DEV012179 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Cranial neural crest defects in Foxd3 mutant embryos. Expression of other NC-specific genes in the pharyngeal arches (PAs) uniformly showed reduced expression in PA1 and PA2. (A) Msx1 expression is strong in the distal tip of a control PA1, whereas expression is present but weaker in the smaller PA1 of a Foxd3 mutant embryo. (B) Dlx5 expression is absent from PA2 and reduced in PA1 in mutant embryos. (C) Crabp expression is reduced in mutant embryos; note especially the stream of NC migrating into PA2. (D) Pdgfa expression is also absent from PA2 and reduced in PA1 in Foxd3 mutant embryos. (E) Pdgfc expression is not greatly altered in the nasal prominence of mutant embryos, but in this example PA1 and PA2 are almost completely absent. Expression sometimes appears slightly upregulated in the cleft between PA1 and the nasal prominence (arrowhead). (F) Using Alizarin Red to stain the skeleton highlighted the bones of the inner ear, revealing that the retrotympanic otic process of the squamosal bone was malformed in the mutants. The tympanic ring was present but sometimes slightly misshapen. PA, pharyngeal arch; rtp, retrotympanic otic process; tp, tympanic ring.

• Supplemental Figure S2 -

  Fig. S2. Minimal cardiac neural crest phenotype. (A,B) A transverse section of a control embryo shows a normal septum between the ventricles (A), whereas a frontal section (B) shows the septum (arrow) and both lobes of the developing thymus (arrowheads). (C,D) A transverse section of a Foxd3 mutant embryo shows a normal septum between the ventricles (C), whereas a frontal section (D) shows a normal septum (arrow) but, in contrast to the control, the two lobes of the developing thymus are not symmetrical (arrowheads). Examination of nearby sections revealed that the left lobe was the same size as the control, but was not descended caudally into the normal position (data not shown). Adjacent sections revealed much smaller salivary glands in the mutants, another NC-derived structure (data not shown). Histological analysis of eight mutant and control littermates was performed and no septal defects were observed. (E-H) Whole-mount preparations of intact hearts from 17.5 dpc embryos. Samples shown in F and H are lineage labeled with β-galactosidase activity from the activated ROSA26R locus. The ductus arteriosis is still open as these preparations are embryonic. Control embryos show normal outflow tracts and arrangement of the great vessels (E,F). Foxd3 mutant embryos are indistinguishable from controls. Lineage labeling revealed that NC contributes to the aortic arch and associated vessels.

• Supplemental Figure S3 -

  Fig. S3. Quantification of cardiac NC in control and Foxd3 mutant embryos. Lineage-labeled 12.5 dpc embryos were stained for β-galactosidase activity with X-Gal in whole-mount, processed and embedded in paraffin. Serial sections (12 µm) were processed for phosphohistone H3 (pH3) immunohistochemistry followed by counterstaining with Nuclear Fast Red. Images of matched serial sections encompassing the entire outflow tract were captured and the area of X-Gal staining-positive tissue was quantified using ImageJ software. Representative images are shown in Fig. 6O-R. (A,B) Screen capture of control and mutant outflow tracts with the X-Gal staining-positive area outlined in yellow with ImageJ software. X-Gal staining/pH3 double-positive cells were counted for each section and statistics were calculated using Excel software. (C) Bar chart showing area of NC along the rostral to caudal extent of the outflow tract. Foxd3 mutant NC cells were detectible throughout the length of the outflow tracts and migrated to caudal levels comparable to control embryos. Cardiac defects were not present in the mutant embryos analyzed, suggesting that reduced NC can mediate aorticopulmonary septation if the level of migration is normal. (D) Bar chart showing the average number of proliferating NC cells per area of cardiac NC. No difference was detected in the ratio of proliferating NC in control and mutant embryos. Error bars were calculated as standard error of the mean for all sections containing X-Gal staining-positive cells. (E) The volume of cardiac NC was greatly reduced in mutant embryos (0.7 mm³) as compared with a littermate control (4.0 mm³). Error bars in D and E represent the standard deviation between areas calculated by two unbiased observers. One control and one mutant embryo were analyzed in this detail, but multiple embryos were examined at 12.5 dpc and 11.5 dpc and all followed the same trend (data not shown). (F) Similarly, proliferation was unchanged in the facial mesenchyme of 12.5 dpc embryos. (G,H) Representative images of lineage-labeled facial mesenchyme. Arrowheads indicate X-Gal staining/pH3 double-positive cells.