Nie S , Kee Y , Bronner-Fraser M .

DE017911 NIDCR NIH HHS, HD037105 NICHD NIH HHS , P01 HD037105 NICHD NIH HHS , R01 DE017911 NIDCR NIH HHS

	Figure 1: Sequence alignment of the Xenopus CaD and other CaD isoforms. Amino acid sequences of CaD isoforms from Xenopus CaD, chicken nonmuscle CaD (M59762), mouse CaD 1 (NM_145575), and human CaD (M83216) were aligned using the Clustal W program in DNAstar. Major binding domains for myosin, tropomyosin, actin, and Ca2+- calmodulin are highlighted in the green, yellow, red, and blue boxes, respectively. Xenopus CaD is highly conserved relative to those from other species in the binding domains, with 82, 91, 93, and 100% identity at the amino acid level, respectively.
	Figure 2: CaD is expressed in neural crest tissue during early Xenopus development. Expression of CaD begins at late gastrula stage (stage 13) at the borders of the forming neural plate, the site of initial neural crest induction. Its expression increases in the CNC region by neurula stages (stage 15) and continues in the migrating neural crest cells as they populate the branchial arches (stage 20 onward). By tailbud stages, the expression decreases and only is present in part of the ganglia and somite at low levels (stage 26). All embryos are oriented with anterior to the left.
	Figure 3: CaD is not required for neural crest induction. Embryos were injected with control-MO or CaD-MO (10 ng) in one cell at the two-cell stage, together with a lineage tracer (nβGal, stained with Red-Gal, bottom). No change in expression of neural crest genes twist and sox10 was observed by in situ hybridization at neurula stages, suggesting that they were not affected by CaD-MO. All embryos were in dorsal view, with anterior to the left.
	Figure 4: CaD-MO disrupted the expression of neural crest genes at tailbud stages. Embryos receiving control-MO or CaD-MO (10 ng) in one cell at the two-cell stage were collected at tailbud stages and analyzed for neural crest gene expression by in situ hybridization. The injected side for each embryo is marked by an asterisk and the control side of the same embryo is placed below serving as internal control. Whereas control-MO did not affect the expression of general neural crest markers twist and sox10 or the third branchial arch marker EphA4, the expression of all three genes was disrupted by CaD-MO (arrows). Marker analysis suggests that neural crest cells failed to extend into the lateral portion of the branchial arches and to separate into clear streams in the cases of twist and sox10. Simultaneous injection of CaD (100–200 pg) rescued the expression of neural crest genes. All embryos were oriented with anterior to the left and dorsal up.
	Figure 5: CaD-MO inhibited the formation of cranial cartilage. Ventral view of cartilage from stage 45+ tadpoles stained with Alcian blue. Embryo halves received 10 ng of control-MO or CaD-MO on the left (marked by an asterisk). Cartilage on the CaD-MO–injected side was often reduced in size, distorted, or missing completely, whereas coexpression of CaD rescued the cartilage formation effectively. CB, ceratobranchial cartilage (from third and fourth branchial arch streams); CH, ceratohyal cartilage (from hyoid stream); M, Meckel’s cartilage (from mandibular stream).
	Figure 6: CaD is required for CNC explants to spread and segregate on FN matrix. (A) CNC explants were dissected from early neurula embryos injected with control-MO or CaD-MO (10 ng) and plated on FN. By 10 h, control-MO–expressing cells spread and migrated extensively and segregated into three streams. However, cells from CaD-MO–expressing explants failed to spread efficiently or separate into streams. Coinjection of 100–200 pg of CaD largely rescued the spread and segregation of the explants. Arrows with numbers marked the segregated lobes. High-magnification differential interference contrast imaging of the explants (B, 40×) and fluorescence imaging of cells with membrane labeling (C, 20×) show that whereas control-MO–expressing cells extended multiple membrane protrusions, CaD-MO–expressing cells remained unpolarized and were often rounded. (D) The cells were tracked for 2 h and trajectories plotted. Although the speed of migration was largely unaffected, the directionality of migration was significantly impaired by CaD-MO. Black traces indicate that the ratio of the final distance to the entire route covered is >0.3, and red indicates that it was <0.3.
	Figure 7: CaD-MO disrupted CNC migration in vivo. (A) GFP-labeled CNC grafts receiving control-MO or CaD-MO (10 ng) were transplanted into unlabeled host embryos at neurula stages and imaged at tailbud stages. Fluorescent and merged images are shown side by side with anterior to the left. Whereas control-MO–receiving grafts migrated laterally into separate branchial arches, CaD-MO–expressing grafts migrated less far and failed to segregate into streams. Coexpression of a low-dose CaD restored the migration of CNC transplants. (B) The embryos were binned into four categories on the basis of the migratory behaviors. Whereas 79% of control grafts migrated and segregated normally (n = 61), 18% of CaD-MO–expressing grafts failed to migrate at all. Another 67% of the grafts migrated shorter distances, over half of which did not segregate (n = 45). Coexpression of CaD (100–200 pg) rescued the segregation (83%) and migration (44%, n = 18) remarkably.
	Figure 8: CaD-MO disrupts the organization of actin filament. (A) CNC explants receiving membrane-tethered EGFP together with control-MO or CaD-MO at 16- to 32-cell stages were fixed on FN and stained with rhodamine-conjugated phalloidin. Whereas actin associated with protrusions and stress fibers in control-MO–expressing cells, actin distributed in a relatively ubiquitous manner in CaD-MO–expressing cells. Arrows indicate injected cells, and arrowheads mark uninjected cells in the same explant. Right, actin staining alone in dissociated cells. (B) Phalloidin staining in cells expressing 0.1 ng of EGFP-CaD revealed colocalization of CaD with actin filaments in membrane protrusions and stress fibers (arrows). Images were taken at 40×. Scale bars, 20 μm.
	Figure 9: Overexpression of various CaD constructs affects actin-associated structures. Phalloidin staining was performed on CNC explants receiving 0.2 ng of EGFP-CaD, CaD39-AB, CaD39-6F, and CaD39-PAKA. To distinguish cells receiving the RNA, H2B-EGFP was coinjected with the mutant constructs. Whereas overexpression of EGFP-CaD produced abundant ectopic actin-rich protrusions, CaD39-AB increased stress fiber formation. CaD39-6F and CaD39- PAKA caused enrichment of short actin bundles around the cell periphery to different extents. Arrows indicate actin-rich structures. Images were taken using 40× objectives. Scale bars, 20 μm.
	Supplementary Figure 1. CaD-MO efficiently inhibits CaD protein translation. Protein extracted from uninjected and CaD-MO injected CNC tissue was resolved on SDS page gel and blot against CaD antibody (Santa Cruz Biotechnology, sc-15374). No CaD protein was detected from CaD-MO expressing tissue indicating CaD-MO efficiently blocked CaD translation. Blotting against α-tubulin antibody served as loading control.
	Supplementary Figure 2. Expression of CaD in preplacodal region. Anterior views of stage 13-14 embryos show that CaD is expressed in anterior and lateral borders of the rostral neural plate, where prospective placodes and cranial neural crest will arise. Dorsal is oriented to the top.
	Supplementary Figure 3. CaD-MO disrupts CNC cell adhesion. (A) Cell-cell adhesion examined by dissociation/reaggregation experiments. While control-MO expressing CNC cells reaggregated into one big cell clump at 6 hours, CaD-MO expressing cells only grouped to medium sized aggregates. (B) Cell adhesion on FN matrix. Dissociated cells were plated on FN-coated dish for an hour and unattached cells were washed away by flipping over the dish. Much fewer cells remained on the FN plate in CaD-MO treated cells.
	cald1 (caldesmon 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 15, dorsal view, anterior left.
	cald1 (caldesmon 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 21, dorsal view, anterior left.
	cald1 (caldesmon 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 26, lateral view, dorsal up, anterior left.
	FIGURE 1:. Sequence alignment of the Xenopus CaD and other CaD isoforms. Amino acid sequences of CaD isoforms from Xenopus CaD, chicken nonmuscle CaD (M59762), mouse CaD 1 (NM_145575), and human CaD (M83216) were aligned using the Clustal W program in DNAstar. Major binding domains for myosin, tropomyosin, actin, and Ca2+-calmodulin are highlighted in the green, yellow, red, and blue boxes, respectively. Xenopus CaD is highly conserved relative to those from other species in the binding domains, with 82, 91, 93, and 100% identity at the amino acid level, respectively.
	FIGURE 2:. CaD is expressed in neural crest tissue during early Xenopus development. Expression of CaD begins at late gastrula stage (stage 13) at the borders of the forming neural plate, the site of initial neural crest induction. Its expression increases in the CNC region by neurula stages (stage 15) and continues in the migrating neural crest cells as they populate the branchial arches (stage 20 onward). By tailbud stages, the expression decreases and only is present in part of the ganglia and somite at low levels (stage 26). All embryos are oriented with anterior to the left.
	FIGURE 3:. CaD is not required for neural crest induction. Embryos were injected with control-MO or CaD-MO (10 ng) in one cell at the two-cell stage, together with a lineage tracer (nβGal, stained with Red-Gal, bottom). No change in expression of neural crest genes twist and sox10 was observed by in situ hybridization at neurula stages, suggesting that they were not affected by CaD-MO. All embryos were in dorsal view, with anterior to the left.
	FIGURE 4:. CaD-MO disrupted the expression of neural crest genes at tailbud stages. Embryos receiving control-MO or CaD-MO (10 ng) in one cell at the two-cell stage were collected at tailbud stages and analyzed for neural crest gene expression by in situ hybridization. The injected side for each embryo is marked by an asterisk and the control side of the same embryo is placed below serving as internal control. Whereas control-MO did not affect the expression of general neural crest markers twist and sox10 or the third branchial arch marker EphA4, the expression of all three genes was disrupted by CaD-MO (arrows). Marker analysis suggests that neural crest cells failed to extend into the lateral portion of the branchial arches and to separate into clear streams in the cases of twist and sox10. Simultaneous injection of CaD (100–200 pg) rescued the expression of neural crest genes. All embryos were oriented with anterior to the left and dorsal up.
	FIGURE 5:. CaD-MO inhibited the formation of cranial cartilage. Ventral view of cartilage from stage 45+ tadpoles stained with Alcian blue. Embryo halves received 10 ng of control-MO or CaD-MO on the left (marked by an asterisk). Cartilage on the CaD-MO–injected side was often reduced in size, distorted, or missing completely, whereas coexpression of CaD rescued the cartilage formation effectively. CB, ceratobranchial cartilage (from third and fourth branchial arch streams); CH, ceratohyal cartilage (from hyoid stream); M, Meckel's cartilage (from mandibular stream).
	FIGURE 6:. CaD is required for CNC explants to spread and segregate on FN matrix. (A) CNC explants were dissected from early neurula embryos injected with control-MO or CaD-MO (10 ng) and plated on FN. By 10 h, control-MO–expressing cells spread and migrated extensively and segregated into three streams. However, cells from CaD-MO–expressing explants failed to spread efficiently or separate into streams. Coinjection of 100–200 pg of CaD largely rescued the spread and segregation of the explants. Arrows with numbers marked the segregated lobes. High-magnification differential interference contrast imaging of the explants (B, 40×) and fluorescence imaging of cells with membrane labeling (C, 20×) show that whereas control-MO–expressing cells extended multiple membrane protrusions, CaD-MO–expressing cells remained unpolarized and were often rounded. (D) The cells were tracked for 2 h and trajectories plotted. Although the speed of migration was largely unaffected, the directionality of migration was significantly impaired by CaD-MO. Black traces indicate that the ratio of the final distance to the entire route covered is >0.3, and red indicates that it was <0.3.
	FIGURE 7:. CaD-MO disrupted CNC migration in vivo. (A) GFP-labeled CNC grafts receiving control-MO or CaD-MO (10 ng) were transplanted into unlabeled host embryos at neurula stages and imaged at tailbud stages. Fluorescent and merged images are shown side by side with anterior to the left. Whereas control-MO–receiving grafts migrated laterally into separate branchial arches, CaD-MO–expressing grafts migrated less far and failed to segregate into streams. Coexpression of a low-dose CaD restored the migration of CNC transplants. (B) The embryos were binned into four categories on the basis of the migratory behaviors. Whereas 79% of control grafts migrated and segregated normally (n = 61), 18% of CaD-MO–expressing grafts failed to migrate at all. Another 67% of the grafts migrated shorter distances, over half of which did not segregate (n = 45). Coexpression of CaD (100–200 pg) rescued the segregation (83%) and migration (44%, n = 18) remarkably.
	FIGURE 8:. CaD-MO disrupts the organization of actin filament. (A) CNC explants receiving membrane-tethered EGFP together with control-MO or CaD-MO at 16- to 32-cell stages were fixed on FN and stained with rhodamine-conjugated phalloidin. Whereas actin associated with protrusions and stress fibers in control-MO–expressing cells, actin distributed in a relatively ubiquitous manner in CaD-MO–expressing cells. Arrows indicate injected cells, and arrowheads mark uninjected cells in the same explant. Right, actin staining alone in dissociated cells. (B) Phalloidin staining in cells expressing 0.1 ng of EGFP-CaD revealed colocalization of CaD with actin filaments in membrane protrusions and stress fibers (arrows). Images were taken at 40×. Scale bars, 20 μm.
	FIGURE 9:. Overexpression of various CaD constructs affects actin-associated structures. Phalloidin staining was performed on CNC explants receiving 0.2 ng of EGFP-CaD, CaD39-AB, CaD39-6F, and CaD39-PAKA. To distinguish cells receiving the RNA, H2B-EGFP was coinjected with the mutant constructs. Whereas overexpression of EGFP-CaD produced abundant ectopic actin-rich protrusions, CaD39-AB increased stress fiber formation. CaD39-6F and CaD39-PAKA caused enrichment of short actin bundles around the cell periphery to different extents. Arrows indicate actin-rich structures. Images were taken using 40× objectives. Scale bars, 20 μm.

Alfandari, Integrin alpha5beta1 supports the migration of Xenopus cranial neural crest on fibronectin. 2003, Pubmed, Xenbase

Alfandari, Integrin alpha5beta1 supports the migration of Xenopus cranial neural crest on fibronectin. 2003, Pubmed , Xenbase
Borchers, Xenopus cadherin-11 restrains cranial neural crest migration and influences neural crest specification. 2001, Pubmed , Xenbase
Borchers, An assay system to study migratory behavior of cranial neural crest cells in Xenopus. 2000, Pubmed , Xenbase
Carmona-Fontaine, Contact inhibition of locomotion in vivo controls neural crest directional migration. 2008, Pubmed , Xenbase
Chalovich, Kinetics of binding of caldesmon to actin. 1995, Pubmed
Chang, A Xenopus type I activin receptor mediates mesodermal but not neural specification during embryogenesis. 1997, Pubmed , Xenbase
Childs, Phosphorylation of smooth muscle caldesmon by mitogen-activated protein (MAP) kinase and expression of MAP kinase in differentiated smooth muscle cells. 1992, Pubmed
Dabrowska, Modulation of gelsolin-induced actin-filament severing by caldesmon and tropomyosin and the effect of these proteins on the actin activation of myosin Mg(2+)-ATPase activity. 1996, Pubmed
De Calisto, Essential role of non-canonical Wnt signalling in neural crest migration. 2005, Pubmed , Xenbase
DeSimone, The Xenopus embryo as a model system for studies of cell migration. 2005, Pubmed , Xenbase
Eppinga, Requirement of reversible caldesmon phosphorylation at P21-activated kinase-responsive sites for lamellipodia extensions during cell migration. 2006, Pubmed
Eves, Caldesmon is an integral component of podosomes in smooth muscle cells. 2006, Pubmed
Foster, Modes of caldesmon binding to actin: sites of caldesmon contact and modulation of interactions by phosphorylation. 2004, Pubmed
Foster, Phosphorylation of caldesmon by p21-activated kinase. Implications for the Ca(2+) sensitivity of smooth muscle contraction. 2000, Pubmed
Fukumoto, Detrimental effects of glucocorticoids on neuronal migration during brain development. 2009, Pubmed
Goncharova, Actomyosin cross-linking by caldesmon in non-muscle cells. 2001, Pubmed
Grosheva, Caldesmon effects on the actin cytoskeleton and cell adhesion in cultured HTM cells. 2006, Pubmed
Gu, Erk1/2 MAPK and caldesmon differentially regulate podosome dynamics in A7r5 vascular smooth muscle cells. 2007, Pubmed
Helbling, Requirement for EphA receptor signaling in the segregation of Xenopus third and fourth arch neural crest cells. 1998, Pubmed , Xenbase
Helfman, Caldesmon inhibits nonmuscle cell contractility and interferes with the formation of focal adhesions. 1999, Pubmed
Hemric, Caldesmon enhances the binding of myosin to the cytoskeleton during platelet activation. 1994, Pubmed
Huang, Caldesmon binding to actin is regulated by calmodulin and phosphorylation via different mechanisms. 2003, Pubmed
Ishikawa, Differential modulation of actin-severing activity of gelsolin by multiple isoforms of cultured rat cell tropomyosin. Potentiation of protective ability of tropomyosins by 83-kDa nonmuscle caldesmon. 1989, Pubmed
Ishikawa, Regulation of actin binding and actin bundling activities of fascin by caldesmon coupled with tropomyosin. 1998, Pubmed
Ishikawa, Annealing of gelsolin-severed actin fragments by tropomyosin in the presence of Ca2+. Potentiation of the annealing process by caldesmon. 1989, Pubmed
Kashef, Cadherin-11 regulates protrusive activity in Xenopus cranial neural crest cells upstream of Trio and the small GTPases. 2009, Pubmed , Xenbase
Kordowska, Phosphorylated l-caldesmon is involved in disassembly of actin stress fibers and postmitotic spreading. 2006, Pubmed
Li, Caldesmon mutant defective in Ca(2+)-calmodulin binding interferes with assembly of stress fibers and affects cell morphology, growth and motility. 2004, Pubmed
Lin, Chapter 1: roles of caldesmon in cell motility and actin cytoskeleton remodeling. 2009, Pubmed
Mak, Phosphorylation of caldesmon by p34cdc2 kinase. Identification of phosphorylation sites. 1991, Pubmed
Matthews, Wnt11r is required for cranial neural crest migration. 2008, Pubmed , Xenbase
Matthews, Directional migration of neural crest cells in vivo is regulated by Syndecan-4/Rac1 and non-canonical Wnt signaling/RhoA. 2008, Pubmed , Xenbase
Mayanagi, Glucocorticoid receptor-mediated expression of caldesmon regulates cell migration via the reorganization of the actin cytoskeleton. 2008, Pubmed
McFawn, Calcium-independent contraction and sensitization of airway smooth muscle by p21-activated protein kinase. 2003, Pubmed
Mellitzer, Eph receptors and ephrins restrict cell intermingling and communication. 1999, Pubmed
Nie, Myosin-X is critical for migratory ability of Xenopus cranial neural crest cells. 2009, Pubmed , Xenbase
Nomura, The role of tropomyosin in the interactions of F-actin with caldesmon and actin-binding protein (or filamin). 1987, Pubmed
Novy, Characterization of cDNA clones encoding a human fibroblast caldesmon isoform and analysis of caldesmon expression in normal and transformed cells. 1991, Pubmed
Numaguchi, Caldesmon-dependent switching between capillary endothelial cell growth and apoptosis through modulation of cell shape and contractility. 2003, Pubmed
Paul, Embryonic chicken gizzard: expression of the smooth muscle regulatory proteins caldesmon and myosin light chain kinase. 1995, Pubmed
Rangarajan, PCNS: a novel protocadherin required for cranial neural crest migration and somite morphogenesis in Xenopus. 2006, Pubmed , Xenbase
Robinson, Roles of Eph receptors and ephrins in neural crest pathfinding. 1997, Pubmed
Sadaghiani, Neural crest development in the Xenopus laevis embryo, studied by interspecific transplantation and scanning electron microscopy. 1987, Pubmed , Xenbase
Santiago, Ephrin-B ligands play a dual role in the control of neural crest cell migration. 2002, Pubmed
Smith, The EphA4 and EphB1 receptor tyrosine kinases and ephrin-B2 ligand regulate targeted migration of branchial neural crest cells. 1997, Pubmed , Xenbase
Sobue, Caldesmon, a novel regulatory protein in smooth muscle and nonmuscle actomyosin systems. 1991, Pubmed
Tanaka, Morphological and biochemical analyses of contractile proteins (actin, myosin, caldesmon and tropomyosin) in normal and transformed cells. 1993, Pubmed
Theveneau, Collective chemotaxis requires contact-dependent cell polarity. 2010, Pubmed , Xenbase
Vallin, Xenopus cadherin-11 is expressed in different populations of migrating neural crest cells. 1998, Pubmed , Xenbase
Van Eyk, Different molecular mechanisms for Rho family GTPase-dependent, Ca2+-independent contraction of smooth muscle. 1998, Pubmed
Warren, Overexpression of microfilament-stabilizing human caldesmon fragment, CaD39, affects cell attachment, spreading, and cytokinesis. 1996, Pubmed
Winklbauer, Cell interaction and its role in mesoderm cell migration during Xenopus gastrulation. 1992, Pubmed , Xenbase
Yamakita, Caldesmon inhibits Arp2/3-mediated actin nucleation. 2003, Pubmed
Yamashiro, Caldesmon: possible functions in microfilament reorganization during mitosis and cell transformation. 1994, Pubmed
Yamashiro, Mutant Caldesmon lacking cdc2 phosphorylation sites delays M-phase entry and inhibits cytokinesis. 2001, Pubmed , Xenbase
Yamashiro, Phosphorylation of non-muscle caldesmon by p34cdc2 kinase during mitosis. 1991, Pubmed
Yamashiro, Mitosis-specific phosphorylation causes 83K non-muscle caldesmon to dissociate from microfilaments. 1990, Pubmed
Yoshio, Caldesmon suppresses cancer cell invasion by regulating podosome/invadopodium formation. 2007, Pubmed
Zheng, A crucial role of caldesmon in vascular development in vivo. 2009, Pubmed
Zheng, Caldesmon is essential for cardiac morphogenesis and function: in vivo study using a zebrafish model. 2009, Pubmed