Expression of forkhead box transcription factor genes Foxp1 and Foxp2 during jaw development

Abstract

Development of the face is regulated by a large number of genes that are expressed in temporally and spatially specific patterns. While significant progress has been made on characterizing the genes that operate in the oral region of the face, those regulating development of the aboral (lateral) region remain largely unknown. Recently, we discovered that transcription factors LIM homeobox (LHX) 6 and LHX8, which are key regulators of oral development, repressed the expression of the genes encoding forkhead box transcription factors, Foxp1 and Foxp2, in the oral region. To gain insights into the potential role of the Foxp genes in region-specific development of the face, we examined their expression patterns in the first pharyngeal arch (primordium for the jaw) of mouse embryos at a high spatial and temporal resolution. Foxp1 and Foxp2 were preferentially expressed in the aboral and posterior parts of the first pharyngeal arch, including the developing temporomandibular joint. Through double immunofluorescence and double fluorescent RNA in situ hybridization, we found that Foxp1 was expressed in the progenitor cells for the muscle, bone, and connective tissue. Foxp2 was expressed in subsets of bone and connective tissue progenitors but not in the myoblasts. Neither gene was expressed in the dental mesenchyme nor in the oral half of the palatal shelf undergoing extensive growth and morphogenesis. Together, we demonstrated for the first time that Foxp1 and Foxp2 are expressed during craniofacial development. Our data suggest that the Foxp genes may regulate development of the aboral and posterior regions of the jaw.

Keywords: Craniofacial development; Foxp1; Foxp2; Mouse; Pharyngeal arch; Temporomandibular joint.

Figure 1. Complementary patterns of expression between Lhx and Foxp genes in PA1

(A-C) Lateral views of the head of E10.5 embryos processed by whole mount RNA in situ hybridization. (D-O) Coronal sections of the head of an E11.5 mouse embryo processed by RNA in situ hybridization. Only the right half of the face is shown. The anterior-posterior axis, medial-lateral axis, and the dorsal-ventral axis are indicated in panels (A), (D), and (E), respectively. Ey, eye; Md, mandibular arch; Mx, maxillary arch; OC, oral cavity; PA2, second pharyngeal arch; To, tongue. Bar, 0.2 mm.

Figure 2. Expression of Foxp1 and Foxp2 in PA1 at E12.5

Coronal sections of the head of an E12.5 mouse embryo processed by RNA in situ hybridization. Only the right half of the face is shown. The arrow in G points to the faint extension of Foxp2 expression. Developing bone, tendon, and muscle are labeled by Runx2, Scx, and MyoD1, respectively. The dotted boxes in F and G indicate the areas shown in Figure 3. De, dentary bone; LM, lower molar; Ma, masseter muscle; MC, Meckel’s cartilage; Pa, palatine bone; PS, palatal shelf; TgG, trigeminal ganglion; UM, upper molar; Zy, zygomatic arch.

Figure 3. Co-expression analyses for the Foxp genes and the markers of the developing bone, tendon, and muscle at E12.5

(A-L) and (P-U) correspond to the boxed area in Fig. 2F. (M-O) correspond to the boxed area in Fig. 2G. (A-F) and (J-R) are the results of double immunofluorescence. (G-I) and (S-U) are the results of double fluorescent RNA in situ hybridization. Each channel is shown in the first two columns, and then the merged image is shown in the third column. The arrows in (C) and (L) point to the expression of FOXP1 and FOXP2 in the dorsal part of the developing zygomatic arch bone. The arrow in (O) points to the expression of FOXP2 in the palatine bone. The insets in (I) and (U) are the enlargements of the dotted box in each panel.

Figure 4. Expression of Foxp1 and Foxp2 in PA1 at E13.5

(A-R) Coronal sections of the head of E13.5 mouse embryos processed by RNA in situ hybridization. Only the right half of the face is shown. The dotted boxes in (G) and (H) indicate the areas shown in Figure 5. (S-l) Sagittal sections of the head of an E13.5 mouse embryo processed by RNA in situ hybridization. LI, lower incisor; Pt, pterygoid muscle; Te, temporalis muscle; UI, upper incisor. Bar, 0.5 mm.

Figure 5. Co-expression analyses for the Foxp genes and the markers of the developing bone and tendon at E13.5

(A-O) correspond to the boxed area in Fig. 4G. (P-U) correspond to the boxed area in Fig. 4H. (A-F) and (J-U) are the results of double immunofluorescence. (G-I) are the results of double fluorescent RNA in situ hybridization. Each channel is shown in the first two columns, and then the merged image is shown in the third column. The inset in (I) is the enlargement of the dotted box in this panel.

Figure 6. Expression of Foxp1 and Foxp2 in PA1 at E14.5

(A-T) Coronal sections of the head of an E14.5 mouse embryo processed by RNA in situ hybridization. The arrowheads in (P,Q,S) point to the expression in the prospective articular disc of TMJ. Co, condyle; GF, glenoid fossa; LPt, lateral pterygoid muscle; MPt, medial pterygoid muscle.

Figure 7. Expression of Foxp1 and Foxp2 in PA1 at E15.5

Coronal sections of the head of an E15.5 mouse embryo stained with cresyl violet (A,D,G) or processed by RNA in situ hybridization (B,C,E,F,H,I). The arrows in B and H point to the expression of Foxp1 in the tendons. The arrowheads in (H,I) point to the expression in the nascent articular disc of TMJ.