during development of the mouse embryo , melanoblasts derive from neural crest cells following closure of the neural tube .
they migrate through the dermis and cross the basement membrane into the epidermis at around embryonic day 12.5 ( e12.5 )   before homing to hair follicles [ 2 , 3 ] .
melanoblasts use rac1 to drive actin assembly and extension of pseudopods , which are stabilized by the actin - bundling protein fascin [ 4 , 5 ] .
here we explored the role of cdc42 in melanoblast migration and population of mouse embryo skin .
our insight into cdc42 function in multicellular organisms is still limited , but generally supports a role in epithelial polarity and actin dynamics .
cdc42 is important for epithelial morphogenesis , early cell polarity , and spindle orientation during the development of drosophila melanogaster and caenorhabditis elegans [ 8 , 9 ] .
knockout  of  cdc42 in the murine neural crest , using wnt-1 cre , allowed survival until e13.5 with severe craniofacial and cardiac abnormalities .
these defects were attributed at least in part to aberrant actin dynamics , altered cell migration , and bone morphogenetic protein 2 signaling .
however , loss of cdc42 or rac1 did not prevent neural crest cells from reaching their targets by e10.5 or growing out of the neural tube in culture .
thus , cdc42 is implicated in development , but its role in migration in  vivo is not clear . here
we describe a distinct role for cdc42 in the regulation of pseudopod dynamics and adhesion during melanoblast migration .
cdc42 null melanoblasts extended long blebbing pseudopods , which were not very dynamic . despite their static nature ,
cdc42 null pseudopods showed regular bursts of actin assembly and elevated levels of rac signal activation but ineffective protrusion .
loss of cdc42 also caused a severe defect in focal adhesion assembly and dynamics and a de - localization of active myosin .
thus , we propose that in addition to a strong role in cell proliferation , cdc42 has a coordinating role in melanocytic cell migration , impacting on multiple systems that need to work together for effective cell translocation .
cdc42 was removed from the melanocyte lineage by crossing mice in a c57bl/6 background carrying two floxed alleles of cdc42 ( cdc42 f / f )   with c57bl/6 mice expressing cre recombinase under the control of the tyrosinase promoter ( tyr::creb+ )   ( figure  1a ) .
they were culled between postpartum day 14 ( p14 ) and p30 , as they were slightly shaky and runted , similar to rac1 f / f tyr::cre mice . all cdc42 f / f ; tyr::creb+ mice ( n  = 20 ) displayed a white patch running down the ventral midline , covering half to most of the underside ( figure  1b ) .
small white patches often appeared along the dorsal midline ( figure  1b , yellow arrows ) .
pigmented areas contained frequent white hairs , and the paws and tails were hypopigmented ( figure  1b ) .
skin cross - sections from p13 mice revealed a lack of melanocytes in hair follicles of white patches but not pigmented areas in both control ( ctr ) and cdc42 f / f ; tyr::creb+ mice ( figure  1c , pink stain ) .
thus , cdc42 nulls had fewer melanocytes in hair follicles in areas distal to the neural tube but fairly normal melanocyte distribution on the back , similar to rac1 deletion .
in contrast to the dramatic phenotype of cdc42 f / f ; tyr::creb+ mice , deletion of rhoa using tyr::creb+ had no overt effect on coat color in mice up to p14 ( figure  s1a ) .
rhoa f / f ; tyr::creb+ mice were born at the expected mendelian ratio and showed no coat color defects , shakiness , or runty phenotype , suggesting that rhoa is dispensable for melanocyte development . to investigate the cellular mechanisms driving pigmentation defects , cdc42
tyrosinase expression begins around e10.5 [ 14 , 15 ] , when melanoblasts appear around the neural tube in the migration staging area .
control and cdc42 f / f ; tyr::creb+ ; dct::lacz ( cdc42 f / f ) embryos had similar numbers of melanoblasts at e11.5 ( figures 2a and 2b ) . at e13.5 , the leading wave of melanoblasts in controls and cdc42 flox heterozygotes cdc42 f/+
; tyr::creb+ had reached midway around the embryo and part of the way down the developing limbs , whereas cdc42 f / f ; tyr::creb+ embryos consistently showed a deficit of melanoblasts in all areas ( boxes , figures 2c2f and figure  s1b ) .
by e15.5 , melanoblasts in control embryos had spread around the entire embryo and down the developing limbs , but in cdc42 f / f ; tyr::creb+ embryos , melanoblasts had only populated partway around the belly and halfway down the developing limb ( figures 2 g and 2i ; figure  s1c ) . cdc42
null cells accumulated around / in hair follicles in places but less efficiently than controls ( figure  2i ) .
thus , cdc42 is a major controller of melanoblast proliferation and spread throughout the skin in  vivo .
although loss of cdc42 caused severe defects in melanoblast population of the skin , there is also striking resiliency within the system , as some of the knockout cells could clearly populate hair follicles and give rise to pigmentation of areas of the mouse skin .
knockout cells also traversed the basement membrane from the dermis to the epidermis normally , with around 20% of  cells still in the dermis at e13.5 and > 90% of cells in the epidermis by e15.5 ( figure  s2 ) . because cdc42 mutant embryos showed a paucity of melanoblasts , we investigated possible defects in cell - cycle progression and cytokinesis . staining for ki67 revealed normal proportions of cdc42 f / f cells in cycle ( figure  3a ; yellow arrows ,  + ki67 ; white arrow , ki67 ) , but brdu staining at 2 and 24 
hr revealed slower cell - cycle progression in cdc42 f / f ( figures 3b and 3c ; yellow arrows ,  + brdu ; white arrows , brdu ) .
we saw no cleaved caspase-3-positive ( apoptotic ) cells in control or cdc42 null skins .
we developed immortalized melanocyte cell lines cultured from p1 pups with cdc42 f / f ; rosa26::cre - er ; cdkn2 to allow conditional deletion of cdc42 with oht ( 4-hydroxytamoxifen ) in culture .
hereafter , we refer to these lines as ew1 , ew2.1 , ew2.2 , and ew7 .
these lines behaved similarly , and many experiments were repeated with at least two lines .
addition of oht induced a loss of cdc42 protein over 5  days ( figure  3d ) .
cdc42 null melanocytes had an increased doubling time in  vitro ( figure  3e ) and accumulated in g1 ( figures 3f and 3 g ) .
thus , cell - cycle progression defects likely contribute to the paucity of melanoblasts in cdc42 f / f ; tyr::creb+ embryos .
in addition , time - lapse imaging of live cells in embryo skin explants from cdc42 f / f ; tyr::creb+ ; z / eg ( driving melanoblast - specific expression of gfp )   revealed that cdc42 null melanoblasts have an approximately 3-fold increased division time ( figures s3a and s3b ; movie s1 ) from cleavage furrow formation ( figure  s3a , green boxes ) to separation of daughter cells ( figure  s3a , red boxes ; figure  s3c ) .
both ew1 and ew7 also spent 2.5 times longer from rounding up to cytokinesis ( figures s3d and s3e ; movie s2 ) .
thus , cdc42 null melanoblasts struggle to  physically separate from each other after division .
these problems likely compound the reduction in numbers of melanoblasts in embryos and contribute to the coat color defects of adults . to investigate whether cdc42 null melanoblasts had migration defects , we imaged melanoblasts migrating in ex  vivo skin sections from cdc42 f / f ; tyr::creb+ ; z / eg . control melanoblasts in e15.5 embryo skin displayed multiple , highly dynamic pseudopods that extended between surrounding keratinocytes ( figure  4a , yellow arrows ) .
cdc42 null melanoblasts , in contrast , displayed long wavy non - dynamic pseudopods ( figure  4a , red arrow ; movie s3 ) .
cdc42 null melanoblasts showed an increase of around 40% spread area ( figure  4b ) . additionally , cdc42 null cells frequently had only one or two pseudopods , with 56% appearing bipolar ( figure  4a , orange arrow ; figure  4c )
. the length - to - width ratio of cdc42 nulls was increased ( figure  4d ) , as was pseudopod length ( figure  4e ) .
we were unable to reliably measure persistence , as the density of melanoblasts in wild - type skin was too high .
cdc42 null cells showed very long lived pseudopods , with 10% lasting the entirety of the 4-hr time - lapse movie , compared with 580  min for controls ( figure  4f ) .
normally , elongation of cells is a hallmark of rapid migration , but cdc42 null cells displayed a 2-fold decrease in speed from around 0.4 to 0.2  m / min ( figure  4 g ) .
thus , cdc42 null cells are elongated , but move only slowly in epidermis . to determine whether a loss of actin dynamics might be responsible for the slow motility , we crossed cdc42 ; tyr::creb+ with the lifeact - gfp transgene   to observe filamentous actin dynamics . bursts of actin occurred at the tips of both control and knockout pseudopods , driving them forward ( figure  4h ; movie s4 ) .
there was a small but significant increase in the time between actin bursts in knockout pseudopods from 9.0   0.8  min sem ( ctr ) to 11.6   0.7  min sem ( cdc42 f / f ) ( figure  4i ) , and cells appeared not to translocate as often or as rapidly following a burst ( movie s4 ) .
actin bursts were usually accompanied by protrusion of the pseudopod , but this was impaired in cdc42 nulls ( figure  4j ) .
there was a linear correlation between the lifetime of a pseudopod and the number of actin bursts ( figure  4k ) .
thus , the frequency of actin bursting was not greatly affected by the loss of cdc42 , but the coupling with protrusion was lost .
all cultured melanocyte lines were confirmed to be over 90% pure by staining with the melanocyte lineage factor mitf ( figure  s4a shows ew7 as an example ) .
control cells formed large lamellipodia on fibronectin surfaces , with a length - to - width ratio close to 1 , and moved at an average speed of 0.4  m / min .
following deletion of cdc42 ( oht ) , cells were almost stationary , with an elongated spindle shape and an average speed of 0.014  m / min ( figures 5a5c ) . as in skin explants , these thin , long protrusions were very stable , with the majority lasting over 600  min ( figure  5d ) .
the increased length - to - width ratio was rescued through transient expression of cdc42-yfp in knockout cells ( figures s4b and s4c ) .
thus , cultured melanocytes behaved similar in  vitro to live skin explants and to cdc42-depleted fibroblast cell lines .
cdc42 has a well - described role in mediating the polarization of the golgi complex in front of the nucleus in a moving cell , so we tested whether cdc42 null melanocytes retained this ability .
when cells were allowed to migrate out of a crowded space ( see supplemental experimental procedures ) , around 80% of the control cells reoriented their golgi to face the direction of migration .
however , golgi orientation was random in cdc42 null cells ( figures 5e and 5f ) .
additionally , the golgi appeared more diffuse , and frequently spread over multiple segments of  a 120 divided mask placed over the cell ( figure  5 g ) .
thus , cdc42 regulates golgi polarity during motility of melanocytes , as previously described in other cell types [ 13 , 18 ] .
in addition to their polarity defects , cdc42 null melanoblasts resembled blebbistatin - treated cells in embryo skin explants   with blebby static pseudopods .
cdc42 is linked to myosin activation via the myotonic dystrophy kinase - related mrck kinases ( reviewed in ) , suggesting that pseudopod retraction defects may be due to reduced activation of myosin light chain  ( mlc ) .
however , levels of mlc phosphorylation at t18/s19 were unaltered in cdc42 null melanocytes ( figures s4d and  s4e ) . despite this , localization of p - mlc was abnormally concentrated toward the cell body of cdc42 null cells , rather than peripheral in the lamellar region , as in the controls ( figures s4f and s4 g ) .
we also performed a blebbistatin wash - out experiment to test the sensitivity of cdc42 null cells to inhibition of myosin - ii ( figures s4h and s4i ) .
control cells ( cdc42 wt [ wild - type ] ) became elongated and resembled cdc42 null cells before blebbistatin treatment .
cdc42 nulls , however , showed very little change in shape , indicating that their myosin - ii was already not  engaged .
thus , although overall levels of p - mlc were not dramatically altered in cdc42 nulls , p - mlc was mislocalized and cdc42 null cells resembled blebbistatin - treated cells and showed very little response to blebbistatin treatment .
we next investigated whether loss of cdc42 affected the major actin organizers scar / wave and arp2/3 complex as well as the cdc42 target formin fmnl2 . both arp2/3 and
wave2 localized in a broad band in lamellipodia of control cells ( figure  5h ) , but in cdc42 null cells they formed small patches near the cell edge ( figure  5i ) , in protrusions ( figure  5i , insets ) . because fmnl2 is a cdc42 target regulating lamellipodial actin , we tested whether constitutively active fmnl2 might rescue the loss of cdc42 .
endogenous fmnl2 localized prominently in lamellipodia and filopodia of both normal and cdc42 null melanocytes ( figure  6a ) .
cells expressing constitutively active fmnl2 showed increased spreading but also a more fenestrated appearance ( figures 6b6e ) .
in particular , mutant fmnl2-gfp constructs fmnl2-dad - gfp and fmnl2-a272e - gfp ( see supplemental experimental procedures ) , which both lack the autoinhibitory regulation provided by the diaphanous autoinhibitory domain ( dad ) region , rescued the length - to - width ratio ( figure  6c ) .
we scored cells in shape categories ( see the legend of figure  6 ) .
expression of active fmnl2-gfp increased the proportion of cells with a wider lamellipodial shape and decreased the proportion classified as elongated ( figure  6d ) .
wild - type fmnl2-gfp   and both active constructs gave a partial rescue of roundness ( figure  6e ) .
thus , a major part of the cell shape change that we observe upon loss of cdc42 appears to be due to the inability of cells to activate fmnl2 .
we next used fluorescence lifetime imaging microscopy ( flim ) imaging of a rac1flare dual - chain ( dc ) biosensor to determine whether loss of cdc42 decreased rac1 activation signals .
lifetime heatmaps of control dturquoise ( donor alone ) or rac1flare.dc showed a decreased lifetime , demonstrating frster resonance energy transfer ( fret ) ( figure  s4j ) .
the percentage fret efficiency in cdc42 knockout cells was to our surprise higher , indicating an increase in rac1 signaling activity ( figure  s4j ) .
we also performed a biochemical pull - down assay ( figures s4k and s4l ) , which revealed no significant difference in rac1 signaling activity between control and cdc42 null cells .
the fret assay is more sensitive , so this likely means that there is a small or localized increase in rac1 activity detectable only by fret .
thus , the pathways that regulate rac1 activation were active in cdc42 null cells , despite cells being unable to use their actin dynamics productively for migration .
taken together , cdc42 null cells still showed active rac signaling but a failure to organize arp2/3 , scar / wave , fmnl2 , and active myosin - ii in their protrusions ; thus , the various motility systems seemed intact , but uncoupled from movement .
adhesion controls the shape and migratory properties of cells , as well as their growth state .
global rna sequencing was performed on cdc42 f / f ; rosa26:cre - er ; cdkn2 melanocyte lines ( ew2.1 and ew2.2 , isolated from littermate pups ) to probe pathways affected by cdc42 loss ( figure  s5a ; data s1 ) .
biological replicates were consistent by principal component analysis ( pca ) of the data ( figure  s5b ) .
0.05 ) downregulated ( 290 ) or upregulated ( 343 ) genes identified lysosomal pathways as significantly upregulated , and hierarchical clustering identified top consistent hits ( figures s5c and s5d ) .  additionally , 
pathways in cancer ,   focal adhesion ,  and  regulation of the actin cytoskeleton  ( figure  s5e )
hierarchical clustering analysis of kegg ( kyoto encyclopedia of genes and genomes )  focal adhesion  genes showed consistency between ew2.1 and ew2.2 ( figure  s5f ) .
4 integrin showed the largest fold change ( 2.93   0.5 ) , which we confirmed at the protein level ( figures s5 g and s5h ) .
additionally , 3 integrin was decreased at the protein level ( figures s5i and s5j ) , despite an increase at the rna level ( + 2.01   0.7 , padjusted  = 0.2 ; data s1 ) .
the expression levels of classical rho - gtpases remained relatively unchanged in cdc42 knockout cells ( figure  s5k ) .
as suggested by global rna - sequencing analysis and cell - shape abnormalities , cdc42 null cells showed adhesion defects .
control melanocytes displayed a belt of multiple large adhesions underneath their lamellipodia that were rich in vinculin ( figure  6f ) , phospho - paxillin ( figure  s6a ) , and 1 integrin ( figure  s6e ) , whereas knockout melanocytes displayed only a few small adhesions with the same components ( figure  6f ; figures s6a and s6e ) .
overall , knockout melanocytes had fewer adhesions per cell , a smaller average adhesion size , and fewer adhesions per cell area ( figures 6g6i ; figures s6b 
s6d ) . because fmnl2 partially rescued the cell - shape defects of cdc42 null cells , we asked whether it affected focal adhesions .
indeed , active fmnl2 expression increased the number of adhesions per cell ( figure  s6f ) and area of adhesions ( figure  s6 g ) but not the number of adhesions per cell area , as fmnl2 expression increased the spread area quite significantly ( figure  s6h ) .
these changes were modest and might have complex reasons , but expression of fmnl2 appeared to give a partial rescue .
live imaging of gfp - paxillin or mapple - vinculin revealed that control melanocytes dynamically extended lamellipodia supported by nascent adhesions , whereas adhesions of cdc42 nulls were smaller and less dynamic .
adhesions in knockout melanocytes showed a slower assembly rate with gfp - paxillin and a slower disassembly rate with both gfp - paxillin and mapple - vinculin , indicating reduced adhesion dynamics in cdc42 knockout cells ( figures 7a7e ; figure  s7a ) .
re - expression of 4 integrin in cdc42 null cells gave a partial rescue ( figure  7f ) .
the length - to - width ratio and number of adhesions per cell did not change significantly , but the roundness and total cell area increased modestly ( figures 7g7j ) .
thus , 4 integrin levels account for part of the phenotype of cdc42 null melanocytes .
cdc42 nulls also showed spreading defects on fibronectin or laminin but not concanavalin a , a lectin mediating integrin - independent adhesion   ( figures 7k7 m ) and formation of fewer adhesions ( figures s7b , s7c , and s7e ) . because filopodia - like protrusions during cell spreading are linked with nascent 1 integrin adhesions , we measured filopodia - like structures ( flss ) in cells spreading on fibronectin .
knockout melanocytes formed fewer , shorter flss ( figures s7d , s7f , and s7 g ) and showed a thicker band of cortical actin than controls ( figure  s7d , white arrows ) , possibly reflecting less dynamic actin .
thus , cdc42 null melanoblasts are defective in adhesion - dependent spreading , with fewer dynamic adhesions and misregulation of both localization and expression of focal adhesion components .
loss of cdc42 in the melanocyte lineage gave a phenotype of similar severity to loss of rac1 , but with multiple unique features revealing cdc42 as a complex organizer of motility systems .
both gtpases appear important for melanoblast proliferation and migration during embryogenesis , but cdc42 null cells were exaggeratedly elongated in  vivo , rather than rounded , like rac1 nulls .
cell shape has been proposed as a potential predictor of metastasis in melanoma [ 23 , 24 , 25 , 26 ] , but in the case of control by rho - gtpases in the melanocyte lineage , we found no obvious correlation between shape and migration properties .
similar to rac1 , cdc42 also plays a key role in promoting g1-to - s phase transition in melanocytic cells in  vivo ( figure  3 ) .
rac1 and cdc42 have a well - established role in cell - cycle progression in cultured cells .
cdc42 has a more direct role in cytokinesis than rac , as it localizes on the spindle and kinetochores during mitosis .
our observations that cdc42 impacts on localization of phosphorylated myosin light chain suggest possible implications for cytokinesis . curiously ,
proliferation and migration defects are generally coupled in melanoblasts in  vivo [ 4 , 5 , 28 ] , perhaps due to a metabolizable signal in the skin or coupling of the mitotic spindle to the cell cortex by motility proteins . at the cellular level , loss of cdc42 had a pleiotropic effect , causing defects in the organization and coordination of actin dynamics , contractile activity , and adhesion .
despite evidence of a hierarchy whereby rac1 is activated downstream of cdc42 in some systems , melanocytic cdc42 null cells showed elevated rac1 activation but only small lamellipodia .
their pseudopod extensions were not dynamic in  vivo despite actin bursts visualized with lifeact - gfp , and they failed to assemble broad lamellipodia driven by the formin fmnl2 , scar / wave , and arp2/3 in  vitro .
myosin - ii was still phosphorylated at normal levels , indicating signaling to activation , but it was not localized properly and cells resembled the blebbistatin - treated state .
previous studies connected cdc42 and actin polymerization with phosphoinositides , p21-activated kinase ( pak ) kinases , and n - wasp . however , neither n - wasp nor pak kinases affected pseudopod dynamics or migration of melanoblasts in skin explants .
null melanocytes showed adhesion defects , unlike cdc42 null mouse embryonic fibroblasts ( mefs ) , which spread normally and had normal ( 1 or 3 , or 1 , 2 , 5 , 6 , or v ) integrin subunit expression levels .
in contrast , cdc42 null melanocytes showed reduced adhesion dynamics only partially rescued by re - expression of 4 integrin or fmnl2 active mutants . despite adhesion and spreading defects in  vitro , melanoblasts null for cdc42 could still cross and interact with the epidermal basement membrane in  vivo ( figure  s2 ) .
in addition to adhesion changes , several genes in a kegg lysosomal pathway were upregulated at the rna level in cdc42 null melanocytes , including several subunits of atp6vo , a vacuolar atpase ( v - atpase ) required for proton pumping in acidic organelles such as the lysosome ( figure  s5 ) .
v - atpase is linked to anoikis resistance of cancer cells   due to its ability to degrade internalized integrins .
thus , cdc42 null cells may have increased ability to degrade internalized integrins , which would also contribute to diminished levels of 4 and 3 integrins .
mouse melanocytes express cdc42 , rac1 , rhoa , and rhoc at high levels and rhob , rhog , rhoj , and rhoq at lower levels ( figure  s5k ; data s1 ) , which were not affected by deletion of cdc42 .
rhog has some overlapping functions with rac1 and cdc42 , but clearly it can not compensate fully for the loss of rac1 or cdc42 in melanoblasts . in summary , cdc42 is an important regulator of cell migration and proliferation of melanocytic cells ; its loss caused failure of multiple aspects of migration , which were not rescued by re - activation or expression of any single component .
all other mouse strains used were previously described , and all antibody and chemical reagents are detailed in supplemental experimental procedures .
primary melanocytes were isolated and purified from the skin of 1-day - old pups and imaged according to methods previously described   and in supplemental experimental procedures .
. , e.g. , and s.w.g.t . performed experiments ; c.h.b . and l.l . provided reagents and advice ; a.h . and w.c .
performed rna sequencing and analysis ; f.k . constructed the fmnl2 mutants ; d.j.m . and k.m.h .
provided the rac1flare.dc construct and advice ; r.h.i . contributed to the experimental design and direction ; and l.m.m . supervised the study and wrote the manuscript with e.f.w .
 summarythe individual molecular pathways downstream of  cdc42 , rac , and rho gtpases are well documented , but we know surprisingly little about how these pathways are coordinated when cells move in a complex environment in  vivo . in the developing embryo , melanoblasts originating from the neural crest must traverse the dermis to reach the epidermis of the skin and hair follicles . 
 we previously established that rac1 signals via scar / wave and arp2/3 to effect pseudopod extension and migration of melanoblasts in skin . here 
 we show that rhoa is  redundant in the melanocyte lineage but that cdc42 coordinates multiple motility systems independent of rac1 . 
 similar to rac1 knockouts , cdc42 null mice displayed a severe loss of pigmentation , and melanoblasts showed cell - cycle progression , migration , and cytokinesis defects . 
 however , unlike rac1 knockouts , cdc42 null melanoblasts were elongated and displayed large , bulky pseudopods with dynamic actin bursts . despite assuming an elongated shape usually associated with fast mesenchymal motility , cdc42 knockout melanoblasts migrated slowly and inefficiently in the epidermis , with nearly static pseudopods . 
 although much of the basic actin machinery was intact , cdc42 null cells lacked the ability to polarize their golgi and coordinate motility systems for efficient movement . 
 loss of cdc42 de - coupled three main systems : actin assembly via the formin fmnl2 and arp2/3 , active myosin - ii localization , and integrin - based adhesion dynamics . 
Introduction
Results
Discussion
Experimental Procedures
Author Contributions