Loss of CXCL12/CXCR4 signalling impacts several aspects of cardiovascular development but does not exacerbate Tbx1 haploinsufficiency

The CXCL12-CXCR4 pathway has crucial roles in stem cell homing and maintenance, neuronal guidance, cancer progression, inflammation, remote-conditioning, cell migration and development. Recently, work in chick suggested that signalling via CXCR4 in neural crest cells (NCCs) has a role in the 22q11.2 deletion syndrome (22q11.2DS), a disorder where haploinsufficiency of the transcription factor TBX1 is responsible for the major structural defects. We tested this idea in mouse models. Our analysis of genes with altered expression in Tbx1 mutant mouse models showed down-regulation of Cxcl12 in pharyngeal surface ectoderm and rostral mesoderm, both tissues with the potential to signal to migrating NCCs. Conditional mutagenesis of Tbx1 in the pharyngeal surface ectoderm is associated with hypo/aplasia of the 4th pharyngeal arch artery (PAA) and interruption of the aortic arch type B (IAA-B), the cardiovascular defect most typical of 22q11.2DS. We therefore analysed constitutive mouse mutants of the ligand (CXCL12) and receptor (CXCR4) components of the pathway, in addition to ectodermal conditionals of Cxcl12 and NCC conditionals of Cxcr4. However, none of these typical 22q11.2DS features were detected in constitutively or conditionally mutant embryos. Instead, duplicated carotid arteries were observed, a phenotype recapitulated in Tie-2Cre (endothelial) conditional knock outs of Cxcr4. Previous studies have demonstrated genetic interaction between signalling pathways and Tbx1 haploinsufficiency e.g. FGF, WNT, SMAD-dependent. We therefore tested for possible epistasis between Tbx1 and the CXCL12 signalling axis by examining Tbx1 and Cxcl12 double heterozygotes as well as Tbx1/Cxcl12/Cxcr4 triple heterozygotes, but failed to identify any exacerbation of the Tbx1 haploinsufficient arch artery phenotype. We conclude that CXCL12 signalling via NCC/CXCR4 has no major role in the genesis of the Tbx1 loss of function phenotype. Instead, the pathway has a distinct effect on remodelling of head vessels and interventricular septation mediated via CXCL12 signalling from the pharyngeal surface ectoderm and second heart field to endothelial cells.

Introduction TBX1 haploinsufficiency is the major contributing factor in the development of congenital cardiovascular defects in the 22q11.2 deletion syndrome (22q11.2DS). Conditional mutagenesis experiments have determined the tissue specific and temporal requirements for this transcription factor in the mouse (reviewed in [1]). Notably, Tbx1 is required in the pharyngeal surface ectoderm for the formation and remodelling of the embryonic pharyngeal arch artery (PAA) system into the great vessels.
Defects of the aortic arch and right subclavian artery (RSA) are prominent e.g. retro-oesophageal RSA. The defects observed in mice correlate well with abnormalities observed in human patients. In particular, interrupted artic arch type B (IAA-B), which represents a left 4 th pharyngeal arch artery (PAA) abnormality is quite specific for 22q11.2 deletion syndrome in that~50% of patients presenting with this defect will test positive for the 22q11.2 deletion [2].
Furthermore, Tbx1 nulls all have a common arterial trunk (CAT) [3,4]; Tbx1 is required in the second heart field for septation of the outflow tract, atrial and ventricular septation (specifically, closure of the membranous part of the septum) and correct alignment of the outflow tract with the ventricles.
We and others have identified defects of neural crest cell (NCC) patterning in Tbx1 null and heterozygous embryos [5,6]. Importantly, Tbx1 is not expressed in NCCs and therefore such abnormalities of NCC patterning must be the result of defective signalling downstream of TBX1. We have attempted to identify pathways downstream of TBX1 by using a combination of dissection and FACS microarrays, comparing wild type with mutant tissue. For instance Gbx2 expression in the pharyngeal surface ectoderm was shown to be dependent upon TBX1 and in turn Slit2 signalling was affected when both Tbx1 and Gbx2 were deleted from the pharyngeal surface ectoderm [5]. Slit signalling is required for inter-ventricular septation [7]. However, such links cannot fully explain defects seen in Tbx1 +/or Tbx1 -/mouse models. We therefore interrogated our existing data set [8] and identified Cxcl12 as encoding a candidate ligand for transmitting cell non-autonomous effects of Tbx1.
In non-mammalian models, CXCL12, acting via CXCR4 has been shown to be a major player in directing cell migration involving contact inhibition of locomotion (CIL) in NCCs [9,10], and in the zebrafish lateral line it forms a self-directed gradient for cell migration [11][12][13]. In frog, disruption of CXCL12 signalling causes craniofacial defects [14]. In chick, the use of viral interference of CXCR4 activity in NCC (more specifically, the cardiac neural crest) led to various heart defects seen in 22q11.2DS [15]. The authors of this paper proposed that Cxcl12/Cxcr4 are genetically downstream of Tbx1 during pharyngeal NCC development and that reduction of CXCR4 signalling causes misrouting of pharyngeal NCCs in chick.
In this work we test the above hypothesis in mice by examination of Cxcl12 expression in control and Tbx1 mutant embryos, and phenotypic examination of several Cxcl12/Cxcr4 mutants. We show that loss of Tbx1 is associated with reduction in the level of Cxcl12 expression within the pharyngeal surface ectoderm and craniofacial mesoderm. However, Cxcl12 null mutants do not have the 4 th PAA defects (i.e. retro-oesophageal RSA and aortic arch interruptions) or great vessel septation defects typical of Tbx1 haploinsufficiency. Instead we identified apparently duplicated carotids and abnormalities of the subclavian arteries. Internal cardiac defects did include ventricular septal defects (VSDs) and occasional outflow alignment abnormalities, as previously reported, but no outflow tract septation defects were observed. No significant genetic interaction between Tbx1, and Cxcl12 /Cxcr4 heterozygosity was detected in terms of 4 th PAA development. Conditional mutagenesis revealed requirements for expression of Cxcl12 in pharyngeal surface ectoderm and second heart field mesoderm. Conditional mutation of Cxcr4 in endothelial cells recapitulated the Cxcr4 null phenotype whilst conditional mutation of Cxcr4 in NCCs did not recapitulate the neck vessel defects, but partially penetrant VSD was detected. We conclude that, in mice, the CXCL12-CXCR4 pathway is at most a minor player downstream of Tbx1 loss of function phenotypes.

Tbx1 mutation is associated with reduced Cxcl12 expression in the pharyngeal apparatus
Our previous microarray analysis was aimed at identifying signalling processes downstream of TBX1 that might contribute to the Tbx1 loss of function phenotype. Cxcl12 was a product of this screen and its expression in Tbx1 -/embryos was analysed by in situ hybridization. In addition to previously described expression in the outflow tract, we detected Cxcl12 transcripts in the pharyngeal surface ectoderm of E9.0 embryos i.e. at the time when signalling from the pharyngeal surface ectoderm would be most likely to impact the underlying, migrating NCCs ( Fig  1A and 1C). Cxcl12 was expressed to a lesser extent in the rostral mesenchyme. Expression of Cxcl12 was reduced, but not abolished, from these regions in Tbx1 -/littermates (Fig 1C and  1D).
In agreement with previous observations in the chick [15,16], CXCR4 is expressed in cardiac NCCs at E9.5 ( Fig 1E-1F'), predominantly in delaminating cells and at a lower level in the migrating streams of NCCs (labelled with AP2α antibody). Interestingly, the majority of NCCs in the pharyngeal arches (PAs) themselves at E9.5 (as lineage-traced using Pax3-Cre crossed with ROSA26R eYFP homozygotes) did not express CXCR4 (Fig 1G-1H'). In contrast to the chick experiments, NCC migration did not appear to be disrupted in Cxcl12 null mouse embryos as shown by analysis of AP2α and Sox10 expression (Fig 1I-1M). NCCs were localised normally within PAA1-3 in the nulls at E9.5 (compare Fig 1J to Fig 1I) and consistent with this, migrating streams of NCCs also appeared normal (Fig 1L and 1M). Thus there appear to be species differences pertaining to the role of CXCL12 signalling in NCC migration.

Cardiovascular defects in CXCL12/CXCR4 Nulls are not those typical of 22q11.2DS or Tbx1 haploinsufficiency
Previous studies of Cxcl12 and Cxcr4 mutant mice have shown intracardiac defects, predominantly VSDs and abnormalities of the coronary arteries [17][18][19][20][21]. In this study we have focussed upon the cardiovascular defect most specifically associated with Tbx1 haploinsufficiency-the morphogenesis of 4 th PAA derivatives. While IAA-B has been observed in several mouse models, examples of these defects in heterozygous as opposed to homozygous mutants are rare (Chd7 heterozygosity being one example [22]). Defects of fourth arch structures are the most sensitive to reduced Tbx1 dosage [23] and therefore, if CXCL12 is implicated downstream of TBX1 in arch morphogenesis, should be most sensitive to loss of CXCL12 signalling. We also specifically looked for retro-oesophageal RSA, a 4 th PAA defect more frequently observed in Tbx1 heterozygotes than IAA-B [5,22,24], and a common arterial trunk (CAT) which is seen in all Tbx1 null embryos (and some 22q11.2DS patients) [3,4], as well as other previously reported heart abnormalities. Defects are summarized in Table 1.
In summary, while all Cxcl12 and Cxcr4 (not shown) constitutively null embryos had a membranous VSD at E15.5 we found no examples of CAT, IAA-B or retro-oesophageal RSA, nor did we see any examples of pulmonary atresia (part of the tetralogy of Fallot spectrum). However, all Cxcl12 and Cxcr4 null mutant embryos had defects of the subclavian arteries on at least one side, and both sides were affected in the majority of mutants. Importantly, these defects were distinct from the defects observed in Tbx1 mutants. On the right side, the Tissue-specific requirements for CXCL12-CXCR4 signalling during murine cardiovascular development subclavian artery was frequently not visible until sectioned ( Fig 2B, blue arrowhead in 2F and 2G), or where visible lacked arterial branches including the vertebral artery ( Fig 2C). On the left side the subclavian was generally positioned abnormally distally and also lacked arterial branches ( Fig 2B, 2C and 2I). Other vessel defects not reported in Tbx1 mutants were also detected, predominantly ectopic vessels that appeared as duplicated carotid arteries (arrows in Fig 2B, 2C and 2G) or vessels arising from other parts of the aortic arch (arrowhead in Fig 2B). In the more distal part of the LCCs duplication was only evident upon sectioning, which showed two lumen existing within the same structure ( Fig 2G). However, ink injections at E10.5 showed normal formation of the PAAs in Cxcl12 mutants (Fig 2J and 2K). Right-sided arch and alignment defects such as over-riding aorta and double outlet right ventricle (DORV) were also found in Cxcl12 mutants ( Fig 2D, 2M and 2N).
In situ hybridisations in E12.5 embryos showed expression of Cxcr4 in the vertebral artery, a branch of the subclavian artery, and confirmed the absence of this vessel in Cxcl12 mutants ( Fig 2O). As the distal portion of the right subclavian (RSA) and the entirety of the left subclavian (LSA) are derived from the seventh intersomitic arteries (proximal portions of the RSA are derived from the right 4 th PAA and the right dorsal aorta) [25,26], we wanted to analyse the intersomitic arteries in Cxcl12 mutants at E11.5 to see if defective development of these vessels could explain the mutant phenotype. Confocal analysis revealed thinner vessels with fewer anastomoses in the Cxcl12 nulls ( Fig 2P-2Q'). Malformation of the vertebral artery was also observed, along with failure of some anterior intersomitic arteries to regress (Fig 2Q and  2Q'). CXCR4 expression was apparent in the developing intersomitic arteries from E9.5 onwards, (S1A-S1F Fig), with strong expression of Cxcl12 in the surrounding mesenchymal tissue at E11.5 (S1G and S1H Fig, serial sections). However, the intersomitic arteries appeared normal in Cxcl12 mutants at E9.5-10. 5 (not shown) showing that CXCL12/CXCR4 signalling is not required for the formation or early development of these vessels.
While IAA-B, retro-oesophageal RSA and CAT were not observed following loss of Cxcl12 or Cxcr4 we reasoned that it was still possible that diminished CXCL12-CXCR4 signalling might interact with TBX1-dependent developmental pathways. We therefore examined Tbx1 +/-;Cxcl12 +/double heterozygote embryos for evidence of synergistic effect of these alleles upon aortic arch phenotypes ( Table 2, Fig 2R and 2S). No significant interaction was observed. In order to impact the signalling pathway further we examined Tbx1 +/-;Cxcl12 +/-;Cxcr4 +/embryos ( Table 2, Fig 2T and 2U). Simplistically, if there is half the amount of ligand and half Table 1. Cardiovascular anomalies in Cxcl12 and Cxcr4 mutants at E15.5/E18.5.

Lineage specific requirements for CXCL12-CXCR4 signalling
These data suggest that absence of Cxcl12 or Cxcr4 is in itself insufficient to cause defects typical of Tbx1 haploinsufficiency. To support this conclusion and to test whether absence of CXCR4 in NCC contributes to abnormal subclavian artery morphogenesis or other cardiovascular defect(s), we undertook conditional mutagenesis using the well characterised Wnt1Cre driver. Of six Wnt1Cre;Cxcr4 fl/fl embryos examined at E15.5, none had great vessel defects, head/neck vessel defects or VSDs (Table 3). It has been noted that Pax3Cre may be active in a slightly earlier NCC population and can give a phenotype where a Wnt1Cre driver does not [27,28]. Of 17 Pax3Cre;Cxcr4 fl/embryos again none had aortic arch defects, although 4 had a membranous VSD (Fig 3B and 3E). No outflow tract defects were observed (Table 3). No vessel defects or VSDs were found in 8 Pax3-Cre +/controls. Interpretation of the presence of VSDs is somewhat complicated by possible interaction between Pax3 and Cxcr4: Pax3 has a known haploinsufficiency in the NCC and the Cre used is a knock in allele [29][30][31]. In contrast, deletion of Cxcr4 from the endothelial lineage with Tie2-Cre yielded frequent abnormalities of the carotid and/or subclavians (Table 3 and Fig 3C), with defects found in 76% of conditional mutants (n = 21) compared with 18 Tie2-Cre;Cxcr4 fl/+ controls which were all normal. Membranous VSDs (including one over-riding aorta) were found in 57% of Tie2-Cre  Tissue-specific requirements for CXCL12-CXCR4 signalling during murine cardiovascular development conditionals examined (n = 14) ( Table 3, Fig 3F). Thus, allowing for reduced penetrance using this Cre driver [17], loss of Cxcr4 from endothelial cells substantially recapitulated the constitutively null phenotype. We next sought to identify which source(s) of CXCL12 was (were) required for mediating the effects seen following loss of Cxcr4 from endothelial cells. We used Ap2α-Cre [32] to target Cxcl12 in the pharyngeal surface ectoderm (this transgene is also active in the NCC, however a recent study reported an entirely normal cardiovascular system following Wnt1Cre ablation of Cxcl12 [33]). 12 Ap2α-Cre;Cxcl12 fl/fl embryos were examined and 5 showed vessel defects, albeit considerably milder than observed in constitutive Cxcl12 nulls. However, small ectopic vessels emerging from the aorta were present (Table 3, Fig 3H and 3I). None of these embryos had a VSD. No defects were found in a total of 11 littermate control embryos (Ap2α-Cre; Cxcl12 fl/+ and Cxcl12 fl/+ ) examined concurrently. Conversely, of 11 AHFMef2cCre;Cxcl12 fl/fl mutants, 4 had a membranous VSD, but none had a vessel defect (Table 3, Fig 3J and 3L); this Cre is active in the second heart field [34]. Littermate controls AHFMef2c-Cre;Cxcl12 fl/+ (n = 6) and Cxcl12 fl/+ (n = 6) had no defects. Thus, CXCL12 is required in pharyngeal surface ectoderm for aspects of normal neck vessel development and the second heart field for membranous interventricular septation.

Discussion
TBX1 has cell autonomous and non-cell autonomous effects that are essential for normal cardiovascular morphogenesis. Here we addressed the recent hypothesis that a non-autonomous effect on cardiac NCCs via the ligand CXCL12 acting through the receptor CXCR4 has a role in this regard. Certainly, results from other model organisms demonstrate a role for CXCL12-CXCR4 signalling in NCC migration. Of most relevance is work in the chick where dominant negative inhibition of CXCR4 activity in cardiac NCC gave rise to cardiac defects (although no arch artery or aortic arch analyses were specifically mentioned) [35]. Following up on previous work, we established that Cxcl12 expression in pharyngeal surface ectoderm and rostral mesoderm is reduced in the absence of Tbx1. We then asked three main questions with regard to the potential role of the CXCL12:CXCR4 pathway in 22q11DS: to what extent do the defects in CXCL12 signalling mutants recapitulate Tbx1 mutation? Is CXCL12 signalling from the pharyngeal surface ectoderm to the cardiac NCC responsible for heart and vessel  We showed that, in concordance with a recent study [33], while homozygous mutation of either Cxcl12 or Cxcr4 yielded intracardiac defects, we found neither retro-oesophageal RSA, IAA-B, nor CAT (fully penetrant in Tbx1 nulls) in these mice. Previous work has identified that Tbx1 expression in pharyngeal surface ectoderm is required for normal NCC patterning and formation of the fourth pharyngeal arch arteries. However, loss of CXCL12 from the pharyngeal surface ectoderm produced only minor ectopic vessel phenotypes in the mutant embryos, whilst formation of PAAs including the 4 th PAA was normal, and no intracardiac defects were found. Loss of CXCL12 from the AHFMef2c lineage was sufficient to cause intracardiac defects. We therefore conclude that CXCL12 from two (at least) independent sources is required for cardiovascular morphogenesis. The duplicated carotid phenotype and rightsided arch were not extensively recapitulated in these experiments; it may be that the recombination of the conditional allele was insufficiently efficient, or that CXCL12 from remaining source lineage(s) is sufficient to prevent these abnormalities arising. In addition, the Cre drivers used would not be expected to target expression of Cxcl12 in the mesenchyme surrounding the ISAs, accounting for the lack of subclavian defects in the conditional mutants. Of interest, Nkx2.5Cre;Cxcr4 fl/mutants survived normally through adulthood with no apparent deficiencies [36]. As this driver is active in the first and, to a lesser extent, second heart fields, targeting atrial and ventricular endocardium and myocardium, but not most vascular endothelium, this is consistent with our interpretation that multi-source CXCL12 signalling to CXCR4-expressing rostral endothelial cells is required for cardiovascular development.
Murine Tbx1 heterozygosity has been shown to interact with heterozygosity of a number of other genes with a resultant increase in the penetrance and or severity of defects e.g. Fgf8 [6], Wnt5a [37], Smad7 [24], Gbx2 [5], and Chd7 [22]. We were unable to detect any exacerbation of the Tbx1 +/phenotype on a background of Cxcl12 heterozygosity, with or without concomitant heterozygosity for Cxcr4. While a larger study might have uncovered a small effect the triple heterozygote study in particular suggests that down-regulation of the CXCL12-CXCR4 pathway does not have a major role in mediating Tbx1 +/haploinsufficient phenotypes.
Taken together, our data are consistent with a role for CXCL12 signalling from pharyngeal surface ectoderm and mesenchyme to endothelial cells and NCCs via CXCR4, with endothelial cells being the more important cell type in the context of cardiovascular morphogenesis. This is consistent with a recent report that demonstrated a requirement for CXCL12 signalling to CXCR4 positive endothelial cells during development of the lung vasculature (not examined in our study) as well as neck vessel development [33]. Both this study and our own work detected subclavian artery and vertebral artery defects in Cxcl12 null embryos. These arteries have contributions from remodelling ISAs. While ISAs were normal at E9.5, from around E11 onwards we observed thinner intersomitic arteries with fewer anastomoses in Cxcl12 nulls. Thus, the phenotypes we observe in neck vessels are likely to be associated with defective remodelling of the intersomitic arteries rather than PAAs.
There are differences between mouse and chick which might underlie the distinct loss of function phenotypes. In chick, CXCR4 down-regulation by miRNA led to NCC apoptosis which could explain the defects seen in that organism [15]. As right-sided arch was observed in approximately 20% of Cxcl12 null embryos, but in not double or triple heterozygotes; it is feasible that chick is more sensitive than mouse to down-regulation of CXCL12. Speculatively, CXCR7 (or another receptor) could compensate in mouse, but not chick. However, such compensation would not be possible in the Cxcl12 constitutive nulls as ligand would be entirely lacking. More generally, it is now accepted that for several genes mouse knockouts fail to show phenotypes analogous to those seen in fish, frog or chick manipulations [38]. Whether humans are closer to mice or non-mammals is of course untested, but it seems likely the two mammals are more closely related from the developmental perspective. Finally, from the point of view of defective aortic arch morphogenesis in Tbx1 heterozygotes, we note that the relevant signalling mechanism(s) originating in pharyngeal surface ectoderm, downstream of TBX1 and communicated to NCCs remains to be discovered.
In this study animal sacrifice was carried out by exposure to rising levels of carbon dioxide followed by cervical dislocation, or by cervical dislocation followed by decapitation. Animal work was carried out according to UK Home Office regulations under project license number PPL 70 6875 2518. The project licence application was approved by the UCL Animal Welfare and Ethical Review Body (AWERB) before submission to the Home Office.

Histology
Embryonic hearts were dehydrated through an ethanol series, embedded in paraffin wax, sectioned and stained with haematoxylin and eosin using standard methods.

Optical projection tomography
OPT was carried out as previously described [48]. Briefly, E15.5 embryos were dissected out, and after chilling for at least 1-2 hours at 4˚C, the trunks were opened and cartilage from the rib cage was removed. They were then fixed overnight in 4% PFA/PBS and mounted in lowmelting agarose (Life Technologies). Samples were trimmed to remove excess agarose and washed in 100% methanol followed by clearing in BABB. Scanning was undertaken using a Bioptonics OPT Scanner 3001M (MRC Technology, Edinburgh, UK). NRecon software (Skyscan NV) was used for image reconstruction from projections using a back-projection algorithm.

Ink injections
Ink injection was performed on E10.5 embryos fixed in 4% paraformaldehyde overnight at 4˚C by targeting the outflow tact with a microinjection needle filled with Indian ink.

In situ hybridisation
In situ hybridisation was carried out on wholemount embryos or 10μm paraffin sections based on standard methods [49] and using digoxygenin-labelled probes for Cxcl12, Cxcr4 and Sox10 as described previously [5,17]. Paraffin sections were de-waxed (Histoclear, National Diagnostics) and re-hydrated through an ethanol series. Sections were permeabilised using Proteinase K (Sigma) at 20μg/ml for 8 min. This was followed by glycine at 2mg/ml for 5 min, washing in PBS and post-fixing in 4% paraformaldehyde for 20 min. After rinsing in PBS, sections were pre-hybridised in hybridisation buffer (50% de-ionised formamide (Promega), 5xSSC pH 5.0, 50μg/ml yeast tRNA (Sigma), 1% SDS and 50μg/ml heparin (Sigma) for one hour at 70˚C. Probe incubation was carried out at 70˚C overnight followed by washes at 65˚C as follows: 3x 15 min in 50% formamide/5x SSC/1% SDS and 2 x 15 min in 50% formamide/2x SSC. The samples were allowed to cool to room temperature then washed twice for 10 min each in MABT (100mM maleic acid, 150mM NaCl, 0.1% Tween-20, pH 7.5) with 2mM tetramisole hydrochloride (Levamisol, Sigma). Blocking was carried out in 2% Blocking Reagent (Roche Life Science)/5% sheep serum/5% goat serum (in MABT) for one hour at room temperature before incubating with anti-Digoxygenin-AP antibody at 1:2000 (4˚C overnight). After removal of the antibody the sections were washed in MABT/levamisol and equilibrated in alkaline phosphatase buffer (2 x 5 min washes) before staining with BM Purple solution (Roche Life Science).

Immunolabelling
Immunolabelling of 10μ frozen sections (in OCT) was carried out according to standard protocols. Briefly, sections were permeabilised in 0.5% Triton X-100 (Sigma) for 5 minutes, rinsed in PBS twice, then incubated in blocking buffer for one hour at room temperature (PBS/10% BSA/10% goat serum/0.1% Triton X-100) followed by primary antibody in blocking buffer at 4˚C overnight. After 3x 5 minute washes in PBS, secondary antibodies in blocking buffer were applied for one hour at room temperature; DNA was counterstained using DAPI (Sigma). Fluorescent images were captured on a Zeiss Axio Lumar.V12 stereomicroscope.
For whole-mount immunolabelling, hearts were permeabilised in PBST (PBS/0.1% Tween-20), blocked for one hour in PBST/10% goat serum, and incubated overnight at 4˚C with primary antibody in blocking buffer. Hearts were washed several times in PBST (one hour washes) before incubating overnight at 4˚C with secondary antibody. After several PBST washes, the immunolabelled embryos were dehydrated through a methanol series before clearing with BABB.