Identifying Regulators of Morphogenesis Common to Vertebrate Neural Tube Closure and Caenorhabditis elegans Gastrulation

Neural tube defects including spina bifida are common and severe congenital disorders. In mice, mutations in more than 200 genes can result in neural tube defects. We hypothesized that this large gene set might include genes whose homologs contribute to morphogenesis in diverse animals. To test this hypothesis, we screened a set of Caenorhabditis elegans homologs for roles in gastrulation, a topologically similar process to vertebrate neural tube closure. Both C. elegans gastrulation and vertebrate neural tube closure involve the internalization of surface cells, requiring tissue-specific gene regulation, actomyosin-driven apical constriction, and establishment and maintenance of adhesions between specific cells. Our screen identified several neural tube defect gene homologs that are required for gastrulation in C. elegans, including the transcription factor sptf-3. Disruption of sptf-3 in C. elegans reduced the expression of early endodermally expressed genes as well as genes expressed in other early cell lineages, establishing sptf-3 as a key contributor to multiple well-studied C. elegans cell fate specification pathways. We also identified members of the actin regulatory WAVE complex (wve-1, gex-2, gex-3, abi-1, and nuo-3a). Disruption of WAVE complex members reduced the narrowing of endodermal cells’ apical surfaces. Although WAVE complex members are expressed broadly in C. elegans, we found that expression of a vertebrate WAVE complex member, nckap1, is enriched in the developing neural tube of Xenopus. We show that nckap1 contributes to neural tube closure in Xenopus. This work identifies in vivo roles for homologs of mammalian neural tube defect genes in two manipulable genetic model systems

Sentin expression is required for the Orbit-Msps interaction, but its localization to microtubules is not regulated by Rac.

<p>(A-C) Sentin-GFP was co-expressed with tRFP-α-tubulin in control cells (A), in cells depleted of Rac1/Rac2/Mtl using RNAi (B), or together with CA-Rac1 (C) Tubulin images are shown as insets. (D) Changes in co-localization of Msps were measured using the Mander’s coefficient, n = 90 from three experiments. (E-J) The localization of overexpressed GFP-Orbit, endogenous Msps, and α-tubulin is shown for cells treated with control (E-G) or Sentin (H-J) dsRNAs.</p

TOG Proteins Are Spatially Regulated by Rac-GSK3β to Control Interphase Microtubule Dynamics

<div><p>Microtubules are regulated by a diverse set of proteins that localize to microtubule plus ends (+TIPs) where they regulate dynamic instability and mediate interactions with the cell cortex, actin filaments, and organelles. Although individual +TIPs have been studied in depth and we understand their basic contributions to microtubule dynamics, there is a growing body of evidence that these proteins exhibit cross-talk and likely function to collectively integrate microtubule behavior and upstream signaling pathways. In this study, we have identified a novel protein-protein interaction between the XMAP215 homologue in <i>Drosophila</i>, Mini spindles (Msps), and the CLASP homologue, Orbit. These proteins have been shown to promote and suppress microtubule dynamics, respectively. We show that microtubule dynamics are regionally controlled in cells by Rac acting to suppress GSK3β in the peripheral lamellae/lamellipodium. Phosphorylation of Orbit by GSK3β triggers a relocalization of Msps from the microtubule plus end to the lattice. Mutation of the Msps-Orbit binding site revealed that this interaction is required for regulating microtubule dynamic instability in the cell periphery. Based on our findings, we propose that Msps is a novel Rac effector that acts, in partnership with Orbit, to regionally regulate microtubule dynamics.</p></div

Measurement of microtubule dynamics.

<p>(A) Representative image of a cell with microtubules that were tracked. Each number represents a separate track. 10 microtubules per cell were tracked at the periphery of the cell for at least 30 seconds. 30 cells per condition were tracked, the experiment was repeated three times, with 10 cells per replicate. Between 5–10 microtubules were tracked per cell. (B) Representative schematic of a diamond plot with the rates on the x-axis, growth (right) and shrinkage (left) and frequency on the y-axis, rescue (up) and catastrophe (down). (C-R) Diamond graphs of microtubule dynamics under each condition. Diamond graphs are jointly normalized. (S) Graphs showing percentage of time microtubules spent in growth, shrinkage and pause with each condition.</p

Orbit is phosphorylated by GSK3β in the linker region between TOG2 and TOG3.

<p>(A) Domain structure of Orbit with a Clustal alignment of the GSK3β phosphorylation region. Serines phosphorylated in <i>Homo sapiens</i> (H.S.) CLASP2 denoted by plus marks (+), serines conserved in <i>Drosophila Melanogaster</i> (D.M.) Orbit denoted by red stars. (B-C) GFP-Orbit 2S->A was expressed in cells with a dual expression vector containing tRFP-α-tubulin alone (B) or with CA-GSK3β (C). (D) Endogenous Msps and α-tubulin were stained in cells transfected with 2S->A. (E-F) GFP-Orbit 3S->A is expressed in cells with a dual expression vector containing α-tubulin-tRFP alone (E) or with CA-GSK3β (F). (G) Endogenous Msps and α-tubulin were stained in cells transfected with 2S->A. (H-I) GFP-Orbit 5S->A was expressed in cells with a dual expression vector containing tRFP-α-tubulin alone (H) or with CA-GSK3β (I). (J) Endogenous Msps and α-tubulin were stained in cells transfected with 5S->A. Tubulin images are shown as insets. (K) Changes in co-localization of Orbit (left) and endogenous Msps (right) were measured using the Mander’s coefficient, n = 90 cells from two (endogenous Msps) or three (GFP-Orbit) experiments. *** p<0.0001.</p

Orbit and GSK3β levels regulate Msps recruitment to the microtubule lattice.

<p>(A) Orbit coimmunoprecipitates with Msps from cells depleted of GSK3β using RNAi. GSK3β depletion was assessed using β-catenin levels, with tubulin as a loading control. (B) Msps coimmunoprecipitates with non-phosphorylatable mutants of Orbit. Immunoprecipitations were performed from cells depleted of endogenous Orbit using dsRNA targeting the 5'UTR of the gene and rescued with the indicated GFP-tagged Orbit constructs. (C-D) Msps-GFP was coexpressed with tRFP-α-tubulin in cells treated with control (C) or Orbit (D) dsRNA. Tubulin images are shown as insets. (E) Changes in co-localization of Msps were measured using the Mander’s coefficient, n = 90 cells from three experiments. *** p<0.0001 (F) GFP-Orbit was overexpressed in cells and stained for endogenous Msps (G). (H) Msps-GFP was coexpressed with a dual expression vector encoding tRFP-α-tubulin and CA-Rac1 in cells depleted of Orbit using RNAi.</p

Model. The interaction between Msps and Orbit is required for Msps lattice association.

<p>(1) At the plus end of a growing microtubule (green) in the cortex of the cell, Msps (purple) can localize to the plus end through its interaction with Sentin (red), which can bind EB1 (black). Msps cannot interact with the lattice and we speculate it is in a folded conformation in which its lattice binding domains are masked. Orbit (grey) is phosphorylated by GSK3β and can interact with the plus end by binding either EB1 directly or Sentin. (2) At the plus end of the microtubule in the periphery, both Msps and Orbit bind to the plus end through the same associations as in the cell cortex. Orbit is no longer phosphorylated by GSK3β and can interact with Msps. The two proteins interact through their C-termini and this allows Msps to bind to the lattice further back from the plus end, possibly by changing the conformation of Msps to expose its lattice binding domains.</p

Msps 1406–1506 interacts with Orbit.

<p>(A-C) MspsCT-GFP was expressed in cells with a dual expression vector containing tRFP-α-tubulin alone (A) or with CA-Rac1 (B) and also in cells with Rac1/Rac2/Mtl depletion (C). (D-F) Msps L4-CT-GFP was expressed in cells with a dual expression vector containing α-tubulin-tRFP alone (D) or with CA-Rac1 (E) and also in cells with Rac1/Rac2/Mtl depletion (F). Tubulin images are shown as insets. (G) Changes in co-localization of Msps were measured using the Mander’s coefficient, n = 90 cells from three experiments. *** p<0.0001 (H) Alignment of sequences contained in Msps Linker4-TOG5, Linker2-TOG3 and 1406–1596, residue number is indicated to the right and left of the residue. (I) Changes in co-localization of endogenous Msps and microtubules due to competition with Msps C-terminal and mutant fragments, n = 90 cells from two experiments. *** p<0.0001 (J) mCherry-tagged Msps 1406–1596 coimmunoprecipitates with C-terminal Orbit residues 1071–1492. (K) mCherry-tagged Msps 1406–1596 mutants fail to coimmunoprecipitate with C-terminal Orbit residues 1071–1492. (L-M) Msps-GFP 3A was expressed in cells with a dual expression vector containing tRFP-α-tubulin alone (L) or with CA-Rac1 (M). (N-O) Msps-GFP 3K3A was expressed in cells with a dual expression vector containing tRFP-α-tubulin alone (N) or with CA-Rac1 (O). Tubulin images are shown as insets. (P) Co-localization between Msps and microtubules were measured using the Mander’s coefficient, n = 90 cells from three experiments. *** p<0.0001 (Q) Localization of wild-type Msps, insets represented with a rectangle highlighting the periphery. (R) Localization of Msps 3K3A, insets represented with a rectangle highlighting the periphery.</p

Identifying Regulators of Morphogenesis Common to Vertebrate Neural Tube Closure and Caenorhabditis elegans

Neural tube defects including spina bifida are common and severe congenital disorders. In mice, mutations in more than 200 genes can result in neural tube defects. We hypothesized that this large gene set might include genes whose homologs contribute to morphogenesis in diverse animals. To test this hypothesis, we screened a set of Caenorhabditis elegans homologs for roles in gastrulation, a topologically similar process to vertebrate neural tube closure. Both C. elegans gastrulation and vertebrate neural tube closure involve the internalization of surface cells, requiring tissue-specific gene regulation, actomyosin-driven apical constriction, and establishment and maintenance of adhesions between specific cells. Our screen identified several neural tube defect gene homologs that are required for gastrulation in C. elegans, including the transcription factor sptf-3. Disruption of sptf-3 in C. elegans reduced the expression of early endodermally expressed genes as well as genes expressed in other early cell lineages, establishing sptf-3 as a key contributor to multiple well-studied C. elegans cell fate specification pathways. We also identified members of the actin regulatory WAVE complex (wve-1, gex-2, gex-3, abi-1, and nuo-3a). Disruption of WAVE complex members reduced the narrowing of endodermal cells’ apical surfaces. Although WAVE complex members are expressed broadly in C. elegans, we found that expression of a vertebrate WAVE complex member, nckap1, is enriched in the developing neural tube of Xenopus. We show that nckap1 contributes to neural tube closure in Xenopus. This work identifies in vivo roles for homologs of mammalian neural tube defect genes in two manipulable genetic model systems