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Abstract
Neural crest cells are responsible for craniofacial formation, pharyngeal arch, artery remodeling, and
cardiac outflow tract septation during vertebrae development.
Cdc42 (cell division cycle 42) is a small GTP-binding protein in Rho family that regulate cytoskeleton
remodeling and stablish cell polarity at a molecular level.
To investigate the role of Cdc42 in NCCs during embryonic development, they deleted Cdc42 in NCCs by
crossing Cdc42 flox (sandwiching a DNA sequence between 2 lox P sites, allowing it to be deleted –
knocked out, translocated or inverted in process) mice with Wnt-cre mice.
- Inactivation of Cdc42 in NCCs caused embryonic lethality with craniofacial deformities and
cardiovascular development defects
o Cdc42 knockout embryos had fully penetrant (characteristic effects in the phenotypes of
individuals possessing it) cleft lips and short snouts
Alcian blue and Alzarin red staining of cranium exhibited unfused nasal capsule and palatine in mutant
embryos
India ink intracardiac injection analysis showed a spectrum of cardiovascular defects including persistent
truncus arteriosus, hypomorphic pulmonary trunks, interrupted aortic arches, and right sided arches
To understand underlying mechanisms of Cdc42 in the formation of great blood vessels, they generated
Wnt1Cre-Cdc42-Rosa26 reporter mice. Beta-galactosidase staining showed a subpopulation of Cdc42-
null NCCs that halted migration midway from the pharyngeal arches to the conotruncal cushions.
Phalloidin staining showed dispersed, shorter and disoriented stress fibers in Cdc42-null NCCs.
Inactivation of Cdc42 in NCCs impaired bone morphogenetic protein (BMP2), which induced NCC
cytoskeleton remodeling and migration.
➔ Cdc42 plays an essential role in NCC migration, inactivating Cdc42 in NCCs impairs craniofacial and
cardiovascular development in mice.
Introduction
Craniofacial and cardiovascular malformations comprise more than 1/3 of human congenital (present
from birth) diseases.
Their structure formations depend of specific interactions between NCCs and its surrounding cells that
are derived from ectoderm, endoderm, and splanchnic mesoderm.
NCCs originate from border between neural tube and surface ectoderm. After induction (give rise), NCCs
undergo epithelial to mesenchymal transition, and then migrate to diverse locations. Then they
differentiate into multiple cell types at their destinations including craniofacial skeletons, vasculature
and smooth cells in the conotruncal structures of the heart.
Cardiac NCCs (subpopulation of cranial NCCs) emigrate from the region between otic placode and their
caudal border of somite three, are necessary for proper septation of the cardiac outflow tract and
correct alignment of aortic arch arteries.
- During embryogenesis, cardiac NCCs ventrolaterally migrate into pharyngeal arch 3, 4, and 6,
and a subpopulation of cells continue migration cardiac outflow tract cushions.
- Interference of NCC functions results in persistent truncus arterosus (PTA) and a shortened
outflow tract that causes malalignment of the outflow tract and cardiac loop defects.
- Molecular signals that operate in NCCs during craniofacial and cardiovascular formation remain
to be addressed.
- GTP-binding proteins act as molecular switches that regulate many cellular activities and
biological responses.
- Cdc42 with Rac and RhoA are members of Rho subfamily in the Ras superfamily of GTPases.
Cdc42 play an essential role in regulation cytoskeleton reorganization, formation of filopodia,
and focal adhesion complexes, the establishment of microtubule-dependent cell polarity, gene
transcription, intracellular trafficking, and endocytosis.
o Cdc42 is a critical regulator of many cellular functions for cell cycle progression,
migration, differentiation, and apoptosis (death of cells that occurs as a normal and
controlled part of an organism’s development).
- GFD1 (Rho GEF and pH domain-containing protein 1) is a Cdc42 putative (considered to be)
guanine nucleotide-exchange factor, and mutations in the human FGD1 gene have been shown
to cause faciogenital dysplasia (wide spaced eyes, front-facing nostrils, broad upper lip,
malformed scrotum, protruding umbilicus, laxity of ligaments, flat feet, hyper extensible
fingers).
- Over 20 target proteins for Cdc42 have been identified in mammalian cells: PAK, Cool-1 (cloned
out of library 1), WASP (Wiskott-Aldrich syndrome protein), and IQGAP.
o Overexpression of dominant negative PAK1 inhibits NCC migration
Cdc42 is activated by integrins and focal adhesion kinase (FAK), and loss of FAK in NCCs results in
craniofacial and cardiovascular development defects
- Cdc42 regulate growth factor-initiated signal transduction pathways, including bone
morphogenetic proteins (BMPs), fibroblast growth factors (FGFs), vascular endothelial growth
factors (VEGFs), and critical functions of these growth factors in NCCs are well accepted.
➔ All of this indicates that Cdc42 may be a critical regulator in NCCs during craniofacial and
cardiovascular development.
BMPs are transforming growth factor-beta (TFG-beta) superfamily members and are among the first
factors identified in the regulation of NCC development.
- This is found from studying genetically modified mice demonstrating that BMP2 is one of the most
important growth factors in NCC migration.
- Also, overexpression of Xnoggin (BMP2) inhibitor blocked NCC migration.
- NCC-specific deletion of BMP2 receptors (ActRIA or BMPR1A) brought defective formation of cardiac
outflow tract, including a shortened outflow tract.
Exposure of C2C12 cells (mouse myoblast) to BMP2 enhanced actin cytoskeleton reorganization and
migration, and these effects were abolished when Cdc42 function was inhibited.
Total deletion of Cdc42 caused embryonic lethality with abnormal actin cytoskeleton organization. Using
tissue-specific gene knockout technology, Cdc42 has been implicated to play a critical role in neuron
apical progenitor cell renewal and cerebral hemispheres separation.
- But functions of Cdc42 in craniofacial and cardiovascular development are not known very well
because knockout embryos died before these structures were formed. So to analyze the
molecular mechanisms of Cdc42 in craniofacial and cardiovascular in detail, they generated a
NCC-specific Cdc42 knockout mouse line by crossing Cdc42 flox mice with Wnt1-Cre mice.
o Found deletion of Cdc42 in NCCs induced embryonic lethality with craniofacial
morphogenetic defects.
o Inactivation of Cdc42 in NCCs caused abnormal great vessel patterns and aortic and
pulmonary septation defects.
o Cellular function analysis showed that Cdc42 is crucial for BMP2-induced cytoskeleton
remodeling and migration.
Materials and Methods
(Wnt1-cre transgenic mouse line is used to study the development of the neural crest and its derivatives
and the midbrain. Wnt1 gene has important development roles in formation of midbrain-hindbrain
boundary, regulation of midbrain size, and neurogenesis of ventral midbrain dopaminergic neurons)
Generation of NCC Cdc42-specific knockout mice
Cdc42/flox mice were crossed with Wnt1-cre mice to inactivate Cdc42 in NCCs.
- LacZ enzyme was introduced into Cdc42 NCC knockout mice by crossing Cdc42 and Wnt1-Cre double
heterozygous mice with R26R-STOP-LacZ (carry a reporter transgene – LacZ, which encodes for bacterial
beta-galactosidase, allowing for distinction from mouse cells) mice.
- Expected sizes of wild-type allele and floxed alleles were 206bp and 412bp respectively
- They were housed in pathogen-free facility (all protocols were approved by institutional annual
care and use committee)
Skeleton staining
E18.5 embryos were de-skinned, gutted and fixed in ethanol. Then they were stained in Alcian blue
staining solution for 3 days. After washing it with ethanol, Alizarin red solution was added, and the
embryos were stained for another 2 days. Afterward, they were cleared in KOH/glycerol solution and
stored for imaging.
Histological analysis and immunofluorescence staining
Embryos were harvested at stages from E9.5 to E18.5 and fixed in paraformaldehyde. Then they were
sectioned and stained with eosin.
Whole-mount X-gal staining
Embryos were dissected and rinsed, and stained with Staining Solution, which comprises of MgCl2,
deoxycholic acid, Igepal CA-630, sodium phosphate, potassium ferricyanide.
