shotgun
E-cadherin and development The cell adhesion molecule E-cadherin mediates the compaction process of mouse preimplantation embryos and is important for the maintenance and function of epithelial cell layers. To determine precisely the role of E-cadherin in epithelial biogenesis the developmental potential was monitored of embryos homozygously negative for E-cadherin that were derived from E-cadherin heterozygous transgenic mice. The homozygous negative embryos died around the time of implantation, although they did undergo compaction like their littermate controls, largely due to the presence of residual maternal E-cadherin. At the blastocyst stage, E-cadherin-negative embryos failed to form a trophectodermal epithelium or a blastocyst cavity. These results demonstrate the pivotal role of E-cadherin in one of the most basic morphogenetic events in the development of multicellular organisms, the biogenesis of an epithelium (Larue, 1994).
The Ca(2+)-dependent cell adhesion molecule E-cadherin functions in the establishment and maintenance of epithelial cell morphology during embryogenesis and adulthood. Downregulation or complete shut-down of E-cadherin expression and mutation of the gene are observed during the progression of tumors of epithelial origin (carcinomas) and correlate with the metastatic potential. A targeted mutation was introduced into the E-cadherin gene by homologous recombination in mouse embryonic stem cells. The mutation removes E-cadherin sequences essential for Ca2+ binding and for adhesive function. These embryonic stem cells were used to generate mice carrying the mutation. Heterozygous mutant animals appear normal and are fertile. However, the homozygous mutation is not compatible with life: E-cadherin -/- embryos show severe abnormalities before implantation. Particularly, the adhesive cells of the morula dissociate shortly after compaction has occurred, and their morphological polarization is then destroyed. Interestingly, the blastomers are still able to form desmosomes and tight junctions at sites of distorted cell-cell contact. Thus, maternal E-cadherin suffices for initial compaction of the morula but not for further preimplantation development to occur (Riethmacher, 1995).
Trophectoderm epithelium formation, the first visible differentiation process during mouse embryonic
development, is affected in embryos lacking the cell adhesion molecule E-cadherin. Membrane localization of alpha- and beta-catenin, and ZO-1 (all three are tight junction proteins), as well as
cortical actin filament organization are abnormal in E-cadherin-negative embryos, and the expression
levels of alpha- and beta-catenin are dramatically reduced, all suggesting a regulatory role for
E-cadherin in forming the cadherin-catenin complex. In contrast, the membrane localization of
plakoglobin, occludin (a tight junction protein), desmoglein1 (a desmosomal transmembrane protein), connexin43 (a gap junction protein), and cytokeratin filaments appear unaltered. The unusual morphogenesis in E-cadherin-negative embryos apparently reflects defects in the molecular
architecture of a supermolecular assembly involving zonulae adherens, tight junctions, and cortical actin
filament organization, although the individual structures still appear normal in electron microscopical
analysis. It is likely that maternal E-cadherin is sufficient for the course of development of junctions. The lack of zygotic E-cadherin might reduce the necessary accumulation of E-cadherin-catenin complexes. Lack of zygotic E-cadherin evidently prevents formation of the blastocyst and enlargement of trophectoderm-like cells and causes leakiness in vectorial fluid transport during blastocyst formation (Ohsugi, 1997).
Two-way interactions between the blastocyst trophectoderm and the uterine luminal epithelium are
essential for implantation. The key events of this process are cell-cell contact of trophectoderm cells
with uterine luminal epithelial cells; controlled invasion of trophoblast cells through the luminal
epithelium and the basement membrane; transformation of uterine stromal cells surrounding the
blastocyst into decidual cells, and protection of the 'semiallogenic' embryo from the mother's
immunological responses. Because cell-cell contact between the trophectoderm epithelium and the
luminal epithelium is essential for implantation in the mouse uterus, the expression of zonula occludens-1
(ZO-1) and E-cadherin, two molecules associated with epithelial cell junctions,
was examined during the periimplantation period. Preimplantation uterine epithelial cells express both ZO-1 and
E-cadherin. With the initiation and progression of implantation, ZO-1 and E-cadherin are expressed in
stromal cells of the primary decidual zone (PDZ). As trophoblast invasion progresses, these two
molecules are expressed in stroma in advance of the invading trophoblast cells. These results suggest
that expression of these adherence and tight junction molecules in the PDZ functions as a
permeability barrier to regulate access of immunologically competent maternal cells and/or molecules
to the embryo and provides homotypic guidance of trophoblast cells in the endometrium (Paria, 1999).
Mouse primordial germ cells and fetal germ cells at certain stages of differentiation express
E-cadherin and alpha and beta catenins. The formation of germ cell aggregates, which rapidly occurs
when monodispersed germ cell populations are released from embryonic gonads in culture, is E-cadherin mediated,
developmentally regulated, and dependent on the sex of the germ cells. Immunoblotting analyses indicate that the lower
ability to form aggregates of primordial germ cells in comparison to fetal germ cells is not due to gross changes in E-cadherin
expression, altered association with betacatenin, or changes in betacatenin phosphorylation. Investigating possible functions of
E-cadherin-mediated adhesion in primordial germ cell development, it was found that E-cadherin-mediated adhesion may
stimulate the motility of primordial germ cells. Moreover, treatment of primordial germ cells cultured on STO cell
monolayers with an anti-E-cadherin antibody causes a significant decrease in their number and markedly reduces their
ability to form colonies in vitro. The same in vitro treatment of explanted undifferentiated gonadal ridges cultured for 4 days
results in decreased numbers and altered localization of the germ cell inside the gonads. Taken together these results suggest
that E-cadherin plays an important role in primordial germ cell migration and homing and may act as a modulator of
primordial germ cell development (Carlo, 2000).
The oocyte to embryo transition in metazoans depends on maternal proteins and transcripts to ensure the successful initiation of development, and the correct and timely activation of the embryonic genome. The maternal gene encoding the cell adhesion molecule E-cadherin was conditionally eliminated and the ß-catenin gene was partially eliminated from the mouse oocyte. Oocytes lacking E-cadherin, or expressing a truncated allele of ß-catenin without the N-terminal part of the protein, give rise to embryos whose blastomeres do not adhere. Blastomere adhesion is restored after translation of protein from the wild-type paternal alleles: at the morula stage in embryos lacking maternal E-cadherin, and at the late four-cell stage in embryos expressing truncated ß-catenin. This suggests that adhesion per se is not essential in the early cleavage stage embryos; that embryos develop normally if compaction does not occur until the morula stage, and that the zona pellucida suffices to maintain blastomere proximity. Although maternal E-cadherin is not essential for the completion of the oocyte-to-embryo transition, absence of wild-type ß-catenin in oocytes does statistically compromise developmental success rates. This developmental deficit is alleviated by the simultaneous absence of maternal E-cadherin, suggesting that E-cadherin regulates nuclear ß-catenin availability during embryonic genome activation (de Vries, 2004).
Although FGF signaling plays an integral role in the migration and patterning of mesoderm at gastrulation, the mechanism and downstream targets of FGF activity have remained elusive. FGFR1 orchestrates the epithelial to mesenchymal transition and morphogenesis of mesoderm at the primitive streak by controlling Snail and E-cadherin expression. Furthermore, FGFR1 functions in mesoderm cell fate specification by positively regulating Brachyury and Tbx6 expression. Finally, evidence is provided that the attenuation of Wnt3a signaling observed in Fgfr1-/- embryos can be rescued by lowering E-cadherin levels.
It is proposed that modulation of cytoplasmic ß-catenin levels, associated with FGF-induced downregulation of E-cadherin, provides a molecular link between FGF and Wnt signaling pathways at the streak (Ciruna, 2001).
Results from the Fgfr1 mutant expression analyses, chimeric studies, and in vitro explant experiments can be assembled into a minimal model for FGFR1 function at gastrulation. This study has defined a specific region of the primitive streak that requires FGFR1 signaling activity; this domain encompasses the paraxial and posterior embryonic mesoderm populations, but excludes the node, axial, and extraembryonic mesoderm. In the context of this domain, it is proposed that FGFR1 signaling orchestrates both the morphogenetic movement and cell fate specification events of gastrulation (Ciruna, 2001).
FGFR1 regulates the morphogenesis and migration of mesodermal cells by differentially regulating intercellular adhesion properties of progenitor populations in the primitive streak. More specifically, FGFR1 signaling is required for the expression of mSnail, a key mediator of epithelial to mesenchymal transitions in development. Furthermore, it is proposed that mSnail expression downstream of FGFR1 is required for the normal downregulation of E-cadherin. Given the morphoregulatory roles for differential cell adhesion during embryogenesis, ectopic E-cadherin expression at the primitive streak of Fgfr1 mutants provides a molecular explanation for the observed defects in epithelial to mesenchymal transition (EMT), progenitor cell migration, and the sorting of Fgfr1-/- from WT cells during gastrulation (Ciruna, 2001).
In addition, it is proposed that FGFR1 signaling indirectly regulates Wnt signal transduction at the primitive streak. In Fgfr1 -/- embryos, although Wnt3a is expressed in the late primitive streak, direct targets of Wnt signaling (i.e., Brachyury and the T-lacZ reporter transgene) are not activated. It is suggested that ectopic E-cadherin expression in Fgfr1 mutants attenuates Wnt3a signaling by sequestering free ß-catenin from its intracellular signaling pool, and demonstrates that forced downregulation of E-cadherin in Fgfr1 -/- explants can rescue endogenous Wnt signaling at the primitive streak. Evidence that cadherins act as regulators of ß-catenin signaling is well documented. E-Cadherin and LEF-1 bind to partially overlapping sites in the central region of ß-catenin; consequently, LEF-1 and E-cadherin form mutually exclusive complexes with ß-catenin and compete for the same intracellular signaling pool. Furthermore, overexpression of cadherins during Drosophila and Xenopus embryogenesis has been shown to phenocopy Wnt/ß-catenin signaling mutants (Ciruna, 2001).
It is well established that Wnt signaling stabilizes cytosolic levels of ß-catenin by inhibiting its GSK3ß-mediated phosphorylation and degradation. At gastrulation, loss of E-cadherin expression downstream of FGFR1 may also facilitate a rapid intracellular transfer of membrane-bound ß-catenin to the cytosolic 'signaling' pool. Since downregulation of E-cadherin alone is not sufficient to induce ectopic activation of T-lacZ and Brachyury expression in WT primitive streak cultures, signaling through the ß-catenin pathway is still dependent on the activity of localized Wnt signals. However, FGF-mediated changes in cadherin levels and ß-catenin localization could still regulate the threshold for and/or speed of Wnt signaling responses at gastrulation. It is therefore proposed that normal downregulation of E-cadherin at the primitive streak not only regulates the EMT and migration of mesoderm progenitor cells at gastrulation, but also permits the rapid and uninhibited accumulation of cytosolic ß-catenin levels in response to localized Wnt signals. This competition for and opposing influences on the intracellular localization and function of ß-catenin thus establishes a molecular link between the FGF and Wnt signaling pathways at gastrulation. Consequently, FGFR1 activity plays an indirect but permissive role in the propagation of Wnt signaling responses at the primitive streak. The fundamental interregulation of cell adhesion, morphogenesis, and cell fate determination, as demonstrated in this analysis of FGFR1 function, serves to underscore the interdependent nature of morphogenesis and patterning at gastrulation and the intricate network of inductive interactions that pattern and shape the developing embryo (Ciruna, 2001).
Convergence extension movements are conserved tissue rearrangements implicated in multiple morphogenetic events. While many of the cell behaviors involved in convergent extension are known, the molecular interactions required for this process remain elusive. However, past evidence suggests that regulation of cell adhesion molecule function is a key step in the progression of these behaviors. Antibody blocking of fibronectin (FN) adhesion or dominant-negative inhibition of integrin ß1 function alters cadherin-mediated cell adhesion, promotes cell-sorting behaviors in reaggregation assays, and inhibits medial-lateral cell intercalation and axial extension in gastrulating embryos and explants. Embryo explants were used to demonstrate that normal integrin signaling is required for morphogenetic movements within defined regions but not for cell fate specification. The binding of soluble RGD-containing fragments of fibronectin to integrins promotes the reintegration of dissociated single cells into intact tissues. The changes in adhesion observed are independent of cadherin or integrin expression levels. It is concluded that integrin modulation of cadherin adhesion influences cell intercalation behaviors within boundaries defined by extracellular matrix. It is proposed that this represents a fundamental mechanism promoting localized cell rearrangements throughout development (Marsden, 2003).
During early chick heart development the expression pattern of N-cadherin, a calcium-dependent cell
adhesion molecule, suggests its involvement in morphoregulation and the stabilization of cardiomyocyte
differentiation. N-cadherin's adhesive activity is dependent upon its interaction with the intracellular
catenins. N-cadherin's association with alpha-catenin and beta-catenin is also believed to be involved in cell
signaling. The restriction of N-cadherin/catenin localization at stage 5+
from a uniform pattern in vivo to specific cell clusters that demarcate areas where mesoderm
separation is initiated, suggests that the N-cadherin/catenin complex is involved in boundary formation
and in the subsequent cell sorting. The latter two processes lead to the specification, separation, and formation of
the somatic and cardiac splanchnic mesoderm. Cells which form patches of N-cadherin/ß-catenin demarcate the dorsal boundary of the cardiac compartment, separating this compartment from the overlying dorsal mesoderm. N-cadherin colocalizes with alpha- and beta-catenin at
the cell membrane before and during the time that its expression becomes restricted to the lateral
mesoderm and continues cephalocaudally into stage 8. These proteins continue to colocalize in the
myocardium of the tubular heart (Linask, 1997).
Wingless is known to be required for induction of cardiac mesoderm in Drosophila, but the function of Wnt family proteins, vertebrate homologs of wingless, in cardiac myocytes remains unknown. When medium conditioned by HEK293 cells overexpressing Wnt-3a or -5a is applied to cultured neonatal cardiac myocytes, Wnt proteins induce myocyte aggregation in the presence of fibroblasts, concomitant with increases in ß-catenin and N-cadherin in the myocytes and with E- and M-cadherins in the fibroblasts. The aggregation is inhibited by anti-N-cadherin antibody and induced by constitutively active ß-catenin. Thus, increased stabilization of complexed cadherin-ß-catenin in both cell types appears crucial for the morphological effect of Wnt on cardiac myocytes. Furthermore, myocytes overexpressing a dominant negative frizzled-2, but not a dominant negative frizzled-4, fail to aggregate in response to Wnt, indicating frizzled-2 to be the predominant receptor mediating aggregation. By contrast, analysis of bromodeoxyuridine incorporation and transcription of various cardiogenetic markers show Wnt to have little or no impact on cell proliferation or differentiation. These findings suggest that a Wnt-frizzled-2 signaling pathway is centrally involved in the morphological arrangement of cardiac myocytes in neonatal heart through stabilization of complexed cadherin-ß-catenin (Toyofuku, 2000).
Using a gene trap strategy, a mouse
mutation for the gene encoding alpha-E-catenin was isolated. This form of alpha-catenin
appears frequently coexpressed with E-cadherin in epithelial cell types. The mutation
obtained eliminates the carboxyl-terminal third of the protein but nevertheless
provokes a complete loss-of-function phenotype. Homozygous mutants show
disruption of the trophoblast epithelium (the first differentiated embryonic tissue), and
development is consequently blocked at the blastocyst stage. This phenotype parallels
the defects observed in E-cadherin mutant embryos. These results show the requirement
of the alpha-E-catenin carboxy terminus for its function and represent evidence of the
role of alpha-E-catenin in vivo, identifying this molecule as the natural partner of
the E-cadherin in trophoblast epithelium (Torres, 1997).
Cells with the potential to form skeletal muscle are present in the chick embryo prior to gastrulation.
Muscle differentiation begins after gastrulation within the somites. The role of cadherin-mediated
adhesion in the commitment and differentiation of skeletal muscle precursor cells was examined by
analyzing the expression of cell-cell adhesion molecules in cultures of epiblast, segmental plate, and
somite cells and by determining the effects of adhesion-perturbing antibodies on the accumulation of
MyoD and sarcomeric myosin. Cultured primitive streak stage epiblast cells downregulate E-cadherin
and upregulate N-cadherin. This switch in cadherin expression also occurs in vivo as epiblast cells
enter the primitive streak. Although MyoD protein is present in cells with N- or E-cadherin, only cells
with N-cadherin differentiate into skeletal muscle. In contrast to the primitive streak stage epiblast
cells, prestreak epiblast cells maintain the expression of E-cadherin in vitro. While the majority of
prestreak cells contain MyoD, only a few synthesize myosin. Treatment of primitive streak stage
epiblast cells with function-perturbing antibodies to N-cadherin results in an inhibition of myosin
accumulation and a decrease in the percentage of cells with MyoD. Segmental plate and somite cells
are similar to primitive streak stage epiblast cells in that most differentiate into skeletal muscle when
cultured in serum-free medium. While function-perturbing antibodies to N-cadherin inhibit the
accumulation of myosin in these mesoderm cells, the number of MyoD positive cells is unaffected in
somite cultures and only partially reduced in segmental plate cultures. These results suggest that
N-cadherin-mediated cell-cell adhesion is involved in both the commitment of muscle precursors and
their terminal differentiation (George-Weinstein, 1997).
Using
immunocytochemistry and in situ hybridization, the expression patterns of murine cadherins in the embryonic kidney have been examined during the time when
mesenchymal cells are phenotypically converted to epithelium and the pattern of the developing
nephrons is established. At the time of mesenchymal induction, cadherin-11 is expressed in the
mesenchyme but not in the ureteric bud epithelium, which expresses E-cadherin. The newly formed
epithelium of the renal vesicle expresses E-cadherin near the ureteric bud tips and cadherin-6 more
distally, suggesting that this primitive epithelium is already patterned with respect to progenitor cell
types. In the s-shaped body, the cadherin expression patterns reflect the developmental fate of each
region. The proximal tubule progenitors express cadherin-6, the distal tubule cells express E-cadherin,
whereas the glomeruli express P-cadherin. Ultimately, cadherin-6 is down-regulated whereas
E-cadherin expression remains in most, if not all, of the tubular epithelium. Antibodies generated
against the extracellular domain of cadherin-6 inhibit aggregation of induced mesenchyme and the
formation of mesenchyme-derived epithelium but do not disrupt ureteric bud branching in vitro. These
data suggest that cadherin-6 function is required for the early aggregation of induced mesenchymal
cells and their subsequent conversion to epithelium. The ability of cells to undergo cadherin switching may be a potent driving force for morphogenesis (Cho, 1998).
The contribution of the cytoplasmic domain of E-cadherin to adhesion, signaling, and differentiation during
murine mammary gland development was studied by in vivo expression of a gene encoding a truncated form of E-cadherin lacking the extracellular domain. The
expression of this gene in mammary epithelial cells during pregnancy induces precocious lobular epithelial morphogenesis associated with morphological
differentiation and the early synthesis of various molecules (advanced milk fat globule appearance and milk protein production). After delivery, when a fully
differentiated and secretory epithelium is required for lactation, the cytoplasmic domain of E-cadherin has a dominant-negative effect on cell-cell adhesion
and affects the structure and function of the epithelium. This also leads to the partial loss of epithelial polarization and changes in the basement membrane,
both important in malignancy. Thus, the cytoplasmic domain of E-cadherin induces epithelial morphogenesis, but also alters the cohesiveness of the fully
differentiated epithelium (Delmas, 1999).
Cell-cell adhesion mediated by some members of the
cadherin family is essential for embryonic survival. The N-cadherin-null embryo dies during mid-gestation, with
multiple developmental defects. N-cadherin-null
embryos expressing cadherins using muscle-specific
promoters, alpha- or beta-myosin heavy chain, are partially
rescued. Somewhat surprisingly, either N-cadherin or
E-cadherin is effective in rescuing the embryos. The
rescued embryos exhibit an increased number of somites,
branchial arches and the presence of forelimb buds;
however, in contrast, brain development is severely
impaired. In rescued animals, the aberrant yolk sac
morphology seen in N-cadherin-null embryos is
corrected, demonstrating that this phenotype is
secondary to the cardiac defect. Dye injection studies and
analysis of chimeric animals that have both wild-type
and N-cadherin-null cells support the conclusion that
obstruction of the cardiac outflow tract represents a major
defect that is likely to be the primary cause of pericardial
swelling seen in null embryos. Although rescued embryos
are more developed than null embryos, they are smaller
than wild-type embryos, even though the integrity of the
cardiovascular system appears normal. The smaller size
of rescued embryos may be due, at least in part, to
increased apoptosis observed in tissues not rescued by
transgene expression, indicating that N-cadherin-mediated
cell adhesion provides an essential survival signal for
embryonic cells. These data provide in vivo evidence that
cadherin adhesion is essential for cell survival and for
normal heart development. These data also show that E-cadherin
can functionally substitute for N-cadherin during cardiogenesis, suggesting a critical role for cadherin-mediated cell-cell adhesion, but not cadherin family member-specific signaling, at the looping stage of heart development (Luo, 2001).
Synaptic remodeling has been postulated as a mechanism underlying synaptic plasticity, and cadherin adhesion molecules are thought to be a regulator of such a process. The effects of cadherin blockage on synaptogenesis were examined in cultured hippocampal neurons. This blockade results in alterations of dendritic spine morphology, such as filopodia-like elongation of the spine and bifurcation of its head structure, along with concomitant disruption of the distribution of postsynaptic proteins. The accumulation of synapsin at presynaptic sites and synaptic vesicle recycling were also perturbed, although these synaptic responses to the cadherin blockade become less evident upon the maturation of the synapses. These findings suggest that cadherin regulates dendritic spine morphogenesis and related synaptic functions, presumably cooperating with cadherin-independent adhesive mechanisms to maintain spine-axon contacts (Togashi, 2002).
The effects of ubiquitous blocking of classic cadherins on synaptogenesis of cultured hippocampal neurons were examined. Two different methods were employed to inhibit cadherin activity. (1) A dominant-negative cadherin construct was used. At their cytoplasmic tail, the classic cadherins bind to a complex of ß-catenin and alpha-catenin, and the alpha-catenin interacts with cytoskeletal components. Interaction with these cytoplasmic components is crucial for cadherins to exert their full adhesive activity. If cadherin molecules with a deletion in their extracellular domain (which renders them nonfunctional) are introduced into cells, they compete with endogenous cadherins for interactions with the cytoplasmic components, resulting in a blockade of the activity of the endogenous cadherins. (2) The gene for alphaN-catenin, a subtype of alpha-catenin that is expressed specifically in the nervous system, was mutated. Without alpha-catenin, cadherin function is perturbed, with the exception that their homophilic interactions at the extracellular domain can still take place, inducing weak cell-cell associations (Togashi, 2002).
The hippocampus expresses at least three classic cadherins: N-cadherin, cadherin-11, and cadherin-8; all of these molecules can be nonspecifically blocked by the above methods. The results of experiments to block the cadherins in hippocampal neurons show that dendritic spine morphology is dramatically altered as a consequence of the cadherin dysfunction; this phenomenon is accompanied by impairment of synaptic vesicle accumulation and recycling, as well as by the disruption of PSD protein accumulation. These observations support the ideas that this family of adhesion molecules plays a critical role in the formation of synapses and that they may function as regulators of synaptic plasticity through their activity of modulating spine morphology. These findings are also consistent with a recent report that Drosophila N-cadherin mutation perturbs synaptic organization in the Drosophila visual system (Togashi, 2002).
The germ cell lineage segregates from the somatic cell lineages in early embryos. Germ cell determination in mice is not regulated by maternally inherited germplasm, but is initiated within
the embryo during gastrulation. However, the mechanisms of germ cell specification in mice remain unknown. Precursors to primordial germ cells (PGCs) have been located within early embryos, and
cell-cell interaction among these precursors is shown to be required for germ cell specification. The expression of a calcium-dependent cell adhesion molecule, E-cadherin, is restricted to the
proximal region of extra-embryonic mesoderm that contains PGC precursors, and blocking the functions of E-cadherin with an antibody inhibits PGC formation in vitro. These results showed that
E-cadherin-mediated cell-cell interaction among cells containing PGC precursors is essential to directing such cells to the germ cell fate (Okamura, 2004).
Several mechanisms by which E-cadherin regulates PGC determination are possible. The simplest model is that E-cadherin itself transmits instructive signals for PGC determination.
Alternatively, E-cadherin might play more permissive roles in transmitting signals for PGC determination. E-cadherin may simply permit close enough contact for other independent instructive
signals such as juxtacrine signals or signals via other adhesion molecules. In this regard, E-cadherin could also function as an anchor that settles precursor cells within niches for PGC
differentiation. Cell adhesion mediated by E-cadherin is required in the Drosophila ovary to anchor germline stem cells in niches for their renewal. In mouse extra-embryonic mesoderm, E-cadherin
expression is restricted to the proximal region that is adjacent to the epiblast, which also expresses E-cadherin, and is not expressed in the distal portion of extra-embryonic mesoderm or in
the allantois. This spatial distribution of E-cadherin prompts the notion that homophilic E-cadherin interactions prevent precursors from moving to the distal part of the extra-embryonic
mesoderm, where the allantois differentiates. The continuous expression of E-cadherin in the proximal extra-embryonic mesoderm might thus protect PGC precursors from allantois differentiation
(Okamura, 2004).
Gene silencing via intracytoplasmic microinjections of morpholino-modified antisense oligonucleotides is an effective and reproducible method to study both maternal and zygotic gene functions during early and late stages of mouse preimplantation development. The zygotic expression of the ß-geo transgene in the ROSA26 mouse strain can be inhibited until at least the early blastula stages. Thus morpholino-triggered gene inactivation appears to be a useful method to study the functional role of genes in preimplantation development. Using this approach, a potential role of maternal expression of Cdh1, the gene encoding the cell-adhesion molecule E-cadherin, was investigated. Inhibition of translation of maternal E-cadherin mRNA causes a developmental arrest at the two-cell stage. BrUTP incorporation assays indicate that this developmental defect cannot be explained by a general failure in transcriptional activity. This defect is reversible since E-cadherin mRNA can rescue the affected embryos, suggesting that a functional adhesion complex, present at the junction between blastomeres, is a prerequisite for the normal development of the mouse preimplantation embryo. This study thus reveals a previously unanticipated role of maternal E-cadherin during early stages of mouse development (Kanzler, 2003).
Epiboly, the spreading of the blastoderm over the large yolk cell, is the
first morphogenetic movement of the teleost embryo. Examining this movement as
a paradigm of vertebrate morphogenesis, focus was placed on the epiboly arrest
mutant half baked (hab), which segregates as a recessive
lethal, including alleles expressing zygotic-maternal dominant (ZMD) effects.
hab is shown to be a mutation in the zebrafish homolog of the
adhesion protein E-cadherin. Whereas exclusively recessive alleles of
hab produce truncated proteins, dominant alleles all contain
transversions in highly conserved amino acids of the extracellular domains,
suggesting these alleles produce dominant-negative effects. Antisense
oligonucleotides that create specific splicing defects in the hab
mRNA phenocopy the recessive phenotypes and, surprisingly, some of the ZMD
phenotypes as well. In situ analyses show that during late epiboly
hab is expressed in a radial gradient in the non axial epiblast, from
high concentrations in the exterior layer of the epiblast to low
concentrations in the interior layer of the epiblast. During epiboly,
using an asymmetric variant of radial intercalation, epiblast cells from the
interior layer sequentially move into the exterior layer and become restricted
to that layer; there, they participate in subtle cell shape changes that
further expand the blastoderm. In hab mutants, when cells intercalate
into the exterior layer, they tend to neither change cell shape nor become
restricted, and many of these cells 'de-intercalate' and move back into the
interior layer. Cell transplantation showed all these defects to be
cell-autonomous. Hence, as for the expansion of the mammalian trophoblast at a
similar developmental stage, hab/E-cadherin is necessary for the cell
rearrangements that spread the teleost blastoderm over the yolk (Kane, 2005).
E-cadherin is a member of the classical cadherin family and is known to be
involved in cell-cell adhesion and the adhesion-dependent morphogenesis
of various tissues. A zebrafish mutant
(cdh1rk3) was isolated that has a mutation in the
e-cadherin/cdh1 gene. The mutation rk3 is a hypomorphic allele,
and the homozygous mutant embryos displayed variable phenotypes in gastrulation
and tissue morphogenesis. The most severely affected embryos displayed epiboly
delay, decreased convergence and extension movements, and the dissociation of
cells from the embryos, resulting in early embryonic lethality. The less
severely affected embryos survived through the pharyngula stage and showed
flattened anterior neural tissue, abnormal positioning and morphology of the
hatching gland, scattered trigeminal ganglia, and aberrant axon bundles from the
trigeminal ganglia. Maternal-zygotic cdh1rk3 embryos
display epiboly arrest during gastrulation, in which the enveloping layer
(EVL) and the yolk syncytial layer but not the deep cells (DC) complete
epiboly. A similar phenotype was observed in embryos that received antisense
morpholino oligonucleotides (cdh1MO) against E-cadherin, and in zebrafish
epiboly mutants. Complementation analysis with the zebrafish epiboly mutant
weg suggests that cdh1rk3 is allelic to half
baked/weg. Immunohistochemistry with an anti-β-catenin antibody and
electron microscopy revealed that adhesion between the DCs and the EVL is
mostly disrupted but the adhesion between DCs is relatively unaffected in the
MZcdh1rk3 mutant and cdh1 morphant embryos.
These data suggest that E-cadherin-mediated cell adhesion between the DC and EVL
plays a role in the epiboly movement in zebrafish (Shimizu, 2005).
During mammalian embryogenesis the trophectoderm represents the first epithelial structure formed. The cell adhesion molecule E-cadherin is ultimately necessary for the transition from compacted morula to the formation of the blastocyst to ensure correct establishment of adhesion junctions in the trophectoderm. This study analyzed the extent to which E-cadherin confers unique adhesion and signaling properties in trophectoderm formation in vivo. Using a gene replacement approach, N-cadherin cDNA was introduced into the E-cadherin genomic locus. The expression of N-cadherin driven from the E-cadherin locus reflects the expression pattern of endogenous E-cadherin. Heterozygous mice co-expressing E- and N-cadherin are vital and show normal embryonic development. Interestingly, N-cadherin homozygous mutant embryos phenocopy E-cadherin-null mutant embryos. Upon removal of the maternal E-cadherin, N-cadherin is able to provide sufficient cellular adhesion to mediate morula compaction, but is insufficient for the subsequent formation of a fully polarized functional trophectoderm. When ES cells were isolated from N-cadherin homozygous mutant embryos and teratomas were produced, these ES cells differentiated into a large variety of tissue-like structures. Importantly, different epithelial-like structures expressing N-cadherin were formed, including respiratory epithelia, squamous epithelia with signs of keratinization and secretory epithelia with goblet cells. Thus, N-cadherin can maintain epithelia in differentiating ES cells, but not during the formation of the trophectoderm. These results point to a specific and unique function for E-cadherin during mouse preimplantation development (Kan, 2007).
E-cadherin (E-cad; cadherin 1) was conditionally substituted with N-cadherin (N-cad; cadherin 2) during intestine development by generating mice in which an Ncad cDNA was knocked into the Ecad locus. Mutant mice were born, demonstrating that N-cad can structurally replace E-cad and establish proper organ architecture. After birth, mutant mice gradually developed a mutant phenotype in both the small and large intestine and died at ~2-3 weeks of age, probably due to malnutrition during the transition to solid food. Molecular analysis revealed an extended domain of cells from the crypt into the villus region, with nuclear localization of β-catenin (β-cat; Ctnnb1) and enhanced expression of several β-cat target genes. In addition, the BMP signaling pathway was suppressed in the intestinal epithelium of the villi, suggesting that N-cad might interfere with BMP signaling in the intestinal epithelial cell layer. Interestingly, mutant mice developed severe dysplasia and clusters of cells with neoplastic features scattered along the crypt-villus axis in the small and large intestine. This experimental model indicates that, in the absence of E-cad, the sole expression of N-cad in an epithelial environment is sufficient to induce neoplastic transformations (Libusova, 2010).
The cardiovascular system forms during early embryogenesis and adapts to embryonic growth by sprouting angiogenesis and vascular remodeling. These processes require fine-tuning of cell-cell adhesion to maintain and reestablish endothelial contacts, while allowing cell motility. This study compared the contribution of two endothelial cell specific adhesion proteins - VE-cadherin (VE-cad/Cdh5; see Drosophila Shotgun) and Esama (Endothelial cell-selective adhesion molecule a) - during angiogenic sprouting and blood vessel fusion (anastomosis) in the zebrafish embryo by genetic analyses. Different combinations of mutant alleles can be placed into a phenotypic series with increasing defects in filopodial contact formation. Contact formation in esama mutants appear wild-type like, while esama-/-; ve-cad+/- and ve-cad single mutants exhibit intermediate phenotypes. The lack of both proteins interrupts filopodial interaction completely. Furthermore, double mutants do not form a stable endothelial monolayer, display intrajunctional gaps, dislocalization of ZO-1 (see Drosophila Polychaetoid) and defects in apical-basal polarization. In summary, VE-cadherin and Esama have distinct and redundant functions during blood vessel morphogenesis and both adhesion proteins are central to endothelial cell recognition during anastomosis (Sauteur, 2017).
E-cadherin and neural development Migrating neural crest cells adhere to fibronectin in an integrin-dependent manner (See Drosophila Myospheroid) while maintaining
reduced N-cadherin-mediated intercellular contacts. In
these cells, the control of N-cadherin may rely directly on the activity of integrins involved in the
process of cell motion. Prevention of neural crest cell migration using RGD peptides or antibodies to
fibronectin and to beta1 and beta3 integrins causes rapid N-cadherin-mediated cell clustering.
Restoration of stable intercellular contacts results essentially from the recruitment of an intracellular
pool of N-cadherin molecules that accumulate into adherens junctions in tight association with the
cytoskeleton and not from the redistribution of a preexisting pool of surface N-cadherin molecules. Agents that cause elevation of intracellular Ca2+ after entry across the plasma membrane
are potent inhibitors of cell aggregation and reduced the N-cadherin- mediated junctions in the cells.
Elevated serine/ threonine phosphorylation of catenins associated with N-cadherin accompany
the restoration of intercellular contacts. These results indicate that in migrating neural crest cells, beta1
and beta3 integrins are at the origin of a cascade of signaling events that involves transmembrane Ca2+
fluxes, followed by activation of phosphatases and kinases that ultimately control the surface
distribution and activity of N-cadherin. Cell aggreation is correlated with changes in beta-catenin phosphorylation. Such a direct coupling between adhesion receptors by means of
intracellular signals may be significant for the coordinated interplay between cell-cell and
cell-substratum adhesion that occurs during embryonic development, in wound healing, and during
tumor invasion and metastasis (Monier-Gavelle, 1997).
Stable expression of an epitope-tagged cDNA of the
hepatocyte-enriched transcription factor, hepatocyte nuclear factor (HNF)4 (see Drosophila HNF4), in dedifferentiated rat
hepatoma H5 cells is sufficient to provoke reexpression of a set of hepatocyte marker genes. The effects of HNF4 expression extend to the reestablishment of differentiated
epithelial cell morphology and simple epithelial polarity. The acquisition of epithelial morphology occurs
in two steps. First, expression of HNF4 results in reexpression of cytokeratin proteins and partial
reestablishment of E-cadherin production. Only the transfectants are competent to respond to the
synthetic glucocorticoid dexamethasone, which induces the second step of morphogenesis, including
formation of the junctional complex and expression of a polarized cell phenotype. Cell fusion
experiments revealed that the transfectant cells, which show only partial restoration of E-cadherin
expression, produce an extinguisher that is capable of acting in trans to downregulate the E-cadherin
gene of well-differentiated hepatoma cells. Bypass of this repression by stable expression of
E-cadherin in H5 cells is sufficient to establish some epithelial cell characteristics, implying that the
morphogenic potential of HNF4 in hepatic cells acts via activation of the E-cadherin gene. Thus, HNF4
seems to integrate the genetic programs of liver-specific gene expression and epithelial morphogenesis (Spath, 1998).
Melanocytes (Mc) and their progenitors melanoblasts (Mb) are derived from the neural crest and migrate along the dorsolateral pathway to colonize the dermis, the
epidermis, and finally the hair matrix. To examine the involvement of cadherins in the migration of Mc lineage cells, flow cytometric analysis of
dissociated live cells was combined with immunohistochemical staining of tissue sections to quantify the level of cadherin expression on the surface of Mb/Mc. At 11.5 days
postcoitum, Mb are in the dermis and are E-cadherin(-)P-cadherin(-) [E-cad(-)P-cad(-)]. During the next 48 h, a 200-fold increase of E-cadherin expression is
induced on the surface of Mb prior to their entry into the epidermis, thereby forming a homogeneous E-cad(high)P-cad(-/low) population. The cadherin expression
pattern then diversifies, giving rise to three populations: an E-cad(-)P-cad(-) dermal population; E-cad(high)P-cad(low) epidermal population, and
E-cad(-)P-cad(med-high) follicular population. In all three populations, the patterns of expression are region-specific, identical with those of surrounding cells
such as keratinocytes and fibroblasts, and preserved before and after pigmentation. While most of the epidermal Mb/Mc disappear after the neonatal stage in
normal mice, forced expression of steel factor in the epidermis of transgenic mice promotes survival of epidermal Mb/Mc, maintaining epidermal-type cadherin
expression pattern [E-cad(high)P-cad(low)] throughout the postnatal life. These findings indicate the involvement of extrinsic cues in coordinating the cadherin
expression pattern of Mb/Mc and suggest a role for E- and P-cadherins in guiding Mc progenitors to their final destinations (Nishimura, 1999).
The development of neural crest cells involves an epithelial-mesenchymal transition (EMT) associated with the restriction of cadherin 6B expression to the pre-migratory neural crest cells (PMNCCs), as well as a loss of N-cadherin expression. Cadherin 6B, which is highly expressed in PMNCCs, persists in early migrating neural crest cells and is required for their emigration from the neural tube. Cadherin 6B-expressing PMNCCs exhibit a general loss of epithelial junctional polarity and acquire motile properties before their delamination from the neuroepithelium. Cadherin 6B selectively induces the de-epithelialization of PMNCCs, which is mediated by stimulation of BMP signaling, whereas N-cadherin inhibits de-epithelialization and BMP signaling. As BMP signaling also induces cadherin 6B expression and represses N-cadherin, cadherin-regulated BMP signaling may create two opposing feedback loops. Thus, the overall EMT of neural crest cells occurs via two distinct steps: a cadherin 6B and BMP signaling-mediated de-epithelialization, and a subsequent delamination through the basement membrane (Par, 2010).
Cadherin-7 (Cad7) and cadherin-6B (Cad6B) are expressed in early and late phases of cranial motoneuron development, respectively. Cad7 is expressed by cranial motoneurons soon after they are generated, as well as in the environment through which their axons extend. By contrast, Cad6B is expressed by mature cranial motoneurons. In chick these cadherins play distinct roles in cranial motor axon morphology, branching and projection. Using in vitro approaches, it was shown that Cad7 enhances motor axon outgrowth, suppresses the formation of multiple axons and restricts interstitial branching, thus promoting the development of a single unbranched axon characteristic of differentiating motoneurons. Conversely, Cad6B in vitro promotes motor axon branching, a characteristic of mature motoneurons. In vivo gain- and loss-of-function experiments for these cadherins yielded phenotypes consistent with this interpretation. In particular, a loss of cadherin-mediated interactions in vivo led to dysregulation of the cranial motoneuron normal branching programme and caused axon navigation defects. It was also demonstrate that Cad6B functions via the phosphatidylinositol 3-kinase pathway. Together, these data show that Cad7 and Cad6B differentially regulate cranial motoneuron growth, branching and axon guidance (Barnes, 2010).
Cadherin and area identity in the mammalian neocortex The contribution of extrinsic and genetic mechanisms in determining areas of the mammalian neocortex has been a
contested issue. This study analyzes the roles of the regulatory genes Emx2 and Pax6, which are expressed in opposing
gradients in the neocortical ventricular zone. Emx2 is expressed in low rostral to high caudal and low lateral to high
medial gradients, whereas Pax6 is expressed in low caudal to high rostral and low medial to high lateral gradients. Changes in the patterning of molecular markers and
area-specific connections between the cortex and thalamus suggest that arealization of the neocortex is disproportionately altered in Emx2 and Pax6 mutant mice in
opposing manners predicted from their countergradients of expression: rostral areas expand and caudal areas contract in Emx2 mutants, whereas the opposite effect
is seen in Pax6 mutants. These findings suggest that Emx2 and Pax6 cooperate to regulate arealization of the neocortex and to confer area identity to cortical cells (Bishop, 2000).
The type II classical cadherins, Cadherin6
(Cad6) and Cadherin8 (Cad8),
are expressed in areal patterns in the late embryonic mouse neocortex:
Cad8 has been reported to mark the rostrally located motor
area, and Cad6 marks the somatosensory area, located immediately caudal to the motor area, and the auditory area, located in
the caudolateral neocortex. Because the cortex is
reduced in size in both Emx2 and Pax6 mutants, as
compared to wild type, the proportion
of the cortical surface covered by domains of cadherin expression is determined as
well as absolute domain size. No change in proportional sizes of cadherin expression domains would indicate that arealization per se has not been affected, suggesting that the full range of putative area identities is present
in the smaller cortex and that all areas are uniformly reduced in size.
A change in proportional sizes would indicate that areas are
disproportionately affected in the mutant neocortex and therefore that
Emx2 and/or Pax6 has a role in regulating
arealization of the neocortex. This finding and interpretation would be
most strongly supported by a change in absolute sizes of cadherin
expression domains (Bishop, 2000).
The areal pattern of cadherin expression in the Emx2
homozygous mutant cortex is substantially different
from that in wild type. The domain of Cad8
expression is expanded caudally. This expansion appears to be
greater along the medial edge of the cortex than laterally, farther on,
suggesting that Cad8 expression may also be expanded medially. The domain of Cad6 expression is shifted caudally
and medially, as seen on the dorsal and lateral surfaces of the cortex. No changes in the cortical patterns of cadherin expression
are observed in Emx2 heterozygous mice. The domain of Cad8 expression is significantly larger in the
Emx2 homozygous mutant neocortex, in both proportional and
absolute area, as well as in its proportional and absolute linear
extent across the cortical surface. In fact, the
absolute area of the Cad8 expression domain in
Emx2 homozygous mutants is almost double that in wild type,
even though the surface area of the dorsal cortex is reduced by
one-third. In Emx2 homozygous mutants, the domain of
Cad6 expression on the dorsal cortical surface is
significantly increased in proportional area and in both proportional
and absolute width, compared to that in wild type. In
contrast, the area and length of the Cad6 expression domain
on the lateral cortical surface of Emx2 homozygous mutants
each exhibit both proportional and absolute reductions compared to that
in wild type. These results suggest that areas
located in rostral and lateral parts of the neocortex are expanded and shifted caudally and medially in the Emx2 mutant neocortex (Bishop, 2000).
Because Pax6 is expressed in a countergradient to
Emx2, it was predicted that changes in domains of cadherin
expression in Pax6 mutant mice
(Sey/Sey mutants) would be in the opposite
direction of those observed in Emx2 mutants. The domain of Cad8 expression is contracted rostrally in the Sey/Sey cortex compared to that in the wild type. The Cad6 expression domain is contracted both laterally and rostrally in the Sey/Sey cortex. The domains of
Cad8 and Cad6 expression on the dorsal surface of
the Sey/Sey cortex, which is about three-quarters
the size of that in the wild type, show both proportional and absolute
reductions in area. Heterozygous mice (Sey/+) show no changes in cortical cadherin expression compared to wild-type mice. These results suggest that
areas located in rostral and lateral parts of the neocortex contract
rostrally and laterally in Sey/Sey mutants (Bishop, 2000).
Changes in the expression domains of Cad6 and
Cad8 in Emx2 and Pax6 mutants suggest
corresponding changes in neocortical arealization. The primary
neocortical areas (motor, visual, somatosensory, and auditory) receive
area-specific inputs from the principal motor and sensory thalamic
nuclei [ventrolateral (VL), dorsal lateral geniculate (dLG),
ventroposterior (VP), and medial geniculate (MG), respectively].
During normal development, thalamocortical axons target and invade
their neocortical areas in a precise manner. Therefore, as an additional assay for changes in area identity in the Emx2 mutant neocortex, retrograde and anterograde axon tracing were used to map thalamocortical projections. This analysis was not done in
Sey/Sey mutants because thalamic axons do not
reach the cortex in these mice. Retrograde labeling from the cortex of Emx2
homozygous mutants indicates a caudal shift in the border between the
somatosensory and visual areas compared to that of the wild type. Injections confined to the cortical plate of the occipital cortex, the location of the primary visual area, normally backlabel neurons in the dLG nucleus. However, in
Emx2 mutants, similarly placed injections label cells in the
VP nucleus, which normally projects to the primary somatosensory area
located rostral to the visual area. Deeper injections made
into the subplate of the occipital neocortex in Emx2 mutants
backlabel neurons in both dLG and VP nuclei, indicating that dLG
thalamic axons extend through the subplate below the occipital cortex
but fail to invade the overlying cortical plate. Retrograde tracing from anterior and posterior portions of the occipital cortex reveals the expected topography in wild-type mice but again indicates a caudal shift in the border between the somatosensory
and visual areas in Emx2 mutants. In wild-type
mice, injections into the anterior occipital cortex backlabel cells in
the posterior dLG nucleus, and injections into the posterior occipital
cortex backlabel cells in the anterior dLG nucleus. In
contrast, in Emx2 mutants, injections into the anterior
occipital cortex do not label cells in the dLG nucleus but instead
label cells in the VP nucleus; injections in the very posterior
occipital cortex do label cells in the dLG nucleus, but their number is significantly reduced compared to the wild type. These
findings suggest that the visual area in Emx2 mutants is contracted and restricted to the extreme caudal part of the occipital cortex (Bishop, 2000).
Anterograde tracing of thalamocortical projections is
consistent with the retrograde tracing results. Injections into the dLG
nucleus of Emx2 mutants label axons in the subplate beneath the caudal occipital cortex, but in comparison to the wild type, few
invade the cortical plate. Injections into the VP nucleus of
Emx2 mutants label axons that extend farther caudally than
in the wild type and aberrantly invade the cortical plate of occipital
cortex, whereas in the wild type, VP axons invade the cortical plate of
the more rostrally located parietal cortex (the location of the primary
somatosensory area). Thalamocortical projections in
heterozygous Emx2 mutants resemble those in the wild type. Overall, anterograde and retrograde tracing of thalamocortical projections in Emx2 mutants provides evidence for a contraction of the visual area and a caudal shift in the border between the somatosensory and visual areas (Bishop, 2000).
Area-specific connections between thalamic nuclei and
neocortical areas are reciprocal. Injections into thalamic nuclei
backlabel cortical neurons in wild-type mice and Emx2
mutants. Injections into the dLG nucleus backlabel
significantly fewer cells in the occipital cortex of Emx2
mutants compared to the wild type. The few labeled cells in Emx2
mutants are restricted to the very caudal cortex. Injections
into the VP nucleus in wild-type mice backlabel cells in the parietal cortex but backlabel none in the occipital cortex. In
contrast, VP injections in Emx2 mutants backlabel a
substantial number of cells in the occipital cortex. These findings suggest that, in Emx2 mutants, corticothalamic neurons located in the occipital cortex have acquired a somatosensory area identity instead of their usual visual area identity. These changes in area-specific corticothalamic projections in Emx2
mutants are consistent with the changes observed in area-specific
thalamocortical projections. Together, they suggest that the
primary visual area is substantially reduced and restricted to the very
caudal part of the neocortex, with a corresponding caudal shift in the
border between visual and somatosensory areas (Bishop, 2000).
Emx2 is reported to be expressed in a small
patch of neuroepithelium in the ventral-most part of the dorsal
thalamus. Although this part does not generate cells of
the principal sensory and motor thalamic nuclei, several markers were used to confirm the normal development and organization
of the dorsal thalamus in Emx2 mutants.
Patterns of acetylcholinesterase (AChE) staining and Gbx2
expression and general morphology revealed by nuclear
4',6-diamidino-2-phenylindole staining are all normal in the dorsal
thalamus of Emx2 mutants. Thus,
alterations in thalamocortical and corticothalamic projections in
Emx2 mutants can be presumed to be due to changes intrinsic
to the neocortex (Bishop, 2000).
These findings implicate Emx2 and
Pax6 in the genetic control of neocortical arealization.
They cannot be explained by a potential delay in neocortical
development, nor are the expansions and contractions of
the cadherin expression domains secondary to a loss of thalamocortical
input. In addition, the observed changes in cadherin
expression domains are not simply a by-product of the reduced overall
size of the cerebral cortex in Emx2 and Pax6
mutants, because the changes are disproportionate in each mutant and
opposing in the two mutants as predicted from the countergradients of
Emx2 and Pax6 expression. Similarly, the observed
changes in thalamocortical and corticothalamic projections in the
Emx2 mutants are not due simply to a caudal truncation of
the neocortex with an associated loss of the visual area. Arguing
against this possibility are the changes in cadherin expression in
Emx2 mutants, especially the expansion of the
Cad8 expression domain in the frontal cortex (the motor
area), in both proportional and absolute size. Instead, the findings
presented here indicate a disproportional, but orderly, arealization of the Emx2 mutant neocortex reflected by an expansion of
rostral areas and a contraction of caudal areas, and an opposite effect on arealization in the Pax6 mutant neocortex (Bishop, 2000).
Changes in the areal expression patterns of the
cadherins and the area-specific distribution of corticothalamic neurons
in the mutants suggest that Emx2 and Pax6 confer
area identities to cortical cells, including projection neurons. The
changes in cadherin expression and, presumably, receptors for axon
guidance molecules that control corticothalamic axon targeting
may be indicative of a direct role for Emx2 and
Pax6 in their regulation, or they may be an indirect effect
of the regulation of area identity. Similarly, the changes in
area-specific thalamocortical projections suggest that
Emx2 and Pax6 are involved either directly or
indirectly in the regulation of axon guidance molecules within the
cortex that control thalamocortical axon targeting. The restricted
cortical expression of Eph receptor tyrosine kinases and their ligands, the ephrins, which act as axon guidance molecules in several systems, makes these candidates for controlling the development of area-specific projections between the thalamus and cortex (Bishop, 2000).
Emx2 and Pax6 may act
independently or in a combinatorial manner (possibly with other
transcription factor genes) to specify neocortical areas. Because areas
in the neocortex have sharp borders, it is likely (but not required)
that the graded expression patterns of Emx2 and
Pax6 are translated to regulate some downstream genes in
restricted patterns with abrupt borders that relate to areas. Although the downstream targets of
Emx2 and Pax6 in the cortex have yet to be
identified, transcription factors such as T-brain1 and
Id2 are expressed in the neocortex in discrete patterns with abrupt borders that may be controlled by upstream regulatory genes expressed in gradients (Bishop, 2000).
Emx2 and Pax6 appear to be
independently regulated. The opposing gradients of
Emx2 and Pax6 may be induced by signals secreted
from the poles of the cortex. Several secreted proteins are candidates
for these inductive signals, including the BMP, WNT (2b, 3a, 5a, and
7a), and FGF8 proteins. In addition, cortical
expression of the transcription factor Gli3 is required for
Emx2 expression. Thus, combinations of
inductive signals and upstream transcription factors may specify gradients of Emx2 and Pax6. A better
understanding of the roles of Emx2 and Pax6 in
regulating neocortical arealization will require identifying the
patterning mechanisms that establish their differential expression,
identifying downstream targets, and defining the mechanisms by which
they, in combination with other factors, intrinsic and extrinsic,
control the process of arealization of the neocortex (Bishop, 2000).
The differentiation of areas of the mammalian neocortex has been hypothesized to
be controlled by intrinsic genetic programs and extrinsic influences such as
those mediated by thalamocortical afferents (TCAs). To address the interplay
between these intrinsic and extrinsic mechanisms in the process of arealization,
the requirement of TCAs in establishing or maintaining graded
or areal patterns of gene expression in the developing mouse neocortex has been analyzed. The differential expression of Lhx2, SCIP, and Emx1, representatives of
three different classes of transcription factors, is described as well as the type II classical cadherins Cad6, Cad8, and Cad11, which are expressed in the cortical plate in graded or areal patterns, as well as layer-specific patterns. The
differential expression of Lhx2, SCIP, Emx1, and Cad8 in the cortical plate is
not evident until after TCAs reach the cortex, whereas Cad6 and Cad11 show
subtle graded patterns of expression before the arrival of TCAs, which later
become stronger. These genes exhibit normal-appearing graded or
areal expression patterns in Mash-1 mutant mice that fail to develop a TCA
projection. These findings show that TCAs are not required for the establishment
or maintenance of the graded and areal expression patterns of these genes and
strongly suggest that their regulation is intrinsic to the developing neocortex (Nakagawa, 1999).
Slow-twitch muscle fibers of the zebrafish myotome undergo a unique set of morphogenetic cell movements. During embryogenesis, slow-twitch muscle derives from the adaxial cells, a layer of paraxial mesoderm that differentiates medially within the myotome, immediately adjacent to the notochord. Subsequently, slow-twitch muscle cells migrate through the entire myotome, coming to lie at its most lateral surface. The cellular and molecular basis for slow-twitch muscle cell migration has been examined. Slow-twitch muscle cell morphogenesis is marked by behaviors typical of cells influenced by differential cell adhesion. Dynamic and reciprocal waves of N-cadherin and M-cadherin expression within the myotome, that correlate precisely with cell migration, generate differential adhesive environments that drive slow-twitch muscle cell migration through the myotome. Removing or altering the expression of either protein within the myotome perturbs migration. These results provide a definitive example of homophilic cell adhesion shaping cellular behavior during vertebrate development (Cortés, 2003).
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