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First published online 26 March 2008
doi: 10.1242/dev.012179

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Requirement for Foxd3 in the maintenance of neural crest progenitors

Lu Teng^1,*,, Nathan A. Mundell^2,4,, Audrey Y. Frist^3,4, Qiaohong Wang¹ and Patricia A. Labosky^1,2,3,4,

¹ Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6058, USA.
² Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, TN 37232-0494, USA.
³ Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN 37232-0494, USA.
⁴ Center for Stem Cell Biology, Vanderbilt University School of Medicine, Nashville, TN 37232-0494, USA.

View larger version (104K): [in this window] [in a new window]   Fig. 1. Generation of a NC-specific deletion in Foxd3. (A) The mouse Foxd3 locus, targeting vector and resulting alleles. Purple box indicates the single coding exon. (B) Southern blot analysis using the 3' probe shown in A with BamHI-digested genomic DNA; 6.5 kb wild-type band and correctly targeted 4 kb band are indicated. (C) Genotyping mice with PCR primers (black arrows in A). PCR product from Foxd3 floxed allele is 220 bp, from wild-type allele 130 bp. (D-K) Foxd3 immunohistochemistry. In a control embryo, NC cells express Foxd3 at 8 dpc in the headfolds (D), at 9.5 dpc in migrating NC (F, black arrow), at 13.5 in cranial nerves (H, white arrow) and in the gut coils (J, white arrow). Foxd3 protein is detected in a few cells in headfolds of a mutant 8 dpc embryo (E, black arrow). By contrast, no Foxd3 expression is detected at 9.5 dpc in the trunk of a Foxd3 mutant embryo (G) or in the cranial nerves (I) or gut coils (K) at 13.5 dpc. Foxd3-expressing ventral interneurons are detected in the neural tube of control and mutant embryos (red arrows in F,G). olf, olfactory epithelium.

View larger version (97K): [in this window] [in a new window]   Fig. 2. Craniofacial dysmorphogenesis, skeletal abnormalities and PA malformations in Foxd3flox/-; Wnt1-Cre mice and embryos. (A) Newborn Foxd3 mutant mice were found with a severe cleft face and palate. (B) Alizarin Red and Alcian Blue staining of the head skeleton and cartilage, lateral and dorsal views. The frontal bone and nasal capsule are missing in mutants and the parietal and interparietal bones are greatly reduced in size. The basioccipital bone and premaxila are present but smaller, and the mandible is shortened and thickened. (C) Side and front views of embryos at 16.5 dpc show these defects at midgestation. (D) Side view of lineage-labeled (R26R) embryos showing the contribution of NC to the face at 13.5 dpc in control and Foxd3 mutant embryos. bo, basioccipital bone; fb, frontal bone; ip, interparietal bone; md, mandible; mx, maxilla; n, nostrils; nb, nasal bone; nsc, nasal capsule; PA1md, mandibular prominence of pharyngeal arch 1; PA1mx, maxillary prominence of pharyngeal arch 1; PA2, pharyngeal arch 2; pb, parietal bone; pmx, premaxilla; t, tongue; wp, whisker pads.

View larger version (135K): [in this window] [in a new window]   Fig. 3. Pharyngeal arch defects in Foxd3flox/-; Wnt1-Cre embryos. (A) β-galactosidase activity as detected by X-Gal staining in control and mutant mouse embryos at 9.5 dpc. PA1 and PA2 are present but smaller in Foxd3 mutants and there is a paucity of cells migrating into PAs 3-4 and into the developing cardiac region (arrowheads in enlarged view below). (B) Scanning electron micrographs of 10.5 dpc embryos showing PA deficiency. Embryos were matched for somite number. (C) Whole-mount antibody staining of neurofilament protein. Cranial nerves are present in mutants but smaller and slightly misdirected. In the trunk (lower panels), the mutant embryo has smaller dorsal root gangia (DRG) and thinner spinal nerves than the control. Note that these embryos have been cleared so that both left and right nerves are visible. (D) Whole-mount in situ hybridization of embryos for Sox10. In Foxd3 mutant embryos, Sox10 mRNA is not detected in the glossopharyngeal (IX) and vagus (X) ganglia, although it is detected at reduced levels in the trigeminal (V), facial (VII) and vestibulocochlear (VIII) ganglia. Sox10 signal is also missing in the foregut of mutants (arrowheads); signal in the otic vesicle (circled) is background. drg, dorsal root ganglion; v, trigeminal ganglion; vii, facial ganglion; viii vestibulocochlear ganglion; ix, glossopharyngeal ganglion; x, vagus ganglion; PA, pharyngeal arch; sn, spinal nerves.

View larger version (114K): [in this window] [in a new window]   Fig. 4. Peripheral and enteric nervous system defects in Foxd3flox/-; Wnt1-Cre embryos. (A-H) β-galactosidase activity detected by X-Gal staining in control and mutant mouse embryos. (A,B) Lateral view of hindlimbs of 14.5 dpc embryos. In control embryos, development of cutaneous nerves is obvious and the developing sciatic nerve is visible (A). None of these structures is seen in Foxd3 mutant embryos (B). (C,D) Dorsal view of the trunk of a control embryo at 14.5 dpc shows the segmented development of spinal nerves radiating out from the developing spinal cord (C). This is not seen in mutant embryos (D). (E,F) Lateral view of a 14.5 dpc embryo shows coils of the developing gastrointestinal tract. In the control embryo, cells of the ENS stain blue on the outside of the gut coils (E). In the Foxd3 mutant embryo, no blue staining of the ENS is visible and the absence of cutaneous peripheral nerves is apparent (F). (G,H) Dissected gastrointestinal tracts from 17.5 dpc embryos. In the control, the entire extent of the gut is ensheathed in NC-derived cells making up the ENS (G). In the mutant, there are no β-gal-positive cells around the outside of the gut (H). Blue staining inside the lumen is background (arrowhead). (I,J) Immunofluorescence for PGP9.5, a protein enriched in neurons, shows the location of the ENS neurons in cross-sections of the intestine in the control embryo (I), whereas no PGP9.5 signal is detected in the mutant (J). PGP9.5 signal is red; sections were counterstained with DAPI (blue) to reveal nuclei. cn, cutaneous nerves; ENS, enteric nervous system; sc, sciatic nerve; sn, spinal nerves; st, stomach.

View larger version (74K): [in this window] [in a new window]   Fig. 5. Deletion of Foxd3 in the NC has subtle infrequent effects on heart development. (A) A section through a lineage-labeled control mouse embryo at 9.5 dpc shows NC entering the developing heart (arrows). Box indicates area enlarged in inset. (B) In a control embryo at 10.5 dpc, NC cells are located in PAs 3 and 4 (arrow) and have migrated into the heart (between arrowheads). (C) At 9.5 dpc, a section through a Foxd3 mutant embryo shows NC entering the developing heart (arrows). Asterisk marks blood inside the heart. (D) In a mutant embryo at 10.5 dpc, very few NC cells are migrating into the heart (arrowhead) and very little NC is detected in PAs 3 and 4 (arrow). (E-H') Corrosion casts (E-H) and corresponding traces (E'-H') of 16.5 dpc cardiac outflow tract and associated vessels. The majority of Foxd3 mutants (13 of 17) are indistinguishable from controls (E,F). A few mutants had a duplicated left carotid artery (3 of 17) (G) and one mutant had a PTA (H). Ao, aorta; bca, brachiocephalic artery; dAo, dorsal aorta; DCA, duplicated carotid artery; lca, left carotid; lsa, left subclavian artery; PT, pulmonary trunk; PTA, persistent truncus arteriosus; rca, right carotid artery; rsc, right subclavian artery; *, ductus arteriosus.

View larger version (102K): [in this window] [in a new window]   Fig. 6. Cell migration, survival and proliferation effects in Foxd3 mutants. (A,B) Sections through lineage-labeled 9.5 dpc control and mutant mouse embryos show that NC cells have migrated ventrally to form the DRG. Samples stained as in Fig. 5. (C,D) A more ventral view of the same region pictured in A and B shows NC populating the foregut in the control embryo (arrow) but halting migration near the dorsal aorta in the Foxd3 mutant (arrowhead). (E-H) Control and mutant DRG contain differentiated neurons and glia at 14.5 dpc as indicated by β III tubulin (Tuj1) and Fabp7 (B-FABP) expression. (I,J) Marked changes in cell death occur in the dorsal spinal cord of 9.5 dpc Foxd3 mutant embryos (arrows). (K-N) Whole 9.0 dpc embryos incubated in LysoTracker Red to indicate dying cells. Note pronounced cell death in the mutant hindbrain and migrating NC (arrowheads). At 10.5 dpc (M,N), the mutant has fewer apoptotic cells in the distal region of the PAs (arrowheads), but increased apoptotic cells in the tail (arrows). The overall loss of PA tissue is apparent. (O-R) Matched sections from control and mutant rostral (O,Q) and caudal (P,R) outflow tracts with lineage label (blue) and pH3 immunostaining (brown) showing reduced NC in the Foxd3 mutant outflow tract. Box indicates area enlarged in inset. Arrowheads in inset indicate pH3-positive NC cells.

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