Tgfbr2 regulates the maintenance of boundaries in the axial skeleton

Abstract

Previously, we showed that deletion of the TGF-beta type II receptor (Tgfbr2) in Type II Collagen (Col2a) expressing cells results in defects in the development of the axial skeleton. Defects included a reduction in size and alterations in the shape of specific vertebral elements. Anterior lateral and dorsal elements of the vertebrae were missing or irregularly shaped. Vertebral bodies were only mildly affected, but the intervertebral disc (IVD) was reduced or missing. In this manuscript, we show that alterations in the initiation or proliferation of cartilage are not detected in the axial skeleton. However, the expression domain of Fibromodulin (Fmod), a marker of the IVD, was reduced and the area of the future IVD contained peanut agglutinin (PNA) staining cartilage. Next, we show that the expression domains of Pax1 and Pax9, which are preferentially expressed in the caudal sclerotome, are expanded over the entire rostral to caudal length of the sclerotome segment. Dorsal-ventral patterning was not affected in these mice as accessed by expression of Pax1, Pax9, and Msx1. Proliferation was modestly reduced in the loose cells of the sclerotome. The results suggest that signaling through Tgfbr2 regulates the maintenance of boundaries in the sclerotome and developing axial skeleton.

Fig. 1

Effects of Tgfbr2 deletion on Sox9 expression. Sagittal sections from control (A) and Col2acre^+/−;Tgfbr^loxP/loxP (B) E12.5-day embryos were hybridized to an ³⁵S-labeled antisense Sox9 riboprobe. Sox 9 mRNA was detected by autoradiography and is seen as bright white grains on the dark field images shown. The developing vertebrae (V) and lung epithelium (Lu) were stained.

Fig. 2

Effects of Tgfbr2 deletion on the IVD. Sagittal sections from the upper thoracic region of E14.5-day control (A) and Col2acre^+/−;Tgfbr^loxP/loxP (B) embryos were stained with H&E. In the control, the developing vertebrae (V) and intervertebral disc (ivd) were clearly demarcated (A). Cells in the annulus fibrosus of the IVD were spindle shaped in the controls (inset A). Cells in the annulus of Col2acre^+/−;Tgfbr^loxP/loxP embryos had a round morphology (inset B). Ventral is to the top, dorsal to the bottom, rostral to the left, and caudal to the right in figures A and B. Fmod expression was determined by radioactive in situ hybridization to sagittal (C, D) and transverse (E, F) sections through the thoracic vertebrae of control (C, E) and Col2acre^+/−;Tgfbr^loxP/loxP (D, F) mice. Transverse sections were taken through the area of the IVD as denoted by the nucleus pulposus (np). Both bright field (left) and dark field (right) images are shown. Areas of Fmod expression are seen as bright white grains in the darkfield images. Fmod expression is greatly reduced in Col2acre^+/−;Tgfbr^loxP/loxP embryos.

Fig. 3

PNA staining in the E14.5 day axial skeleton. Sagittal sections from the thoracic (A, B) and cervical (C, D) regions of control (A, C) and Col2acre^+/−; Tgfbr^loxP/loxP (B, D) E14.5-day embryos were stained with rhodamine conjugated PNA (red). The nuclei were counterstained with YoPro (green). The merged images are also shown. IVD and vertebrae are denoted with brackets. PNA staining is reduced or excluded from the IVD region in control embryos, whereas PNA stained cartilage is detected in the IVD region of Col2acre^+/−;Tgfbr^loxP/loxP mice. Ventral is to the top, dorsal to the bottom, rostral to the left, and caudal to the right.

Fig. 4

PNA staining in the E12.5 day axial skeleton. Sagittal sections from the thoracic (A–D) and cervical (E, F) regions of control (A, C, E) and Col2acre^+/−; Tgfbr^loxP/loxP (B, D, F) E12.5-day embryos are shown. Subdomains of the sclerotome (brackets, 1–3) were observed in H&E stained sections of control (A) and Col2acre^+/−;Tgfbr^loxP/loxP (B) embryos. Domain 3 was not observed in Col2acre^+/−;Tgfbr^loxP/loxP mice (B). Insets show high magnification images of the area between domains 1 and 3 in control (A inset) and mutant (B inset) mice. (C–F) Sections were stained with rhodamine conjugated PNA (red; C–F). The nuclei were counterstained with YoPro (green). The merged images are also shown. A sharp boundary is observed at the caudal domain of PNA staining in control samples (arrows; C, E). The caudal boundary of PNA staining is blurred in Col2acre^+/−;Tgfbr^loxP/loxP embryos (arrows D, F). Subdomains of the sclerotome (brackets, 1–3) were also defined by PNA staining. Domain 3, a dark area at the caudal end of the PNA staining domain in control mice (C, E merged), was not observed in Col2acre^+/−; Tgfbr^loxP/loxP mice (D, F merged). Ventral is to the top, dorsal to the bottom, rostral to the left, and caudal to the right.

Fig. 5

Role of Tgfbr2 in dorsal–ventral patterning. The expression domains of Pax9 (A, B) and Msx1 (C–F) were determined in control (A, C, E) and Col2acre^+/−; Tgfbr^loxP/loxP (B, D, F) E11.5day embryos by whole-mount (A, B, E, F) or E12.5-day embryos by radioactive in situ hybridization (C, D). Embryos were hybridized to digoxigenin labeled probes for whole-mount in situ hybridization and visualized using a colorimetric assay so that areas of expression are dark purple. Expression is seen as bright white grains on the dark field image for radioactive in situ hybridization. The expression domain of the ventral marker, Pax9, and dorsal marker, Msx1, were similar in control and Col2acre^+/−;Tgfbr^loxP/loxP mice (arrows).

Fig. 6

Effect of Tgfbr2 deletion on the expression of caudal markers. The expression domains of Pax1 (A, B) and Pax 9 (C, D) were determined in control (A, C) and Col2acre^+/−;Tgfbr^loxP/loxP (B, D) E11.5-day embryos by whole-mount in situ hybridization. Areas of expression are seen as dark purple staining. In control embryos, Pax1 and Pax9 mRNAs are preferentially expressed in the caudal domain of the sclerotome (A, C). Expression is expanded rostrally through the entire sclerotome in Col2acre^+/−;Tgfbr^loxP/loxP embryos (B, D). Insets show a close-up of the region near the hindlimb.

Fig. 7

Effect of Tgfbr2 deletion on the expression of rostral markers. Lateral sagittal sections from the thoracic region of E12.5-day control (A) and Col2acre^+/−; Tgfbr^loxP/loxP (B) embryos were stained with H&E and the segmentation of the dorsal root ganglion (drg) in relation to the vertebrae (V) were examined. Drg was segmented properly in control and Col2acre^+/−;Tgfbr^loxP/loxP embryos. The expression domain of Tbx18 (C, D) was determined in control (C) and Col2acre^+/−; Tgfbr^loxP/loxP (D) at E12.5 day by radioactive in situ hybridization. Expression is seen as bright white grains in the dark field images. Tbx18 mRNA is restricted to the rostral domain of the sclerotome in both control and Col2acre^+/−;Tgfbr^loxP/loxP embryos. An area near the hindlimb is shown. Rostral (R) and caudal (C) domains are indicated with brackets. Ventral is to the top, dorsal to the bottom, rostral to the left, and caudal to the right.