Inactivation of Fam20b in the neural crest-derived mesenchyme of mouse causes multiple craniofacial defects

Abstract

The glycosaminoglycan (GAG) chains attached to the core proteins of proteoglycans (PGs) exert multiple roles, such as enriching signal molecules and regulating the binding of ligands to the corresponding receptors. Family with sequence similarity 20 member B (FAM20B), a newly identified kinase, is essential for the formation of GAG chains. FAM20B phosphorylates the initial xylose on the side chain of a serine residue in the protein. Although the GAG chains of PGs are believed to be indispensable during craniofacial development, there were few reports on their exact functions in craniofacial organogenesis. In this study, by mating the Wnt1-cre mice with Fam20b floxed mice (Fam20b^f/f mice) mice, we created Wnt1-Cre;Fam20b^f/f mice in which Fam20b was ablated in the neural crest-derived mesenchyme. The Wnt1-Cre;Fam20b^f/f mice died immediately after birth due to complete cleft palates. In addition to cleft palates, Wnt1-Cre;Fam20b^f/f mice also manifested tongue elevation, micrognathia, microcephaly, suture widening and reduced mineralization in the calvarium, facial bones and temporal-mandibular joint (TMJ) bones. These findings indicate that the PGs formed through the catalysis of FAM20B are essential to the morphogenesis and mineralization of the craniofacial complex.

Proteoglycans (PGs) are made of core proteins and one or several glycosaminoglycan (GAG) chains; the core proteins and GAG chains may perform distinctive functions (1). The GAG chains not only provide physical elasticity and mechanical supports for tissues or organs, but also regulate intracellular signaling by modulating the affinity of growth factors to receptors (2, 3). The synthesis of the GAG chain is initiated by linking a xylose to the serine residue of a core protein via O-linkage (4). After the subsequent addition of two galactoses onto the xylose, the phosphorylation of the xylose is required for the addition of a gluconic acid (GlcA) to form the tetrasaccharide linkage (GlcAβ1-3Galβ1-3Galβ1-4Xylβ1-O-Ser), which connects GAG chains (disaccharide repeats) to core proteins (5). Loss of phosphorylation in the initial xylose leads to a premature termination of the tetrasaccharide linkage, which impaired the assembly of GAG chains (5, 6). Family with sequence similarity 20 member B (FAM20B) is identified as a kinase responsible for phosphorylating the initial xylose in the tetrasaccharide linkage of GAG chains (7, 8). The Fam20b-dependent phosphorylation on the initial xylose is so critical for the formation of tetrasaccharide linkage that the conventional loss-of-function of Fam20b results in embryonic lethality in mouse (9), and severe deformities in the cartilages and bones of zebrafish (10). Although we previously reported that inactivation of Fam20b in oral ectoderm led to the supernumerary incisors along with the compromised enamel (11), the roles of Fam20b in the development of craniofacial mesenchyme remain largely unknown.

All procedures for animal experiments in this study followed the protocol approved by the Animal Care and Use Committee at Dalian Medical University. To create Wnt1-cre;Fam20b^f/f mice, we first mated the Wnt1-cre mice with Fam20b^f/f mice to produce Wnt1-cre;Fam20b^f/+. Then, we generated Wnt1-cre;Fam20b^f/f mice by crossing Wnt1-cre;Fam20b^f/+ mice with Fam20b^f/+or Fam20b^f/f mice. Genotyping was done as previously described (11). We observed that all the Wnt1-cre;Fam20b^f/f mice died immediately after birth. The Wnt1-cre;Fam20b^f/+ littermates showed no apparent difference from wild type littermates. All the Wnt1-cre;Fam20b^f/f mice exhibited a reduction of cranial size (microcephaly) with the particularly shortened mandible (micrognathia) and nose (Fig. 1A&B). As a result of micrognathia, the fronto-naso-mental (FNM) angle in Wnt1-cre;Fam20b^f/f newborns was reduced to 92⁰ (SD=2⁰) compared to the angle of 107⁰ (SD=5⁰; p<0.05) in the normal littermates (Fig. 1C&D). The complete cleft palates were detected in all the Wnt1-cre;Fam20b^f/f newborn mice, which was believed to be the cause of lethality (Fig. 1E&F). Histological analysis further revealed that the tongue position of the E14.5 Wnt1-cre;Fam20b^f/f mouse was higher than in the normal controls, particularly in the posterior portion (Fig. 1G&H). Our previous study showed that when Fam20b was inactivated in the palatal mesenchyme, but not in the mandibular bones by Osr2-cre, the palatogenesis in Osr2-cre;Fam20b^f/f mice was normal (12). Therefore, we speculate that the cleft palates in Wnt1-cre;Fam20b^f/f newborns were most likely secondary to the micrognathia. Taken into account of the tongue elevation, which seemed to have blocked the elevation of the palatal shelves, there was a strong indication that Pierre Robin Sequence had occurred in the Wnt1-cre;Fam20b^f/f mice. At the E14.5, although the mesenchyme of the presumptive Wnt1-cre;Fam20b^f/f tempomandibular joint (TMJ) was evidently condensed, but smaller in size (Fig. 2A&B). The presumptive disc of the Wnt1-cre;Fam20b^f/f TMJ was hard to be distinguished from the underlying condyle, compared to the wild type (WT) controls (Fig. 2A&B). At E16.5, when the disc was separated from the condyle in the WT control, the Wnt1-cre;Fam20b^f/f mouse disc was still connected to the condyle by the sustained fibrous tissues (Fig. 2C&D). In addition, the hypertrophic chondrocytes in the Wnt1-cre;Fam20b^f/f condylar cartilage were almost absent, indicating the maturation of the chondrocytes was impaired by the deficiency of Fam20b. Von Kossa staining showed that the TMJ of P0 Wnt1-cre;Fam20b^f/f mouse displayed not only a decreased mineralization in the condyle, but also a thinner disc although the disk was separated from the condyle (Fig. 2E&F). Since a previous study reported that the abrogation of Fam20b in the mesoderm-derived joint cartilage resulted in over-proliferation, poor differentiation and impaired maturation of the chondrocytes (12), our findings in this study suggested that the differentiation and maturation of the chondrocytes in tempomandibular joint, which were derived from neural crest mesenchyme, were also impaired due to Fam20b deficiency.

Fig. 1.

Craniofacial morphology of Wnt1-cre;Fam20b^f/f mice. The gross lateral views for the heads of P0 wild-type (A) and Wnt1-cre;Fam20b^f/f mice (B), indicating the micrognathia in Wnt1-cre;Fam20b^f/f mice (arrowhead in B). The bone and cartilage staining showed the micrognathia (arrowhead) and the reduced FNM angle (dashed lines) in the P0 Wnt1-cre;Fam20b^f/f head (D) compared with the wild-type head (C). The gross ventral view of the upper jaw exhibited a complete cleft palate in P0 Wnt1-cre;Fam20b^f/f mice (arrows in F) and the integrated palatal shelves in wild-type littermates (E). (G-N) When the palatal shelves in E14.5 wild-type mouse fused together at both anterior (G) and posterior (I), the palatal shelves of Wnt1-cre;Fam20b^f/f mouse still separated from each other at anterior (H) and posterior (J). Similarly, compared with the fused E17.5 anterior (K) and posterior palate (M) in wild-type mouse, both the anterior (L) and posterior palatal shelves (N) of Wnt1-cre;Fam20b^f/f mouse kept vertically. Moreover, the Wnt1-cre;Fam20b^f/f tongue (J, N) was significantly higher than the wild-type tongue (L, M) at both E14.5 and E17.5. Scale bar, 500um. Three Wnt1-cre;Fam20b^f/f mouse heads at P0, E14.5 and E17.5, respectively, with the corresponding littermates were used in each experiment.

Fig. 2.

The defects in Wnt1-cre;Fam20b^f/f TMJ. At E14.5, the wild-type mouse displayed a larger presumptive TMJ condensation (A) and a distinct presumptive disc (arrow in A) compared with Wnt1-cre;Fam20b^f/f mouse (B). At E16.5, the condyle in wild-type mouse separated from the disc (arrow in C) and possessed hypertrophic chondrocytes (H in C), while the Wnt1-cre;Fam20b^f/f condyle was connected to the disc by fibrous tissue (arrow in D) and devoid of hypertrophic chondrocytes. At P0, although separated, the Wnt1-cre;Fam20b^f/f disc (arrow in F) was thinner than that in wild-type (E). As shown in Von Kossa staining, the mineralization in wild-type extended into the condyle (E). In contrast, the mineralization area just surrounded the Wnt1-cre;Fam20b^f/f condyle (F). Scale bar, 200um. Three E14.5 and P0 Wnt1-cre;Fam20b^f/f mouse heads were used, respectively, with their littermates in the corresponding experiments. While four E16.5 Wnt1-cre;Fam20b^f/f heads were employed together with the littermates in the experiments for this figure.

Although microcephaly was evident with a complete penetrance in Wnt1-cre;Fam20b^f/f newborns, craniosynostosis, characterized with the premature closure of cranial sutures, was not observed. On the contrary, in the Wnt1-cre;Fam20b^f/f cranium, the anterior and posterior fontanels were connected together by the broadened sagittal suture (Supplemental Fig. 1A&B). Von Kossa Staining confirmed that the sagittal suture between the Wnt1-cre;Fam20b^f/f parietal bones became enlarged rather than premature closure (Supplemental Fig. 1C&D). Similarly, the mineralized mandibular and palatine bones in the Wnt1-cre;Fam20b^f/f mice showed the reduced volume, although the morphology of tooth germs looked normal compared to the normal controls (Supplemental Fig. 1E&F). These results indicated that the loss of Fam20b could impair the differentiation, maturation and/or mineralization of the osteoblasts during intramembranous ossification.

In this study, we demonstrated that the loss of Fam20b impaired the morphogenesis of mandible, the differentiation and maturation of the osteoblasts and chondrocytes derived from neural crest. Since the bones and cartilages in craniofacial region are derived from the neural-crest mesenchyme (13), it is not surprising that abrogation of Fam20b in the neural crest cells causes the deformities in craniofacial complex. PGs are distributed throughout cell surface, intracellular, pericellular and extracellular spaces (16, 17), and capable of modulating BMP, HH, FGF and Wnt signaling during development through the GAG chains, which sequester and enrich the factors, and/or facilitate the binding of the factors to the receptors (18, 19, 20, 21). Because the inactivation of Fam20b may incapacitate the elongation and assembly of GAG chains in many types of PGs (6, 8), the signaling pathways affected by Fam20b loss in various tissues may be different. Therefore, it is hard to conclude that a specific signaling pathway is affected by the loss of Fam20b in all deformities. Further studies are needed to clarify how Fam20b deficiency results in different types of developmental defects.