily dependent on and initiated by homophilic binding of matching isoforms present on sister neurites (Hattori et al., 2009; He et al., 2014; Wojtowicz et al., 2007). The results reported here provide evidence that Dscam1-Dscam1 interactions in axonal growth cones are subject to branch-specific modulation by extrinsic cues. Binding of the ligand Slit to Dscam1 can locally enhance cis-interactions with the receptor tyrosine phosphatase RPTP69D as well as the dephosphorylation of Dscam1. While homophilic Dscam1 interactions can be considered playing an initial permissive role in all neurite-neurite interactions in a sprouting growth cone, the spatial restriction of an extrinsic Dscam1 ligand likely initiates functional disparity of Dscam1 signaling across different growth cone compartments.

Dscam1 is a RPTP69D substrate and specific phosphorylation events regulate Dscam1 activity in vivo

The biochemical data support the notion that RPTP69D directly dephosphorylates Dscam1 at specific cytoplasmic tyrosines. We identified three candidate tyrosines for the regulation of Dscam1 phosphorylation: Y1857, Y1890, Y1981. Two of the tyrosine residues, Y1857 and Y1890, are part of consensus SH2 binding sites and therefore likely involved in regulating recruitment of SH2-domain containing adapter molecules. Given that these mutations diminish the Dscam1 GOF effects, it seems reasonable to speculate that they are required for downstream signaling and/or receptor turn-over or trafficking. Surprisingly, the single Y1981F mutation causes strong dominant interference with axon branching where the phenotypic effects are qualitatively indistinguishable from a loss of Dscam1 isoform diversity (He et al. 2014), which is thought to increase the probability of matching isoform interactions (i.e. GOF activity). The primary amino acid sequence surrounding Y1981 does not reveal any distinct signaling motif. However, in silico 3-D protein modeling based on structural predictions suggests that phosphorylation of Y1981 could directly result in structural changes of the Dscam1 cytoplasmic domain and thereby influence Dscam1 activity (M-L. E. and D.S., data not shown).

Slit is a ligand for Dscam1 in a Robo1-3 independent pathway

Biochemical results suggest that Slit can enhance Dscam1-RPTP69D complex formation and Dscam1 de-phosphorylation. Furthermore, Slit-N can directly bind to the N-terminal Ig-domains of Dscam1 (Ig1-4) with an affinity comparable to other guidance cue/receptor interactions, suggesting that Slit-N can function as a bona fide Dscam1 ligand. Numerous studies have shown that the repellent as well as the branch-promoting function of vertebrate Slit require the function of Robo receptors (Brose et al., 1999b; Ma and Tessier-Lavigne, 2007; Wang et al., 1999). Our results show that for the formation of specific axon collaterals of Drosophila ms-neurons Slit functions via Dscam1 in a Robo1-3 independent pathway.

Slit is one of the best-characterized “axon repellent” cues and but also contributes to axon branching (Chedotal, 2007). Imaging single ms-axons and growth cone branching, we find that in Slit mutant animals only filopodia or micropodia with a midline-directed growth direction are reduced consistent with a positive role of Slit in promoting the extension of specific branches. In contrast, branch point initiation in ms-neurons is likely independent of Slit or RPTP69D.

Slit drives spatial specificity of Dscam1-RPTP69D interactions

Given that high Slit protein concentrations are likely only encountered by filopodia- or micropodia-like extensions that reach the midline proximity, the Slit-Dscam1-RPTP69D interactions are likey only occuring in a spatially restricted sub-compartment of the branching growth cone. We envision that the Dscam1-RPTP69D interactions in ms-axons constitute a molecular selection process, which depends on Dscam1-RPTP69D complex formation in a subset of axonal processes that encounter sufficient Slit protein (Figure S7). As a result, Dscam1 de-phosphorylation by RPTP69D is increased locally and triggers a response by either promoting axon branch extension or blocking repulsion.

The loss of only a subset of axon branches in RPTP69D/Slit mutants suggests that there are multiple molecular control pathways accounting for the selection of different axon collaterals or the extension of the main axon shaft. Although this study has focused on RPTP69D and Slit, it is most likely that other co-receptors and extracellular cues control the activity of Dscam1 in growth cones.

EXPERIMENTAL PROCEDURES

Cell culture

S2 cells, Slit-expressing cells and BG3C2 cells were obtained from DGRC. S2 cells were grown in SF-900 Insect Medium (Life Technologies) and BG3C2 cells in Schneiders medium (Life Technologies) (10% FBS (Life Technologies), BPYE (Difco) and Insulin (10mg/ml) (Sigma)).

Stable cell lines were grown in the presence of Blasticidin (Life Technologies) or Hygromycin (Sigma-Aldrich). Expression was induced using CuSO4 (Sigma-Aldrich) (750 µM final concentration). The Met-Dscam1 receptor was activated by addition of HGF (ebioscience) to the culture medium supplemented with FBS. Conditioned media were harvested after 2 days. For experiments testing the effect of Slit on Dscam1 complex formation or phosphorylation we added concentrated Slit-conditioned medium, at a Slit concentration >10 µg/ml. For controls cell were incubated with conditioned medium from WT S2 cells.

Cells were transfected using the Amaxa Nucleofector II device (Lonza) with 1 to 2µg of a given plasmid/dsRNA (solution V, program D023).

For AP-Slit production PCR amplified Slit-N fragment was used to generate a recombinant bacmid in the Bac to Bac HBM expression system (Life Technologies). Viral particles were produced in Sf9 cells for infection of High Five cells. Protein concentrations were determined using AP Assay Reagent A (GenHunter).

IPs

Cells were harvested post-transfection (day 2 for S2 cells; day 5 for BG3C2 cells) and lysed in RIPA buffer containing protease inhibitors (Pierce) and if necessary phosphatase inhibitors (Sigma-Aldrich). Lysates were incubated with beads coated with IP antibody. Antibodies used in this study are described in SI. Proteins were detected using Pierce ECL Substrate and analyzed with Chemi-Doc-IT imager with Vision-Works-LS software (UVP).

Reagents

Buffers, constructs, Taqman probes, qPCR reagents, dsRNA and protocols are described in PhD dissertation of M-L.E and available upon request.

Kinetic Binding Studies

Media containing the Dscam EC4-FC fusion protein (Wotjowicz et al., 2004) were incubated with Protein G Dynabeads for one hour at RT and then washed with high salt binding buffer (100 mM phosphate buffer with 1M NaCl, 10 U/ml Heparin, 1mM DTT, 0.2mM PMSF and 1/1000 proteinase inhibitor cocktail). AP-Slit was added to the Dscam-EC4 beads, incubated for 1 hour, washed with high salt binding buffer. Amount of bound AP-Slit-N was determined using AP Assay Reagent A. Absorbance was measured at 405 nm using a microplate spectrophotometer. For negative controls, supernatant from High five cells only or human anti-IgG Fc fragment were used. Data was plotted with KaleidaGraph (Synergy software) and fitted to a Michaelis Menton curve to generate the Kd value.

Genetics

Alleles and transgenes are provided in SI. RNAi lines were obtained from the VDRC and TriP collections. P-element and ϕC31-directed integration was performed by Genetic Services (Boston, USA) and Rainbow transgenics (California).

All crosses were performed at 25°C, except for RPTP69D hypomorphs where a shift to 18°C at mid-pupal stage was used (Desai and Purdy, 2003).

Single-cell labeling and transgene expression were performed as described (Urwyler et al. 2015).

Immunohistochemistry

Immunostaining of adult brains and ventral nerve cords was done as described (Pfeiffer et al., 2008). Antibodies/concentrations used in this study are listed in SI.

Image acquisition/analysis

Images were taken using a Zeiss LSM 710 confocal microscope and processed using FIJI and Adobe PhotoShop software. Z-stacks confocal images were flattened and despeckled. Intensity levels and orientation of images were adjusted for better comparisons.

Statistical analysis was performed using Prism 6 software.

Supplementary Material

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Acknowledgments

This work was supported by NIH (2RO1NS046747-05A1) (D.S.); FWO (G059611N) (D.S.); FWO (G078913N) (D.S.); FWO (G077013N) (D.S.); BELSPO IUAP VII-20 “WIBRAIN” (D.S.); VIB funding; and fellowships of FWO (D. D.), Boehringer Ingelheim Fonds (M.E. and S.S.), EMBO (M.P.), JSPS & HFSP (Y.K.), SNSF (O. U.).

Work of T.K and M.S. was supported by NSF (IOS-1052555), the National Center for Research Resources (P20RR016464, 5P20RR024210) and the NIGM (8 P20 GM103554) from the NIH.

We thank Tineke Breynaert for technical help (cell culture). We thank members of the lab, Paul Garrity (Brandeis University), Fritz Rathjen (MDC, Berlin), Bassem Hassan, Matthew Holt, Georg Halder (VIB) for critical reading, discussions and insightful comments. We would like to thank the Clemens, Bashaw, Dickson, Zinn, Suzuki, Treisman, Lee, Hassan, Kidd, and Bogdan labs for fly stocks, antibodies and plasmids.

Footnotes

AUTHOR CONTRIBUTIONS

Design and planning of experiments: D.D., M-L.E. and D.S.; Genetics and phenotype analysis: D.D. with additional help from R.B. A.I., O.U, F.T. M. P., Y.K., H.H., D. A.; Biochemistry Experiments Dscam1/RPTP69D/Slit: M-L.E., with help from S.S., D.D.; Experiments Slit/Dscam1 affinity measurements: M.S., T.K.; Experiments single growth cone analysis: A.I. and D.D. with additional help from O.U., D.S.; Writing of Manuscript: D.S. and D.D., M-L.E., with additional help from O.U.; Supervision: D.S. and T.K.

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supp fig 1
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supp fig 2
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supp fig 3
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supp fig 4
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supp fig 5
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supp fig 6
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supp fig 7
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supp info
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supp legends
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