Conditional Deletion of the Foxd3 Locus in NC
A targeting vector was designed so that after homologous recombination in ES cells the Foxd3 coding region ( purple box) would be flanked with loxP sequences ( orange triangles). Correctly targeted ES cells were used to generate mice carrying a Foxd3flox allele. Mice carrying two Foxd3flox alleles (Foxd3flox/flox mice) appeared normal and healthy. To test whether Cre-mediated deletion of the Foxd3 locus produced a null allele efficiently, we crossed Foxd3flox/flox mice with Foxd3flox/+; EIIa-Cre mice. Timed pregnant females from this cross were dissected at 6.5 days post coitum (dpc) and 25% of the resulting embryos had a phenotype identical to the original loss of function allele (data not shown). There was no difference in results observed with or without the neo selection cassette (data not shown).
To selectively delete Foxd3
in the NC, we used the well-characterized Wnt1-Cre
transgenic mouse line in which Cre recombinase is expressed in migrating NC starting at 8.0–8.5 dpc (Danielian et al., 1998
). We used reciprocal matings between Foxd3flox/flox
mice to obtain Foxd3flox/−
(mutant) embryos. At the four-somite stage (~8.0 dpc), Foxd3 protein is detected in migrating NC in the head folds (). In contrast, we see a severe reduction in Foxd3 protein in head folds of mutant embryos, detecting only one or two cells that are weakly positive for Foxd3 protein expression (, arrow). By 9.5 dpc, no Foxd3 protein is detected in the mutant dorsal neural tube or migratory path of NC (compare ). Foxd3 expression is not maintained in the facial mesenchyme at 13.5 dpc, expression is limited to forming cranial ganglia () and this expression is missing in mutant embryos. In the ENS, Foxd3 protein is expressed in the gut coils at 13.5 dpc and is missing in mutant embryos (). These results demonstrate that Foxd3
is deleted specifically in the NC by Wnt1-Cre.
Maintenance of Cranial NC is Dependent on Foxd3
Initially, litters were allowed to progress to term and no mutant mice were found at weaning (21 days). However, we found one dead mutant newborn pup (pictured in ) and began monitoring litters before the mice were born (). While the majority of 18.5 dpc littermates appeared normal and could survive being delivered by a caesarian section, the mutant mice attempted to breathe several times but then expired. summarizes the number of offspring and embryos from both Foxd3flox/+ × Foxd3+/−; Wnt1-Cre and Foxd3flox/flox × Foxd3+/−; Wnt1-Cre crosses. Embryos were found in the expected ratios at all times in development and no mutant mice survived more than a few hours.
Craniofacial dysmorphogenesis, skeletal abnormalities and PA malformations in Foxd3flox/−; Wnt1-Cre mice and embryos
All mutant mice had a severe cleft face and palate incompatible with survival and their inability to breathe is presumably the cause of their death immediately after birth. These defects occurred with 100% penetrance although the width of the cleft sometimes varied in size. Nostrils and whisker pads were present but the facial midline never fused and the eyelids were partially open. Much of the skull vault is derived from cranial NC so we analyzed skeletons using Alcian Blue and Alizarin Red staining. Comparison of the control and mutant skulls in revealed that the frontalnasal bone and nasal cartilage were missing in the mutants and the interparietal, parietal and basal occipital bones were greatly reduced in size. The maxilla and premaxila were shortened and the mandible was shortened and thickened. These cranial defects were obvious at midgestation stages ().
To monitor NC and their derivatives, we introduced the ROSA26R (R26R)
conditional reporter allele into the mutant background (Soriano, 1999
). Upon expression of Cre protein, all cells and their descendants transcribe the lacZ
gene, and therefore NC cell fate can be monitored directly with and without Foxd3 function. Note that we are comparing Foxd3flox/−
; Wnt1-Cre; R26R
(mutant) embryos to Foxd3flox/+
; Wnt1-Cre; R26R
(control) embryos so that the Cre protein will be recombining two sets of loxP sites; one to delete the Foxd3
coding region and one to delete the stop sequence in the ROSA26
locus. Xgal-positive tissue is present in the frontalnasal mesenchyme in both control and mutant embryos indicating that NC cells contribute to these tissues in the absence of Foxd3 (, Sup. ).
Pharyngeal Arch Defects in Foxd3flox/−; Wnt1-Cre embryos
Pharyngeal Arch Defects
At 9.5 dpc, a comparison of lineage-mapped embryos revealed that NC have migrated into pharyngeal arches (PA) 1 and 2 in both mutants and controls (). However, by 10.5 dpc morphological differences in the PAs were apparent by SEM (). The maxillary and the mandibular prominences of PA1 were present but reduced in size. There is a striking decrease of NC in PA 3 and 4. Immunohistochemistry for Neurofilament protein showed all cranial ganglia and nerves are present but smaller than normal. Some cranial nerves are slightly misdirected (the glossopharyngeal nerve in ) or have not extended as far as the control (the facial and vestibulocochlear nerves). Spinal nerves along the trunk of the mutant embryos were thinner. Examining expression of Sox10, a marker of early NC, revealed reduced expression in the developing trigeminal, facial and vestibulocochlear ganglia and no expression in the glossopharyngeal and vagus ganglia. Sox10 expression was also reduced along the trunk of the embryo (). Expression of other NC specific genes in the PAs uniformly showed reduced expression in PA 1 and 2 (Sup. ). Together, these data suggest that the initial specification of NC occurs, but there is an overall failure to maintain NC progenitors as demonstrated by a loss of PA-derived structures and a concomitant decrease in expression of NC specific genes.
Reduction of Peripheral Nervous System and Loss of Enteric Nervous System
NC migrate into the limbs during development giving rise to neurons, Schwann cells and terminal glia cells. Cutaneous nerves and the developing sciatic nerve are highlighted in a hindlimb from a 14.5 dpc control embryo by Xgal lineage staining (). All of these NC-derived cells are missing in the mutant embryos (). A dorsal view of the same pair of embryos reveals the segmented pattern of spinal nerves exiting the dorsal root ganglia (DRG) and projecting ventrally in the control (); the spinal nerves are absent in mutant embryos ().
Peripheral and Enteric Nervous System Defects in Foxd3flox/−; Wnt1-Cre embryos
NC that migrate ventrally from the dorsal neural tube contribute to the ENS: the neurons and glia that innervate the entire gastrointestinal tract. Using the ROSA26R reporter allele, NC contribution to the ENS is easily visualized. In control midgestation embryos (14.5 dpc) the intestines are ensheathed in a plexus of NC-derived Xgal-positive cells while in a mutant littermate the gut is completely lacking these cells (). When the gastrointestinal tract is dissected out of a 17.5dpc embryo, the control sample is surrounded in X-gal-positive cells along the entire anterior to posterior axis while the mutant sample has no NC-derived cells around the outside of the gut tube (). Expression of PGP9.5 is readily detectable in neurons of the ENS in control embryos; there are no PGP9.5-positive cells in mutant embryos (). Similar results were obtained with Tuj1 antiserum to detect neurons and FABP7 antiserum to detect glia (data not shown).
Subtle Cardiac Neural Crest Defects
NC from the mid-otic placode to somite 4 migrate through rhombomeres 6–8, into PAs 3, 4 and 6, contributing to the aortic arch arteries and the aorticopulmonary septum of the cardiac outflow tract. In control embryos, NC enter PAs 3,4 and 6 () and the developing heart field by 9.5 dpc ( arrows) and continue migrating at 10.5 dpc (). We observed a striking decrease in the cardiac NC of mutant embryos. Fewer cells entered the heart field at 9.5 dpc (). At 10.5 dpc, there is a paucity of NC in mutant PAs (, arrow) and greatly reduced number of cells entering the heart (, arrowhead).
Deletion of Foxd3 in the NC has subtle infrequent effects on heart development
To further investigate development of the cardiac NC we examined patterning of vascular NC derivatives using corrosion casting (). We observed normal patterning of the aortic arch in the majority of Foxd3
mutants (13/17, 76%) between 15.5 dpc until birth. However, in 3 out of 17 Foxd3
mutant embryos examined, we observed a duplication of the left common carotid artery (). One mutant embryo had severe cardiac NC defects including type A2 persistent truncus arteriosis (PTA) in which the septation of the outflow tract did not occur and the ductus arteriosis was absent () (Van Praagh and Van Praagh, 1965
). We examined the NC lineage in both control and mutant embryos with the ROSA26R
reporter allele and saw no differences in NC contribution to the cardiovascular system at 17.5 dpc (n = 7 mutants, Sup. ). Histological analysis of eight mutant and control littermates was performed and no septal defects were observed (Sup. ), however, while in the process of performing resin casts, 1/17 (the sample shown in ) displayed a ventricular septal defect.
NC Cell Migration, Differentiation, Death, and Proliferation
NC must maintain a delicate balance of proliferation and migration, and timing of differentiation is crucial for their final tissue contributions in the adult. Lineage tracing revealed that DRGs were formed () but were smaller than normal. The size of the DRGs varies with the angle of section, but in whole mount, mutant DRGs are consistently smaller than controls (). However, a more ventral field of the same region revealed that while control NC enter the foregut to eventually populate the ENS (), mutant NC are fewer in number and halt their migration near the dorsal aorta in a position consistent with the final location of sympathetic ganglia (). No mutant NC enter the developing gut anywhere along the anterior-posterior axis.
Cell migration, survival and proliferation effects
Terminal differentiation of NC occurs as cells reach their final destination. We detected expression of neural and glial markers in control and mutant DRGs. At 9.5 and 14.5 dpc, the neural marker beta III tubulin and the glial marker FABP7 (also called B-FABP) are expressed in both control and mutant DRGs ( and data not shown).
We examined cell death in control and mutant embryos and detected an increase in TUNEL-positive apoptotic cells in the dorsal spinal cord while there were few, if any, TUNEL-positive cells in the spinal cord of control embryos (). Confirming this with LysoTracker® Red dye, at 9.0 dpc, mutant embryos showed increased apoptosis in the hindbrain and by 10.5 dpc, apoptosis was more pronounced in the posterior tail of the embryo (). The absence of apoptosis in distal PAs of mutant embryo at 10.5 dpc () is likely due to the large deficit in NC cell numbers (note morphology of PAs in and lineage label in ).
To more closely examine the curious recovery of most of the cardiac derivates we analyzed cell proliferation in the cardiac NC using a combination of lineage labeling and immunohistochemistry for phosphorylated histone H3 protein indicating cells in mitosis. Septation is complete by 13.0 dpc so we chose to examine embryos at 12.5 dpc (Hiruma et al., 2002
). Representative sections of 12.5 dpc control and mutant embryos are shown in clearly demonstrating the paucity of NC present in the outflow tract at most rostral and caudal levels. The volume of the outflow tract control and mutant embryos was calculated as 4 mm3
and 0.7 mm3
, respectively. The proportion of pH3-positive NC cells was not different between controls and mutants (details in Sup. ). These results suggest that although loss of Foxd3 reduced the number of cardiac NC, these cells are still able to pattern the outflow tract and give rise to smooth muscle cells.