To investigate the timing and kinetics of E- to N-cadherin switch during early neural development, we analyzed the expression profile of their proteins and messages by immunofluorescence and in situ hybridization on serial sections of chick embryos as well as on whole embryos, between stages 3 and 19 of Hamburger and Hamilton (HH) (Hamburger and Hamilton, 1951), i.e., between the 12th and the 72nd hour of incubation. In parallel, we analyzed the distribution patterns of their main transcriptional regulators, i.e., Snail-1, Snail-2, Zeb-2 (also known as SIP-1), and Twist-1, acting as repressors of E-cadherin, and Sox-2, known as an activator of N-cadherin transcription (Nieto, 2002; Matsumata et al., 2005; Peinado et al., 2007). Sox-2 was also used as a marker for definitive neural tissues (Uchikawa et al., 2003, 2011; Linker and Stern, 2004).
Timing and Kinetics of E- to N-Cadherin Switch During Early Neural Induction
To determine first whether the switch from E- to N-cadherin during neural development occurs as a consequence of neural induction and is involved in the morphological transformation of neural epithelial cells (Takeichi, 1988; Halbleib and Nelson, 2006; Suzuki and Takeichi, 2008), we analyzed their expression patterns throughout the process of neural induction in the anterior region of the embryo. In the chick, neural induction starts during gastrulation at stage HH3 and becomes apparent at stage HH4 by expression of Sox-2 in the neural plate, in a horse-shoe-shaped area surrounding the Hensen's node (Uchikawa et al., 2003, 2011). At this stage (Fig. 1A, B), immunostaining for E-cadherin was strong on the whole surface of all superficial epithelial cells both in the nascent neural plate and in the non-induced epiblast. No E-cadherin was found in the mesoderm, but, as shown previously (Nakaya et al., 2008; Hardy et al., 2011), it was detectable in the primitive streak, in both ingressing and early migrating mesodermal cells. N-cadherin exhibited a reciprocal pattern to E-cadherin, with a strong expression in the mesoderm and a complete exclusion from the neural plate and the epiblast. In the primitive streak, it was codistributed with E-cadherin in the ingressing mesodermal cells. In situ hybridizations on whole embryos (Fig. 1C) revealed that E-cadherin messages were uniformly expressed in the superficial layer of the whole blastoderm, with a slight decrease in the primitive streak and neural plate. N-cadherin, in contrast, was conspicuous in the primitive streak and mesoderm, but neither in the neural plate nor the non-induced epiblast. Of interest, its expression pattern matched precisely that of Brachyury, a T-box transcription factor involved in mesoderm formation (Kispert et al., 1995). While they were not detected at stage HH3 (not shown, but see Sheng et al., 2003; Uchikawa et al., 2003, 2011), Sox-2 and Zeb-2 became both induced in conjunction at stage HH4 in the neural plate. Snail-2, in contrast, was absent from this region, being essentially confined to the primitive streak (see also Sefton et al., 1998; Acloque et al., 2011). Finally, among the other transcriptional repressors of E-cadherin tested, neither Twist-1 nor Snail-1 was expressed in the neural plate during early neural induction as well as at later stages during neurulation (Figs. 1C, 2B–D; see also Sefton et al., 1998; Tavares et al., 2001).
Figure 1. Expression patterns of E- and N-cadherins and their transcriptional regulators during early neural induction.
A,B: Immunostainings for E-cadherin (E-cad), N-cadherin (N-cad), fibronectin (Fn), and Sox-2 on cross-sections through the neural plate (A) and the primitive streak (B) of a stage-HH4 chick embryo. Cells nuclei were stained with DAPI. ep, epiblast; m, mesoderm; np, neural plate; ps, primitive streak. C: In situ hybridizations for E-cadherin, N-cadherin, Sox-2, Zeb-2, Snail-2, Snail-1, and Brachyury on whole embryos at stage HH4/4+. Hn, Hensen's node; np, neural plate; ps, primitive streak.
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Figure 2. Expression patterns of E- and N-cadherins and their transcriptional regulators during late neural induction.
A: Immunostainings for E-cadherin (E-cad) and N-cadherin (N-cad) on cross-sections through the neural plate of a stage-HH7 chick embryo. Cell nuclei were stained with DAPI. m, mesoderm; n, notochord; np, neural plate. B–D: In situ hybridizations for E-cadherin, N-cadherin, Sox-2, Zeb-2, Snail-2, Snail-1, Twist-1, and Brachyury on whole embryos at stages HH7 (1–2 somites), HH8 (4–6 somites), and HH10 (9–11 somites). hb, hindbrain; Hn, Hensen's node; mb, midbrain; n, notochord; np, neural plate; ps, primitive streak; s, somite; tnt, trunk neural tube.
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By stage HH7 (Fig. 2A, B), during formation of the head fold, while E-cadherin remained conspicuous in the entire neural plate, expression of N-cadherin transcripts was initiated in its anterior-most portion. However, no N-cadherin protein could be detected in neural epithelial cells by immunofluorescence at this stage. At stage HH8 (Fig. 2C), N-cadherin expression became significantly more pronounced in the anterior neural plate up to the first somites, and this was accompanied by gradual accumulation of the protein on the cell surface (see Fig. 4A–C). In contrast, E-cadherin persisted in the anterior neural plate until stage HH10 (Fig. 2C, D), and its complete repression was observed only at stage HH11 (see Figs. 4, 5). Between stages HH4 and HH8, Sox-2 and Zeb-2 expressions were markedly increased in the entire neural plate ahead of the regressing node, with the notable exception of the notochord, while Snail-2 remained limited to the primitive streak as earlier.
Therefore, our results show that, at the onset of neural induction at stage HH3, cells of the prospective neural plate were undistinguishable from the rest of the epiblast in their shape and cadherin content, and expressed none of the known transcriptional regulators of E- and N-cadherins. During progression of neural induction at stage HH4, neural epithelial cells started to acquire their morphological traits, in particular their elongated, spindle-like shape. However, this was not accompanied by an immediate and significant modification in cadherin expression. Changes in cadherin expression occurred only gradually over time, first with increase in N-cadherin messages well after neural induction and formation of the neural plate, then with repression of E-cadherin even later (Fig. 3A). These observations fully support previous genetic studies in the mouse and Zebrafish showing that, in the absence of N-cadherin, onset of formation of the neural epithelium develops normally. The first signs of neural tube malformations are detected relatively late, in the mouse, by the appearance of undulations along the neural tube after closure (Radice et al., 1997) and, in the fish, by the absence of cavitation due to alterations in movements of convergence and intercalation (Hong and Brewster, 2006). Therefore, rather than a prerequisite to the morphological changes, cadherin switch appears merely as a secondary event during neurulation.
Figure 3. A: Schematic representation of the timing of expression of E- and N-cadherins and of Sox-2 and Zeb-2 during neurulation in relation to the morphological changes of neural epithelial cells from cuboidal to elongated and pseudo-stratified. B: Tentative fate map of epiblast cells in the chick blastoderm at stage HH4. Three distinct territories have been defined that differ in cell fates, expression of Sox-2, Zeb-2, Snail-2, and Brachyury transcription factors, and in the ability of cells to execute E- to N-cadherin switch and EMT. The territory fated to give rise to ectodermal cells (yellow) expresses no cadherin-regulating transcription factor, and undergoes neither an E- to N-cadherin switch nor an EMT. In contrast, the territory from which the mesoderm develops (green) expresses both Snail-2 and Brachyury, and undergoes an E- to N-cadherin switch and an EMT followed by extensive cell migration. Finally, the neural plate territory (blue) that expresses both Sox-2 and Zeb-2 executes a gradual switch from E- to N-cadherin but this is not accompanied by EMT.
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Of the transcriptional regulators of cadherin examined here, only Sox-2 and Zeb-2 appeared to exhibit a spatial expression pattern consistent with the cadherin switch in neural epithelial cells, as they were uniformly expressed throughout the entire neural plate and absent from the ectoderm. Previous genetic studies in the mouse showed that embryos lacking Zeb-2 exhibit persistent expression of E-cadherin in the neural tube, thereby revealing that Zeb-2 is necessary for E-cadherin repression in the neural epithelium (Van de Putte et al., 2003). On the other hand, misexpression of Sox-2 in the cranial ectoderm of early chick embryos has been found to cause ectopic N-cadherin expression, indicating that Sox-2 is sufficient to drive expression of N-cadherin in neurectodermal tissues (Matsumata et al., 2005). In addition, in agreement with the fact that both factors have been ascribed critical roles in neural induction in chick and Xenopus (Eisaki et al., 2000; Sheng et al., 2003; Uchikawa et al., 2003, 2011; Takemoto et al., 2006), we also found that Sox-2 and Zeb-2 are induced almost coincidently in neural plate cells at an early step of neural induction. These observations therefore suggest that Sox-2 and Zeb-2, in conjunction, are key players in the spatiotemporal control of the E- to N-cadherin switch in the nervous system. However, rather unexpectedly, we observed that induction of N-cadherin and complete extinction of E-cadherin occurred long after onset of expression of Sox-2 and Zeb-2 in the neural plate (Fig. 3A). While the 6-hr delay between Sox-2 and N-cadherin expression in the neural plate is consistent with the 4-hr delay reported previously for the sensory placodes (Matsumata et al., 2005), repression of cadherin expression more than 12 hr after induction of Zeb-2 suggests that, albeit necessary, this factor may not be sufficient to modify transcription of the E-cadherin gene in neural epithelial cells and that additional co-factors required for complete repression may be induced only at late stages during neural induction. Additionally, it cannot be excluded that post-transcriptional regulatory events may account for the delay between the timing of expression of Sox-2 and Zeb-2 and the changes in surface expression of E- and N-cadherin in neural epithelial cells. Indeed, both E- and N-cadherins may be subjected to post-transcriptional regulations, such as proteolytic cleavage or cytoplasmic trafficking (Halbleib and Nelson, 2006), that may locally modulate their activity. However, the overall consistent expression of E- and N-cadherin messages and proteins throughout neurulation suggests that cadherin activity in neural epithelial cells is primarily regulated at the transcriptional level. Moreover, we found no major difference in immunostainings using antibodies directed against the cytoplasmic tail or the external part of E- and N-cadherin (not shown).
The slow replacement of E-cadherin by N-cadherin in the neural plate contrasts strikingly with the situation found in the primitive streak, where ingressing mesodermal cells express Snail-2 and Brachyury and immediately execute a complete EMT program with breakdown of the basement membrane, loss of cell polarity, and a rapid E- to N-cadherin switch (Sefton et al., 1998; Nakaya et al., 2008; Acloque et al., 2011). Beside Snail-1, Snail-2 is considered as the prototype of EMT inducers. It is often aberrantly overexpressed in metastatic tumor cells and is commonly recruited in tissues normally undergoing rapid and massive EMT (Thiery and Sleeman, 2006; Peinado et al., 2007; Thiery et al., 2009). Moreover, functional studies in the chick have demonstrated that it is sufficient to induce ectopic cell delamination in the epiblast during gastrulation (Acloque et al., 2011). Concerning Brachyury, although it has been regarded essentially as an inducer of mesodermal identity, it is also necessary for mesodermal cell movements during gastrulation as shown by clonal analyses in the mouse embryo (Wilson and Beddington, 1997), and recent findings revealed that it can trigger EMT in epithelial cell lines both directly and indirectly through Snail-1/2 (Fernando et al., 2010). Thus, in the early chick gastrula, Snail-2 in possible conjunction with Brachyury may promote a rapid switch between E- and N-cadherin in primitive streak cells, associated with complete EMT. On the other hand, although Zeb-2 has been found to perform the same roles as Snail-1/2 in epithelial cells maintained in vitro (Thiery and Sleeman, 2006; Peinado et al., 2007; Vandewalle et al., 2009), our data show that, during neural induction, its expression does not correlate with events of EMT and previous functional studies revealed that, rather than inducing disintegration of the epithelial structure, its ectopic expression in the epiblast at the gastrula stage prevents EMT and cell movements through the primitive streak (Sheng et al., 2003). Moreover, Zeb-2 is also known to be a potent repressor of Brachyury (Papin et al., 2002; Sheng et al., 2003). Intriguingly enough, ectopic expression of Snail-2 in the neural tube at a time when it also expresses Zeb-2 is not sufficient to provoke EMT of neural epithelial cells (del Barrio and Nieto, 2002; Cheung et al., 2005; Théveneau et al., 2007). All together, these observations suggest possible antagonistic roles for Snail-2 and Zeb-2 in the control of EMT in neurectodermal cells, irrespective of their function as cadherin regulators. Thus, it can be hypothesized that Snail-2 and Brachyury, on the one side, and Zeb-2 and Sox-2, on the other side, may define two distinct territories in the chick blastoderm with divergent fates, but both characterized by a switch from E- to N-cadherin (Fig. 3B). Cells expressing Snail-2 and Brachyury may become fated to become mesodermal cells and undergo a rapid E- to N-cadherin switch followed by EMT and ingression through the primitive streak whereas cells expressing Sox-2 and Zeb-2 would acquire a neural identity and execute a slow transition between E- and N-cadherin while being prevented from losing their epithelial character and ingressing in the underlying space.
Timing and Kinetics of E- to N-Cadherin Switch During Primary Neurulation
To determine whether the switch from E- to N-cadherin in the neural epithelium correlates with the movements of neurulation and neural tube closure (Takeichi, 1988; Bronner-Fraser et al., 1992; Redies, 1995; Halbleib and Nelson, 2006), we studied their expression patterns during the whole process of neurulation at various defined positions along the neural axis, from the midbrain up to the posterior trunk. In the chick, the neural tube is formed by two independent mechanisms involving different morphogenetitc movements and termed primary and secondary neurulations (Colas and Schoenwolf, 2001; Lowery and Sive, 2004). Primary neurulation occurs in the head and upper trunk by rolling of the neural plate and formation of neural folds that then fuse dorsally, resulting in the typical hollow shape of the neural tube.
Analysis of the expression patterns of cadherins at different levels of the neural tube formed by primary neurulation revealed essentially two regions that differ in the timing and kinetics of the E- to N-cadherin switch: the first one encompasses the midbrain and anterior hindbrain and the second one extends from the posterior hindbrain to the anterior trunk. At the midbrain and anterior hindbrain levels, movements of neurulation start early at stage HH7. At this stage (Fig. 4A), both the neural plate and the ectoderm expressed uniformly high amounts of E-cadherin but no N-cadherin. During elevation of the neural folds at stage HH8 (Fig. 4B), while E-cadherin remained conspicuous over the entire cell surface of all neural epithelial cells, a faint, punctate staining for N-cadherin could be detected at the apical side of cells situated close to the midline (arrowhead in Fig. 4B). At stages HH8+ and HH9, during apposition of the neural folds (Fig. 4C, D), staining for N-cadherin became gradually stronger, but still concentrated at the apical side of cells, in the entire neural tube, with the notable exception of the prospective neural crest cells in the folds. Coincidently, staining for E-cadherin declined slowly and became markedly less intense than in the ectoderm. In contrast to N-cadherin staining, which propagated gradually from the midline up to the lateral (then dorsal) sides of the neural tube, E-cadherin staining decreased uniformly throughout the neural tube, with no obvious converse gradient from lateral to medial. After fusion of the folds and at the time of crest cell emigration from the neural tube at stage HH10 (Fig. 4E), N-cadherin staining became prominent over the entire surface of all neural epithelial cells. However, although it was completely closed and separated from the ectoderm, the neural tube still expressed detectable amounts of E-cadherin. Strikingly, neural crest cells expressed no N-cadherin but retained E-cadherin expression throughout delamination, as well as at the initial steps of migration. At stage HH11 (Fig. 4F), after completion of neural tube closure and neural crest cell emigration, E-cadherin staining has totally disappeared from the neural tube, which instead expressed uniformly high levels of N-cadherin.
Figure 4. Expression patterns of E- and N-cadherins during neurulation at the midbrain level. Immunostainings for E-cadherin (E-cad), N-cadherin (N-cad), and fibronectin (Fn in A–D) on cross-sections through the mesencephalon of chick embryos at stages HH7 (1 somite), HH8− (3 somites), HH8+ (5 somites), HH9 (7 somites), HH10 (9 somites), and HH11 (12 somites). Cell nuclei were stained with DAPI. cm, cranial mesoderm; e, ectoderm; n, notochord; nc, neural crest; ne, neural epithelium; nf, neural fold.
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In the posterior hindbrain and anterior trunk, movements of neurulation commence between stage HH8 and HH9 when the paraxial mesoderm is still unsegmented <zaq 4>(Fig. 5A, B). In this region, although the neural plate was initially organized as a flat epithelial layer in continuity with the ectoderm as in the midbrain, it clearly differed from the more anterior levels by the spatial distribution of cadherins over its surface. E-cadherin showed a latero-medial gradient in the neural plate with strong and uniform expression in the ectoderm and neural folds, weaker and apically-polarized expression in the intermediate neural tube, and a very faint, barely detectable expression in the median part. N-cadherin exhibited an almost exactly complementary pattern in the neural plate, being present medially, though at low levels, and absent laterally. Of note, unlike more anteriorly, codistribution of E-and N-cadherins was limited both spatially and temporally to cells of the intermediate portion of the neural tube. During elevation and apposition of the neural folds at stage HH10 (Fig. 5C, D), expression of E-cadherin disappeared almost entirely from the neural tube to become restricted to a few cells in the dorsal midline. Conversely, N-cadherin was expressed by all neural tube cells with the notable exception of the prospective neural crest population. By stage HH11 (Fig. 5E), after fusion of the neural folds and healing of the ectoderm, E-cadherin has entirely disappeared from all neural tube cells. Most remarkably, neural crest cells that have not yet started delaminating remained negative for N-cadherin. At stage HH12 (Fig. 5F), neural crest cell migration has just began and neither E-cadherin nor N-cadherin was expressed on their surface during delamination and early migration. As at more anterior levels, cessation of neural crest cell delamination was accompanied by a gradual increase in N-cadherin expression in the dorsal midline (not shown).
Figure 5. Expression patterns of E- and N-cadherins during neurulation at the anterior trunk (cervical) level.
Immunostainings for E-cadherin (E-cad), N-cadherin (N-cad), and fibronectin (Fn) on cross-sections through the anterior trunk (level of somites 7–10) of chick embryos at stages HH8 (3 somites), HH9 (6 somites), HH10− (9 somites), HH10+ (11 somites), HH11 (13 somites), and HH12 (15 somites). Cells nuclei were stained with DAPI. e, ectoderm; n, notochord; np, neural plate; ne, neural epithelium; nf, neural fold; pm, paraxial mesoderm.
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Figure 6. Expression patterns of E- and N-cadherins and their transcriptional regulators during neurulation at the midbrain level. In situ hybridizations for E-cadherin, N-cadherin, Sox-2, Zeb-2, Snail-2, and Claudin-1 on cross-sections through the mesencephalon of chick embryos at stages HH8 (4 somites), HH9 (7 somites), HH10 (9 somites), and HH11 (12 somites). cm, cranial mesoderm; e, ectoderm; n, notochord; nc, neural crest; ne, neural epithelium; nf, neural fold.
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In situ hybridization analyses revealed that, at both head and upper-trunk levels, expression of E- and N-cadherin transcripts matched approximately the spatio-temporal profile of their corresponding proteins (Figs. 2, 6, 7). In addition, Sox-2 exhibited the same expression pattern as N-cadherin in cells derived from the neurectoderm: it was abundant in neural tube cells, but absent from the ectoderm and progenitors of the neural crest in the folds (see also Wakamatsu et al., 2004). As amply described previously (for a review, see Duband, 2006), Snail-2 was restricted to prospective neural crest cells until they became segregated entirely from the neural tube. Immunostaining using antibodies to Snail-2 gave essentially the same pattern (not shown). In contrast to Snail-2 and Sox-2, which marked distinct cell populations in the neural tube, Zeb-2 was equally expressed in neural tube and neural crest cells during neural tube closure and it was excluded from the ectoderm. Interestingly, at posterior hindbrain and truncal levels, while Zeb-2 was uniform throughout the neural tube prior to neural crest cell emigration, it became markedly enhanced in crest cells as soon as they commenced their migration (Fig. 7D).
Figure 7. Expression patterns of E- and N-cadherins and their transcriptional regulators during primary neurulation at the anterior trunk (cervical) level. In situ hybridizations for E-cadherin, N-cadherin, Sox-2, Zeb-2, Snail-2, and Claudin-1 on cross-sections through the anterior trunk (level of somites 7–10) of chick embryos at stages HH8 (5 somites), HH10 (9 somites), HH11 (14 somites), and HH14 (21 somites). e, ectoderm; n, notochord; ne, neural epithelium; nf, neural fold; pm, paraxial mesoderm.
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These observations, therefore, reveal that, in the rostral half of the embryo, from the midbrain up to the anterior trunk, neural epithelial cells underwent a complete switch from E- to N-cadherin during neurulation. However, both the timing and the kinetics of replacement of E-cadherin by N-cadherin varied significantly along the embryonic axis (Fig. 8). At the cranial level, the E- to N-cadherin switch occurred only gradually with a relatively long time period where both molecules were coexpressed in cells, and the complete disappearance of E-cadherin from the neural epithelium was complete only after neural tube closure and neural crest cell emigration. In the anterior trunk, the switch also occurred gradually, but much earlier during the process of neural tube closure than in the midbrain. It started before bending of the neural plate and was complete at apposition of the neural folds. In addition, in contrast to the midbrain level where the domain of overlap between E- and N-cadherins extended over the entire neural tube, in the trunk, this domain was restricted to the intermediate zone of the neural tube.
Figure 8. Schematic representation of the timing and kinetics of the switch from E- to N-cadherin in neural epithelial cells in the intermediate zone of the neural plate during movements of neurulation at different axial levels along the anterior half of the embryo.
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By comparing the time of complete E-cadherin repression in neural epithelial cells with that of completion of neural tube closure at different axial levels from the midbrain to the anterior trunk, we found that loss of E-cadherin occurred approximately at a single time point at all levels (between stages HH10 and HH11), whereas closure of the neural tube was effective in these regions in a sequential manner from stage HH10 to HH12 (Fig. 8). This indicates that the timing and kinetics of E- to N-cadherin switch are not synchronized with the movements of neurulation. Analysis of the expression patterns of E- and N-cadherin transcripts in whole embryos at stages HH7–11 (Fig. 2B–D) revealed large domains along the anteroposterior axis in which cadherin expression evolves in a coordinated manner within each domain and differently from the other domains. Thus, three regions could be identified, encompassing the whole brain up to the otic placode for the first one, the anterior trunk covering approximately the first 10–15 somites for the second one, and the rest of the trunk for the third one. Surprisingly, Sox-2 and Zeb-2 displayed uniform expression domains showing no obvious correspondence with these domains, suggesting that additional, yet undefined co-regulators may contribute to the fine tuning of cadherin expression in the neural tube. It should be stressed, however, that although Sox-2 expression in the early embryonic nervous system appears uniform, it is subdivided into several domains by multiple enhancers with distinct spatio-temporal specificities (Uchikawa et al., 2003). Given that the regulatory sequences of the N-cadherin gene itself contains several region-specific enhancers that all rely on Sox-2 for their activity (Matsumata et al., 2005), it is conceivable that the spatial control of N-cadherin expression in the nervous system also depends on Sox-2 activation.
At the onset of neurulation, the neurectoderm comprises exclusively E-cadherin-expressing cells, while at its completion, at least three populations with distinct cadherin repertoires are segregated spatially: laterally, the ectoderm in which E-cadherin expression persists; medially, the neural tube at the origin of the central nervous system in which E-cadherin is substituted for N-cadherin; and at the connection between both compartments, the neural crest progenitors in which E-cadherin is lost but not replaced by N-cadherin. These domains are also characterized by different repertoires of cadherin regulators: none in the ectoderm, Sox-2 and Zeb-2 in the neural tube, and Zeb-2 and Snail-2 in the neural crest. Besides cell adhesion, one of the major roles of cadherins is to mediate cell recognition and segregation. Using transfected cell lines (Duguay et al., 2003) showed that sorting depends on both the type of cadherin expressed by the cells and its relative amount on the cell surface. Therefore, it is tempting to propose that the timing and kinetics of E- to N-cadherin switch during neurulation reflects the spatial segregation of the different lineages arising from the neurectoderm along the medio-lateral axis.
Timing and Kinetics of E- to N-Cadherin Switch During Secondary Neurulation in the Caudal Trunk
Secondary neurulation starts at closure of the posterior blastopore at stage HH13, and concerns a large portion of the trunk extending beyond the 28th somite and corresponding to the future lumbosacral region. In this region, the neural tube derives from the tail bud, a mesenchymal population of cells that coalesce into a rod and transform into an epithelium. The lumen is formed secondarily by cavitation to produce a hollow tube (Colas and Schoenwolf, 2001).
In the caudal extremity of the embryo at stages HH14–18, undifferentiated tail bud cells formed a uniform solid cord in which early neural progenitors expressing low levels of Sox-2 were morphologically undistinguishable from the paraxial mesoderm. At this level, while E-cadherin was confined to the overlying superficial ectoderm, N-cadherin was uniformly expressed in all tail bud cells (Fig. 9A). During epithelialization of the neural blastema and cavitation, N-cadherin became preferentially accumulated in the apical side of the Sox-2-positive neural epithelial cells, while it remained uniform in the mesenchymal paraxial mesoderm (Fig. 9B–E). In situ hybridizations studies (Fig. 10) confirmed that the expression profiles of E- and N-cadherins showed no region of overlap. E-cadherin expression was restricted to the ectoderm and to the caudal tip of the tail bud, corresponding to the remnants of the primitive streak (Ohta et al., 2007), abutting precisely the region where N-cadherin is expressed. The expression domain of N-cadherin appeared significantly larger than that of Sox-2 in the tail bud, suggesting that, contrary to more anterior levels, Sox-2 plays no role in the initiation of N-cadherin expression in the caudal neural tube. Rather than Sox-2, Tbx-6L, a close relative to Brachyury, exhibited an expression profile matching precisely with that of N-cadherin in the tail bud. More anteriorly, however, in the region where the neural tube is being formed, Tbx-6L was replaced by Sox-2 in the N-cadherin-expressing cells of the neural tube (see also Takemoto et al., 2011). Finally, reflecting the absence of E-cadherin in neural progenitors throughout the course of secondary neurulation, Zeb-2 was not found in the caudal neural tube.
Figure 9. Expression patterns of E- and N-cadherins during secondary neurulation at the posterior trunk (lumbar) level. Immunostainings for E-cadherin (E-cad), N-cadherin (N-cad), and Sox-2 on consecutive cross-sections through the posterior trunk of a chick embryo at stage HH13/14 (19–23 somites). A is most posterior and E most anterior. Cell nuclei were stained with DAPI. e, ectoderm; n, notochord; ne, neural epithelium; pm, paraxial mesoderm; tb, tail bud.
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Figure 10. Expression patterns of E- and N-cadherins and their transcriptional regulators during secondary neurulation at the posterior trunk (lumbar) level. A: In situ hybridizations for E-cadherin, N-cadherin, Sox-2, and Zeb-2 on whole embryos at stage HH14 (21 somites), showing the caudal part of the embryo. B–D: In situ hybridizations for E-cadherin, N-cadherin, Sox-2, Zeb-2, and Snail-2 on consecutive cross-sections through the posterior trunk of a chick embryo at stage HH14 (22 somites). B is most posterior and D most anterior. e, ectoderm; n, notochord; nb, neural blastema; ne, neural epithelium; pm, paraxial mesoderm.
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These observations, therefore, indicate that, during secondary neurulation, elaboration of the neural tube does not involve an E- to N-cadherin switch consecutive to transition from a neuroectodermal progenitor to a neural fate. Instead, the neural tube forms from a pool of cells at the origin of the paraxial mesoderm and caudal neural tube (Wilson et al., 2009). These cell populations express N-cadherin from the very beginning on and become gradually segregated probably as a result of selective adhesion. However, we observed no striking difference in the relative cadherin content over time between the neural and mesodermal precursors, indicating that differential level of expression of N-cadherin does not constitute the triggering signal for cell segregation. Yet, expression of cadherin on the surface of the neural cells gradually evolved from uniform to polarized, most likely accompanying the transition from a mesenchymal organization to an epithelial structure.
Cadherin Switch During Node Regression and Formation of the Notochord
Neurulation is accompanied by the formation of the notochord, which is laid down during regression of the Hensen's node. To investigate whether cadherins are involved in notochord segregation, we analyzed the expression patterns of E- and N-cadherins in the node at different stages of its regression during primary neurulation. At stages HH6 to HH8, node regression generates the notochord corresponding to the head and anterior trunk levels, while at stage HH10 it lays down the notochord of the mid-trunk. At stages HH6–8, node cells were organized as a compact mass of Sox-2-negative cells, distributed over the midline and splitting the neural plate into two lateral parts (Fig. 11A, B). These cells could be easily visualized by their high N-cadherin and low E-cadherin content, contrasting with both neural plate cells that expressed exclusively E-cadherin and primitive streak cells that co-expressed both E- and N-cadherins. At stage HH10, the neural plate is no longer organized as a flat, uniform epithelium along the plan of the embryo; rather, it is composed of two epithelial sheets facing each other and oriented along the dorso-ventral axis. At this level, the node is located deeply at the junction between the two neural plate sheets (Fig. 11C). It is often referred to as chordo-neural hinge. As in more proximal regions, node cells could be discerned from the neighboring neural cells by the lack of Sox-2 expression and their strong and uniform staining for N-cadherin. These observations, therefore, suggest that notochord progenitors may segregate from the rest of the neural epithelium owing to their high N-cadherin content. Interestingly, of the various transcriptional regulators of cadherins studied, neither Sox-2, Zeb-2, Snail-1, Snail-2, nor Twist-1 exhibited a spatiotemporal pattern consistent with the selective increase of N-cadherin in node cells. Brachyury, in contrast, was conspicuous at all stages in the node and in notochord (Figs. 1C, 2B–D). The recent demonstration that Brachyury may control cadherin expression and EMT (Fernando et al., 2010) raises the intriguing possibility that it may up-regulate N-cadherin in nodal cells, allowing their segregation from the rest of the neural plate.
Figure 11. Expression patterns of E- and N-cadherins during node regression and formation of the notochord. Immunostainings for E-cadherin (E-cad), N-cadherin (N-cad), fibronectin (Fn), and Sox-2 on cross-sections through the node of chick embryo at stage HH7 (A), and at stage HH8 (B), and through the chordo-neural hinge at stage HH10 (C). Cell nuclei were stained with DAPI. cnh, chordo-neural hinge; e, ectoderm; Hn, Hensen's node; m, mesoderm; np, neural plate.
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