Marivin A , Morozova V , Walawalkar I , Leyme A , Kretov DA , Cifuentes D , Dominguez I , Garcia-Marcos M .

R01 GM108733 NIGMS NIH HHS , R01 GM130120 NIGMS NIH HHS , R00 HD071968 NICHD NIH HHS , R01 GM098367 NIGMS NIH HHS

	Figure 1. DAPLE localizes to apical cell junctions and promotes apical cell constriction. (A) Confocal fluorescence microscopy pictures of MDCK cells sparsely expressing ectopic MYC-hDAPLE costained for MYC (red), ZO-1 (green), and E-cadherin (blue). Top panels correspond to a view on the monolayer from the top, and panels on the bottom are optical cross sections of the monolayer. a and b segments correspond to the width of the apical and basal cell membrane domains. (B and C) Confocal fluorescence microscopy pictures of MDCK (B) or EpH4 (C) cell monolayers stained for DAPLE (Sigma-Aldrich or Millipore antibody, red), ZO-1 (green), and E-cadherin (blue) as indicated. The top panels correspond to views on the cell monolayers from the top, and panels on the bottom are optical cross sections of the monolayers. (D) Quantification of the relative apical area of DAPLE-transfected cells compared with neighboring, untransfected cells shows that hDAPLE WT, but not hydrocephalus-associated mutants (H1 and H2), cause apical constriction in MDCK cells. Results are presented as box-and-whiskers plots (minimum to maximum) of n = 5–9 independent experiments per condition. ***, P < 0.001 using the Mann–Whitney U test. (E) Fluorescence microscopy pictures of MDCK cells sparsely expressing the indicated MYC-hDAPLE constructs and costained for MYC (magenta) and ZO-1 (green) show that H1 and H2 mutants are not enriched at apical cell junctions like WT. Scale bars, 10 µm.
	Figure 2. Both the PBM and GBA motif of DAPLE are required to promote apical cell constriction. (A) Quantification of the relative apical area of DAPLE-transfected cells compared with neighboring, untransfected cells shows that hDAPLE ΔPBM and hDAPLE GBA* mutants fail to promote apical constriction in MDCK (left) or EpH4 (right) cells. Results are presented as box-and-whiskers plots (error bars indicate minimum to maximum range) of n = 4–9 independent experiments per condition. *, P < 0.05; **, P < 0.01 using the Mann–Whitney U test. (B) Confocal fluorescence microscopy pictures of MDCK cells sparsely expressing the indicated MYC-hDAPLE constructs and costained for MYC (magenta) and ZO-1 (green) show that hDAPLE GBA* mutant is enriched at apical junctions like WT, while hDAPLE ΔPBM mutant is not. Scale bars, 10 µm. (C) Diagram depicting the different functions of the PBM and the GBA motif in DAPLE-induced apical cell constriction. A possible effector pathway to promote apical cell constriction though G protein activation is shown on the right, along with the treatments used in D to test it (green). (D) Box-and-whiskers plots (error bars indicate minimum to maximum range) for the quantification of relative apical area show that DAPLE-mediated apical constriction requires the activity of myosin (inhibited by blebbistatin), ROCK (inhibited by Y-27632), free Gβγ (inhibited by Gallein, but not its inactive analogue, fluorescein) and p114RhoGEF (inhibited by siRNA). n = 4 independent experiments per condition. *, P < 0.05 using the Mann–Whitney U test. The immunoblot (IB) on the bottom right shows the reduction of p114RhoGEF expression upon siRNA treatments.
	Figure 3. Loss of DAPLE delays NT closure in Xenopus. (A) Quantification of DAPLE mRNA abundance in whole Xenopus embryos at different stages by RNA sequencing showing induction during neurulation. Extracted from Peshkin et al. (2015). (B) Left: Whole-mount RNA in situ Hybridization for xDAPLE showing restricted expression in neural tissues from the onset of neurulation, but not at earlier stages (st). Right: anterior transversal section on the right shows specific expression in neuroepithelial cells. (C) xDAPLE morphants at different stages of development showing a delay in the closure of the neural plate compared with controls. Graph at the bottom shows a quantification of the distance between neural folds from 10 embryos at the indicated stages (average ± SEM). ***, P < 0.001 using the t test (two-tailed, unpaired). (D) Quantification of neural plate bending defects in embryos unilaterally injected with xDAPLE MOs and/or xDAPLE mRNA. n = 50–100 embryos per condition analyzed at stage 17. ***, P < 0.001 using the χ2 test. Validation of xDAPLE MOs is shown in Fig. S2. All scale bars represent 250 µm, except the one in the transversal section in B, which represents 50 µm.
	Figure 4. Loss of DAPLE in Xenopus causes apical constriction defects during neurulation. (A) Whole-mount F-actin staining (magenta) of Xenopus embryos unilaterally coinjected with xDAPLE MO and a lineage tracer (mRFP or GFP-CAAX, green) showing enlarged apical surface of DAPLE-depleted neuroepithelial cells compared with uninjected control sides at stages 15 and 16. (B) Transversal cryosection stained for β-catenin (magenta) of the anterior neural plate of an embryo at stage 16 unilaterally coinjected with xDAPLE MO and a lineage tracer (GFP-CAAX, green). Outlines of cell borders are depicted in the bottom to show the lack of wedge shape morphology in the outer layer of neuroepithelial cells depleted of DAPLE. (C–F) Whole-mount pMLC2 (C), ZO-1 (D), GFP (E), and Vangl2 (F) staining (magenta) of Xenopus embryos unilaterally coinjected with xDAPLE MO and a lineage tracer (GFP-CAAX or mRFP; green). In E, embryos were bilaterally injected with Crb3-GFP (top) or GFP-Lgl2 (bottom). In F, staining for the lineage tracer (mRFP) is not shown for clarity, and an immunoblot from dissected neural plates is shown in the bottom. xDAPLE depleted sides show defective staining for actomyosin contractility and PCP markers at stage 15, while markers of apical cell junctions or apicobasal polarity are not changed. All images presented in this figure are representative results of n ≥ 3 experiments. All scale bars represent 25 µm, except those in A, which represent 50 µm.
	Figure 5. xDAPLE promotes G protein signaling and apical cell constriction via two GBA motifs in tandem. (A) Comparison of domain composition of hDAPLE and xDAPLE, and alignment of xDAPLE GBA1 and GBA2 with other GBA motifs. ce, C. elegans; h, Homo sapiens. (B) Full-length xDAPLE from HEK293T cell lysates binds to immobilized GST-Gαi3 when the G protein is loaded with GDP (inactive), but not when it is loaded with GDP-AlF4−. (C) Steady-state GTPase (black) and GTPγS binding (red) experiments showing that purified His-xDAPLE-CT accelerates nucleotide exchange of purified His-Gαi3. Results are the average ± SEM of n = 3 experiments. (D) xDAPLE-CT and hDAPLE-CT activate G protein signaling in a yeast-based β-galactosidase reporter assay. Diagram on the left depicts the pathway activated by DAPLE in yeast lacking the cognate GPCR and with the endogenous G protein replaced by human Gαi3. Results on the right are the average ± SEM of n = 3 experiments. (E) Protein–protein binding experiment showing that purified His-Gαi3 binds to both GBA1 and GBA2 of xDAPLE. Diagram on the top shows a detail of the sequence of the purified GST-fused xDAPLE constructs used in the experiment and the position of FA point mutations (in red). (F) G protein activity assays in yeast (as in D) show that both GBA1 and GBA2 have to be mutated simultaneously to abolish xDAPLE-mediated activation. Results are the average ± SEM of n = 5 experiments. (G) Steady-state GTPase experiments showing that activation of purified His-Gαi3 by GST-xDAPLE FA1+2 (red) is impaired compared GST-xDAPLE WT (black). Results are the average ± SEM of n = 3 experiments. Basal activity = 0.16 mol Pi/mol Gαi3/15 min. (H) Coimmunoprecipitation (IP) experiments showing that xDAPLE WT, but not xDAPLE FA1+2 mutant, binds to Gαi3-FLAG when expressed in HEK293T cells. Immunoblots (IB) of the FLAG IPs are shown on the top and equal aliquots of the starting lysates used for it are shown on the bottom. (I) Box-and-whiskers plots (minimum to maximum) for the quantification of relative apical area show xDAPLE ΔPBM and xDAPLE GBA** (FA1+2) mutants fail to promote apical constriction in MDCK cells compared with xDAPLE WT. Results are from n = 4–9 independent experiments. *, P < 0.05; **, P < 0.01 using the Mann–Whitney U test. (J) Fluorescence microscopy pictures of MDCK cells sparsely expressing the indicated MYC-xDAPLE constructs and costained for MYC (magenta) and ZO-1 (green) show that xDAPLE GBA** mutant is enriched at apical junctions like WT, while xDAPLE ΔPBM mutant is not. Scale bars, 10 µm.
	Figure 6. Activation of G protein signaling by xDAPLE is required for apical cell constriction during neurulation. (A) Quantification of neural plate bending defects in embryos unilaterally injected with xDAPLE MO and/or xDAPLE mRNAs as in Fig. 3 D shows that xDAPLE WT, but neither ΔPBM (ΔP) nor GBA** (G**), rescues neural plate bending defects upon xDAPLE depletion. The number (n) of embryos per condition in indicated above the bars. ***, P < 0.001 or not significant (ns) using the χ2 test. (B) Confocal fluorescence microscopy pictures of the neural plate of Xenopus embryos unilaterally injected with MYC-xDAPLE mRNA and costained for MYC (green) and β-catenin (magenta). The left and middle panels correspond to a view on the neuroepithelium from the top, and panels on the right are optical cross sections. The yellow dotted line separates the injected from the uninjected side of the neuroepithelium. Scale bars, 20 µm. (C) Morphology of Xenopus embryos at stage 16 after treatment with the Gβγ inhibitor M158C or its inactive analogue, M158D. Scale bars, 250 µm. (D) Scatterplot for the quantification of the distance between neural folds from 90 embryos treated with M158D or M158C (average ± SEM). ***, P < 0.001 using the t test (two-tailed, unpaired). (E) Whole-mount F-actin staining of Xenopus embryos at stage 17 after treatment with the Gβγ inhibitor M158C or its inactive analogue, M158D. Red boxes in the top panels are magnified in the bottom panels to show the enlarged area of the neuroepithelial cells treated with M158D compared with M158C. Scale bars, 50 µm. (F) Quantification of neural plate bending defects in embryos unilaterally injected with p114RhoGEF MO1 (splicing-interfering, validated in Fig. S5) and p114RhoGEF MO2 (translation blocking). n = 80 embryos analyzed at stage 17. ***, P < 0.001 using the Fisher exact test. Scale bars, 250 µm. (G) Whole-mount F-actin staining (green) of Xenopus embryos unilaterally coinjected with p114RhoGEF MO1 and a lineage tracer (GFP-CAAX, magenta). The red box in the left panel is magnified in the panels on the right to show the enlarged area of the neuroepithelial cells depleted on p114RhoGEF compared with the control sides. Scale bars, 50 µm. All images presented in this figure are representative results of n ≥ 3 experiments.
	Figure 7. Mechanism of G protein–mediated regulation of apical cell constriction during neurulation by the non-GPCR protein DAPLE. (A) Expression of DAPLE is specifically induced during neurulation. Upon expression, DAPLE localizes to apical cell junctions of neuroepithelial cells, where it triggers G protein activation that leads to apical cell constriction and the subsequent bending of the neural plate. (B) Theme and variations of G protein–regulated apical cell constriction during epithelial tissue morphogenesis in vertebrates versus invertebrates. Heterotrimeric G proteins are part of a conserved ubiquitous machinery that controls actomyosin contractility, but they are regulated differently across species. In vertebrates, DAPLE fulfills the role performed by GPCRs in invertebrates as tissue-specific activators of signaling that drives apical cell constriction. (C) The G protein regulatory function of DAPLE (i.e., its GBA motif) was acquired during evolution in the transition from invertebrates to vertebrates, suggesting that the unconventional mechanism of G protein activation described here is an evolutionary innovation for epithelial remodeling in vertebrates.

Aittaleb, Structure and function of heterotrimeric G protein-regulated Rho guanine nucleotide exchange factors. 2010, Pubmed

Aittaleb, Structure and function of heterotrimeric G protein-regulated Rho guanine nucleotide exchange factors. 2010, Pubmed
Aznar, Daple is a novel non-receptor GEF required for trimeric G protein activation in Wnt signaling. 2015, Pubmed
Barrett, The Rho GTPase and a putative RhoGEF mediate a signaling pathway for the cell shape changes in Drosophila gastrulation. 1997, Pubmed
Blum, Morpholinos: Antisense and Sensibility. 2015, Pubmed , Xenbase
Bonacci, Differential targeting of Gbetagamma-subunit signaling with small molecules. 2006, Pubmed
Butler, Spatial and temporal analysis of PCP protein dynamics during neural tube closure. 2018, Pubmed , Xenbase
Cabrita, A family of E. coli expression vectors for laboratory scale and high throughput soluble protein production. 2006, Pubmed
Camerer, Local protease signaling contributes to neural tube closure in the mouse embryo. 2010, Pubmed
Cismowski, Genetic screens in yeast to identify mammalian nonreceptor modulators of G-protein signaling. 1999, Pubmed
Coleman, Evolutionary Conservation of a GPCR-Independent Mechanism of Trimeric G Protein Activation. 2016, Pubmed
Costa, A putative cell signal encoded by the folded gastrulation gene coordinates cell shape changes during Drosophila gastrulation. 1994, Pubmed
de Opakua, Molecular mechanism of Gαi activation by non-GPCR proteins with a Gα-Binding and Activating motif. 2017, Pubmed
DiGiacomo, When Heterotrimeric G Proteins Are Not Activated by G Protein-Coupled Receptors: Structural Insights and Evolutionary Conservation. 2018, Pubmed
Drielsma, Two novel CCDC88C mutations confirm the role of DAPLE in autosomal recessive congenital hydrocephalus. 2012, Pubmed
Ekici, Disturbed Wnt Signalling due to a Mutation in CCDC88C Causes an Autosomal Recessive Non-Syndromic Hydrocephalus with Medial Diverticulum. 2010, Pubmed
Fuentealba, Expression profiles of the Gα subunits during Xenopus tropicalis embryonic development. 2016, Pubmed , Xenbase
Garcia-Marcos, G Protein binding sites on Calnuc (nucleobindin 1) and NUCB2 (nucleobindin 2) define a new class of G(alpha)i-regulatory motifs. 2011, Pubmed
Garcia-Marcos, GIV is a nonreceptor GEF for G alpha i with a unique motif that regulates Akt signaling. 2009, Pubmed
Garcia-Marcos, A structural determinant that renders G alpha(i) sensitive to activation by GIV/girdin is required to promote cell migration. 2010, Pubmed
Ghosh, A G{alpha}i-GIV molecular complex binds epidermal growth factor receptor and determines whether cells migrate or proliferate. 2010, Pubmed
Gilman, G proteins: transducers of receptor-generated signals. 1987, Pubmed
Gilmour, From morphogen to morphogenesis and back. 2017, Pubmed
Haigo, Shroom induces apical constriction and is required for hingepoint formation during neural tube closure. 2003, Pubmed , Xenbase
Heer, Tension, contraction and tissue morphogenesis. 2017, Pubmed
Jao, Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system. 2013, Pubmed
Kanesaki, Heterotrimeric G protein signaling governs the cortical stability during apical constriction in Drosophila gastrulation. 2013, Pubmed
Keller, Shaping the vertebrate body plan by polarized embryonic cell movements. 2002, Pubmed
Kerridge, Modular activation of Rho1 by GPCR signalling imparts polarized myosin II activation during morphogenesis. 2016, Pubmed
Kobayashi, Novel Daple-like protein positively regulates both the Wnt/beta-catenin pathway and the Wnt/JNK pathway in Xenopus. 2005, Pubmed , Xenbase
Kölsch, Control of Drosophila gastrulation by apical localization of adherens junctions and RhoGEF2. 2007, Pubmed
Lee, Coactivation of G protein signaling by cell-surface receptors and an intracellular exchange factor. 2008, Pubmed
Lee, Using 32-cell stage Xenopus embryos to probe PCP signaling. 2012, Pubmed , Xenbase
Leyme, Integrins activate trimeric G proteins via the nonreceptor protein GIV/Girdin. 2015, Pubmed
Leyme, Specific inhibition of GPCR-independent G protein signaling by a rationally engineered protein. 2017, Pubmed , Xenbase
Lin, Essential roles of G{alpha}12/13 signaling in distinct cell behaviors driving zebrafish convergence and extension gastrulation movements. 2005, Pubmed
Lin, Structural basis for activation of trimeric Gi proteins by multiple growth factor receptors via GIV/Girdin. 2014, Pubmed
Lopez-Sanchez, GIV/Girdin is a central hub for profibrogenic signalling networks during liver fibrosis. 2014, Pubmed
Lowery, Strategies of vertebrate neurulation and a re-evaluation of teleost neural tube formation. 2004, Pubmed
Lowery, Totally tubular: the mystery behind function and origin of the brain ventricular system. 2009, Pubmed , Xenbase
Manning, Regulation of epithelial morphogenesis by the G protein-coupled receptor mist and its ligand fog. 2013, Pubmed
Manning, The Fog signaling pathway: insights into signaling in morphogenesis. 2014, Pubmed
Martin, Apical constriction: themes and variations on a cellular mechanism driving morphogenesis. 2014, Pubmed
Maziarz, A biochemical and genetic discovery pipeline identifies PLCδ4b as a nonreceptor activator of heterotrimeric G-proteins. 2018, Pubmed
Meeker, Method for isolation of PCR-ready genomic DNA from zebrafish tissues. 2007, Pubmed
Midde, Multimodular biosensors reveal a novel platform for activation of G proteins by growth factor receptors. 2015, Pubmed
Moreno-Mateos, CRISPRscan: designing highly efficient sgRNAs for CRISPR-Cas9 targeting in vivo. 2015, Pubmed , Xenbase
Nakajima, Lulu2 regulates the circumferential actomyosin tensile system in epithelial cells through p114RhoGEF. 2011, Pubmed
Nakata, Xenopus Zic3, a primary regulator both in neural and neural crest development. 1997, Pubmed , Xenbase
Newport, A major developmental transition in early Xenopus embryos: I. characterization and timing of cellular changes at the midblastula stage. 1982, Pubmed , Xenbase
Nikolopoulou, Neural tube closure: cellular, molecular and biomechanical mechanisms. 2017, Pubmed
Nishimura, Planar cell polarity links axes of spatial dynamics in neural-tube closure. 2012, Pubmed
Niu, G Protein betagamma subunits stimulate p114RhoGEF, a guanine nucleotide exchange factor for RhoA and Rac1: regulation of cell shape and reactive oxygen species production. 2003, Pubmed
Offermanns, Embryonic cardiomyocyte hypoplasia and craniofacial defects in G alpha q/G alpha 11-mutant mice. 1998, Pubmed
Okae, Neural tube defects and impaired neural progenitor cell proliferation in Gbeta1-deficient mice. 2010, Pubmed
Oshita, Identification and characterization of a novel Dvl-binding protein that suppresses Wnt signalling pathway. 2003, Pubmed , Xenbase
Ossipova, Planar polarization of Vangl2 in the vertebrate neural plate is controlled by Wnt and Myosin II signaling. 2015, Pubmed , Xenbase
Parag-Sharma, Membrane Recruitment of the Non-receptor Protein GIV/Girdin (Gα-interacting, Vesicle-associated Protein/Girdin) Is Sufficient for Activating Heterotrimeric G Protein Signaling. 2016, Pubmed
Parks, The Drosophila gastrulation gene concertina encodes a G alpha-like protein. 1991, Pubmed
Peshkin, On the Relationship of Protein and mRNA Dynamics in Vertebrate Embryonic Development. 2015, Pubmed , Xenbase
Plummer, Development of the mammalian axial skeleton requires signaling through the Gα(i) subfamily of heterotrimeric G proteins. 2012, Pubmed
Ruggeri, Bi-allelic mutations of CCDC88C are a rare cause of severe congenital hydrocephalus. 2018, Pubmed
Siletti, Daple coordinates organ-wide and cell-intrinsic polarity to pattern inner-ear hair bundles. 2017, Pubmed
Smrcka, Molecular targeting of Gα and Gβγ subunits: a potential approach for cancer therapeutics. 2013, Pubmed
Sokol, Spatial and temporal aspects of Wnt signaling and planar cell polarity during vertebrate embryonic development. 2015, Pubmed , Xenbase
Sokol, Mechanotransduction During Vertebrate Neurulation. 2016, Pubmed , Xenbase
Stols, A new vector for high-throughput, ligation-independent cloning encoding a tobacco etch virus protease cleavage site. 2002, Pubmed
Takagishi, Daple Coordinates Planar Polarized Microtubule Dynamics in Ependymal Cells and Contributes to Hydrocephalus. 2017, Pubmed
Tall, Mammalian Ric-8A (synembryn) is a heterotrimeric Galpha protein guanine nucleotide exchange factor. 2003, Pubmed
Terry, Spatially restricted activation of RhoA signalling at epithelial junctions by p114RhoGEF drives junction formation and morphogenesis. 2011, Pubmed
Thisse, High-resolution in situ hybridization to whole-mount zebrafish embryos. 2008, Pubmed
Wallingford, Neural tube closure requires Dishevelled-dependent convergent extension of the midline. 2002, Pubmed , Xenbase
Wallingford, Low-magnification live imaging of Xenopus embryos for cell and developmental biology. 2010, Pubmed , Xenbase
Wallis, Surprisingly good outcome in antenatal diagnosis of severe hydrocephalus related to CCDC88C deficiency. 2018, Pubmed
Wang, Par6b regulates the dynamics of apicobasal polarity during development of the stratified Xenopus epidermis. 2013, Pubmed , Xenbase
Wettschureck, Mouse models to study G-protein-mediated signaling. 2004, Pubmed
Yu, Variable and tissue-specific hormone resistance in heterotrimeric Gs protein alpha-subunit (Gsalpha) knockout mice is due to tissue-specific imprinting of the gsalpha gene. 1998, Pubmed
Zhang, Polycystin 1 loss of function is directly linked to an imbalance in G-protein signaling in the kidney. 2018, Pubmed , Xenbase
Zwaveling-Soonawala, Clues for Polygenic Inheritance of Pituitary Stalk Interruption Syndrome From Exome Sequencing in 20 Patients. 2018, Pubmed