UNC-40/DCC, SAX-3/Robo, and VAB-1/Eph polarize F-actin during embryonic morphogenesis by regulating the WAVE/SCAR actin nucleation complex

Abstract

Many cells in a developing embryo, including neurons and their axons and growth cones, must integrate multiple guidance cues to undergo directed growth and migration. The UNC-6/netrin, SLT-1/slit, and VAB-2/Ephrin guidance cues, and their receptors, UNC-40/DCC, SAX-3/Robo, and VAB-1/Eph, are known to be major regulators of cellular growth and migration. One important area of research is identifying the molecules that interpret this guidance information downstream of the guidance receptors to reorganize the actin cytoskeleton. However, how guidance cues regulate the actin cytoskeleton is not well understood. We report here that UNC-40/DCC, SAX-3/Robo, and VAB-1/Eph differentially regulate the abundance and subcellular localization of the WAVE/SCAR actin nucleation complex and its activator, Rac1/CED-10, in the Caenorhabditis elegans embryonic epidermis. Loss of any of these three pathways results in embryos that fail embryonic morphogenesis. Similar defects in epidermal enclosure have been observed when CED-10/Rac1 or the WAVE/SCAR actin nucleation complex are missing during embryonic development in C. elegans. Genetic and molecular experiments demonstrate that in fact, these three axonal guidance proteins differentially regulate the levels and membrane enrichment of the WAVE/SCAR complex and its activator, Rac1/CED-10, in the epidermis. Live imaging of filamentous actin (F-actin) in embryos developing in the absence of individual guidance receptors shows that high levels of F-actin are not essential for polarized cell migrations, but that properly polarized distribution of F-actin is essential. These results suggest that proper membrane recruitment and activation of CED-10/Rac1 and of WAVE/SCAR by signals at the plasma membrane result in polarized F-actin that permits directed movements and suggest how multiple guidance cues can result in distinct changes in actin nucleation during morphogenesis.

Figure 1. Axonal guidance receptors are required for embryonic epidermal morphogenesis.

(A) Comparison of morphogenesis in embryos with wild type, Partial Gex or Full Gex phenotypes. In wild type the epidermal cells (blue) enclose the embryo by ∼400 minutes after first cleavage at 20°C. By ∼600 minutes the embryos elongate to the three-fold stage. In Partial Gex embryos epidermal cells do not fully enclose the embryo by 400 minutes, and by 600 minutes the internal organs (green = pharynx, red = intestine) partially extrude through epidermal gaps. In Full Gex embryos the epidermal cells completely fail cell migration and by 600 minutes the internal organs are fully extruded to the surface of the embryo. (B) Embryonic morphogenesis defects visualized using DIC optics and the dlg-1::gfp (xnIs16) transgene that marks junctions of epithelial tissues. Embryos are oriented with anterior to the left. Representative images from the ventral (left columns) or lateral (right columns) view and corresponding ventral and lateral images of DLG-1::GFP are shown. DIC images. Arrows: anterior pharynx. Block arrows: anterior intestine. White brackets: extruded internal organs. DLG-1::GFP images. Asterisks: ventral gap between epidermal cells. Open arrows in right panels: leading edge of epidermis. Dots outline the unenclosed regions of embryos. Alleles shown are putative nulls, including deletion mutations in ced-10, unc-6, sax-3 and vab-1, with the exception of ced-10(n1993) a hypomorph , , , . (C) Morphogenesis failure in genetic doubles of axonal guidance mutants. Labeled as in (B). Most unc-40(e1430); vab-1(dx31) and vab-1(e2); sax-3(ky123) doubles die with the Partial Gex phenotype. unc-40(e1430); sax-3(ky200ts) doubles show a synergistic increase in the number of Full Gex embryos. (D) Summary of the proposed contribution of the axonal guidance receptors, the CED-10/Rac1 GTPase, the WAVE/SCAR complex and the Arp2/3 complex to embryonic viability (% lethality) and epidermal morphogenesis (% Full Gex).

Figure 2. Guidance pathway proteins regulate F-actin organization and levels in migrating embryonic cells.

Embryos are oriented with anterior to the left. (A) Polymerized actin visualized in 4D using the plin26::vab10ActinBindingDomain::gfp transgene (mcIs51) , . Embryos imaged at 2-minute intervals for 2 hours, beginning at 240 minutes, at 23°C. Embryos at Early, Middle, and Late stages, as related to F-actin-dependent events (B), are shown. t = minutes after first cleavage. Dots outline unenclosed regions of the embryo. Leading Cells (LCs) are boxed and magnified. White dashed line: leading edge. Embryos were pseudo-colored using GLOWormJ “Fire” setting, from low (blue) to high (yellow) intensity. (B) Time intervals between actin-dependent events. Time “0” corresponds to the first appearance of epidermal pocket cells protrusions, at approximately 250 minutes after first cleavage in wild type and mutants. (C) The intensity of F-actin in the LCs analyzed at the time of actin enrichment at the leading edge. The two LCs (yellow boxed region shown in A) were compared. Additional details about F-actin measurements in Materials and Methods. (D). Distribution of actin peaks in the ventral half compared with the dorsal half of LCs. Close-ups of representative embryos at Late stages (∼290 min.) are shown. V = Ventral, bottom. D = Dorsal, top. A ventral to dorsal line was drawn through the cell using the Plot Profile tool in GLOWormJ and fluorescent intensity was measured. Peaks, defined as regions at least 10 fluorescent units higher than the background, were counted and are marked by asterisks. Dashed red lines mark half the cell's length. (E) Ratios of ventral to dorsal actin distribution based on the actin peaks measured as in D during 40 minutes beginning with the enrichment of actin at the leading edge. Error bars show SEM. Asterisks mark statistical significance, * = p<.05, *** = p<0.001 as determined by a One-way Anova test followed by the Tukey test.

Figure 3. The dynamic turnover of F-actin protrusions is altered in morphogenesis mutants.

F-actin protrusions produced by the two Leading Cells (LCs) on one side during epidermal cell migration were analyzed using the plin26::vab10 Actin Binding Domain::gfp transgene (mcIs51) , . Micrographs are close-ups of migrating LCs. Arrows point to protrusions. Asterisks denote site of protrusion retraction. Four consecutive time points are shown with an arbitrary timing of t0 set as 2 minutes before protrusion formation. Bar graph shows the average duration of the F-actin protrusions in minutes. Error bars show SEM. Asterisks mark statistical significance, * = p<.05, *** = p<0.001 as determined by a One-way Anova test followed by the Tukey test.

Figure 4. Guidance receptors affect subcellular distribution and levels of the GTPase CED-10/Rac1.

All embryos are oriented with anterior to the left and dorsal up. Boxed regions are amplified and enhanced equally for contrast. Number of embryos that showed the represented phenotype are indicated. (A) Embryos carrying an integrated rescuing ced-10::gfp (pjIs4) transgene were double-stained with antibodies to GFP (Abcam, ab6556) and to endogenous AJM-1 to indicate the adherens junctions , . Dotted lines outline the basolateral region of the epidermal cells if the region is discernable. (B) Embryos carrying an integrated rescuing ced-10::gfp transgene were double stained with antibodies to GFP and to endogenous basolaterally localized UNC-70/beta spectrin . Readings were taken across two cell junctions using the line tool in ImageJ for both the UNC-70 and the GFP signal. 5 readings were taken per cell and averaged, and plotted in IPad Prism. SEM is shown. (C) Total levels of CED-10::GFP measured with antibody to GFP (Abcam, ab6556). Numbers below each lane are the levels of GFP normalized to HSP90 (Abcam ab13492) as loading control, and relative to wild type, averaged from 4 blots from two sets of lysates. (D) Genetic interactions of WAVE/SCAR and guidance receptor mutants with the integrated rescuing CED-10::GFP transgene. “Early Arrest” refers to embryonic arrest before morphogenesis begins. Asterisks indicate significant change in the phenotypes compared to the single mutants. * = p<.05, *** = p<0.001 as determined by a One-way Anova test followed by the Tukey test.

Figure 5. Guidance receptors affect subcellular distribution and levels of WAVE/SCAR.

Embryos are oriented with anterior to the left and dorsal up. (A) Embryos carrying the integrated, rescuing gfp::wve-1 (pjIs1) transgene were double-stained with antibodies to GFP (Abcam, ab6556, polyclonal) and AJM-1. Boxed regions are amplified and enhanced equally for contrast. Number of embryos that showed the represented phenotype are indicated. Dotted line outlines the basolateral region of the epidermal cells where the region is discernable. (B) Embryos carrying the rescuing integrated gfp::wve-1 transgene were double stained with mAb to GFP and basolaterally localized UNC-70/beta spectrin . Readings were taken across two cell junctions using the line tool in ImageJ for both the UNC-70 and the GFP signal. 5 readings were taken per cell and averaged and plotted in IPad Prism. SEM is shown. (C) Total levels of endogenous WVE-1 in whole worm lysates, or embryonic lysates (Materials and Methods) measured with a polyclonal antibody to WVE-1 . Levels of WVE-1 normalized to tubulin and relative to WT are shown below the graph, based on the average of 4 blots from 3 sets of lysates. (D) Subcellular distribution of WVE-1 in fractionated lysates, measured using an antibody to endogenous WVE-1. Lysates were spun at increasing speeds and duration. Pellets were resuspended to match the volume of their partner Supernatant fraction. Equal volumes of each S and P fraction were loaded so that relative amounts of protein in the S vs. P fraction could be compared (See Materials and Methods). 10 µl of each fraction were loaded. Numbers below each band represent the relative percentage of total protein found in each fraction. Numbers represent average of three blots (one set of lysates). S = supernatant, P = pellet. Graph shows average of total protein in S1 and P1 based on 3 blots. SEM is shown. Red rectangles indicate fractions with most significant changes compared to WT.

Figure 6. Post-embryonic genetic interactions of WAVE/SCAR genes and axonal guidance genes during neuronal migrations.

(A) WAVE/SCAR and Arp2/3 are required for ventral axonal guidance of the AVM neuron. The mec-4::gfp (zdIs5) transgene is expressed in 6 mechanosensory neurons . Micrographs show representative ventral migration patterns. The cartoons below each micrograph show the path of the AVM axon (green) with ALM (black) for reference. In wild type animals the AVM axon migrates ventrally, then anteriorly towards the head. Anterior is on the left. Control RNAi: nematodes were fed the RNAi empty vector, L4440 in HT115 bacteria. (B) Loss of WAVE/SCAR or Arp2/3 partially suppresses the AVM ventral migration defect caused by misexpression of Slt/Robo. The nematode strains myo-3::slt-1 (kyIs218), myr::vab-1(quIs5) and myr::unc-40::gfp (lqIs131), carry transgenes that cause gain of function phenotypes in three axonal guidance receptors. gof = gain-of-function. (C) Summary of AVM defects: ventral migration defects, abnormal cell bodies and ectopic projections. Animals were cultured at 20°C. Asterisks indicate significant change in the phenotypes compared to the single mutants. ** = p<.01, *** = p<0.001 as determined by a One-way Anova test followed by the Tukey test.

Figure 7. Model for the regulation of the CED-10/Rac1 – WAVE/SCAR – Arp2/3 pathway by axonal guidance receptors.

UNC-40 activity supports the transport of inactive CED-10/Rac1 (Rac1-GDP, grey circles) to endosomes where it becomes activated (Rac1-GTP, red circles). Activated CED-10/Rac1 is then targeted to the plasma membrane where it is recruited by SAX-3/Robo. Activated CED-10/Rac1 can then recruit and help assemble the WAVE/SCAR complex (blue ovals), which leads to robust branched actin polymerization through the Arp2/3 complex (yellow oval). VAB-1/Ephrin inhibits the targeting of activated CED-10/Rac to the membrane thereby modulating branched actin polymerization.