DEV004242 Supplementary Material

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• Supplemental Figure S1 -

  Fig. S1. Characterization of an antibody that recognizes DCas, and DCas mutants exhibit normal muscle formation and neuronal cell fate determination. (A,B) Characterization of an antibody generated against a highly conserved mammalian p130Cas peptide that recognizes DCas. We took several different approaches to characterize an antibody that recognized Drosophila Cas. We first attempted to generate antibodies against Cas by immunizing mice with a Drosophila Cas fusion protein. As a complementary approach, since regions of Drosophila Cas were highly conserved with vertebrate Cas and a number of Drosophila proteins often cross-react with mammalian antibodies (e.g. Colville et al.; Miller and Benzer, 1983), we obtained all published vertebrate Cas antibodies that recognized an epitope highly conserved in Drosophila Cas. We then took our Myc-tagged full-length DCas, subcloned it into a mammalian expression vector (pRK5), overexpressed it in tissue culture cells, and examined our DCas antisera and the vertebrate Cas antibodies for their ability to recognize DCas on a western blot. We tried nine different commercially available antibodies and our own antisera. Although our Drosophila Cas antisera did not recognize Drosophila Cas, we identified one antibody that recognized overexpressed Drosophila ^MycDCas when expressed in 293 cells. (A) Lysates from 293 cells transfected with ^MycDCas were examined for the presence of an immunostained band corresponding to the same size as the ^MycDCas protein as visualized with an antibody against Myc (lane 1). A band (arrow) was present in lysates from 293 cells transfected with ^MycDCas (lane 3) that was absent in control 293 cells (lane 2) when blotted with the phospho410-p130Cas (Cas) antibody. Bands that are presumably endogenous mammalian Cas proteins are also observed. (B) Drosophila embryos expressing DCas (^MycDCas) or DCas lacking an SH3 domain (^Myc-ΔSH3DCas; unpublished) using the ELAV-GAL4 driver or the DCas^P1- GAL4 LOF mutant line were genotyped and subjected to western analysis with antibodies generated against Myc or Cas (phospho410-p130Cas). The Myc antibody recognizes ^MycDCas expressed in all neurons using the ELAV-GAL4 driver (lane 1). The phospho410-p130Cas antibody recognizes a band of similar size when ^MycDCas is expressed in all neurons (lane 2). The Myc antibody recognizes Myc-DCas expressed in a DCas^P1 mutant background using the DCas^P1-GAL4 driver (lane 3). The phospho410-p130Cas antibody recognizes ^MycDCas expressed in a DCas^P1 mutant background using the DCas^P1-GAL4 driver (lane 4). The Myc antibody recognizes ^Myc-ΔSH3DCas expressed in a DCas^P1 mutant background using the DCas^P1-GAL4 driver (lane 5). The phospho410-p130Cas antibody recognizes ^Myc-ΔSH3DCas expressed in a DCas^P1 mutant background using the DCas^P1-GAL4 driver (lane 6). Molecular weight is given in kDa. (C-E) Characterization of an enhancer trap line expressing DCas in its endogenous pattern. To further characterize DCas protein distribution, we took advantage of a P-element enhancer trap line in which a Gal4 promoter element is inserted in the DCas gene downstream of the translational start site (DCas^P1-Gal4, see Fig. 1A). The location of DCas^P1-Gal4 suggested that Gal4 might be expressed under the control of the endogenous DCas promoter, allowing for assessment of DCas protein distribution. To test this idea, we generated a Myc-tagged full-length DCas transgene under the control of the UAS promoter (UAS-^MycDCas). Using the Gal4-UAS system, we first determined that when UAS-^MycDCas was expressed in all neurons using the neuron-specific driver Elav-Gal4, ^MycDCas immunostaining was observed in CNS and motor axons all along their axonal trajectories and at sites of muscle innervation. (C) A stage 16/17 filleted preparation of an embryo expressing ^MycDCas under control of the pan-neuronal ELAV-GAL4 promoter (ELAV- GAL4/+; UAS-^MycDCas/+) immunostained with Myc antibodies shows that driving ^MycDCas expression in all neurons results in ^MycDCas localization in motor axons (asterisks) and within the ISNb (black arrows) and SNa (open arrows) nerves. (D,E) We crossed our UAS-^MycDCas line with the DCas^P1-Gal4 line and observed ^MycDCas immunostaining in commissural and longitudinal CNS axons, in motor axons and at their sites of muscle innervation, and at segment boundaries in muscle attachment sites. (D) A stage 15 embryo expressing ^MycDCas under the endogenous DCas promoter, as driven by the DCas^P1 P-element GAL4 enhancer trap (DCas^P1 -GAL4/+; UAS- ^MycDCas/+), immunostained with an antibody against Myc. ^MycDCas expression is seen in groups of CNS neurons (n), within axons occupying the anterior (A) and posterior (P) commissures, the longitudinal connectives (L) and within motor nerve roots (asterisks). (E) ^MycDCas expression driven by the endogenous DCas promoter is also observed at muscle attachment sites (arrowheads) and in the ISNb nerve innervating muscles 6 and 7 (arrow). These results show that ^MycDCas expressed in the developing CNS localizes to CNS and motor axon projections, and strongly support the idea that endogenous DCas is expressed in neurons during development. (F-L) Normal muscle formation and neuronal cell fate determination in DCas mutants. β1 integrin (mys) null mutations are embryonic lethal and result in complete detachment of muscles (Leptin et al., 1989; Wright, 1969). In α2 integrin (if) mutants, muscles develop normally through stage 17, past the point when the embryonic motor and CNS axon guidance events we are assessing have taken place, and then detach from their muscle attachment sites but still remained stretched (Hoang and Chiba, 1998; Prokop et al., 1998). We see similar results on occasion we saw defects in the underlying musculature as judged by Nomarski optics (e.g. in α2 integrin mutants); these segments were not included in our analysis for axon guidance defects. In α1 integrin (mew) mutants, muscles develop and attach normally during embryogenesis, and the mutants die during larval stages (Brower et al., 1995; Roote and Zusman, 1995). Since DCas is highly expressed at muscle attachment sites in addition to the nervous system, we examined whether defects in muscle organization and/or neuronal cell fate determination might contribute to the axon guidance defects we see in DCas mutants. Wild-type Drosophila embryos exhibit a segmentally repeated pattern of muscles that can be visualized with muscle myosin antibody as they attach to epidermis at muscle attachment sites (Bate, 1993). (F-I) The organization of muscle fibers in Drosophila embryos as visualized with an antibody (FMM5) (Kiehart and Feghali, 1986) specific for muscle myosin in which selected muscle attachment sites (between arrowheads) are indicated. (F-G) The normal organization of muscle fibers as visualized in two hemisegments of a wild-type embryo (F), and at higher power revealing muscles 6, 7, 12 and 13 (G). (H-I) In DCas LOF mutants, muscle integrity as viewed with Nomarski optics shows that muscle formation was not disrupted (see Figs 3 and 4). A normal pattern of muscle fibers is also seen in DCas LOF mutant (DCas^{Df(3L)Exel6083}/DCas^{Df(3L)Exel6083}) embryos stained with the muscle myosin antibody (H), including the morphology and attachment of muscles 6,7, 12 and 13 (I). These results are consistent with our ability to rescue the axon guidance defects seen in DCas LOF mutants with neuron-specific expression of DCas (see Fig. 3I, Fig. 4J and Fig. S2 in the supplementary material). (J-L) Axon guidance defects can also result from a loss of neurons in the ventral nerve cord or changes in neuronal differentiation or specification (Landgraf et al., 1999; Thor et al., 1999). Therefore, we examined neuronal organization of the ventral nerve cord in DCas mutants. No abnormalities in the cellular morphology of the nerve cord were observed in the DCas mutants using the Fas2 antibody and Nomarski imaging (data not shown). We next examined DCas mutants for the presence or absence of specific motoneurons. In particular, since we see axon guidance defects in DCas mutants where ISNb axons remain fasciculated with ISN axons, we asked whether these axon guidance defects are the result of a conversion of ISNb neurons to ISN neurons. The Even-skipped (Eve) protein is an excellent marker for ISN neurons since it is expressed in approximately 16 cells per abdominal hemisegment, including six of the neurons (one aCC, one RP2, four CQ/U neurons) that give rise to axons that form the ISN nerve (Doe et al., 1988; Landgraf et al., 1997; Landgraf et al., 1999; Patel et al., 1989). Altering the levels of Eve affects the axon trajectories of these neurons (Doe et al., 1988; Landgraf et al., 1999). (J) Two of these ISN neurons (aCC, red; RP2, green) are shown schematically in three segments of a wild-type Drosophila embryo (arrowheads). (K) A normal complement of aCC (red arrowhead) and RP2 (green arrowhead) neurons are found in DCas mutants (DCas^{Df(3L)Exel6083}/DCas^{Df(3L)Exel6083}). (L) Embryos in which high levels (+++) of ^MycDCas are expressed in all neurons using the ELAV-GAL4 driver in a wild-type background (Neuronal DCas GOF; see Fig. 5, Fig. S2 in the supplementary material, and Table 1) also show the normal complement of aCC (red arrowhead) and RP2 (green arrowhead) neurons. Scale bars: 20 μm in C; 13 μm in D; 7μm in E; in G, 10 μm for F-I; in J, 5 μm for J-L.

• Supplemental Figure S2 -

  Fig. S2. Cas is highly expressed in the mammalian spinal cord and DCas mutants exhibit CNS axon guidance defects. (A-E) Cas proteins are highly expressed in the mammalian spinal cord. Immunostaining of P0 rat spinal cord was conducted using standard approaches (Terman et al., 2000). P0 rat pups were perfused, the spinal cord and surrounding vertebral column was removed, and sections were cut at 20 μm on a cryostat. Slide-mounted sections were then immunostained with a selected antibody including a monoclonal antibody generated against rat p130Cas (Clone 8G4-E8, 1:400; Upstate Biotechnology), a monoclonal antibody generated against mouse Sin (Clone 13, 1:400, BD Transduction Labs) that also recognizes rat Sin, and polyclonal antibodies specific for several tyrosine phosphorylated forms of p130Cas: phospho-p130Cas (Tyr249), #4014, Lot#1, 1:100, Cell Signaling Technologies; phospho-p130Cas (Tyr410), #4011, Lot#1, 1:200, Cell Signaling Technologies). Additional antibodies generated against members of the Cas family of proteins including mouse monoclonal anti-p130Cas (Clone 21, 1:400, BD Transduction Labs), mouse monoclonal anti-p130Cas (CAS-14, 1:400, Chemicon International), rabbit polyclonal anti-Cas (N-17, Lot#I101, 1:500; Santa Cruz Biotechnology), rabbit polyclonal anti-phospho-p130Cas phospho-p130Cas (Tyr165), #4015, Lot# 1, 1:100, Cell Signaling Technologies and goat polyclonal anti-p130Cas (S-20, Lot#B282, 1:100, Santa Cruz Biotechnology) were found to give similar results. (A-D) Adjacent sections taken at lumbo-sacral levels of a P0 rat spinal cord immunostained with antibodies that recognize different Cas family members. DF, dorsal funiculus; dLF, dorsolateral funiculus; vLF, ventrolateral funiculus; VF, ventral funiculus; cc, central canal; dr, dorsal root; nr, nerve root. (A) A rat monoclonal antibody that recognizes all three rat Cas family members (Clone 8G4-E8) shows that Cas proteins are strongly expressed in the grey and white matter of the rat spinal cord. Immunostained neurons were present in all of Rexed’s laminae. (B) High power view of the region outlined in A in an adjacent section immunostained with a polyclonal antibody (phospho249-p130Cas) directed against a phosphorylated form of p130Cas. Labeled neurons (arrowheads) and their processes (arrows) are indicated. (C) High-power view of the spinal cord region outlined in A from an adjacent section immunostained with a different polyclonal antibody (phospho410-p130Cas) directed against a phosphorylated form of p130Cas. Lamina IX is shown and contains what are very likely to be, based on location and cell body size, immunostained motoneuron cell bodies (within circle of black dots) and their axons (arrows). (D) High-power view of the spinal cord region outlined in A from an adjacent section immunostained with a monoclonal antibody specific for Efs/Sin (Clone 13). Strong immunostaining is seen in the ventral funiculus and selected immunostained neurons are indicated (arrowheads; white arrow, CNS midline). (E) Rat superior cervical ganglion (SCG) neurons were removed, dissociated and cultured using standard techniques (Qian et al., 1998). Lysates from these SCG neurons were then collected and run on SDS-PAGE and western analysis was performed using standard approaches. The western blot was then stained using antibodies that recognize p130Cas, CasL, and Sin (Cas, N-17, 1:500; Santa Cruz Biotechnology). Cas, CasL and Efs/Sin (arrows) are all expressed in SCG neurons. (F-P) DCas LOF and GOF mutants exhibit CNS axon guidance defects. (F) In wild-type and DCas^{Df(3L)Exel6083}/+ embryos, three (labeled 1, 2, 3 in A) major longitudinal axon tracts are detected with the 1D4 antibody. These tracts are normally evenly spaced and of relatively uniform thickness (open arrows in F,I and J point to the third longitudinal connective). (G) Axons contributing to the third longitudinal in DCas LOF mutant embryos are thinner than normal, discontinuous and often not present (arrow). (H) Axons that constitute the third longitudinal in α1 integrin (mew^M6) mutants are either abnormal in appearance or often absent (arrow). (I,J) Expression of DCas rescues the CNS defects observed in DCas mutants. (I) Expression of ^MycDCas under the DCas^P1-GAL4 driver restores the third longitudinal connective in DCas mutants (UAS- ^MycDCas/+; Df(3L)ED201/DCas^P1) (open arrows). (J) Neuronal expression of ^MycDCas using the ELAV-GAL4 driver also restores the third longitudinal connective in DCas mutants (ELAV-GAL4/UAS- ^MycDCas; DCas^{Df(3L)Exel6083}/DCas^{Df(3L)Exel6083}) (open arrows). (K-P) Overexpression of ^MycDCas in all neurons using the pan-neuronal ELAV-GAL4 driver dramatically affects CNS axonal trajectories. Three different levels of ^MycDCas overexpression were examined: overexpression utilizing one copy of both the ^MycDCas reporter and ELAV-GAL4 driver (+; Table 1), overexpression of two copies of both ^MycDCas and ELAV-GAL4 (++), and overexpression of two copies of both ^MycDCas and ELAV-GAL4 at elevated temperature (29°C) to induce higher ^MycDCas expression levels (+++). (K-L) Examination of CNS axons in embryos overexpressing DCas in all neurons using a marker that labels all commissural and longitudinal axons, BP102 (Seeger et al., 1993), reveals a marked difference from the normal ladder-like appearance of CNS axons observed in wild-type embryos. Ectopic neuronal expression of DCas results in disrupted longitudinal connectives and abnormal commissures, a phenotype quite similar to that observed in the absence of the Slit receptor Roundabout (Robo) or increased signaling by the Netrin receptor Frazzled/DCC (Bashaw and Goodman, 1999; Kidd et al., 1998; Seeger et al., 1993). (K) In a wild-type embryo, axons cross the midline in the anterior (A) and posterior (P) commissures, here visualized with the BP102 monoclonal antibody. (L) Overexpression of ^MycDCas in all neurons in a wild-type background using the ELAV-GAL4 driver at 29^oC (+++), results in increased and abnormal crossing of commissural axons (arrows) and also thin and disrupted longitudinal (L) connectives. (M-P) Examination of Fasciclin II-expressing, 1D4-positive, CNS longitudinal connectives in DCas GOF mutants shows that, as in DCas LOF mutants, the outermost 1D4-positive longitudinal connective is thinner in some segments, discontinuous in others, and in some cases fused with the middle 1D4-positive longitudinal fascicle. In addition, we found that longitudinal connectives, which are normally evenly spaced on either side of the CNS midline, often converged upon this repulsive midline barrier. We further observed that axons of the medial longitudinal connective often aberrantly crossed and recrossed the CNS midline, and also that the three longitudinal connectives on occasion bundled into a single fascicle. Stalling of axons also occurred within the longitudinal connectives and as axons exited the ventral nerve cord, resulting in abnormal fused projections extending away from the CNS. Therefore, DCas GOF results in extensive CNS axon pathfinding defects, most of which are consistent with hyperfasciculation. (M-P) In particular, following elevated neuronal expression of ^MycDCas (++ or +++), axons within the three 1D4-positive longitudinal connectives were abnormally fasciculated with one another. These defects included the absence or disruption of axons within the third longitudinal connectives (M, arrows), abnormal crossing of axons at the midline (M, open arrowhead), large abnormally fasciculated bundles of axons (N-O, small arrows), and stalling of hyperfasciculated axons in the CNS (O, arrowhead) or the periphery (N, P, arrowheads). Scale bars: 40 μm in A; 20 μm in B; 20 μm in C, for C,D; 6 μm in F, for F-J; 13 μm in K, for K-L;15 μm in M, for M-P.

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