Fig 1.
Tbx1 and Foxi3 genetically interact for thymus and parathyroid gland development.
(A-B) Whole mount in situ hybridization (WMISH) using an antisense Foxi3 probe on wildtype (WT; A) and Tbx1-/- mouse embryos at E9.5 (B); n = 3 for each genotype. Individual pharyngeal arches, PA1, PA2 and PA3 are indicated and the arrows point to the corresponding arch. (C-H) WMISH using an antisense Tbx1 probe on whole mount and sagittal sections of control (Foxi3+/-; C-E) and Foxi3-/- (F-H) mouse embryos at E9.5 (n = 3 for each genotype). Black arrow indicates the core mesoderm and blue arrow indicates the endoderm. Zoomed in image of the pharyngeal pouch (E, H) next to the blue arrow (D, G), to demonstrate that Tbx1 is still expressed in the control and mutant embryos, respectively. Pharyngeal endoderm is indicated as PE. (I) Table summarizing defects found in each embryo with the genotype listed in the first column on the left. The second column indicates the number of embryos. The rest of the columns indicate the number and percent (in parentheses) with various defects as determined by histological analysis at E15.5. Absent thymus and parathyroid glands were observed in all Tbx1-/-, Foxi3-/- and Sox172A-iCre/+;Foxi3f/f embryos. Abbreviations: interrupted aortic arch type B (IAAB), retro-esophageal right subclavian artery (RRSA) and ventricular septal defect (VSD). (J-K) Bar graphs summarizing parathyroid (J) and thymus (K) defects found in Tbx1+/-, Foxi3+/-, and Tbx1+/-;Foxi3+/- embryos versus controls. Fisher’s exact two tailed test was used to determine significance between defects observed in Tbx1+/- and Tbx1+/-;Foxi3+/- embryos. Defects observed in Tbx1+/-;Foxi3+/- embryos include ectopic or absent parathyroid glands as well as ectopic and hypoplastic or absent thymus glands. (L-S) Histological sections of control (WT) embryos (L-O) and Foxi3-/- embryos (P-S) at E15.5. Foxi3-/- embryo with absent thymus glands and RRSA is shown (Q); IAAB is also present (R) as well as a VSD (S). Abbreviations: right subclavian artery (R. sub), thymus (Thy), aorta (Ao), pulmonary trunk (PT), right atrium (RA), left atrium (LA), right ventricle (RV), left ventricle (LV), ventricular septum (VS). More examples of defects that occurred in other mutant embryos are shown in S1 and S4 Figs.
Fig 2.
Thymus and parathyroid gland defects in Tbx1+/-;Foxi3+/- and Tbx1Cre/+;Foxi3f/f embryos.
(A-F) Transverse histology sections stained with H&E of WT (A-B), Tbx1+/-;Foxi3+/- (C-D) and Tbx1Cre/+;Foxi3f/f (E-F) embryos at E15.5. Abbreviations: thyroid (T) and thymus (Thy). Asterisks in A indicate parathyroid glands. Asterisks in F indicate absent thymus glands. Numbers include: WT, n = 16; Tbx1+/-;Foxi3+/-, n = 13; Tbx1Cre/+;Foxi3f/f, n = 14. Normal parathyroid glands are located adjacent to thyroid glands (A) as compared to when they are not present adjacent to the thyroid glands in mutant embryos (C, E). Thymus glands are located at the branchpoint between the innominate and right carotid artery (B) but are more rostrally located and smaller (D) or absent (E) in mutant embryos. (G-I) RNAscope in situ hybridization with mRNA probes for Gcm2 (green) and Foxn1 (red) probes on sagittal sections in WT (G), Tbx1+/-;Foxi3+/- (H) and Tbx1Cre/+;Foxi3f/f (I) embryos at E11.5. Gcm2 marks parathyroid gland precursor cells and Foxn1 marks thymus gland precursor cells; n = 3 for each genotype. (J) Quantification of RNAscope experiments. Number of mRNA signal dots was quantified in proportion to number of cells present in each tissue section. P-values were determined using the t-test. Two stars indicate a P-value <0.005 and one star indicates a P-value <0.05. (K-L) H&E coronal histology sections of Tbx1Cre/+;Foxi3f/+ (K), and Tbx1Cre/+;Foxi3f/f (L) embryos at E10.5. Arrows indicate the third pharyngeal pouch and morphology defects. Third pouch is absent in L. Tbx1Cre/+;Foxi3f/+, n = 6 and Tbx1Cre/+;Foxi3f/f, n = 8.
Fig 3.
Foxi3 and Tbx1 expression in embryonic pharyngeal pouches and Tbx1Cre and Sox172A-iCre lineages.
(A-D) RNAscope in situ hybridization with mRNA probes for Foxi3 and Tbx1 on coronal sections from a WT embryo at E9.5. Tbx1 expression is shown in red and Foxi3 expression is shown in green. Abbreviations: Pharyngeal endoderm (PE); pharyngeal pouch 2 (PP2); pharyngeal cleft 2 (PC2); pharyngeal pouch 3 (PP3); pharyngeal cleft 3 (PC3); and cardiopharyngeal mesoderm (CPM). Total of n = 3 embryos. (E-H) Lineage tracing of Tbx1Cre/+;Rosa26GFPf/+ embryos. GFP expression is visible in whole mount embryos and the corresponding coronal sections at E8.5 (E-F) and at E9.5 (G-H); n = 3 for each genotype and stage. (I-L) Lineage tracing of Sox172A-iCre/+;Rosa26GFPf/+ embryos. GFP expression is visible in whole mount embryos and the corresponding coronal sections at E8.5 (I-J) and E9.5 (K-L); n = 4 for each genotype and stage.
Fig 4.
Inactivation of Foxi3 within the Tbx1Cre/+ lineage disrupts PA3 morphogenesis.
(A-R) DAPI (blue), E-cadherin (green), and ZO-1 (red) antibodies were used for immunofluorescence on coronal sections from embryos at E8.5-E9.0 (n = 3, each), E9.5 (n = 5) and E10.5 (n = 4) in Tbx1Cre/+;Foxi3f/+ (A-D, O-P) or WT (I-K) controls and in Tbx1Cre/+;Foxi3f/f (E-H, L-N, Q-R) mutant embryos. White boxes indicate the location of the higher magnification images. White arrows in E10.5 images (P, R) indicate 3rd pharyngeal pouch in control and absent 3rd pharyngeal pouch in mutant embryos.
Fig 5.
PA segmentation in wildtype, Tbx1 and Foxi3 mutant embryos.
(A-F) To visualize epithelial cells within the PA, DAPI (blue), E-cadherin (green), and ZO-1 (red) antibodies were used for immunofluorescence on WT (A), Tbx1-/- (B), and Foxi3-/- (C) coronal sections at E9.5 (WT, n = 5; Tbx1-/-, n = 4 and Foxi3-/-, n = 5). White boxes (A) indicate where the region was magnified (D-F). (D) Second pouch and cleft where segmentation is complete and epithelial cells formed a dual intercalated layer. (E) Third pouch and clefts are forming at E9.5. The epithelium is stratified. (F) Location of future PA4-PA6, where invagination is initiated, as can be observed by cell projections from the epithelia. PE indicates the pharyngeal endoderm and Ect. indicates the ectoderm for each section. (D’-F’) Cartoon illustrating the segmentation process from images in D-F. (G-R). DAPI (blue), E-cadherin (green), and ZO-1 (red) antibodies were used for immunofluorescence to visualize epithelial cells within the PA in coronal sections from WT E8.5 (G-H), E8.75 (I-K), E9.0 (L-M), E10.0 (O-P), E10.5 (Q-R) embryos. White boxes indicate the location of higher magnification images. PE indicates pharyngeal endoderm; n = 4 for each stage.
Fig 6.
Foxi3-/- and Sox17-2A-iCre/+;Foxi3f/f mutant embryos have defects in PA segmentation.
(A-R) DAPI (blue), E-cadherin (green), and ZO-1 (red) mark epithelial cells on coronal sections from WT (A-F), Foxi3-/- (G-L) and Sox172A-iCre/+;Foxi3f/f (M-R) embryos from E8.5-E10.5 (n = 4, each). White boxes indicate the location of higher magnification images. White arrows indicate where invagination occurs in the PE and ectoderm. (S-T) Quantification of epithelial cells within the PA at E9.5 in WT controls and mutant embryos. Error bars represent standard error. One asterisk indicates P-value <0.05; two asterisks indicate P-value <0.005 (n = 4, each). (U-V) Quantification of proliferation assays includes counting E-cadherin positive epithelial cells within the PA and calculation of the mitotic index, which is the ratio of pH3 positive cells within the E-cadherin positive epithelial cells. Error bars represent standard error, and asterisks indicate P-value <0.05. Pharyngeal endoderm (PE) and ectoderm (Ect) were quantified separately in conditional mutant embryos. At E8.5 and E9.5 a total of n = 6 and n = 4, were examined, respectively, for control and mutant embryos. Controls include WT, Foxi3+/-, and Sox172A-iCre/+;Foxi3f/+ littermates.
Fig 7.
Expression of Notch pathway genes Jag1, Hes1, and Hey1 were downregulated in Foxi3 and Tbx1 null mutant embryos.
(A-I) WMISH with antisense Jag1, Hes1, and Hey1 mRNA probes on whole mount embryos at E9.5. Jag1 antisense probe on WT (A), Foxi3-/- (B) and Tbx1-/- mutant embryos (C); n = 3 of each genotype. Hes1 probe on WT (D), Foxi3-/- (E) and Tbx1-/- (F) mutant embryos; n = 2–3 for each genotype. Hey1 probe on WT (G), Foxi3-/- (H) and Tbx1-/- (I) mutant embryos; n = 2, each. (J-X) RNAscope in situ hybridization with mRNA probes for Jag1, Hes1, and Hey1 on coronal sections of WT (J-N), Foxi3-/- (O-S), and Tbx1-/- (T-X) embryos at E9.5; n = 3, each genotype. Merged channels are shown (J, O, and T); DAPI is shown in blue color (K, P, and U); Jag1 mRNA expression is in green (L, Q, and V); Hes1 mRNA expression is in red (M, R, and W); and Hey1 expression is in white (N, S and X). The sections correspond to the same rostral-caudal location within in each embryo. (Y) Quantification of RNAscope experiments on WT versus Tbx1-/- and Foxi3-/- embryos at E9.5. Nuclei and mRNA were quantified for each section and probe. The total number of mRNA signal dots was divided by the total number of cells for each replicate. Graph represents the average ratio. Asterisks indicate P-values < 0.05.
Fig 8.
Alcam and Fibronectin expression is reduced in epithelial cells within the PA at E9.5 in Foxi3-/-, but not in Tbx1-/- embryos.
(A-H) DAPI (blue), Alcam (green) and Ephrin b2 (red) antibodies were used to examine coronal sections in embryos at E9.0. Alcam localizes to the basal side of epithelium and Ephrin b2 localizes to the apical side. Sections from Foxi3+/- control embryos are shown with all three fluorescence color channels (A) and only DAPI and Alcam (B). Sections from Foxi3-/- embryos are shown with all three channels (C) and only DAPI and Alcam (D). Sections from WT littermates of Tbx1-/- embryos are shown with all three channels (E) and only DAPI and Alcam (F). Tbx1-/- embryos are shown with all three channels (G) and only DAPI and Alcam (H). White arrows indicate where Alcam expression is reduced in Foxi3-/- mutant embryos, but is present in Tbx1-/- embryos in comparison to control embryos. (I-P); DAPI (blue), E-cadherin (E-cad, green) and Fibronectin (Fn1, red) antibodies mark epithelial cells and the intracellular matrix on coronal sections at E9.5. (I-J) Sections from Foxi3+/- control embryos shown with all three fluorescent channels (I) and only the red and blue channels (J). (K-L) Sections from Foxi3-/- embryos showing all three channels (K) and only the red and blue channels (L). White arrows indicate where expression is spotty and inconsistent in Foxi3-/- mutants. (M-P) Sections from WT (M-N) and Tbx1-/- embryos (O-P) showing all three channels (M and O) and only the red and blue channels (N and P). White arrow in N and P indicates where Fibronectin expression is increased in Tbx1-/- mutants. n = 3 for each genotype in each experiment.
Fig 9.
Summary cartoon of Tbx1 and Foxi3 functions in PA segmentation.
Cartoon of epithelial cells during PA segmentation for each arch. Tbx1 and Foxi3 are co-expressed where segmentation to individual pharyngeal arches occurs. When segmentation of the distal PA begins at E8.5, invagination of E-cadherin expressing cells (dark gray) is initiated and a partially stratified multilayer of epithelium forms by E8.75-E9.0, near the point where invagination continues. Tbx1 acts upstream of Foxi3 to promote proper invagination as indicated. The outermost cell layer maintains apical/basal polarity as depicted by ZO-1 expression (red), while the inner layers do not. The epithelial cells begin to project towards each other as shown. Next, a multilayer is juxtaposed where invagination has advanced, and a zippering process ensues from the center of the region, where cells are reorganized. Finally, a dual-layer of intercalated epithelial cells are formed at the mature pouch-cleft junction and both arches are separated. Foxi3 appears to inhibit excess proliferation of PE cells early, while Tbx1 doesn’t alter proliferation. In Tbx1 null mutant embryos more cells are present in the shortened PA. Inactivation of Tbx1 or Foxi3 results in the appearance of excessive layers of endoderm cells, in particular, where invagination is initiating. Therefore, these genes might both promote invagination and restrict excessive multilayers during PA segmentation. We found that Tbx1 and Foxi3 may act in the same pathway upstream of some Notch pathway genes, Pax8 and Pax9, as well as Fgf3. Some of these genes might be required pharyngeal segmentation. It is previously known that Tbx1 and Foxi3 act upstream of Fgf8. While loss of Foxi3 resulted in reduction of Alcam and Fibronectin expression in the extracellular matrix, global loss of Tbx1 did not have the same role. Thus, this data explains, in part, the basis of the genetic interaction between the two genes.