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First published online May 30, 2007
doi: 10.1242/10.1242/dev.004242

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Crk-associated substrate (Cas) signaling protein functions with integrins to specify axon guidance during development

Zhiyu Huang^1,*, Umar Yazdani^1,*, Katherine L. Thompson-Peer^2,, Alex L. Kolodkin^2, and Jonathan R. Terman¹

¹ Center for Basic Neuroscience, Department of Pharmacology, NA4.301/5323 Harry Hines Blvd, The University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
² Solomon H. Snyder Department of Neuroscience, Howard Hughes Medical Institute, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, USA.

View larger version (46K): [in this window] [in a new window]   Fig. 1. Molecular characterization of Drosophila Cas. (A) Organization of the DCas locus (DCasP1: site of GAL4-containing P-element insertion). (B) Domain organization of human (H) and Drosophila (D) Cas proteins, and percentage amino acid identity of human Cas-family SH3-domains compared with DCas. (C) Alignment of the Drosophila and human Cas proteins, with conserved residues highlighted. In addition to the SH3 domain (black), tyrosine residues (blue), including those residing in consensus Crk/Nck SH2-binding sites (YXXP, also blue), and serine/threonine residues (purple) are conserved. Additional conserved regions include an SH3-domain-binding motif (yellow), a dimerization motif (black underline) and a Src-binding site (pYDYV, red underline).

View larger version (104K): [in this window] [in a new window]   Fig. 2. Drosophila Cas is highly expressed in the embryonic nervous system. Localization of (A-D) DCas RNA and (E-H) protein reveals that DCas is highly expressed in the central and peripheral embryonic nervous system. (A,B) Stage 15 whole-mount Drosophila wild-type embryos viewed ventrally (A) and laterally (B) following in situ hybridization with antisense DCas RNA probes. Note the prominent DCas expression in the ventral nerve cord (VNC) and brain. Anterior, left. (C,D) A DCasP1/DCasP1 Drosophila embryo (C) subjected to in situ hybridization exhibits significantly reduced levels of DCas transcript, whereas an embryo deficient for the DCas gene (D) shows little to no DCas transcript (ventral views). (E) Western analysis of lysates obtained from late stage 16/17 embryos using an antibody generated against Cas (see text). The Cas antibody recognizes two prominent bands in wild-type embryo lysates (+) that are absent in DCas mutant lysates (Df). Molecular weight (100 kDa) is indicated. (F,G) A stage 15 wild-type embryo immunostained with Cas, showing Cas in axons within the anterior (A) and posterior (P) commissures, and longitudinal connectives (L) and motor nerves (asterisks). Cas immunostaining is also present at muscle attachment sites (arrows). (H) A stage 15 embryo homozygous for a DCas deficiency exhibits little to no Cas immunostaining (arrowheads, unstained longitudinal connective axons visualized using Nomarski optics). Scale bar: 30 µm in A-D; 10 µm in F-H.

View larger version (109K): [in this window] [in a new window]   Fig. 3. Drosophila Cas and integrin mutants exhibit ISNb motor axon defasciculation defects. (A-I) Abdominal segments of filleted stage 16/17 Drosophila embryos immunostained with the monoclonal antibody 1D4 to visualize all motor axons (Anterior, left, dorsal, up). The intersegmental nerve (ISN), intersegmental nerve b (ISNb), intersegmental nerve d (ISNd), segmental nerve c (SNc), segmental nerve a (SNa) and ventral muscles 6, 7, 12 and 13 are indicated. (A) ISNb motor axons in DCasDf(3L)Exel6083/+ embryos defasciculate normally from the ISN and grow dorsally to innervate muscles 6/7 and 12/13 (open arrows, used similarly in D,E,H,I). (B-E) DCas loss-of-function mutant embryos often fail to innervate their muscle targets (black arrows) and exhibit a range of ISNb guidance phenotypes indicative of an increase in axon-axon fasciculation. These defects include ISNb axons remaining abnormally fasciculated with the ISN (B,C, arrowheads), and ISNb axons abnormally bundled together (C, broad arrow). ISNd axons also do not defasciculate normally to their muscle targets (B, white arrow). (D) ISNb axons in DCas mutants are also abnormally fasciculated with the ISN (arrowhead), where they often separate at inappropriate locations and extend abnormally (small arrows). (E) ISNb axons in a DCas mutant are abnormally fasciculated such that they extend past their muscle targets (arrowhead, data not shown) and eventually take abnormal routes to reach their muscle targets (large black arrow). (F,G) Axons in 1 integrin (mewM6) (F, 1Int) and 2 integrin (ifK27E) (G, 2Int) LOF mutants exhibit axon guidance defects resembling DCas LOF mutants, including abnormal fasciculation of ISNb axons with the ISN (arrowheads), abnormal fasciculation of ISNd axons with the ISN (white arrow, F) and failure of ISNb axons to innervate their muscle targets (black arrows). (H,I) Expression of DCas rescues ISNb defects observed in DCas LOF mutants. Expression of either MycDCas under the DCasP1-GAL4 driver (H, UAS-MycDCas/+; Df(3L)ED201/DCasP1) or MycDCas in all neurons using the ELAV-GAL4 driver (I, ELAV-GAL4/UAS-MycDCas; DCasDf(3L)Exel6083/DCasDf(3L)Exel6083) restores the normal ISNb innervation pattern in DCas mutants (open arrows). Scale bar: 15 µm. (J) Schematic summarizing ISNb motor axon guidance phenotypes.

View larger version (113K): [in this window] [in a new window]   Fig. 4. Drosophila Cas and integrin mutants exhibit SNa motor axon defasciculation defects. (A-J) Immunostaining as in Fig. 3. The intersegmental nerve (ISN) and the dorsal (D) and lateral (L) branch of segmental nerve a (SNa) are indicated. (A) In DCasDf(3L)Exel6083/+ embryos, axons within the SNa defasciculate normally from the SN and extend past the ventral musculature as a single tightly fasciculated bundle. Near the dorsal edge of muscle 12, SNa axons defasciculate (small arrow) to generate a dorsal (D) and a lateral (L) branch. Axons within the dorsal branch extend dorsally between muscles 22 and 23 and then make two characteristic turns (open arrows), continuing dorsally between muscles 23 and 24. (B-F) DCas loss-of-function mutant embryos often fail to reach their muscle targets and exhibit a range of SNa axon guidance phenotypes indicative of defects in axon-axon defasciculation. (B,C) SNa axons in DCas mutants exhibit abnormal fasciculation (arrowheads) of the lateral branch with the dorsal branch (B, arrow), and also of the dorsal branch with the lateral branch (C, arrow). (D,E) In DCas mutants, axons within the dorsal branch of the SNa often do not make their two characteristic turns, extending past their defasciculation choice point (arrow, D) or stalling in the vicinity of this choice point (arrow, E). (F) SNa axons in DCas mutants are also abnormally fasciculated, extending past their defasciculation point (arrow) but eventually taking an abnormal route to reach lateral muscle targets (open arrows). (G,H) SNa axon guidance defects seen in 1 integrin (mewM6) (G, 1Int) and 2 integrin (ifK27E) (H, 2Int) LOF mutants resemble those observed in DCas LOF mutants, including: SNa stalling past the dorsal SNa choice point (large arrow, G), axons fused with the ISN (arrowhead, H), and SNa axons within the dorsal branch growing past their choice point (large arrow, H). (I,J) Expression of DCas rescues SNa defects observed in DCas LOF mutants. Expression of either MycDCas under the DCasP1-GAL4 driver (I, UAS-MycDCas/+; Df(3L)ED201/DCasP1) or MycDCas in all neurons using the ELAV-GAL4 driver (J, ELAV-GAL4/UAS-MycDCas; DCasDf(3L)Exel6083/DCasDf(3L)Exel6083) restores the normal SNa trajectory and innervation pattern in DCas mutants (open arrows). Scale bar: 15 µm. (K) Schematic summary of SNa motor axon guidance phenotypes.

View larger version (137K): [in this window] [in a new window]   Fig. 5. Neuronal DCas overexpression results in axon guidance defects similar to those seen in DCas loss-of-function mutants. Overexpression of MycDCas in all neurons in a wild-type background using the ELAV-GAL4 driver results in ISNb (A-C,G) and SNa (D-G) guidance defects characterized by increased fasciculation of axons and decreased target innervation (A-G, large arrows). (A) Overexpression using one copy (+) each of both the MycDCas reporter and the ELAV-GAL4 driver in all neurons results in a reduction of ISNb muscle target innervation (arrows). (B,C) Overexpression using two copies (++) of both MycDCas and ELAV-GAL4 in all neurons results in ISNb axons remaining fasciculated with the ISN (arrowheads), and sometimes exiting the ISN in abnormal locations and stalling in large clumps of fasciculated axons (C, small arrows). (D) Overexpression of one copy (+) of both MycDCas and ELAV-GAL4 in all neurons results in a reduction of SNa axon target innervation; SNa axons often stalled at the choice point in the dorsal branch of the SNa (D, arrow). (E,F) Overexpression using two copies (++) of both MycDCas and ELAV-GAL4 in all neurons results in SNa axons remaining abnormally fasciculated with the lateral branch of the SNa (E, arrowhead) at the choice point (E, small arrow) and remaining abnormally fasciculated with the ISN (F, arrowheads). (G) Following temperature (29°C)-induced elevated expression using two copies of both UAS-MycDCas and ELAV-GAL4 (+++), many motor axons stalled in the motor roots following exit from the CNS (small arrows), and those ISNb and SNa axons that did extend into the muscle fields often remained fasciculated with the ISN (arrowheads), failing to innervate muscle targets (large arrows). Scale bar: 15 µm in A-F; 7 µm in G.

View larger version (108K): [in this window] [in a new window]   Fig. 6. DCas functions with integrins to regulate axon fasciculation during development. (A-E) Dominant (transheterozygous) genetic LOF interactions between DCas and the ß1, 1 or 2 integrin genes (ß1Int, 1Int or 2Int). (A) ISNb axons in ß1 integrin (mys1) heterozygous embryos (ß1Int/+) innervate muscles 6/7 and 12/13 (open arrows). (B-D) ISNb axons in embryos heterozygous for both ß1 integrin (mys1) (B), or 1 integrin (mewM6) (C), or 2 integrin (ifK27E) (D) and DCasDf(3L)Exel6083 fail to innervate their muscle targets (arrows), exhibiting increased axonal fasciculation (arrowheads). (E) The percentage of defective ISNb, SNa and CNS pathways in embryos heterozygous for either DCas, ß1Int, 1Int or 2Int, or in embryos heterozygous for both DCas and ß1Int, 1Int or 2Int. (F-H) Expression of one copy of MycDCas and one copy of the ELAV-GAL4 pan-neuronal driver results in axon guidance phenotypes that are suppressed by heterozygosity at the ß1 integrin locus. (F) ISNb axon innervation of muscles 6/7 (black arrow) is absent in embryos expressing one copy of MycDCas in all neurons in a wild-type background. (G) Normal ISNb axon innervation of muscles 6/7 and 12/13 (open arrows) in an embryo in which one copy of MycDCas is expressed in all neurons in combination with heterozygosity at ß1 integrin. (H) The percentage of defective ISNb, SNa and CNS pathways in embryos expressing low levels of neuronal DCas (Neuronal DCas GOF: UAS-MycDCas/+, ELAV-GAL4/+), in embryos expressing low levels of neuronal DCas that are also heterozygous for DCas LOF (Neuronal DCas GOF; DCas/+: UAS-MycDCas, ELAV-GAL4/+; DCasDf(3L)Exel6083/+), and in embryos expressing low levels of neuronal DCas that are also heterozygous for ß1 integrin LOF (Neuronal DCas GOF; ß1Int/+: mys1/+; UAS-MycDCas, ELAV-GAL4/+). The differences in the percentage of defective guidance phenotypes observed between Neuronal DCas GOF; DCas/+ or Neuronal DCas GOF; ß1Int/+ and Neuronal DCas GOF for each axon pathway are statistically significant (*, P<0.05; 2 test). Scale bar: 15 µm in A-D; 15 µm in F,G.

View larger version (29K): [in this window] [in a new window]   Fig. 7. Model for integrin/Cas-mediated regulation of axonal fasciculation. (A) During axon pathfinding, integrin/Cas signaling (+) functions to regulate axon-axon fasciculation. In this model, integrin/Cas-mediated adhesive interactions are strongest at choice points, slowing axons so that they can navigate specific defasciculation events. (B) Removal of integrin or Cas adhesive signaling in a loss-of-function (LOF) mutant background prevents axons from defasciculating at their choice points, maintaining fasciculation with the main axon bundle. (C) Neuronal overexpression (gain-of-function, GOF) of Cas increases integrin/Cas adhesive signaling and also prevents axons from defasciculating at their choice points. This model suggests that integrin/Cas adhesive signaling is necessary for axonal defasciculation but is not sufficient, such that other guidance cues, including axonal repellents, coordinately work together with integrin/Cas signaling to direct specific axon defasciculation.

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