Cadherin-mediated adhesion regulates posterior body formation
© Harrington et al; licensee BioMed Central Ltd. 2007
Received: 04 September 2007
Accepted: 28 November 2007
Published: 28 November 2007
The anterior-posterior axis of the vertebrate embryo undergoes a dramatic elongation during early development. Convergence and extension of the mesoderm, occurring during gastrulation, initiates the narrowing and lengthening of the embryo. However the lengthening of the axis continues during post-gastrula stages in the tailbud region, and is thought to involve convergent extension movements as well as other cell behaviors specific to posterior regions.
We demonstrate here, using a semi-dominant N-cadherin allele, that members of the classical cadherin subfamily of cell-cell adhesion molecules are required for tailbud elongation in the zebrafish. In vivo imaging of cell behaviors suggests that the extension of posterior axial mesodermal cells is impaired in embryos that carry the semi-dominant N-cadherin allele. This defect most likely results from a general loss of cell-cell adhesion in the tailbud region. Consistent with these observations, N-cadherin is expressed throughout the tailbud during post-gastrulation stages. In addition, we show that N-cadherin interacts synergistically with vang-like 2, a member of the non-canonical Wnt signaling/planar cell polarity pathway, to mediate tail morphogenesis.
We provide the first evidence here that N-cadherin and other members of the classical cadherin subfamily function in parallel with the planar cell polarity pathway to shape the posterior axis during post-gastrulation stages. These findings further highlight the central role that adhesion molecules play in the cellular rearrangements that drive morphogenesis in vertebrates and identify classical cadherins as major contributors to tail development.
During early embryogenesis, the shape of the vertebrate embryo changes dramatically, as the embryo narrows along the mediolateral axis and simultaneously lengthens along the anterior-posterior (A-P) axis. These morphological changes are brought about by cellular rearrangements, known as convergent extension (CE) movements, that have been studied extensively in the frog and fish embryo (reviewed in [1–3]). In the fish, CE in the mesoderm involves the convergence of lateral mesodermal cells dorsally by directed migration [4, 5]. During mid-gastrulation and continuing into early segmentation, axial mesoderm cells (the precursors of the notochord) undergo mediolateral intercalation to extend the dorsal axis [6, 7]. At a cellular level, directed migration is mediated by several types of cell behaviors that occur within distinct dorso-ventral domains [4, 5]. Axial cells undergoing intercalation at the dorsal midline become tightly packed and polarized along the mediolateral axis . Following gastrulation, the embryonic axis continues to elongate in the posterior (tailbud) region. Kanki and Ho (1997; ) identified four distinct stages of tailbud development: tailbud formation (aggregation of marginal cells to establish the tailbud), tailbud extension (cell movement along the ventral side of the yolk), tailbud protrusion (accumulation of cells in the tailbud, resulting in the formation of an aggregate) and tail eversion (tailbud growth away from the yolk cell). At a cellular level, the mechanisms of posterior body formation in vertebrates involve movements and behaviors that are similar to CE and others that are unique to the posterior body [8–11].
CE movements are mediated, in part, by the non-canonical Wnt signaling pathway, also known as the planar cell polarity (PCP) pathway, which was first identified in Drosophila. In vertebrates this branched pathway is activated by the binding of Wnt11/Silberblick (Slb) and Wnt5/Pipetail (Ppt) to their seven-transmembrane-domain receptor Frizzled (Fz; ) and transduced through the multifunctional protein Dishevelled (Dsh). Another gene in this pathway, Knypek (Kny, or Glypican 4) is thought to potentiate Wnt signaling . Other transducers of the non-canonical Wnt pathway include: Vang-like 2 (Vangl2) (previously known as Strabismus (Stbm) in the zebrafish), a transmembrane protein [14, 15, 5]; Prickle (Pkl), an intracellular protein containing three LIM domains and a conserved "PET" domain [16–18]; Rho Kinase 2; and members of the Rho family of small GTPases (Rho A, Rac1 and Cdc42), that regulate different cellular responses such as cytoskeletal rearrangements and cell adhesion [19–22]. c-jun N-terminal kinase (JNK), although not specific to this pathway, is required downstream of small GTPases for CE [23, 24]. Consistent with their role in CE, vertebrate embryos in which genes in this pathway are either disrupted via mutation/use of dominant-negative constructs or overexpressed, typically exhibit shortened and broadened A-P axes (reviewed in [25, 3]).
There is increasing evidence that the cellular rearrangements that occur during CE also require the regulation of cadherin-based intercellular adhesion, as cells reposition themselves relative to one another [26, 27]. The cadherins constitute a large superfamily that comprises classical cadherins, protocadherins and cadherin-like proteins such as Flamingo (Fmi). Knockdown or overexpression of several members of this family have been shown to disrupt CE movements during gastrulation. Zebrafish flamingo1a and b (fmi1a, b) are expressed in mesodermal cell populations undergoing gastrulation. Morpholino (MO) knockdown of these genes prevents the extension of the entire A-P axis, including the prechordal plate and ventral diencephalic precursors. Fmi1 is thought to function in concert with non-canonical Wnt signaling proteins in these mesodermal populations , in agreement with the established role of Drosophila Fmi (Starry Night) as a PCP pathway component [29, 30]. Wnt11 was recently shown to recruit Frizzled7 (Fz7) and Fmi to the plasma membrane, where the latter is thought to modulate local cell contact persistence in the zebrafish gastrula . Paraxial protocadherin (papc), a member of the protocadherin subfamily expressed in the paraxial mesoderm of the gastrulating embryo, is required for dorsal convergence movements during gastrulation in the Xenopus and zebrafish [32–34]. Consistent with this observation, overexpression of papc RNA triggers gastrulation movements in Xenopus animal cap explants treated with low levels of activin . Xenopus papc was shown to signal through the small GTPases RhoA, Rac1 and JNK, that also function downstream of the non-canonical Wnt pathway. Although papc is able to bind to the Xenopus Fz7 receptor and shares common downstream components with the non-canonical Wnt signaling pathway, papc and Wnt signaling are not redundant for mesodermal morphogenesis [35, 34].
In addition to members of the protocadherin subfamily, classical cadherins, including C-cadherin (C-cad) and E-cadherin (E-cad), are required for mesodermal morphogenesis. In the Xenopus, C-cad is essential for gastrulation movements . Moreover, activin-induced axial elongation is accompanied by decreased C-cad-mediated cell-cell adhesion [37, 38], indicating that the levels of C-cad need to be tightly controlled for proper mesodermal morphogenesis. Interestingly, papc, functioning downstream of activin and independently from non-canonical Wnt signaling, was shown to downregulate the adhesive activity of C-cad in the paraxial mesoderm . E-cad has also been implicated in gastrulation. Following epiboly, zebrafish mesendodermal progenitors upregulate E-cad and become increasingly motile as they migrate along the overlying epiblast towards the animal pole and contribute to axis elongation. When E-cad function is compromised, mesendodermal progenitors fail to elongate and efficiently migrate along the epiblast . Evidence suggests that Wnt11 controls the levels of E-cad on the cell surface and the general cohesiveness of mesendodermal progenitors by regulating E-cad endocytosis via the GTPase Rab5 , highlighting again the dynamic modulation of cell adhesion that accompanies morphogenesis. Finally, it was recently demonstrated that a gradient of adhesive activity established downstream of BMP signaling by a classical cadherin, the identity of which is unknown, drives gastrulation movements in the zebrafish . Several members of the cadherin superfamily thus play a critical role in CE during gastrulation and some appear to interact with the non-canonical Wnt pathway to mediate these movements.
In vertebrates, N-cadherin (N-cad, a classical cadherin) expression is restricted to neural tissues, the notochord, somites, cardiac and skeletal muscle [43, 44]. Loss of N-cad function results in a variety of neural tube defects in vertebrate embryos [44–49]. In contrast to its well-characterized role in the neural tube, there is little evidence that N-cad is required for the morphogenetic events that shape the embryonic axis during gastrulation or post-gastrulation stages. In the mouse and zebrafish, only late mesodermal defects associated with the formation of somites [44, 50–52] or cardiac muscle  have been reported in N-cad null embryos. We demonstrate here using a semi-dominant N-cad allele that N-cad, and possibly other members of the classical cadherin subfamily, are essential for tailbud eversion and to a lesser extent for shaping the mesoderm during gastrulation. Supporting these observations, zebrafish N-cad is expressed in axial and paraxial mesoderm during gastrulation and throughout the tailbud. In addition, we show that N-cad interacts genetically with vangl2, a member of the non-canonical Wnt signaling pathway, to mediate tailbud eversion. We further rule out a role for N-cad in regulating apoptosis during the onset of tailbud eversion. Together these data point to a central role for cadherins in mediating the cellular behaviors that drive the elongation of the posterior body.
N-cadherinmutants display a range of tail phenotypes
N-cad m117 may block the adhesive activity of other classical cadherins
The posterior defects observed in N-cad m117 mutants suggest that N-cad is required for posterior morphogenesis. However, given the more subtle posterior defects observed in presumed N-cad null mutants (Figure 1C), it is also possible that N-cad m117 is a gain of function allele, increasing rather than reducing the adhesive activity of N-cad (and possibly of other cadherins). This possibility seems unlikely given the essential nature of the Trp2 residue in mediating adhesion [60–64] and the cell adhesion defects described below. Alternatively, N-cad m117 may alter the activity of other classical cadherins in a dominant or a semi-dominant negative manner. Consistent with this hypothesis, members of this subfamily are known to interact in trans in both a homophilic and a heterophilic manner [66, 67]. Moreover, several zebrafish classical cadherins, including N-, E- and M-cadherin, are expressed in the mesoderm during embryogenesis [68, 40].
To further investigate the reason underlying the difference in severity of the posterior defects in N-cad mutants, we also examined N-cad protein expression and localization in several N-cad mutants. Interestingly, N-cad protein was mislocalized away from the plasma membrane in N-cad p79emcf mutants (Figure 3D), that harbor a point mutation in the EC5 domain . This finding was confirmed using an antibody against β-catenin, a protein that associates with the cytoplasmic domain of N-cad (data not shown). Consistent with previously published observations, no N-cad protein was detected in N-cad r2.10 homozygous mutants (Figure 3E; ). In contrast, normal or slightly elevated levels of N-cad protein were observed in N-cad m117 mutants (Figure 3B) and in putative N-cad m117 heterozygotes (Figure 3C). Together these observations suggest that N-cad p79emcf may function as a null allele, explaining why the phenotype of N-cad p79emcf mutants is very similar to that of N-cad r2.10 mutants. Furthermore, the presence of N-cad protein in N-cad m117 heterozygotes and homozygotes is consistent with this allele being semi-dominant and altering the adhesive activity of other cadherins by heterophilic interactions.
In summary, loss of N-cad and other classical cadherin function impairs posterior mesoderm morphogenesis and somite boundary formation, in addition to neural convergence. Moreover, the fact that severe brain but only mild posterior defects are observed in embryos carrying N-cad null alleles (Figure 1B, C) suggests that brain development is mostly dependent on N-cad function whereas tail developmental may require several cadherin family members with overlapping functions. Since N-cad m117 /N-cad p79emcf transheterozygotes have an intermediate phenotype, as compared to their homozygous mutant counterparts, this indicates that N-cad does participate in posterior morphogenesis. We focus the rest of this study on the posterior defects.
Loss of cadherinfunction blocks tail elongation
In order to determine whether loss of cadherin function may cause subtle defects in CE movements during gastrulation that were not detectable using morphological criteria, embryos were further analyzed using molecular markers for ectodermal and mesodermal tissues. The extent of axial mesoderm CE was examined at the 1 som stage, using a no tail riboprobe to label the axial mesoderm. N-cad m117 mutant embryos were readily identifiable at this stage of development by co-labeling with dlx3, a marker for the edge of the neural plate. Indeed, a quarter of the embryos from a cross between N-cad m117 heterozygous parents revealed an enlarged neural plate (compare Figures 4I, J), caused by impaired CE in the neural ectoderm. This phenotype is consistent with the expression of N-cad in the neural plate and the known role of N-cad in neural convergence [48, 49]. The width of the notochord in N-cad m117 mutant embryos was comparable to that of WT siblings (Figure 4J). To test for paraxial mesoderm convergence defects, 7 som stage embryos were examined using papc and myoD, markers for presomitic paraxial mesoderm and somites, respectively. Presomitic mesoderm appeared laterally expanded in N-cad m117 homozygous mutants (Figure 4L) but not in N-cad m117 heterozygotes (Additional file 2). These results suggest that loss of cadherin function can cause mild mesodermal convergence defects and confirm that the N-cad m117 allele is not fully dominant. Consistent with the enlarged papc domain, somites were slightly wider in N-cad m117 mutants than in WT siblings (Figure 4N). These phenotypes are similar to, although less pronounced than, those observed in non-canonical Wnt signaling mutants, such as kny and trilobite (tri; which disrupts vangl2). In addition to the lengthening of the somites, myoD-positive cells were observed in ectopic positions in N-cad m117 mutants (Figure 4N), suggesting a loss of cell-cell adhesion. This mixing of mesodermal populations was previously observed in embryos in which papc is disrupted . Dorsal views of 7 som N-cad m117 mutants imaged using Nomarski optics also revealed the presence of scattered, disorganized cells adjacent to a poorly defined notochord and throughout the tailbud, confirming a loss of tissue integrity in the paraxial mesoderm of N-cad m117 mutants (Figure 4P).
Together, these observations suggest that the cell intercalation behaviors that drive the narrowing and elongation of the dorsal axis (notochord) during gastrulation are normal in absence/reduced function of N-cad and other cadherin family members, but the convergence movements in paraxial mesoderm may be slightly defective. In addition, the tailbud fails to undergo proper eversion in N-cad m117 mutants, which most likely accounts for the severe posterior shortening observed in older embryos. This defect may be caused by impaired intercellular adhesion. Given that neural and mesodermal markers are expressed in a regionally appropriate manner in these embryos (; this study and data not shown), cell fate specification is unlikely to be a contributing cause to the posterior defect.
N-cadherinis expressed in the mesoderm during gastrulation and post-gastrulation stages
By mid-somitogenesis, N-cad is present ubiquitously in neural tissue with highest levels in the anterior neural rod (Figure 5C, D). Expression in the presomitic (Figure 5C') and paraxial mesoderm (Figure 5D') is prominent in the trunk region. In the tailbud, N-cad expression is observed throughout the prospective neural tissue and mesoderm but is conspicuously absent from the epidermis and enveloping layer (Figure 5C", D"). By late somitogenesis (18 som), during tailbud protrusion, N-cad expression is very high in anterior neural tissue (Figure 5E), while trunk and tail neural tissue have lower levels (Figure 5E', E"). Trunk somites express N-cad in postmigratory slow cells (PSCs) while the tail somites express N-cad throughout, as previously described by Cortes et al., 2003 (Figure 5E', E").
Increased apoptosis does not contribute to the onset of posterior defects
Apoptosis during tailbud eversion. Number of cells labeled using the TUNEL assay in 18–19 som (A) and 22–23 som (B) embryos is shown as mean ± SD. (1, 2, 3) Tail regions in which the TUNEL-positive cells were counted (refer to figure 6 for illustration). 0.4 ng of vangl2 MO was injected into each embryo where specified
(n = 4)
2.75 ± 1.26
2.75 ± 2.22
1.75 ± 2.22
(n = 3)
11.00 ± 3.46
10.33 ± 2.31
7.33 ± 1.53
WT w/vangl2 MO
(n = 3)
6.67 ± 5.51
9.00 ± 6.93
10.67 ± 10.07
(n = 3)
6.33 ± 0.58
13.33 ± 1.15
1.33 ± 1.53
N-cad p79emcf w/vangl2 MO
(n = 2)
14.00 ± 8.48
6.50 ± 3.54
3.00 ± 2.83
(n = 4)
3.00 ± 3.56
2.00 ± 2.83
3.25 ± 2.36
(n = 7)
3.14 ± 1.77
5.57 ± 3.21
0.29 ± 0.49
WT w/vangl2 MO
(n = 7)
1.29 ± 2.56
0.71 ± 1.25
1.00 ± 1.00
(n = 8)
2.13 ± 2.03
1.63 ± 1.60
0.50 ± 0.53
N-cad p79emcf w/vangl2 MO
(n = 7)
1.86 ± 1.57
0.86 ± 0.69
1.71 ± 1.60
Impaired movement of posterior axial mesodermal cells underlies the tailbud elongation defects
Zebrafish N-cad is known to drive the convergence of neural cells [48, 49] and other zebrafish classical cadherins have been implicated in mesodermal morphogenesis ([68, 40, 70, 71, 42]; reviewed in ). It is therefore likely that N-cad and other members of the cadherin family also participate in the cell movements implicated in posterior body elongation. The tailbud is derived from two cell populations, posterior tailbud cells (originating from the ventral blastoderm margin and fated to become tail paraxial mesoderm) and anterior tailbud cells (derived from the dorsal region of the embryo and fated to become axial mesoderm . Cell tracing experiments were therefore carried out to determine whether cell movements of posterior or anterior tailbud cells are impaired in N-cad m117 mutants.
Anterior tailbud cells are thought to extend into the tail by cell rearrangement and intercalation (a continuation of gastrulation movements, as described in the chick and the mouse, [72, 73]) and by enlargement of notochord cells due to vacuolation. To investigate whether posterior extension of the axial mesoderm is defective, cells immediately anterior to KV were labeled at 4 som (Figure 7D–G) and the position of their progeny scored at the 18 som stage (Figure 7E'–G'). Labeling of WT siblings revealed that cells aligned along the posterior midline and adopted a cuboidal morphology characteristic of notochord cells (Figure 7E'–E", n = 2 out of 2 embryos). In contrast, labeled cells in N-cad m117 mutants were observed in ectopic positions, which mostly coincided with the location of large vacuoles in the tail tip. Cells were found associated with the dorsal region of the tailbud (Figure 7F', n = 3 out of 7 embryos), adjacent to the yolk extension (Figure 7G', n = 3 out 7 embryos) or within the vacuoles (data not shown, n = 1 out of 7 embryos). In addition, labeled cells failed to adopt the characteristic cuboidal morphology (n = 7 out of 7 embryos). Thus, cadherins appear to be necessary for the proper positioning of axial mesoderm cells. It is likely that loss of tissue integrity in the tailbud region, as evidenced by the scattering of cells (Figure 4N, P and Figure 7C"), may be the underlying reason for this defect. Indeed, cell-cell adhesion is known to be essential for cell intercalation movements such as those that drive notochord elongation (reviewed in ).
In situ hybridization on wholemount 30 hpf N-cad m117 mutants using myoD (a marker for ventral-derived cells that differentiate into somatic mesoderm) and no tail (a marker for anterior-derived axial mesoderm cells that differentiate into notochord) confirmed the uncaging data. myoD was expressed throughout the posterior region (although labeled cells did not organize into somites, data not shown), indicating that ventral-derived cells were able to populate the posterior region. In contrast, no tail expression was not observed in the tail tip (data not shown), supporting the finding that axial mesoderm does not extend posteriorly in N-cad m117 mutants.
N-cadherin and vang-like 2interact to regulate posterior body morphogenesis
Together these observations suggest that N-cad and vangl2 interact genetically and that this interaction is primarily required, during post-gastrula stages, for tailbud eversion. Increased apoptosis in posterior regions was ruled out as a likely contributing factor to tail shortening, as the number of apoptotic cells at 18–19 som and 22–23 somites in N-cad p79emcf ; vangl2 MO embryos was roughly equivalent to that observed in controls (WT, WT injected with vangl2 and N-cad p79emcf ; Table 1). These findings suggest that impaired cell movements cause the posterior defects. Moreover, the fact that the phenotypes observed in N-cad m117 mutants and N-cad p79emcf ; vangl2 MO embryos are qualitatively different suggests that N-cad and vangl2 may not interact to regulate cell-cell adhesion.
N-cadherin and vang-like 2are likely to function in parallel pathways
The genetic interaction between N-cad and vangl2 is a priori consistent with these genes functioning either in linear or parallel pathways. To distinguish between these two possibilities, the ability of the vangl2 MO to worsen the N-cad m117 null phenotype was tested. If these genes function in a linear pathway exclusively, then vangl2 MO should have little or no effect on N-cad m117 mutants. vangl2 MO was injected at the same low concentration used in combination with the N-cad p79emcf allele (0.8 ng). 100% (n = 8 out of 8 embryos) of the N-cad m117 mutants injected with this dosage of vangl2 MO had enhanced tail defects at 30 hpf (Figure 8F) relative to uninjected N-cad m117 mutants (Figure 8E), suggesting that N-cad and vangl2 function synergistically and in parallel pathways to regulate CE.
It has recently been reported that Wnt11 controls the general cohesiveness of mesendodermal progenitors, by regulating E-cad endocytosis , indicating that, in some cases, classical cadherins function downstream of the non-canonical Wnt pathway. To further address whether the relation between vangl2 and N-cad is strictly non-linear, N-cad levels and localization were analyzed in two planar cell polarity mutants, tri and kny. Cross sections through the tail region of 18 som embryos did not reveal any overt differences in N-cad labeling between WT and mutants (Additional file 4). These findings are consistent with N-cad and the planar cell polarity pathway functioning synergistically and in parallel to regulate posterior morphogenesis.
Role of N-cadherinin posterior mesoderm and neural tube morphogenesis
Several pieces of evidence point to a primary role of N-cad and possibly other cadherins in regulating posterior mesoderm morphogenesis. Firstly, the length of the A-P axis in N-cad m117 mutants appears normal until 16 som, which coincides with the onset of tailbud eversion . In contrast, embryos in which members of the non-canonical Wnt signaling pathway such as vangl2 and kny have been disrupted, show a dramatic shortening of the axis beginning at mid-somitogenesis stages [13, 15, 5, 11]. The early onset of A-P defects in these mutants reflects the role of vangl2 and kny in mesodermal morphogenesis during gastrulation. Secondly, molecular markers only show a slight impairment in paraxial mesodermal convergence during gastrulation in N-cad m117 mutants. Thirdly, in vivo labeling of targeted cell populations in the tailbud revealed that while the anteriorward movement of ventral-derived cells (the precursors of paraxial mesoderm) appears normal, dorsal-derived cells (notochord precursors) are unable to extend posteriorly. This finding is surprising given the widespread expression of N-cad throughout the tailbud region. However, several lines of evidence indicate that cadherins are essential for cell intercalation behaviors underlying notochord extension (reviewed in ), but may not be required for directed migration of cells, a process that is thought to mostly rely on cell-extracellular matrix interactions mediated by integrins. The extension of the notochord along the yolk ball via cell intercalation is thought to be a contributing force for posterior tail extension  and may therefore explain, at least in part, the tail defects observed in N-cad m117 mutants.
The data presented in this paper has focused on N-cad's role in the mesoderm, as CE in this germ layer is thought to account for most of the elongation of the A-P axis. However, N-cad is also required for the morphogenesis of the neural tube (NT). Fate mapping studies have shown that the posterior spinal cord derives from dorsal cells in the epiblast of the anterior tailbud. These precursor cells are thought to become distributed along the entire length of the developing tail by CE-like movements and cell proliferation . The NT in posterior regions of N-cad m117 mutants and WT embryos injected with high levels of N-cad MO is not only shortened but also has a characteristic "T" shape, indicative of impaired NT closure [48, 49]. Thus, in addition to shaping the mesoderm, N-cad is also required for proper posterior NT morphogenesis, consistent with its broad expression throughout the tailbud.
Why is the N-cad m117 phenotype stronger than that of presumed null mutants?
Several N-cad null alleles have been previously described in the zebrafish . Embryos homozygous for these null alleles exhibit mild posterior defects. We have further characterized another potential null allele, N-cad p79emcf , that carries a point mutation in the EC5 domain . Immunofluorescence studies revealed that the N-cad protein is mislocalized away from the plasma membrane in these mutants. This may be potentially explained by a misfolding of the protein, such that it is not able to properly insert into the membrane. Accordingly, the phenotype of N-cad p79emcf homozygous mutants is very similar to that of confirmed N-cad null mutants. It is possible that the tail curvature defect observed in these embryos may be caused by impaired neurulation but normal mesodermal CE. With stronger N-cad alleles (N-cad m117 ), impaired mesodermal morphogenesis may alleviate this curvature defect while causing a shortened axis.
N-cad m117 mutants have a severe posterior defect that is not observed in N-cad null mutants. We propose that this allele is semi-dominant, as mild neural convergence defects were observed in heterozygous siblings. Since N-cad is known to interact heterophilically with other members of the classical cadherin family [66, 67], N-cad m117 may affect the adhesive activity of these proteins. Alternatively, N-cad m117 may impair the function of other N-cad paralogues, recently identified following sequencing of the zebrafish genome.
N-cadherinmay function as an adhesion or a signaling molecule
At a molecular level, how could N-cad function to promote cellular rearrangement? As a member of the classical cadherin subfamily, N-cad is known to mediate cell-cell adhesion [43, 48, 74, 75]. Morphogenetic movements such as those that occur during CE require a dynamic regulation of adhesion, as contacts between cells have to be constantly broken and re-established in order for cells to exchange neighbors and locally reposition themselves . In this context, the adhesive activity of C-cad is known to play a critical role during Xenopus gastrulation [37, 38]. It was recently shown that C-cad's adhesive activity is regulated by papc, functioning downstream of activin and independently from non-canonical Wnt signaling . This raises the intriguing possibility that a similar relationship exists between N-cad and Papc in the paraxial mesoderm and may be required for proper mesodermal morphogenesis. There is also strong evidence that classical cadherins can function as signaling molecules (reviewed in ). For example, axonal outgrowth in retinal ganglion cells is dependent on the interaction between N-cad and the FGF receptor (FGFR) [77–80]. The invasive activity of N-cad during cancer metastasis also results from a functional interaction with FGFR at the cell surface [81, 82]. Other signaling molecules through which cadherins can function to stimulate cell motility is the Rho family of small GTPases, the steady state activation of which increases in the presence of N-cad  and Retinal cadherin (R-cad)-mediated cell-cell contact . Activation of these GTPases correlates with increased cell motility [84–86].
Thus, experimental data strongly supports a role for N-cad in both adhesion and signaling. Further elucidation of the role of N-cad and other cadherins in promoting posterior morphogenesis will require assays to distinguish between these two functions.
Interaction between N-cadherin and the non-canonical Wnt signaling pathway
A genetic interaction between N-cad and vangl2 (a non-canonical Wnt signaling component) was demonstrated by slightly lowering the levels of vangl2 in embryos carrying the N-cad p79emcf allele. N-cad p79emcf ; vangl2 MO embryos exhibited a dramatically shortened tail, similar to that observed in N-cad m117 mutants. N-cad and vangl2 were interpreted to function synergistically and in parallel pathways, as lowering the levels of vangl2 in N-cad m117 mutants worsened the tail defect in these embryos even further and N-cad levels and localization were not perturbed in tri and kny mutants. These findings suggest that there are other pathways regulating the distribution of N-cad protein in cells undergoing movement. Moreover, the similar yet distinct phenotype of N-cad m117 mutants and N-cad p79emcf ; vangl2 MO embryos suggests that N-cad and Vangl2 may not interact to regulate intercellular adhesion but rather some other cell behavior.
There is increasing evidence that regulation of cell adhesion plays a central role during gastrulation (reviewed in ). Data presented in this paper complements these findings by demonstrating that the role of cadherins extends beyond gastrulation, to orchestrate posterior body formation.
Formation of the vertebrate tail involves a continuation of gastrulation-type movements that shape the head and trunk region and posterior-specific behaviors . While the cadherin superfamily has a well established role in mediating mesodermal morphogenesis during gastrulation, less is known about the function of cadherins in lengthening the posterior body region. We provide here several pieces of evidence that N-cad and other members of the classical cadherin subfamily are essential for eversion of the tailbud and to a lesser extent for shaping the mesoderm during gastrulation. Consistent with these observations, zebrafish N-cad is expressed in axial and paraxial mesoderm during gastrulation and throughout the tailbud. Moreover, N-cad appears to interact synergistically with vangl2, a member of the non-canonical Wnt signaling pathway to mediate tailbud eversion. Together these findings further highlight the central role of members of the cadherin superfamily in the cell behaviors that shape the vertebrate embryo.
Zebrafish maintenance, embryo generation, staging
Zebrafish embryos (Danio rerio) were collected from mated adult fish within 30 minutes post fertilization and maintained at 28.5°C until the desired developmental stage was reached . Offspring from: wild type, AB and TL; N-cad heterozygotes, N-cad p79emcf , N-cad m117 and N-cad r2.10 ; tri m747 heterozygotes and kny hi1688 heterozygotes were used.
Antisense N-cad morpholino oligonucleotides (MO) were generated against the translation initiation start site of zebrafish N-cadherin (Gene Tools; ):
N-cad MO: 5'TCTGTATAAAGAAACCGATAGAGTT-3'
This MO targets N-cad efficiently, as immuno-labeling using an antibody against zebrafish N-cad did not detect any signal in embryos injected with moderate concentrations of MO (Additional file 5) and a genome-wide database search yielded no significant hits other than N-cad.
Antisense vangl2 MO (also called stbm MO) were generated against the translation initiation start site of zebrafish vangl2 as previously described (Gene Tools; ):
vangl2 MO: 5'GTACTGCGACTCGTTATCCATGTC-3'
MO stock solution (10 mg/ml) was diluted to desired concentrations in Danio water. Embryos were injected with N-cad MO (0.8 ng) or vangl2 MO (0.4 ng, 0.48 ng, 0.56 ng, 0.64 ng, 0.72 ng, 0.8 ng) at the 1- to 2- cell stage using a nitrogen-pressured microinjector (Harvard Apparatus). To ensure accuracy of results, MO injections were carried out using the same microinjection needle per injection series. In addition, injections for each series of experiments were done on the same day and comparisons were done between siblings whenever possible. At the appropriate stage, embryos were either imaged live or fixed for in situ hybridization or/and immunocytochemistry.
In situ hybridization was performed as described in Thisse (1993; ). To synthesize antisense digoxigenin RNA probes, N-cad  was linearized with HindIII and transcribed with T7 polymerase, myoD  was linearized with BamHI and transcribed with T7 polymerase, no tail  was linearized with XbaI and transcribed with T7, dlx3  was linearized with SalI and transcribed with T7 polymerase, papc  was linearized with ApaI and transcribed with T3 polymerase.
Antibody and other labeling
Immunocytochemistry was carried out as described in Westerfield (2000; ). Primary antibodies: rabbit α-Sox3C , working concentration of 1:2000 dilution; rabbit α-N-cad , working concentration of 1:100 dilution; and α- β-catenin (BD Transduction Laboratories), working concentration of 1:200. Detection of primary antibodies was carried out using fluorescein-conjugated secondary antibodies: Cy3 α-rabbit (Biomedia), working concentration of 1:200 dilution; Alexa 488 α-rabbit, working concentration of 1:200 dilution; or/and Alexa 488 α-mouse, working concentration of 1:200 dilution. DAPI was used according to the manufacturer's instructions (Molecular Probes).
In situdetection of apoptosis in whole mounts
Detection of apoptotic cells was carried out using the In situ Cell Death Detection Kit, Fluorescein (Roche). Labeling on whole mount embryos was carried out according to Cole and Ross (2001; ) excluding the color reaction. Optical sections of the tail region were taken using a confocal microscope and cells were counted in three different subregions (refer to Table 1 and Figure 6).
Tail length measurements
Tail length measurements were made on Nomarski images of live embryos, using the OpenLab software. Measurements were taken from the beginning of the yolk extension to the tip of the tail.
Cell labeling/movement analysis
Injection and uncaging of fluorescein in cell groups for cell movement analysis was carried out as described by Sepich et al., 2000 , with slight modifications. Anionic dextran DMNB caged fluorescein, 10,000 MW (Molecular Probes, D-3310) was dissolved in 120 mM KCl, 20 mM Hepes pH 7.5 to a final concentration of 1% . Before use, the dye was centrifuged 3 to 5 minutes in a microfuge. A small quantity (1 nl) was injected into the yolk of 1- to 2- cell stage embryos using a nitrogen-pressured microinjector (Harvard Apparatus). Embryos at the 3–4 som stage were dechorionated, anesthetized with tricaine (Sigma), and positioned dorsal side up on a depression slide filled with 3% methycellulose (Sigma). Uncaging was performed with the 440 nm beam from a Photonic Instruments MicroPoint laser system mounted on a Zeiss Axiophot microscope and focused through a 20× objective. Embryos were imaged at 18 hpf using a Zeiss Axioskop 2 microscope.
Imaging and sectioning
Imaging of live specimens – At the desired developmental stage embryos were dechorionated, placed in imaging solution containing 3% methylcellulose (Sigma) with MESAB (4 mg/ml ethyl-m-aminobenzoate methanesulphonate, 1% Na2HPO4) (1:100 dilution) and imaged using a Zeiss Axioskop 2 compound microscope.
Imaging of fixed preparations – Whole-mount embryos or sections labeled using in situ hybridization were mounted in 100% glycerol (whole mounts) or PBS (sections) and imaged using a Zeiss Axioskop 2 compound microscope. Fluorescently labeled specimens, (whole mounts or sections) were placed in Aqua Poly/Mount (Polysciences, Inc.) and imaged using a Zeiss LSM 510-Meta confocal microscope.
Sectioning – Embryos were sectioned using a vibratome (Vibratome, Inc.), as described in Hong and Brewster, 2006 .
We give special thanks to: Qin Liu and to Michael Klymkowsky for the α-N-Cadherin and α-Sox3C antibodies, respectively; to the Solnica-Krezel Laboratory for the tri m747 stock; to the Koester Laboratory for the N-cad r2.10 stock; to the Michael Granato for the N-cad p79emcf stock; to Robyn Goodman, Marnie Halpern and Shannon Fisher for their technical assistance with the uncaging experiments and generous gift of the caged fluorescein; to Mark Van Doren, Linda Harrington, Kavita Chalasani, Pradeepa Jayachandran and Larry Shapiro for discussions or critique of the manuscript and to Dwane Clarke and Pradeepa Jayachandran for technical assistance with some of the experiments.
The WT, N-cad m117 , and kny hi1688 stocks were obtained from the Zebrafish International Resource Center.
This work was funded by a National Science PECASE Award, #0448432, awarded to Rachel Brewster, an NIGMS initiative for minority student development grant, #R25-GM55036, that supported Michael Harrington and a Meyerhoff Undergraduate Scholarship awarded to Oluwafoyinsayo Fasanmi.
- Keller R, Davidson L, Edlund A, Elul T, Ezin M, Shook D, Skoglund P: Mechanisms of convergence and extension by cell intercalation. Philos Trans R Soc Lond B Biol Sci. 2000, 355: 897-922. 10.1098/rstb.2000.0626.PubMed CentralView ArticlePubMedGoogle Scholar
- Solnica-Krezel L, Cooper MS: Cellular and genetic mechanisms of convergence and extension. Results Probl Cell Differ. 2002, 40: 136-165.View ArticlePubMedGoogle Scholar
- Wallingford JB, Fraser SE, Harland RM: Convergent extension: the molecular control of polarized cell movement during embryonic development. Dev Cell. 2002, 2: 695-706. 10.1016/S1534-5807(02)00197-1.View ArticlePubMedGoogle Scholar
- Trinkaus JP, Trinkaus M, Fink RD: On the convergent cell movements of gastrulation in Fundulus. J Exp Zool. 1992, 261: 40-61. 10.1002/jez.1402610107.View ArticlePubMedGoogle Scholar
- Jessen JR, Topczewski J, Bingham S, Sepich DS, Marlow F, Chandrasekhar A, Solnica-Krezel L: Zebrafish trilobite identifies new roles for Strabismus in gastrulation and neuronal movements. Nat Cell Biol. 2002, 4: 610-615.PubMed CentralPubMedGoogle Scholar
- Glickman NS, Kimmel CB, Jones MA, Adams RJ: Shaping the zebrafish notochord. Development. 2003, 130: 873-887. 10.1242/dev.00314.View ArticlePubMedGoogle Scholar
- Henry CA, Crawford BD, Yan YL, Postlethwait J, Cooper MS, Hille MB: Roles for zebrafish focal adhesion kinase in notochord and somite morphogenesis. Dev Biol. 2001, 240: 474-487. 10.1006/dbio.2001.0467.View ArticlePubMedGoogle Scholar
- Kanki JP, Ho RK: The development of the posterior body in zebrafish. Development. 1997, 124: 881-893.PubMedGoogle Scholar
- Schoenwolf GC: Histological and ultrastructural studies of secondary neurulation in mouse embryos. Am J Anat. 1984, 169: 361-376. 10.1002/aja.1001690402.View ArticlePubMedGoogle Scholar
- Griffith CM: The vertebrate tailbud: Three germ layers from one tissue. Anat Embryol. 1992, 185: 101-113. 10.1007/BF00185911.View ArticlePubMedGoogle Scholar
- Marlow F, Gonzalez EM, Yin C, Rojo C, Solnica-Krezel L: No tail co-operates with non-canonical Wnt signaling to regulate posterior body morphogenesis in zebrafish. Development. 2004, 131: 203-216. 10.1242/dev.00915.View ArticlePubMedGoogle Scholar
- Winklbauer R, Medina A, Swain RK, Steinbeisser H: Frizzled-7 signalling controls tissue separation during Xenopus gastrulation. Nature. 2001, 413: 856-860. 10.1038/35101621.View ArticlePubMedGoogle Scholar
- Topczewski J, Sepich DS, Myers DC, Walker C, Amores A, Lele Z, Hammerschmidt M, Postlethwait J, Solnica-Krezel L: The zebrafish glypican knypek controls cell polarity during gastrulation movements of convergent extension. Dev Cell. 2001, 1: 251-264. 10.1016/S1534-5807(01)00005-3.View ArticlePubMedGoogle Scholar
- Wolff T, Rubin GM: Strabismus, a novel gene that regulates tissue polarity and cell fate decisions in Drosophila. Development. 1998, 125: 1149-1159.PubMedGoogle Scholar
- Park M, Moon RT: The planar cell-polarity gene stbm regulates cell behaviour and cell fate in vertebrate embryos. Nat Cell Biol. 2002, 4: 20-25. 10.1038/ncb716.View ArticlePubMedGoogle Scholar
- Gubb D, Green C, Huen D, Coulson D, Johnson G, Tree D, Collier S, Roote J: The balance between isoforms of the prickle LIM domain protein is critical for planar polarity in Drosophila imaginal discs. Genes Dev. 1999, 13: 2315-2327. 10.1101/gad.13.17.2315.PubMed CentralView ArticlePubMedGoogle Scholar
- Wallingford JB, Goto T, Keller R, Harland RM: Cloning and expression of Xenopus Prickle, an orthologue of a Drosophila planar cell polarity gene. Mech Dev. 2002, 116: 183-186. 10.1016/S0925-4773(02)00133-8.View ArticlePubMedGoogle Scholar
- Carreira-Barbosa F, Concha ML, Takeuchi M, Ueno N, Wilson SW, Tada M: Prickle 1 regulates cell movements during gastrulation and neuronal migration in zebrafish. Development. 2003, 130: 4037-4046. 10.1242/dev.00567.View ArticlePubMedGoogle Scholar
- Winter CG, Wang B, Ballew A, Royou A, Karess R, Axelrod JD, Luo L: Drosophila Rho-associated kinase (Drok) links Frizzled-mediated planar cell polarity signaling to the actin cytoskeleton. Cell. 2001, 105: 81-91. 10.1016/S0092-8674(01)00298-7.View ArticlePubMedGoogle Scholar
- Choi SC, Han JK: Xenopus Cdc42 regulates convergent extension movements during gastrulation through Wnt/Ca2+ signaling pathway. Dev Biol. 2002, 244: 342-357. 10.1006/dbio.2002.0602.View ArticlePubMedGoogle Scholar
- Habas R, Dawid IB, He X: Coactivation of Rac and Rho by Wnt/Frizzled signaling is required for vertebrate gastrulation. Genes Dev. 2003, 17: 295-309. 10.1101/gad.1022203.PubMed CentralView ArticlePubMedGoogle Scholar
- Penzo-Mendez A, Umbhauer M, Djiane A, Boucaut JC, Riou JF: Activation of Gbetagamma signaling downstream of Wnt-11/Xfz7 regulates Cdc42 activity during Xenopus gastrulation. Dev Biol. 2003, 257: 302-314. 10.1016/S0012-1606(03)00067-8.View ArticlePubMedGoogle Scholar
- Boutros M, Paricio N, Strutt DI, Mlodzik M: Dishevelled activates JNK and discriminates between JNK pathways in planar polarity and wingless signaling. Cell. 1998, 94: 109-118. 10.1016/S0092-8674(00)81226-X.View ArticlePubMedGoogle Scholar
- Yamanaka H, Moriguchi T, Masuyama N, Kusakabe M, Hanafusa H, Takada R, Takada S, Nishida E: JNK functions in the non-canonical Wnt pathway to regulate convergent extension movements in vertebrates. EMBO Rep. 2002, 3: 69-75. 10.1093/embo-reports/kvf008.PubMed CentralView ArticlePubMedGoogle Scholar
- Myers DC, Sepich DS, Solnica-Krezel L: Convergence and extension in vertebrate gastrulae: cell movements according to or in search of identity?. Trends Genet. 2002, 18: 447-455. 10.1016/S0168-9525(02)02725-7.View ArticlePubMedGoogle Scholar
- Gumbiner BM: Regulation of cadherin-mediated adhesion in morphogenesis. Nat Rev Mol Cell Biol. 2005, 6: 622-634. 10.1038/nrm1699.View ArticlePubMedGoogle Scholar
- Solnica-Krezel L: Gastrulation in zebrafish -- all just about adhesion?. Curr Opin Genet Dev. 2006, 16: 433-441. 10.1016/j.gde.2006.06.009.View ArticlePubMedGoogle Scholar
- Formstone CJ, Mason I: Combinatorial activity of Flamingo proteins directs convergence and extension within the early zebrafish embryo via the planar cell polarity pathway. Dev Biol. 2005, 282: 320-335. 10.1016/j.ydbio.2005.03.026.View ArticlePubMedGoogle Scholar
- Chae J, Kim MJ, Goo JH, Collier S, Gubb D, Charlton J, Adler PN, Park WJ: The Drosophila tissue polarity gene starry night encodes a member of the protocadherin family. Development. 1999, 126: 5421-5429.PubMedGoogle Scholar
- Usui T, Shima Y, Shimada Y, Hirano S, Burgess RW, Schwarz TL, Takeichi M, Uemura T: Flamingo, a seven-pass transmembrane cadherin, regulates planar cell polarity under the control of Frizzled. Cell. 1999, 98: 585-595. 10.1016/S0092-8674(00)80046-X.View ArticlePubMedGoogle Scholar
- Witzel S, Zimyanin V, Carreira-Barbosa F, Tada M, Heisenberg CP: Wnt11 controls cell contact persistence by local accumulation of Frizzled 7 at the plasma membrane. J Cell Biol. 2006, 175: 791-802. 10.1083/jcb.200606017.PubMed CentralView ArticlePubMedGoogle Scholar
- Kim SH, Yamamoto A, Bouwmeester T, Agius E, Robertis EM: The role of paraxial protocadherin in selective adhesion and cell movements of the mesoderm during Xenopus gastrulation. Development. 1998, 125: 4681-4690.PubMedGoogle Scholar
- Yamamoto A, Amacher SL, Kim SH, Geissert D, Kimmel CB, De Robertis EM: Zebrafish paraxial protocadherin is a downstream target of spadetail involved in morphogenesis of gastrula mesoderm. Development. 1998, 125: 3389-3397.PubMed CentralPubMedGoogle Scholar
- Unterseher F, Hefele JA, Giehl K, De Robertis EM, Wedlich D, Schambony A: Paraxial protocadherin coordinates cell polarity during convergent extension via Rho A and JNK. Embo J. 2004, 23: 3259-3269. 10.1038/sj.emboj.7600332.PubMed CentralView ArticlePubMedGoogle Scholar
- Medina A, Swain RK, Kuerner KM, Steinbeisser H: Xenopus paraxial protocadherin has signaling functions and is involved in tissue separation. Embo J. 2004, 23: 3249-3258. 10.1038/sj.emboj.7600329.PubMed CentralView ArticlePubMedGoogle Scholar
- Lee CH, Gumbiner BM: Disruption of gastrulation movements in Xenopus by a dominant-negative mutant for C-cadherin. Dev Biol. 1995, 171: 363-373. 10.1006/dbio.1995.1288.View ArticlePubMedGoogle Scholar
- Brieher WM, Gumbiner BM: Regulation of C-cadherin function during activin induced morphogenesis of Xenopus animal caps. J Cell Biol. 1994, 126: 519-527. 10.1083/jcb.126.2.519.View ArticlePubMedGoogle Scholar
- Zhong Y, Brieher WM, Gumbiner BM: Analysis of C-cadherin regulation during tissue morphogenesis with an activating antibody. J Cell Biol. 1999, 144: 351-359. 10.1083/jcb.144.2.351.PubMed CentralView ArticlePubMedGoogle Scholar
- Chen X, Gumbiner BM: Paraxial protocadherin mediates cell sorting and tissue morphogenesis by regulating C-cadherin adhesion activity. J Cell Biol. 2006, 174: 301-313. 10.1083/jcb.200602062.PubMed CentralView ArticlePubMedGoogle Scholar
- Montero JA, Carvalho L, Wilsch-Brauninger M, Kilian B, Mustafa C, Heisenberg CP: Shield formation at the onset of zebrafish gastrulation. Development. 2005, 132: 1187-1198. 10.1242/dev.01667.View ArticlePubMedGoogle Scholar
- Ulrich F, Krieg M, Schotz EM, Link V, Castanon I, Schnabel V, Taubenberger A, Mueller D, Puech PH, Heisenberg CP: Wnt11 functions in gastrulation by controlling cell cohesion through Rab5c and E-cadherin. Dev Cell. 2005, 9: 555-564. 10.1016/j.devcel.2005.08.011.View ArticlePubMedGoogle Scholar
- von der Hardt S, Bakkers J, Inbal A, Carvalho L, Solnica-Krezel L, Heisenberg CP, Hammerschmidt M: The Bmp gradient of the zebrafish gastrula guides migrating lateral cells by regulating cell-cell adhesion. Curr Biol. 2007, 17: 475-487. 10.1016/j.cub.2007.02.013.View ArticlePubMedGoogle Scholar
- Hatta K, Takeichi M: Expression of N-cadherin adhesion molecules associated with early morphogenetic events in chick development. Nature. 1986, 320: 447-449. 10.1038/320447a0.View ArticlePubMedGoogle Scholar
- Radice GL, Rayburn H, Matsunami H, Knudsen KA, Takeichi M, Hynes RO: Developmental defects in mouse embryos lacking N-cadherin. Dev Biol. 1997, 181: 64-78. 10.1006/dbio.1996.8443.View ArticlePubMedGoogle Scholar
- Bronner-Fraser M, Wolf JJ, Murray BA: Effects of antibodies against N-cadherin and N-CAM on the cranial neural crest and neural tube. Dev Biol. 1992, 153: 291-301. 10.1016/0012-1606(92)90114-V.View ArticlePubMedGoogle Scholar
- Nakagawa S, Takeichi M: N-cadherin is crucial for heart formation in the chick embryo. Dev Growth Differ. 1997, 39: 451-455. 10.1046/j.1440-169X.1997.t01-3-00006.x.View ArticlePubMedGoogle Scholar
- Ganzler-Odenthal SI, Redies C: Blocking N-cadherin function disrupts the epithelial structure of differentiating neural tissue in the embryonic chicken brain. J Neurosci. 1998, 18: 5415-5425.PubMedGoogle Scholar
- Lele Z, Folchert A, Concha M, Rauch GJ, Geisler R, Rosa F, Wilson SW, Hammerschmidt M, Bally-Cuif L: parachute/n-cadherin is required for morphogenesis and maintained integrity of the zebrafish neural tube. Development. 2002, 129: 3281-3294.PubMedGoogle Scholar
- Hong E, Brewster R: N-cadherin is required for the polarized cell behaviors that drive neurulation in the zebrafish. Development. 2006, 133: 3895-3905. 10.1242/dev.02560.View ArticlePubMedGoogle Scholar
- Oberlender SA, Tuan RS: Expression and functional involvement of N-cadherin in embryonic limb chondrogenesis. Development. 1994, 120: 177-187.PubMedGoogle Scholar
- Linask KK, Ludwig C, Han MD, Liu X, Radice GL, Knudsen KA: N-cadherin/catenin-mediated morphoregulation of somite formation. Dev Biol. 1998, 202: 85-102. 10.1006/dbio.1998.9025.View ArticlePubMedGoogle Scholar
- Cinnamon Y: Differential effects of N-cadherin-mediated adhesion on the development of myotomal waves. Development. 2006, 133: 1101-1112. 10.1242/dev.02291.View ArticlePubMedGoogle Scholar
- Linask KK, Knudsen KA, Gui YH: N-cadherin-catenin interaction: necessary component of cardiac cell compartmentalization during early vertebrate heart development. Dev Biol. 1997, 185: 148-164. 10.1006/dbio.1997.8570.View ArticlePubMedGoogle Scholar
- Jiang YJ, Brand M, Heisenberg CP, Beuchle D, Furutani-Seiki M, Kelsh RN, Warga RM, Granato M, Haffter P, Hammerschmidt M, Kane DA, Mullins MC, Odenthal J, van Eeden FJ, Nusslein-Volhard C: Mutations affecting neurogenesis and brain morphology in the zebrafish, Danio rerio. Development. 1996, 123: 205-216.PubMedGoogle Scholar
- Driever W, Solnica-Krezel L, Schier AF, Neuhauss SC, Malicki J, Stemple DL, Stainier DY, Zwartkruis F, Abdelilah S, Rangini Z, Belak J, Boggs C: A genetic screen for mutations affecting embryogenesis in zebrafish. Development. 1996, 123: 37-46.PubMedGoogle Scholar
- Amsterdam A, Nissen RM, Sun Z, Swindell EC, Farrington S, Hopkins N: Identification of 315 genes essential for early zebrafish development. Proc Natl Acad Sci U S A. 2004, 101: 12792-12797. 10.1073/pnas.0403929101.PubMed CentralView ArticlePubMedGoogle Scholar
- Wiellette E, Grinblat Y, Austen M, Hirsinger E, Amsterdam A, Walker C, Westerfield M, Sive H: Combined haploid and insertional mutation screen in the zebrafish. Genesis. 2004, 40: 231-240. 10.1002/gene.20090.View ArticlePubMedGoogle Scholar
- Birely J, Schneider VA, Santana E, Dosch R, Wagner DS, Mullins MC, Granato M: Genetic screens for genes controlling motor nerve-muscle development and interactions. Dev Biol. 2005, 280: 162-176. 10.1016/j.ydbio.2005.01.012.View ArticlePubMedGoogle Scholar
- Malicki J, Jo H, Pujic Z: Zebrafish N-cadherin, encoded by the glass onion locus, plays an essential role in retinal patterning. Dev Biol. 2003, 259: 95-108. 10.1016/S0012-1606(03)00181-7.View ArticlePubMedGoogle Scholar
- Tamura K, Shan WS, Hendrickson WA, Colman DR, Shapiro L: Structure-function analysis of cell adhesion by neural (N-) cadherin. Neuron. 1998, 20: 1153-1163. 10.1016/S0896-6273(00)80496-1.View ArticlePubMedGoogle Scholar
- Pertz O, Bozic D, Koch AW, Fauser C, Brancaccio A, Engel J: A new crystal structure, Ca2+ dependence and mutational analysis reveal molecular details of E-cadherin homoassociation. Embo J. 1999, 18: 1738-1747. 10.1093/emboj/18.7.1738.PubMed CentralView ArticlePubMedGoogle Scholar
- Kitagawa M, Natori M, Murase S, Hirano S, Taketani S, Suzuki ST: Mutation analysis of cadherin-4 reveals amino acid residues of EC1 important for the structure and function. Biochem Biophys Res Commun. 2000, 271: 358-363. 10.1006/bbrc.2000.2636.View ArticlePubMedGoogle Scholar
- Haussinger D, Ahrens T, Sass HJ, Pertz O, Engel J, Grzesiek S: Calcium-dependent homoassociation of E-cadherin by NMR spectroscopy: changes in mobility, conformation and mapping of contact regions. J Mol Biol. 2002, 324: 823-839. 10.1016/S0022-2836(02)01137-3.View ArticlePubMedGoogle Scholar
- Boggon TJ, Murray J, Chappuis-Flament S, Wong E, Gumbiner BM, Shapiro L: C-cadherin ectodomain structure and implications for cell adhesion mechanisms. Science. 2002, 296: 1308-1313. 10.1126/science.1071559.View ArticlePubMedGoogle Scholar
- Pujic Z, Malicki J: Mutation of the zebrafish glass onion locus causes early cell-nonautonomous loss of neuroepithelial integrity followed by severe neuronal patterning defects in the retina. Dev Biol. 2001, 234: 454-469. 10.1006/dbio.2001.0251.View ArticlePubMedGoogle Scholar
- Niessen CM, Gumbiner BM: Cadherin-mediated cell sorting not determined by binding or adhesion specificity. J Cell Biol. 2002, 156: 389-399. 10.1083/jcb.200108040.PubMed CentralView ArticlePubMedGoogle Scholar
- Prakasam AK, Maruthamuthu V, Leckband DE: Similarities between heterophilic and homophilic cadherin adhesion. Proc Natl Acad Sci U S A. 2006, 103: 15434-15439. 10.1073/pnas.0606701103.PubMed CentralView ArticlePubMedGoogle Scholar
- Cortes F, Daggett D, Bryson-Richardson RJ, Neyt C, Maule J, Gautier P, Hollway GE, Keenan D, Currie PD: Cadherin-mediated differential cell adhesion controls slow muscle cell migration in the developing zebrafish myotome. Dev Cell. 2003, 5: 865-876. 10.1016/S1534-5807(03)00362-9.View ArticlePubMedGoogle Scholar
- Murakami T, Hijikata T, Matsukawa M, Ishikawa H, Yorifuji H: Zebrafish protocadherin 10 is involved in paraxial mesoderm development and somitogenesis. Dev Dyn. 2006, 235: 506-514. 10.1002/dvdy.20622.View ArticlePubMedGoogle Scholar
- Shimizu T, Yabe T, Muraoka O, Yonemura S, Aramaki S, Hatta K, Bae YK, Nojima H, Hibi M: E-cadherin is required for gastrulation cell movements in zebrafish. Mech Dev. 2005, 122: 747-763. 10.1016/j.mod.2005.03.008.View ArticlePubMedGoogle Scholar
- Kane DA, McFarland KN, Warga RM: Mutations in half baked/E-cadherin block cell behaviors that are necessary for teleost epiboly. Development. 2005, 132: 1105-1116. 10.1242/dev.01668.View ArticlePubMedGoogle Scholar
- Sausedo RA, Schoenwolf GC: Cell behaviors underlying notochord formation and extension in avian embryos: quantitative and immunocytochemical studies. Anat Rec. 1993, 237: 58-70. 10.1002/ar.1092370107.View ArticlePubMedGoogle Scholar
- Sausedo RA, Schoenwolf GC: Quantitative analyses of cell behaviors underlying notochord formation and extension in mouse embryos. Anat Rec. 1994, 239: 103-112. 10.1002/ar.1092390112.View ArticlePubMedGoogle Scholar
- Takeichi M, Hatta K, Nose A, Nagafuchi A: Identification of a gene family of cadherin cell adhesion molecules. Cell Differ Dev. 1988, 25 Suppl: 91-94. 10.1016/0922-3371(88)90104-9.View ArticlePubMedGoogle Scholar
- Takeichi M: Cadherins: a molecular family important in selective cell-cell adhesion. Annu Rev Biochem. 1990, 59: 237-252. 10.1146/annurev.bi.59.070190.001321.View ArticlePubMedGoogle Scholar
- Derycke LD, Bracke ME: N-cadherin in the spotlight of cell-cell adhesion, differentiation, embryogenesis, invasion and signalling. Int J Dev Biol. 2004, 48: 463-476. 10.1387/ijdb.041793ld.View ArticlePubMedGoogle Scholar
- Williams EJ, Williams G, Howell FV, Skaper SD, Walsh FS, Doherty P: Identification of an N-cadherin motif that can interact with the fibroblast growth factor receptor and is required for axonal growth. J Biol Chem. 2001, 276: 43879-43886. 10.1074/jbc.M105876200.View ArticlePubMedGoogle Scholar
- Lom B, Hopker V, McFarlane S, Bixby JL, Holt CE: Fibroblast growth factor receptor signaling in Xenopus retinal axon extension. J Neurobiol. 1998, 37: 633-641. 10.1002/(SICI)1097-4695(199812)37:4<633::AID-NEU11>3.0.CO;2-L.View ArticlePubMedGoogle Scholar
- Perron JC, Bixby JL: Distinct neurite outgrowth signaling pathways converge on ERK activation. Mol Cell Neurosci. 1999, 13: 362-378. 10.1006/mcne.1999.0753.View ArticlePubMedGoogle Scholar
- Utton MA, Eickholt B, Howell FV, Wallis J, Doherty P: Soluble N-cadherin stimulates fibroblast growth factor receptor dependent neurite outgrowth and N-cadherin and the fibroblast growth factor receptor co-cluster in cells. J Neurochem. 2001, 76: 1421-1430. 10.1046/j.1471-4159.2001.00140.x.View ArticlePubMedGoogle Scholar
- Suyama K, Shapiro I, Guttman M, Hazan RB: A signaling pathway leading to metastasis is controlled by N-cadherin and the FGF receptor. Cancer Cell. 2002, 2: 301-314. 10.1016/S1535-6108(02)00150-2.View ArticlePubMedGoogle Scholar
- Shin EY, Lee BH, Yang JH, Shin KS, Lee GK, Yun HY, Song YJ, Park SC, Kim EG: Up-regulation and co-expression of fibroblast growth factor receptors in human gastric cancer. J Cancer Res Clin Oncol. 2000, 126: 519-528. 10.1007/s004320000128.View ArticlePubMedGoogle Scholar
- Charrasse S, Meriane M, Comunale F, Blangy A, Gauthier-Rouviere C: N-cadherin-dependent cell-cell contact regulates Rho GTPases and beta-catenin localization in mouse C2C12 myoblasts. J Cell Biol. 2002, 158: 953-965. 10.1083/jcb.200202034.PubMed CentralView ArticlePubMedGoogle Scholar
- Johnson E, Theisen CS, Johnson KR, Wheelock MJ: R-cadherin influences cell motility via Rho family GTPases. J Biol Chem. 2004, 279: 31041-31049. 10.1074/jbc.M400024200.View ArticlePubMedGoogle Scholar
- Grosheva I, Shtutman M, Elbaum M, Bershadsky AD: p120 catenin affects cell motility via modulation of activity of Rho-family GTPases: a link between cell-cell contact formation and regulation of cell locomotion. J Cell Sci. 2001, 114: 695-707.PubMedGoogle Scholar
- Yanagisawa M, Anastasiadis PZ: p120 catenin is essential for mesenchymal cadherin-mediated regulation of cell motility and invasiveness. J Cell Biol. 2006, 174: 1087-1096. 10.1083/jcb.200605022.PubMed CentralView ArticlePubMedGoogle Scholar
- Kimmel CB, Ballard WW, Kimmel SR, Ullmann B, Schilling TF: Stages of embryonic development of the zebrafish. Dev Dyn. 1995, 203: 253-310.View ArticlePubMedGoogle Scholar
- Thisse C, Thisse B, Schilling TF, Postlethwait JH: Structure of the zebrafish snail1 gene and its expression in wild-type, spadetail and no tail mutant embryos. Development. 1993, 119: 1203-1215.PubMedGoogle Scholar
- Bitzur S, Kam Z, Geiger B: Structure and distribution of N-cadherin in developing zebrafish embryos: morphogenetic effects of ectopic over-expression. Dev Dyn. 1994, 201: 121-136.View ArticlePubMedGoogle Scholar
- Weinberg ES, Allende ML, Kelly CS, Abdelhamid A, Murakami T, Andermann P, Doerre OG, Grunwald DJ, Riggleman B: Developmental regulation of zebrafish MyoD in wild-type, no tail and spadetail embryos. Development. 1996, 122: 271-280.PubMedGoogle Scholar
- Schulte-Merker S, Hammerschmidt M, Beuchle D, Cho KW, De Robertis EM, Nusslein-Volhard C: Expression of zebrafish goosecoid and no tail gene products in wild-type and mutant no tail embryos. Development. 1994, 120: 843-852.PubMedGoogle Scholar
- Akimenko MA, Ekker M, Wegner J, Lin W, Westerfield M: Combinatorial expression of three zebrafish genes related to distal-less: part of a homeobox gene code for the head. J Neurosci. 1994, 14: 3475-3486.PubMedGoogle Scholar
- Westerfield M: THE ZEBRAFISH BOOK; A guide for the laboratory use of zebrafish (Danio rerio). 2000, Eugene, Oregon Press, 363-Google Scholar
- Zhang C, Basta T, Jensen ED, Klymkowsky MW: The beta-catenin/VegT-regulated early zygotic gene Xnr5 is a direct target of SOX3 regulation. Development. 2003, 130: 5609-5624. 10.1242/dev.00798.View ArticlePubMedGoogle Scholar
- Liu Q, Babb SG, Novince ZM, Doedens AL, Marrs J, Raymond PA: Differential expression of cadherin-2 and cadherin-4 in the developing and adult zebrafish visual system. Vis Neurosci. 2001, 18: 923-933.PubMedGoogle Scholar
- Cole LK, Ross LS: Apoptosis in the developing zebrafish embryo. Dev Biol. 2001, 240: 123-142. 10.1006/dbio.2001.0432.View ArticlePubMedGoogle Scholar
- Sepich DS, Myers DC, Short R, Topczewski J, Marlow F, Solnica-Krezel L: Role of the zebrafish trilobite locus in gastrulation movements of convergence and extension. Genesis. 2000, 27: 159-173. 10.1002/1526-968X(200008)27:4<159::AID-GENE50>3.0.CO;2-T.View ArticlePubMedGoogle Scholar
- Kozlowski DJ, Murakami T, Ho RK, Weinberg ES: Regional cell movement and tissue patterning in the zebrafish embryo revealed by fate mapping with caged fluorescein. Biochem Cell Biol. 1997, 75: 551-562. 10.1139/bcb-75-5-551.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.