By continuing to browse this site you agree to us using cookies as described in About Cookies
Notice: Due to essential maintenance the subscribe/renew pages will be unavailable on Wednesday 26 October between 02:00- 08:00 BST/ 09:00 – 15:00 SGT/ 21:00- 03:00 EDT. Apologies for the inconvenience.
The neural crest is a group of migratory cells induced at the dorsal tip of the neural tube as a consequence of interactions between the neural and non-neural ectoderm (Barembaum and Bronner-Fraser,2005). After induction, these cells undergo an epithelial to mesenchymal transition, delaminate from the neuroectoderm and migrate profusely to populate a wide variety of embryonic structures, where they contribute to many different tissues (Le Douarin and Kalcheim,1999). During the past years, considerable effort has been directed toward understanding the different aspects of neural crest biology, including its induction and delamination from the neuroectoderm, the control of the migratory properties, and the mechanisms of differentiation into specific tissues (Le Douarin and Dupin,2003; Meulemans and Bronner-Fraser,2004; Morales et al.,2005). The emerging picture is quite complex and is further complicated by the apparent differences among different model organisms (Aybar and Mayor,2002).
While many experimental analyses have been performed in chicken, zebrafish, and Xenopus embryos, information relating to the early processes of neural crest biology in the mouse is scarce and it is not clear if the mechanisms that are thought to be relevant in other species also apply to mammals. An additional level of difficulty is that often a function that has been associated to a specific member of a gene family in the chicken is performed by a different member of the family in the mouse. For instance, based on expression analyses, it has been suggested that the function of Snail2 (previously known as Slug) in the chicken neural crest is performed by Snail1 in the mouse (Sefton et al.,1998). However, when this idea was challenged using genetic tools, the results were surprising, as they indicated that neither Snail1 nor Snail2 seemed to be involved in neural crest production in the mouse (Murray and Gridley,2006). This finding suggests that some of the mechanisms of neural crest development are not conserved among vertebrates.
The bone morphogenetic proteins (BMPs) were among the first factors implicated in the earliest processes of neural crest cell development. Early experiments performed in chicken embryos suggested that BMP4 and BMP7 were implicated in neural crest cell induction (Liem et al.,1995). This view was challenged by later results, which showed that the ability of these factors to induce neural crest cells in vitro depends on the concomitant activity of other still not identified factors present in the serum of the culture medium (Garcia-Castro et al.,2002). A different set of studies performed in chicken embryos suggested that BMPs are required for the migration rather than the induction of neural crest cells (Sela-Donenfeld and Kalcheim,1999). In addition, it has been proposed that BMP activity is further modulated by noggin and that the graded inactivation of the expression of this gene controls the onset of neural crest migration (Sela-Donenfeld and Kalcheim,1999). The molecular mechanisms that mediate BMP signaling in neural crest cell delamination have also been studied. In the initial studies, it was proposed that BMP activity was mediated by induction of Cad6B and the GTPase RhoB (Sela-Donenfeld and Kalcheim,1999). Recent data implicate BMP signaling in modulating progression through the cell cycle, an effect that could be mediated, at least in part, by Wnt1 (Burstyn-Cohen et al.,2004). It has also been proposed that the activity of BMP signaling is mediated by the cleavage of N-cadherin into a soluble cytoplasmic form, which would participate, together with the canonical Wnt pathway, in the control of neural crest cell emigration (Shoval et al.,2007).
In the mouse, a variety of genetic studies indicate that BMP signaling is essential at different stages of neural crest development. Bmp5 and Bmp7 have been reported to be required in a partially redundant manner for the survival of postmigratory crest cells, as demonstrated by their increased death in Bmp5;Bmp7 double mutant embryos (Solloway and Robertson,1999). In addition, Bmp2 is essential for the formation of migratory neural crest cells (Kanzler et al.,2000; Ohnemus et al.,2002). Expression analyses in the mouse indicate that Bmp2 is expressed in the surface ectoderm adjacent to the neuroectoderm with a spatiotemporal dynamics that correlates with neural crest cell production (Kanzler et al.,2000). Blocking Bmp2 signaling in vivo in the dorsal neural tube through expression of noggin in transgenic mice, resulted in the absence of migratory neural crest cells and in the lack of the corresponding derivatives (Kanzler et al.,2000; Ohnemus et al.,2002). In addition, preliminary analysis of Bmp2 mutant embryos failed to detect the streams of Crabp1 reactivity associated with the hindbrain characteristic of neural crest cells, which was correlated with an absence of branchial arches (Kanzler et al.,2000). What is not clear from these studies is whether Bmp2 is required for the induction of neural crest cells or for their delamination/migration from the neural tube. In this study, we show that neural crest cells are induced in the Bmp2 mutants. We also show that the absence of migratory neural crest cells in these mutants does not derive from increased death of the premigratory neural crest cells, and thus it is probable that Bmp2 is required for triggering migration of these progenitors. The absence of migration of these cells seems to result in an extra accumulation of cells in the neuroepithelium, which produces abnormal foldings in the anterior–posterior and medial–lateral axes. Our results also suggest that, although BMP signaling is required for neural crest cell migration in chicken and mouse embryos, the molecular mediators may differ among vertebrates.
As reported earlier, Bmp2 mutant embryos do not survive past embryonic day (E) 9.0, which hampers analysis of the formation of neural crest derivatives (Zhang and Bradley,1996; Kanzler et al.,2000). However, living embryos can be recovered at this stage, thus allowing analysis of early stages of neural crest cell development. A morphological analysis of these “late” Bmp2 mutants showed several malformations, in addition to those previously reported (Zhang and Bradley,1996). In particular, the rostral neural tube always failed to close and displayed abnormal foldings both in the anterior–posterior and medial–lateral axes, without a fixed pattern (Fig. 1). In addition, the surface of the neural tube seemed extended medial–laterally in the mutant embryos when compared with their wild-type littermates. As the neural tube fails to close, there are no real dorsal and ventral domains, but rather medial and lateral areas in the neuroectoderm, which is the nomenclature that we will use throughout the text.
Neural Crest Cells Are Induced in the Absence of Bmp2
Previous work indicated that Bmp2 mutant mice have no migratory neural crest cells, but whether this deficiency resulted from a lack of neural crest cell induction or from the inability of these cells to migrate from the neural tube was not investigated (Kanzler et al.,2000). To answer this question, we characterized early events of neural crest biology in Bmp2 mutant mice. Ap2α is a neural crest marker in the mouse (Mitchell et al.,1991) that can be detected in the premigratory neural crest cells at the dorsal tip of the neural tube and in the migratory neural crest cells, most particularly in the cranial area (Fig. 1A,B). Examination of Ap2α expression in Bmp2 mutant embryos revealed no obvious streams of migratory neural crest cells, confirming that no migratory neural crest cells are produced in the absence of Bmp2 (Fig. 1C,D). However, Bmp2 mutant embryos showed a clear Ap2α signal associated with the lateral border of the neural folds. Interestingly, the spatial distribution of the Ap2α transcripts in this area along the rostral–caudal axis was very similar to that observed in wild-type embryos, as it combined areas of strong and weak expression, the latter corresponding in wild-type embryos to rhombomeres (r) 3 and 5 (Fig. 1A,C). Analysis of sections of these embryos confirmed that Ap2α expression in Bmp2 mutant embryos is indeed associated with the dorsal part of the neural tube (Fig. 1D). A careful analysis of the sections revealed that the expression in this area expands medially farther than in wild-type embryos. Indeed, in wild-type embryos, very few cells in the dorsal neural tube were positive for Ap2α, possibly as a result of the migration of cells when expression of this transcription factor is activated. In Bmp2 mutant embryos, Ap2α expression was clearly localized to the lateral borders of the neuroectoderm, which could represent an accumulation of induced neural crest cells. In addition, examination of serial sections covering the whole anterior–posterior axis revealed some Ap2α-positive cells in the mesenchyme adjacent to the neuroectoderm–surface ectoderm interface. These patches of cells were very rare and involved just a few cells (the picture in the embryo shown in Fig. 1D is the best image observed in that particular embryo). This finding suggests that, while the production of migratory neural crest cells is very reduced, some cells do manage to start migration. These results suggest that neural crest cells are induced but fail to migrate in the absence of Bmp2.
In chicken embryos, the early neural crest biology has been extensively characterized and the role that BMP signaling plays in this process has been addressed by several groups (Selleck et al.,1998; Burstyn-Cohen et al.,2004; Shoval et al.,2007). In those studies, several genes have been reported to be downstream of the BMP signaling pathway during early stages of neural crest cell biology (Burstyn-Cohen et al.,2004). Among these, Cad6B and Wnt1 are also neural crest cell markers in the mouse. As in chicken embryos, Cad6B is expressed in the premigratory neural crest of the mouse. However, contrary to what has been reported in chicken (Taneyhill et al.,2007), mouse migratory neural crest is also positive for Cad6B, at least in the cranial region (Fig. 1E,F). In Bmp2 mutant embryos, Cad6B expression was readily detected in the lateral edges of the neuroectoderm (Fig. 1G,H). However, it could not be detected in the domain where migratory neural crest should be present, further supporting the inference of the absence of this cell population. The other potential BMP target in the neural crest, Wnt1, shows an equivalent expression pattern in chicken and mouse embryos, being restricted to the dorsal neural tube but absent from the migratory neural crest population (Wilkinson et al.,1987; Bally-Cuif et al.,1992; Fig. 1I,J). In the mouse, a variety of cell tracing and mutagenesis experiments indicates that, while migratory neural crest cells do not contain transcripts for Wnt1, the premigratory neural crest cells are included within the expression domain of this gene in the dorsal neural tube (Echelard et al.,1994; Jiang et al.,2000). In Bmp2 mutant embryos, the expression domain of Wnt1 in the lateral borders of the neuroectoderm was largely unaffected, including its spatial distribution along the anterior–posterior axis (Fig. 1K,L). These results indicate that, contrary to what has been reported for the chicken system (Burstyn-Cohen et al.,2004), in the mouse neither Cad6B nor Wnt1 seems to be downstream of BMP signaling in early neural crest biology, at least that dependent on Bmp2.
The Snail gene family has been implicated in the delamination of neural crest cells in chicken and Xenopus embryos (Nieto et al.,1994; Aybar et al.,2003) and, while some reports consider these genes BMP-independent (Burstyn-Cohen et al.,2004), others place them downstream of BMP signaling (Selleck et al.,1998). In the mouse, Snail1, instead of Snail2, has been reported to be associated with the neural crest (Sefton et al.,1998). In wild-type embryos, expression of this gene is turned on in cells delaminating from the dorsal neural tube. At least in our hands, it is rarely observed in the premigratory neural crest cells (Fig. 1N), probably because of the immediate delamination of the Snail1-positive neural crest cells, but gives a strong signal in the streams of migratory neural crest cells (Fig. 1M,N). In Bmp2 mutant embryos, no Snail1 expression was observed in the mesenchyme adjacent to the cranial neural tube, which is consistent with the absence of migratory neural crest cells (Fig. 1O,P). However, a clear signal was observed adjacent to the lateral tip of the neural folds, which is also consistent with the presence of neural crest cell progenitors that failed to migrate away of the neural tube. Analysis of sections revealed that, in addition to this signal, cells in the lateral tip of the neuroectoderm are often positive for Snail1 (Fig. 1P), a signal that, as mentioned above, was not observed in wild-type embryos (Fig. 1O). This result is also consistent with the existence of neural crest progenitors in Bmp2 mutant embryos, lending further support for a role for Bmp2 in neural crest migration, rather than induction, in the mouse. Also, these results indicate that expression of the Snail family genes in the neural crest is independent of Bmp2 signaling.
The Id family genes have also been reported to be downstream of the BMP pathway (Hollnagel et al.,1999) and to play a role in neural crest formation, at least in chicken and Xenopus embryos (Martinsen and Bronner-Fraser,1998; Kee and Bronner-Fraser,2005). Of the four Id genes that exist in mice, Id2 seemed to give the clearest expression pattern in the neural crest (our unpublished data). Its expression was already detected in the lateral edges of the neuroectoderm at E8.0, which is compatible with the dynamics of neural crest cell production (Fig. 1R,S; and not shown). Expression was then detected also in the migratory neural crest cells, being stronger in the mesencephalic crest domain, although the levels of Id2 expression in migratory neural crest cells were always very reduced and disappeared after E9.0 (Fig. 1R,S; and not shown). In Bmp2 mutant embryos, Id2 expression in the lateral neural folds was very strong, indicating that, at least in this area, Id2 expression is not under the positive control of Bmp2. Actually, in all Bmp2 mutant embryos analyzed (n = 3), expression of Id2 extended medially to a considerable extent (Fig. 1T,U; and not shown). Nevertheless, if Id2 also labels the premigratory neural crest population in mouse embryos, the expression of this gene in the Bmp2 mutant embryos is also consistent with the conclusion that production of neural crest cells is not affected by the absence of Bmp2.
To further analyze whether neural crest cells are formed in the absence of Bmp2, we used a transgenic approach. It has been described that the HtPA gene contains an enhancer that labels specifically the neural crest cells (Pietri et al.,2003). We used this enhancer to express the RFP gene in transgenic mice (HtPA:RFP mice), thus labeling the neural crest cells with red fluorescent protein (RFP). Analysis of transgenic embryos revealed RFP fluorescence in migratory neural crest cells in the cranial region (Fig. 2A). In keeping with previous reports, only a few RFP-positive premigratory neural crest cells were observed, arguably because these cells delaminate shortly after activation of the enhancer (Pietri et al.,2003). In the Bmp2 mutant, background RFP-positive cells were also readily detected but remained mostly associated with the dorsal neural tube (Fig. 2B). It is possible that a few RFP-positive cells were located in the adjacent mesenchyme rather than in the neuroepithelium, which could indicate the existence of a residual migratory ability of the neural crest cells in the absence of Bmp2. These results are consistent with the data obtained with molecular markers and further support the conclusion that Bmp2 does not have an effect on neural crest cell production.
Apoptosis in the Neural Tube of Bmp2 Mutant Embryos
To understand whether the absence of migratory neural crest cells in Bmp2 mutant embryos resulted from the death of the induced progenitors, we performed terminal deoxynucleotidyl transferase–mediated deoxyuridinetriphosphate nick end-labeling (TUNEL) assays. At E8.5, some TUNEL-positive cells were observed in the dorsal neural tube, with a focal concentration of apoptotic cells in the anterior hindbrain (Fig. 3A,B). Considerable cell death was also evident in the forebrain region. In Bmp2 mutant embryos, no obvious increase of TUNEL-positive cells was detected associated with the lateral borders of the neural tube (Fig. 3C,D), indicating that increased cell death is probably not responsible for the absence of migratory neural crest cells. It should be noted that, in these embryos, focal areas of TUNEL-positive cells were observed in the most anterior part of the neuroepithelium, which corresponds to the forebrain region. In addition, an extra area of increased cell death was observed in Bmp2 mutant embryos in the region that would correspond to the anterior hindbrain–posterior mesencephalon (Fig. 3D), an area where we never observed cell death in control littermates (Fig. 3B). The possible relevance of this observation will be discussed later.
Anterior–Posterior Patterning in the Neural Tube of Bmp2 Mutant Mice
When we analyzed expression of Wnt1 in Bmp2 mutant embryos, we observed that, while expression in the dorsal tip of the neural tube was not compromised, expression in the midbrain–hindbrain boundary could not be detected (Fig. 1K). As the neuroectoderm in the Bmp2 mutant embryos is abnormally folded, the absence of Wnt1 expression in this area could indicate abnormal anterior–posterior patterning of the neural tube in Bmp2 mutant embryos. Therefore, we analyzed expression of a few molecular markers for specific areas of the neural tube in Bmp2 mutant embryos. Engrailed genes are specifically expressed in the midbrain, next to the midbrain–hindbrain boundary, in an area adjacent to the Wnt1 expression domain (Fig. 4A; Joyner and Martin,1987). En2 was expressed in equivalent patterns in control embryos and the Bmp2 mutants (Fig. 4B), indicating that the midbrain–hindbrain border was not globally disturbed. Importantly, the En2 domain was restricted to a specific area of the neural tube and did not extend its most anterior border, suggesting that this area was also correctly patterned. We confirmed this finding with the analysis of Otx2 expression, which is detected in the forebrain and midbrain down to the border with the hindbrain (Fig. 4C,D; Simeone et al.,1992). In addition, the hindbrain also seemed to keep a normal anterior–posterior pattern, as indicated by the restricted expression of hindbrain markers like Hoxb1, which is expressed in r4 (Fig. 4E,F; Frohman et al.,1990). These results indicate that, globally, the neuroectoderm of the Bmp2 mutant embryos is correctly patterned along the anterior posterior axis, although expression of specific markers, most particularly Wnt1, seemed affected.
In this manuscript, we showed that, in the mouse, Bmp2 is not required for the induction of neural crest cells, but it is essential for their migration from the neural tube. It is clear that migratory neural crest production is strongly compromised in the absence of Bmp2, which is in keeping with our previous data using biochemical blockage of BMP signaling in transgenic embryos (Kanzler et al.,2000; Ohnemus et al.,2002). However, all tests for neural crest cell induction, either using a variety of molecular markers or a transgenic approach, turned out positive in the Bmp2 mutant embryos, indicating that Bmp2 is not essential for the induction of this cell population, but rather for its migration from the neural tube. Interestingly, with the limitations inherent to the abnormal neural tube morphology of Bmp2 mutant embryos, the spatial distribution of transcripts for the neural crest cell markers along the anterior–posterior axis was very similar to that observed in wild-type embryos. Thus, there were areas of strong expression, which corresponded to the neural crest-producing domains, and areas with very little or no expression, which corresponded to the neural crest-free areas of the hindbrain (Lumsden et al.,1991). This finding suggests that not only neural crest cell induction occurs in the absence of Bmp2, but also that the basic mechanisms are still in place in Bmp2 mutant embryos. It should be noted that we cannot rule out that specific subsets of neural crest cells fail to be induced in Bmp2 mutant embryos.
The absence of migratory neural crest cells seems not to be a consequence of increased cell death, as no increase in TUNEL-positive cells was detected associated with the lateral borders of the neuroectoderm in Bmp2 null mutant embryos. It is still possible that the elimination of migratory cells occurs at a rate below the levels of detection of our assay. However, apoptotic cells were readily detected in embryos carrying mutations in other genes essential for survival of postmigratory neural crest cells (Solloway and Robertson,1999; Trumpp et al.,1999). Therefore, given the large numbers of neural crest cells produced during the early stages of embryogenesis, an extent of cell death that could account for the absence of this migratory population should most probably be above the level of detection by TUNEL.
An alternative hypothesis for the absence of migratory neural crest cells in Bmp2 mutants is that Bmp2 is involved in triggering the migratory competence of the induced neural crest cells. A role for BMP (Dpp) signaling in the promotion of cell movement has already been reported in other systems (Vincent et al.,1997). Moreover, some of our data can be interpreted according to this hypothesis. In particular, we have observed that cells positive for some neural crest markers, like Ap2α, Snail1, or the RFP in HtPA:RFP transgenics, which are rarely detected in the dorsal neural tube but rather in migratory neural crest population, seem to accumulate in the lateral edges of the neuroectoderm, remaining as premigratory neural crest cells. It will be important to address this problem directly and determine the underlying mechanism.
It could be expected that, in the absence of migration, neural crest cells accumulate in the lateral neural tube of Bmp2 mutant embryos. Although the domain positive for neural crest cell markers in Bmp2 mutants may be slightly extended compared with wild-type embryos, it is clear that there is no overt accumulation of premigratory neural crest cells. Experiments in chicken and amphibian embryos indicate that neural crest cell induction requires interactions between the surface ectoderm and the neuroectoderm (Moury and Jacobson,1990; Selleck and Bronner-Fraser,1995). If the same is true for mouse embryos, the signals involved in this process could have a limited range of activity and, therefore, could not act much further than a few cells in distance. In this context, the induced neural crest cells that fail to migrate could act as a barrier for further neural crest induction in more medial cells within the neuroectoderm, thus maintaining the size of the neural crest compartment within normal limits. Another consequence of the absence of neural crest cell migration and their lack of elimination by death could be an abnormal accumulation of cells in the neuroepithelium. This explanation is consistent with the observation that neuroepithelium of Bmp2 mutant embryos is clearly wider than those of control embryos and is abnormally folded. It should be noted, however, that increased apoptosis was also detected in specific areas of the neuroepithelium, which could contribute (at least partially) to the elimination of the extra cells accumulating as a result of the absence of migration of the induced crest cells. This cell death, of unclear origin, could also account for the lack of Wnt1 expression in the midbrain/hindbrain boundary that we observed in the Bmp2 mutants. Indeed, the observed domain of cell death contains the area of Wnt1 expression in the neuroepithelium.
An important implication of our results is that the role of Bmp2 in mouse neural crest development seems to be different to what has been reported for BMP signaling in other vertebrates. In chicken embryos, Cad6B and Wnt1 are both downstream of BMPs in the signaling cascades that generate migratory neural crest cells (Sela-Donenfeld and Kalcheim,1999; Burstyn-Cohen et al.,2004). Most particularly, it was proposed that Wnt1 was an essential mediator of BMP signaling in the induction of migratory properties to neural crest cells (Burstyn-Cohen et al.,2004). In our case, it is clear that neither of the two genes was affected by the absence of Bmp2, but neural crest cells were still unable to migrate. There are several possible explanations for these inconsistencies. This may reflect another basic difference in the mechanisms that modulate production of migratory neural crest cells in the two vertebrates. The existence of such divergences has been clearly documented for the Snail family of transcription factors. It has been reported that Snail2 (previously known as Slug) is required for the epidermal to mesenchymal transitions involved in the genesis of migratory neural crest cells in chicken embryos (Nieto et al.,1994). However, a recent report has clearly shown that mouse embryos carrying null mutations for both Snail1 and Snail2 still produce migratory neural crest cells, indicating that neither of these two genes is essential for this process in the mouse (Murray and Gridley,2006). In this context, it is important to note that Snail2 expression is maintained in the Bmp2 mutants and neural crest cell delamination is still blocked. It is also clear that canonical Wnt signaling in the mouse cannot play the essential role in neural crest cell delamination reported for chicken embryos, as both Wnt1;Wnt3A double mutants and conditional mutants for β-catenin in the neural crest seem to have no problems in neural crest cell delamination (Ikeya et al.,1997; Brault et al.,2001). In the case of BMP signaling in the neural crest cells, additional differences have already been reported between mouse and chicken embryos. For instance, BMP4 seems to be the relevant molecule for the induction/migration of chicken neural crest cells (although an activity for BMP7 has also been reported), while Bmp2 seemed irrelevant in this process (Liem et al.,1995). Conversely, inactivation of Bmp2 alone is enough to block production of migratory neural crest cells in the mouse, while Bmp4 does not seem to play a major role in the process in this species (Kanzler et al.,2000). Bmp5 and Bmp7 also seem to play a role in the development of neural crest cells, but their function seems to be required at later stages (Solloway and Robertson,1999). Another apparent BMP signaling-related difference between mouse and chicken neural crest is that mutant data in the mouse are not consistent with a role for the BMP inhibitor Noggin in the spatial and temporal control of neural crest cell delamination that has been proposed according to experiments performed in chicken embryos (McMahon et al.,1998; Sela-Donenfeld and Kalcheim,1999).
Another possible explanation for the inconsistency of our results with those obtained for BMP signaling in chicken and Xenopus embryos is that, while most of our analyses have been performed in the cranial region, which is where our phenotypes were most easily scored, experiments performed to evaluate the relevance of BMP signaling in chicken neural crest delamination have been typically performed in the caudal neural tube. This finding would suggest that induction/delamination of cranial and caudal neural crest cells is controlled by different mechanisms. However, as far as we could evaluate from our expression analyses, what we documented for the cranial area seems to apply also for the trunk neural crest cells (our unpublished data).
In conclusion, while BMP signaling seems to be required for neural crest cell migration in various vertebrates, the molecular mechanisms for this process evidently diverge among species.
The Bmp2 mutant mice were described previously (Zhang and Bradley,1996). To generate the HtPA-RFP transgenic mice, the RFP cDNA was cloned downstream of the HtPA enhancer that drives expression in the neural crest (Pietri et al.,2003). The SV40 polyadenylation signal was cloned downstream of the RFP cDNA. The purified construct was used to generate transgenic mice by pronuclear injection according to standard protocols. The HtPA-RFP transgenic animals and embryos were identified by polymerase chain reaction (PCR) using the primers 5′-TCCGAGGACGTCATCAGGGAG-3′ and 5′-ATGGTGTAGTCCTCGTTGTGG-3′.
In Situ Hybridization and Apoptosis
In situ hybridization was performed as previously described (Kanzler et al.,1998). At least three embryos for each genotype were analyzed per probe. The probes used were Ap2α (Mitchell et al.,1991), Snail1 (Sefton et al.,1998), Cad6B (Ionue et al.,1997), Wnt1 (Wilkinson et al.,1987), En2 (Joyner and Martin,1987), and Otx2 (Simeone et al.,1992). The Hoxb1 probe was obtained as a 1.1-kb fragment containing the whole open reading frame by reverse transcriptase-PCR, cloned into the EcoRI and BamHI sites of pKS Bluescript. The probe for Id2 was obtained from IMAGE clone 6515664. Stained embryos were embedded in gelatin/sucrose, sectioned to 30 μm with a Vibratome and photographed under Nomarski optics.
Apoptosis was determined by TUNEL as described in Kanzler et al. (2000) except for the use of an alkaline phosphatase-conjugated instead of horseradish peroxidase-conjugated antifluorescein antibody and subsequent detection of the activity with NBT and BCIP (nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate) as performed in the in situ hybridization protocol. The RFP fluorescence was acquired by confocal microscopy using a Leica TCS SP2 system, and Z-projections of the confocal stacks were processed and colored using Imairs (v5.0.3) and ImageJ (v1.37).
We thank Trish Labosky and Allan Bradley for the Bmp2 mutant mice, Silvye Dufour for the HtPA enhancer, Angela Nieto for the Snail1 probe, Andy McMahon for the Wnt1 probe, Hubert Schorle for the Ap1α probe, Andreas Kispert for the Cad6B probe, José Belo for the En2 and Otx2 probes, Randy Cassada for reading the manuscript, and all members of the Mallo lab for support and discussion. This work was funded by the Centro de Biologia do Desenvolvimento and by FCT.