Wnt1-Cre mediates Bmpr1a ablation from early stages of neural crest development, resulting in mid-gestational lethality
Our strategy for assessing the role of Bmpr1a
in NCC development relied on the use of a conditional allele of Bmpr1a
(Mishina et al., 2002
) in concert with an established Wnt1-Cre
transgene (Danielian et al., 1998
) to drive recombination specifically in NCCs (Brault et al., 2001
; Chai et al., 2000
; Jiang et al., 2000
). To visualize Cre activity in NCCs, Wnt1-Cre
mice were crossed to a Cre recombination reporter line, R26R (Soriano, 1999
). Recombination by Cre at the R26R locus brings a lacZ
transgene under the irreversible control of a ubiquitous promoter. Embryos carrying R26R
in all cells that express active Cre recombinase, and in the descendents of those cells.
Recombination at the dorsal neural folds of the midbrain/hindbrain junction was visible by 4 s (), reflecting a domain of Wnt1
expression required for midbrain development (McMahon and Bradley, 1990
). The first distinct NCCs were seen along the dorsal neural folds of the hindbrain at the 5 s stage; these are more numerous by 6 s (). Recombination extended into the rostral spinal cord by 8 s (). Transverse sections of Wnt-1Cre; R26R
embryos at ~10 s (E8.5) revealed migration from the neural tube to the pharyngeal arches and other target tissues ( and data not shown). Recombination then extended caudally along the dorsal neural tube to include the entire neural crest and its descendents (). Wnt1-Cre
activity in the neural crest at 5 s coincides with the time at which the first NCCs have been detected by cell labeling studies (Serbedzija et al., 1992
). Thus, Wnt1-Cre
allows recombination of Bmpr1a
in the neural crest from very early stages of its development.
Fig. 1 Wnt1-Cre recombination in the neural crest cells is marked by the expression of β-galactosidase in Wnt1-Cre; R26R embryos. (A,B) At 4 somites, Wnt1-Cre mediates loxP recombination in the forming NCCs of the hindbrain, but not the neural tube. (more ...)
Mice carrying the Wnt1-Cre
transgene and a null allele of Bmpr1a
) were crossed to mice homozygous for a conditional allele of Bmpr1a
; ). One quarter of the resulting embryos are of the genotype Wnt1-Cre; Bmpr1aflox/null
, and are presumably unable to signal through BMPRIA in NCCs and their derivatives: we designate these as ‘mutant’. Simultaneous expression of the Wnt1-Cre
transgene and the Bmpr1a
conditional allele results in recombination at the Bmpr1a
locus as intended (; data not shown). Although we are unable to analyze precisely when BMPRIA ceases to function in any given NCC cell, our experimental system results in the removal of BMPRIA during all but the earliest stages of NCC development. Available antibodies against BMPRIA and the phosphorylated form of SMAD1 (indicating BMP signal transduction) proved ineffective in assessing BMPRIA protein and its output at the single-cell level (R.W.S. and J.K., unpublished). However, several previous studies with this conditional allele in a variety of contexts (Ahn et al., 2001
; Gaussin et al., 2002
; Hebert et al., 2002
; Jamin et al., 2002
; Mishina et al., 2002
) have given no reason to suspect significant residual BMPRIA protein or incomplete Bmpr1aflox
recombination. Thus, although ablation of BMPRIA in the neural crest domain will not necessarily happen before the induction of the first NCCs, which must occur prior to 5 s in the hindbrain, the receptor is likely to be absent well before NCC generation is complete at any axial level. This occurs at 11 s at the earliest location, in the rostral hindbrain (Serbedzija et al., 1992
), by which time expression of Cre has been robust for half a day. Moreover, BMPRIA should be absent in NCCs during their migration to target tissues and their differentiation upon arrival.
Fig. 2 Ablation of Bmpr1a in neural crest cells. We have used the Wnt1-Cre transgene to ablate Bmpr1a activity in mouse NCCs. In the mating scheme outlined here (A), one quarter of the resulting embryos cannot transduce signals through BMPRIA in the neural crest. (more ...)
In dissections from the crosses to produce mutant embryos, Wnt1-Cre; Bmpr1aflox/null embryos were fully represented up to E11.5 (), but did not survive beyond E12.5 (n=0/47; ; ). At E.11.0, mutants looked externally normal and had normal circulation (). By contrast, from E11.5, they showed evidence of severe circulatory defects (n>60). For example, yolk sacs appeared to contain no blood () and embryos displayed pooling of blood in major vessels, heart and liver (). The majority of embryos recovered at E12.5 were clearly necrotic (). Thus, embryos lacking Bmpr1a in NCCs die abruptly with symptoms of acute heart failure around E11.5–12.0.
Survival of Wnt1-Cre; Bmpr1aflox/null embryos
Neural crest cells are specified and differentiate in Bmpr1a neural crest mutants
Mutant embryos showed no deficit in NCC specification or migration from the neural tube, as assayed by expression of the neural crest markers Tcfap2a (AP2α: n=4), cadherin 6 (n=4) and Crabp1 (n=3) at E9.5 and E10.5 (). Initial differentiation also appeared normal; for example, an antibody marking neurofilaments of the peripheral nervous system indicated normal formation of sensory neurons (). By including the recombination reporter R26R in our breeding scheme to ablate Bmpr1a in the neural crest, such that each resulting embryo carries one copy of R26R, we simultaneously mark both NCCs and cells lacking Bmpr1a (though we cannot be certain that any specific labeled cell necessarily lacks Bmpr1a, as the loxP sites in R26R and Bmpr1aflox recombine independently). These NCC-labeled embryos showed that mutant cells achieved a normal distribution at E11.0 and integrated into many differentiating tissues and organs, such as pharyngeal arches and dorsal root ganglia (). Although assessing the ability of mutant NCCs to contribute to each neural crest derivative type is well beyond the scope of this study, it is clear that NCCs devoid of Bmpr1a can contribute normally to many tissues up through the time of embryonic death. Our experiments do not allow us to assess the requirement for BMPRIA during initial neural crest specification; nevertheless, the data show that Bmpr1a is not required in NCCs from early somite stages for any ongoing specification, nor for migration or differentiation into many derivative cell types.
Fig. 3 NCCs develop in BMPRIA NCC mutant embryos. (A,C,E) Wild-type, stage-matched control embryos for Wnt1-Cre; Bmpr1aflox/null mutants (B,D,F). (A,B) Expression of Crabp1 and other neural crest markers showed normal NCC specification in mutants. (C,D) Immunostaining (more ...)
Circulatory defects in Bmpr1a neural crest mutants
The appearance of mutant embryos at E11.5 indicated severe cardiovascular defects. Although we observed a loss of yolk sac circulation, we detected no NCC contribution to extra-embryonic tissues such as the yolk sac in either wild-type or mutant embryos (). Moreover, yolk sac vessels appeared normal, as judged by histological analysis and by expression of the endothelial marker Tie2-lacZ
(). Thus, the effect of NCCs on the yolk sac blood flow is indirect. Embryonic peripheral vascular circulation in the mutants also appeared abnormal at E11.5, but did not show a structural defect in endothelium, as marked by the expression of Tie2-lacZ
or by anti-PECAM1 (). NCCs contribute to the supportive smooth muscle layer surrounding peripheral vasculature (Etchevers et al., 2001
; Waldo et al., 1996
; Willette et al., 1999
). However, immunohistochemistry to label vascular smooth muscle cells in either pharyngeal arch arteries () or outflow tract tissue () showed no differences between wild-type and mutant embryos before the onset of embryonic necrosis. These data strongly suggest that the lack of yolk sac circulation and the frequent pooling of blood in the mutant () is a sign of impending death, secondary to a crucial cardiovascular deficit elsewhere in the embryo, such as inadequate blood flow from the heart.
Fig. 4 Normal vasculature in BMPRIA NCC mutants. (A) No Wnt1-Cre expressing cells were observed in yolk sacs (corresponding embryo in A′). Expression of a Tie2-lacz transgene marking endothelial cells showed no structural defect in mutant yolk sacs (B) (more ...)
Ablation of Bmpr1a in neural crest cells leads to outflow tract defects
NCCs are known to have an essential role in heart development: they migrate along the pharyngeal arch arteries to populate the outflow tract (OFT), where they are required for septation of the truncus arteriosus into the aorta and pulmonary artery (Kirby and Waldo, 1995
). All mutant embryos analyzed (14) showed shortened OFTs and right ventricles as early as the 18 s stage when compared with stage-matched control embryos. Further analysis of mutant embryos carrying the R26R
reporter showed reduced NCC contribution to the truncus arteriosus. This reduced NCC population and right ventricle shortening was observed at all stages examined from E9.0 (, ~18 s) to E11.0 (, 36–40 s). Shortened truncus arteriosus and right ventricle were present in mutant embryos past E11.0/40 s but these were excluded from analysis as the defect in the dying embryos could be a result of overall embryonic necrosis (data not shown). Shortened outflow tract tissue with reduced NCCs also lacked pronounced, well-formed endocardial cushions (). Consistent with reduced NCC immigration, the Tie2-lacZ
transgene marked the endothelium of the distinct aorta and pulmonary artery of wild-type embryos (), but revealed a lack of septation of the truncus arteriosus in mutants (arrow in ). Although the embryos observed at E12.0 were beginning to show signs of necrosis, their apparent lack of endocardial cushion formation is consistent with the findings of reduced NCCs and persistent truncus arteriosus.
Fig. 5 BMPRIA mutant embryos show cardiac outflow tract defects. (A–D) Dissections at E9.5 show mutant embryos (B,D) to have shortened outflow tract and future ventricular tissue (rv and lv indicate future right and left ventricle, respectively; broken (more ...)
We considered that a reduction in NCC migration to the OFT might result in occlusion of pharyngeal arch arteries by ectopic NCCs, which could potentially cause embryonic death at mid-gestation due to insufficient blood flow to the body. It is noteworthy that these mutant embryos also occasionally showed hypoplastic aortic arch arteries at E11.5 ( and data not shown). We tested the ability of the heart, great vessels and aortic arch arteries from Bmpr1a
NCC mutants to allow unimpeded blood flow by injecting ink into the ventricles and following its dispersal; we never detected any blockage or impeded flow (). More importantly, we examined mutant and control embryos by serial histological sections of the entire OFT and pharyngeal arches at E11.5, and never observed blocked or occluded vessels (). Even in a clearly necrotic embryo at E12.5 (), sections through the outflow tract show a lack of septation and pooling of blood cells, but no ectopic tissues creating a barrier to blood flow to the peripheral tissues (). OFT septation defects per se are not necessarily lethal until birth in chicks, mice or humans (Conway et al., 1997
; Kirby and Waldo, 1995
; Mair et al., 1992
), and thus seem unlikely to account for the mid-gestational death we observed. These considerations led us to suspect that although the outflow tract defects might exacerbate circulatory deficiency, the primary problem in Bmpr1a
neural crest mutants lies elsewhere.
Thin ventricular wall in Bmpr1a neural crest mutants
To elucidate the cause of the cardiovascular dysfunction and lethality in embryos lacking Bmpr1a in NCCs, we examined whether there might be defects in cardiac tissues not known to involve NCC contributions. Ventricular structure at E10.5 and E11.0 looked normal in mutant embryos () with formation of both the compact myocardium on the periphery of the ventricle, and the trabecular myocardium adjacent to the ventricular cavity. At E11.5, however, both layers were significantly reduced in mutants, showing a severely thinned compact myocardial wall and little expansion of the trabeculated myocardium relative to wild type (). These defects were seen prior to the onset of embryonic necrosis ().
Fig. 6 Embryos lacking BMPRIA in NCCs show defective ventricular myocardium by the 45–50 somite stage. Transverse sections of ventricles from embryos at E11.0 [36–40 somites (A–D)] and E11.5 [45–50 somites (E–H)]. The (more ...)
Such dramatic changes in the morphology of ventricular tissue could be accomplished via a change in the amount of cell death or cell proliferation. Using the TUNEL assay to mark apoptotic cells in sectioned hearts, we detected no increase in myocardial cell death prior to global necrosis of mutant embryos (data not shown). By contrast, cell proliferation assays, using immunohistochemistry for phosphorylated histone H3 to mark cells in metaphase, revealed a dramatic difference in cell proliferation rates in the ventricular myocardium (). Total myocardial proliferation in mutants was decreased 24% at E10.5 (P<0.001) and 21% at E11.5 (P<0.001), relative to stage-matched control embryos. When proliferating cells were tallied as a percentage of total cells in the ventricular myocardium to calculate a myocardial mitotic index, they were significantly reduced by this measure as well (2.93% in controls versus 1.63% in mutants, P<0.015).
A population of neural crest cell derivatives may colonize the epicardium
The mechanism by which the lack of a gene specifically in NCCs could lead to a deficient ventricular myocardium is unclear. To determine whether there might be a previously unknown NCC contribution to the mouse heart, we used Wnt1-Cre; R26R
embryos to assess the distribution of NCC derivatives in and around the ventricles. Embryos at E9.5 showed a few β-galactosidase-positive cells immediately ventral to the heart and in the epicardium (), a thin mesothelial tissue surrounding the heart. The epicardium derives primarily from cells on the posterior side of the septum transversum, which lies just caudal to the primitive ventricles. The epicardium expands to cover the ventricular surface in an epithelial sheet by E10.0 (Komiyama et al., 1987
). Some of the epicardial cells undergo an epithelial-mesenchymal transition and contribute to several lineages within the heart, including connective tissue and coronary vascular elements (Munoz-Chapuli et al., 2002
). In E10.5 and E11.5 Wnt1-Cre; R26R
embryos, progressively more marked cells were seen in the epicardium, and also in the outer ventricular myocardium (). No labeled cells were ever seen in the epicardium (or elsewhere) of R26R
embryos lacking Cre; furthermore, the same distribution of stained cells is seen in Wnt1-Cre; R26R
embryos regardless of whether they were processed for β-galactosidase activity and then sectioned, or sectioned and then processed for activity (). Together, these data eliminate the possibility that the labeled cells are artifacts of β-galactosidase staining. Although an epicardial NCC lineage has not been documented previously using this transgene, all other Wnt1-Cre
lineages detected in our embryos are identical to those shown in previous studies (Jiang et al., 2000
Fig. 7 NCCs populate epicardium and ventricular myocardium. (A–C) Wnt1-Cre; R26R embryos showed blue β-galactosidase-positive cells (arrowheads) immediately caudal to the heart (abutting the septum transversum) and on the cardiac ventral surface, (more ...)
Importantly, in both wild-type and mutant embryos, most cells of the epicardium do not express the NCC lineage marker; the labeled epicardial population never exceeded 12% in any of our specimens. Moreover, we failed to detect labeled cells in the epicardium of some Wnt1-Cre; R26R embryos, despite equivalent staging, fixation and processing. We suspect the failure to observe these cells in all specimens reflects a technical limitation of our detection procedures.
Like other epicardial cells, a few of these lineage-labeled cells then invade the ventricular myocardium (). Because cells of the epicardium populate the coronary arteries, we examined these vessels in late-gestation Wnt1-Cre; R26R embryos for the presence of labeled cells. We observed labeled cells in the coronary arteries in both whole-mount () and sectioned hearts (). Collectively, this distribution of marked cells suggests that a small population of NCCs migrates to the epicardium, where it integrates into the nascent epicardial epithelium and has similar cell fates as neighboring epicardial cells from other origins.
We conducted additional tests to further evaluate the hypothesis that the labeled epicardial cells derive from the neural crest. First, we assessed the distribution of cells labeled by an independent NCC genetic lineage marker, P0-Cre
(Yamauchi et al., 1999
). P0-Cre; R26R
embryos showed a similar contribution of NCCs to the epicardium and myocardium (). We then considered whether these cells might reflect coincidental activity of Wnt1-Cre
transgenes in some novel domain outside the neural crest, such as the proepicardial organ or epicardium itself. To determine whether these cells could result from ectopic Cre
expression outside the neural crest, we assayed tissue ventral to the neural tube, including the heart and all other thoracic tissues, for Wnt1-Cre
expression by reverse transcription coupled to the polymerase chain reaction (RT-PCR). We found no Cre expression in ventral tissues collected from E9.5 and E10.5 Wnt1-Cre
embryos, covering a wide range of somite numbers (0/37; ). We cannot exclude the possibility that there may be a brief burst of short-lived Cre expression in a temporal window not represented in our samples, although this seems unlikely. Instead, these data taken together strongly suggest that a small population of labeled epicardial and myocardial cells derive from the neural crest.
Among several BMPs expressed in the developing heart, BMP2 and BMP4 are known to be ligands for BMPRIA. BMP2 is expressed in the atrioventricular canal from E9 to E11 (Zhang and Bradley, 1996
). Using a Bmp4-lacZ
transgene (Lawson et al., 1999
), we observed that Bmp4
is expressed at high levels in the parietal pericardium of the thoracic body wall from E9.5 through E11.5 (; data not shown). This suggests that at least one BMPRIA ligand, BMP4, is present in a cell population adjacent to potential receiving cells in the epicardium.
Intact epicardium in Bmpr1a neural crest mutants
We then assessed whether the epicardium might show abnormalities in Bmpr1a
neural crest mutants. Examination of Wnt1-Cre; Bmpr1aflox; R26R
embryos revealed an indistinguishable distribution of labeled cells in the epicardium between mutant and wild-type embryos ( compared with ). This is in contrast to the truncus arteriosus, where there is a striking reduction in NCC population in mutants. Although mutant NCC derivatives populate the epicardium, it is conceivable nonetheless that the epicardium is structurally abnormal in the mutants. Physical delay of migration of cells from the proepicardial organ results in a less extensive epicardial covering of the ventricles; this in turn is associated with reduced myocardial proliferation in underlying tissue (Perez-Pomares et al., 2002
). To analyze the structure of the epicardium, we compared ventricular epicardium in sectioned mutant hearts and control littermates, and found no evidence for a disrupted or patchy epicardium (). Moreover, the epicardial cell density in sections was not significantly different (P
>0.100) between stage-matched mutant and wild-type embryos.
Fig. 8 Epicardium appears normal in BMPRIA mutants. (A,B) NCCs appear in the same limited quantities in mutants as in control embryos at E9.5 (A) and E11.5 (B: compare to ). (C–F) Histological analysis (transverse sections) of the epicardium (more ...)
Although mutant embryos displayed a structurally intact epicardium, we assayed molecular markers of the epicardium to determine whether epicardial gene regulation is generally perturbed in the Bmpr1a
neural crest mutants. In the heart, epicardin is expressed specifically in the epicardium (Robb et al., 1998
). We detected no change in the expression of epicardin relative to wild type (). WT1 is also expressed in the epicardium, but unlike epicardin, has been shown to have an important role in epicardial promotion of myocardial maturation (Kreidberg et al., 1993
; Moore et al., 1999
). Immunohistochemistry for the WT1 protein revealed no change in protein levels or localization between control and mutant embryos (). Thus, the epicardium appears to be largely normal in mutant embryos.
Taken together, these data indicate that cells may migrate from the neural crest to the epicardium, where they are potentially exposed to several BMPs, including BMP4. Loss of Bmpr1a in NCCs results in midgestational defects in the underlying ventricular myocardium similar to those seen in epicardial ablations. The epicardial population of NCCs is distributed normally in mutant embryos, and the epicardium is structurally intact. These results therefore suggest a specific compromise in the proliferative influence of epicardium on ventricular myocardium upon loss of BMPRIA in neural crest derivatives.