June 15, 2009;
Cadherin-11 regulates protrusive activity in Xenopus cranial neural crest cells upstream of Trio and the small GTPases.
) is expressed when cranial neural crest
) acquire motility. However, its function in stimulating cell migration is poorly understood. Here, we demonstrate that Xcad-11
initiates filopodia and lamellipodia formation, which is essential for CNC
to populate pharyngeal pouches
. We identified the cytoplasmic tail
as both necessary and sufficient for proper CNC
migration as long as it was linked to the plasma membrane
. Our results showing that guanine nucleotide exchange factor (GEF)-Trio binds to Xcad-11
and can functionally substitute for it like constitutively active forms of RhoA
, and cdc42
unravel a novel cadherin function.
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References [+] :
Figure 1. Depletion of Xcad-11 blocks CNC migration. (A) Wholemount ISH. Lateral view of Xenopus CNC at stage 26, analyzed by expression of twist. (Left column) Noninjected side (nis). (Right column) Injected side (is). Embryos injected with Xcad-11MO (2 pmol) showed twist signal adjacent to the brain but not in pharyngeal pouches (72%), indicating impaired CNC migration. The mandibular population remained unaffected. Coinjection of Xcad-11 mRNA (75 pg) restored the migration phenotype in 82%. CoMOinjected (2 pmol) embryos showed no effect. (B) Transplantation to follow migration in vivo in CNC. Wild-type CNC grafts exhibited normal migration (86%). Grafts taken from Xcad-11-depleted embryos were unable to invade hyoidal and branchial arches (70%; marked by arrows). Mandibular CNC remained unaffected. Single cell tracking demonstrated wild-type CNC migrating into the pharyngeal arches while Xcad-11 depletion stopped migration.
Figure 3. The beta-cat-binding site and the TM are essential for Xcad-11 function. (A) Xcad-11 constructs for reconstitution. (S) Signal peptide (green); (EC) extracellular domains (orange); TM (red); (p120/beta-cat) p120-binding site (blue) or beta-cat-binding site (dark red); intracellular domain (yellow). (B) Whole-mount ISH. Lateral view of CNC at stage 26, analyzed by expression of twist. (Left column)Noninjected side (nis). (Right column) Injected side (is). Embryos injected with Xcad-11MO (2 pmol) together with 50 pg of DELTA-e or DELTA-e/DELTA-p120 showed twist signal in pharyngeal pouches, while coinjection of 75 pg of DELTA-c, 50 pg DELTA-e/DELTA-beta-cat, DELTA-e/DELTA-TM, or DELTA-e/DELTA-IMD did not. (C) Statistics for reconstitution. In the case of Xcad-11MO injection, only 28% of embryos showed normal migration. Coinjection of 75
pg of FLXcad-11 resulted in 82%, 75 pg of DELTA-e in 88%, and 75 pg of DELTA-e/DELTA-p120 in 82% of embryos with CNC present in pharyngeal pouches; in the case of DELTA-c, DELTA-e/DELTA-beta-cat, DELTA-e/DELTA-TM, and DELTA-e/DELTA-IMD coinjection, only 30%, 27%, 28%, and 52%, respectively. (D) CNC explants. Coinjection of FLXcad-11, DELTA-e, or DELTA-e/DELTA-p120 with Xcad-11MO restored formation of lamellipodia and filopodia in CNC; DELTA-e/DELTA-beta-cat coinjection showed blebbing. Bar, 10 mm.
Figure 4. Xcad-11 function is mediated by small GTPases and GEFTrio. (A) Whole-mount ISH. Injection of ca Rac1 and two GEF subunits of hTrio together with Xcad-11MO rescued CNC migration, demonstrated by twist ISH. (B) Statistics of reconstitution experiments with 10 pg of ca cdc42, Rac1, or RhoA. Coinjection with Xcad-11MO rescued CNC migration into pharyngeal pouches in 76%, 62%, or 66%, respectively. Twenty picograms of Trio or single GEF domains restored disturbed invasion in pharyngeal pouches in 52% (Trio), 55% (Gef1), 65% (Gef2), or 88% (Gef 1 + 2). (C) In Xcad-11-depleted CNC explants, coinjection of ca cdc42, Rac1, or RhoA, as well as with Trio or the two GEF subunits, restored formation of lamellipodia and filopodia. Bar, 10 mm. (D) Statistics of gainof-function analysis with soluble Xcad-11 constructs; dn GTPases cdc42, Rac1, and RhoA; as well as a combination of all three GTPases together. Overexpression of the cytoplasmic domain of Xcad-11 with or without the beta-cat-binding site resulted in a strong reduction of CNC migration (30% and 28% migrating CNC). The three dn GTPases cdc42, Rac1, and RhoA showed migration in 65%, 55%, and 44% of the embryos, respectively. A combination of all three dn GTPases together resulted in 46% migrating CNC. (E) Coimmunoprecipitation of Trio with different Xcad-11 constructs. (Bottom left panel) Precipitation of Trio with HA antibody from transfected COS cells. Trio was detected in Western blot. Western blot for Xcad-11 showed successful coprecipitation of Xcad-11 (top left) as well as of Xcad-11 DELTA-e/DELTA-beta-cat (top right). In the case of Xcad-11 DELTA-e/DELTA-IMD, no coprecipitation was detected. (Right half of panels)Input.
Supplement Figure S1: A, Sequence of Xcadherin-11 Morpholino 1 (Xcad-11MO) and 2 (Xcad-11 MO2) and control morpholino in comparison to the Xcadherin-11 sequence. The Xcad-11MO binding sequence spans the start codon while Xcad-11MO2 binds in the 5’ UTR. B, Function of Xcad-11MO. The function of the chosen Xcad-11MO was demonstrated by in vitro transcription and translation. The TNT was performed according to manufacturer’s description (Promega). For detection of synthesized protein S35-labelled methionine was incorporated. The addition of Xcad-11MO to the reaction mix resulted in a strong decrease of detectable protein whereas the addition of control morpholino had no influence (left panel). Xcad-11MO was not able to suppress protein production of murine cadherin-6 or XB-cadherin (right panel). C, Endogenous function of Xcad-11MO and Xcad-11MO2. After injection of 2 pmol Xcad-11MO and Xcad-11MO2 a strong decrease of protein was detected in embryo lysates in comparison to wildtype embryos. Endogenous XB-cadherin was not affected by Xcad-11Mo and Xcad-11MO2 injection. For detection, αCad-11 and 6D5 αXB-cadherin antibody were used. D, Specificity of Xcadherin-11 function. The impaired invasion of pharyngeal pouches caused by injection of Xcad-11MO could neither be rescued by co-injection of the type1 XB-cadherin nor by type II murine cadherin-6 as demonstrated by whole mount in situ hybridizations with the neural crest marker twist. Instead, human Cadherin-11 was able to rescue twist signal in the pharyngeal pouches (up to 90%). Injected side: middle panel, control side: left panel. Right panel: statistics for rescue experiments. In case of Xcad-11MO injection 28%, and in case of 9 pmol Xcad-11MO2 39% of the embryos showed a normal invasion in the pharyngeal pouches. A rescue with 50 pg XB-cadherin or with 100 pg murine cadherin-6 resulted in only 37% or 31% twist positive cells in the branchial arches, respectively.
Xenopus ADAM 13 is a metalloprotease required for cranial neural crest-cell migration.