Mina

The induction of odontogenesis in non-dental mesenchyme combined with early murine mandibular arch epithelium

First and second branchial arches were dissected from mouse embryos of 9-13 days gestational age. The epithelial and mesenchymal components were separated after enzymic digestion. Scanning electron microscopy did not reveal a dental lamina along the dental arches before day 12, after which the lamina was formed locally in the incisor and molar regions. There was no epithelial down-growth in the diastema region. Heterotypic recombinations of mandibular arch and second branchial arch tissues showed that early mandibular arch epithelia, before day 12, have odontogenic potential and can elicit the formation of a dental papilla in non-odontogenic, neural-crest-derived mesenchymal cells of the second arch. However, the mandibular mesenchyme must interact with mandibular epithelium in order to have the competence to induce teeth in non-odontogenic epithelium.

Distinct functions for Bmp signaling in lip and palate fusion in mice

Previous work suggested that cleft lip with or without cleft palate (CL/P) is genetically distinct from isolated cleft secondary palate (CP). Mutations in the Bmp target gene Msx1 in families with both forms of orofacial clefting has implicated Bmp signaling in both pathways. To dissect the function of Bmp signaling in orofacial clefting, we conditionally inactivated the type 1 Bmp receptor Bmpr1a in the facial primordia, using the Nestin cre transgenic line. Nestin cre; Bmpr1a mutants had completely penetrant, bilateral CL/P with arrested tooth formation. The cleft secondary palate of Nestin cre; Bmpr1a mutant embryos was associated with diminished cell proliferation in maxillary process mesenchyme and defective anterior posterior patterning. By contrast, we observed elevated apoptosis in the fusing region of the Nestin cre; Bmpr1a mutant medial nasal process. Moreover, conditional inactivation of the Bmp4 gene using the Nestin cre transgenic line resulted in isolated cleft lip. Our data uncover a Bmp4-Bmpr1a genetic pathway that functions in lip fusion, and reveal that Bmp signaling has distinct roles in lip and palate fusion.

Directing the expression of a green fluorescent protein transgene in differentiated osteoblasts: comparison between rat type I collagen and rat osteocalcin promoters

The osteocalcin (OC) and a 2.3 kb fragment of the collagen promoter (Col2.3) have been used to restrict transgenic expression of a variety of proteins to bone. Transgenic mice carrying a green fluorescent protein (GFP) gene driven by each promoter were generated. Strong GFP expression was detected in OC-GFP mice in a few osteoblastic cells lining the endosteal bone surface and in scattered osteocytes within the bone matrix in long bones from 1-day-old to 6-month-old transgenic animals. Similar findings were noted in the forming tooth in which only individual odontoblasts expressed GFP without detectable expression from the dental pulp. This limited pattern of OC-GFP-positive cells contrasts with the uniform expression in the Col2.3GFP mice in which large proportion of osteoblasts, odontoblasts, and osteocytes strongly expressed the transgene. To assess transgene expression during in vitro differentiation, marrow stromal cell and neonatal calvarial osteoblast cultures were analyzed. The activity of both transgenes was restricted to mineralized nodules but the number of positive cells was lower in the OC-GFP-derived cultures. The different temporal and spatial pattern of each transgene in vivo and in vitro reveals potential advantages and disadvantages of these two transgene models.

Histological Analysis of GFP Expression in Murine Bone

The power for appreciating complex cellular interactions during embryonic development using green fluorescent protein (GFP) as a visual histological marker has not been applied to adult tissues due to loss of GFP signal during paraffin embedding and a high autofluorescent background, particularly in section of bone and bone marrow. Here we demonstrate that the GFP signal is well preserved in frozen sections of adult decalcified bone. Using a tape-transfer system that preserves histological relationships, GFP expression can be related to standard histological stains used in bone biology research. The choice of a dual-filter cube and a strong GFP signal makes it possible to readily distinguish at least four different GFP colors that are distinctly different from the autofluorescent background. An additional advantage of the frozen sections is better preservation of immunological epitopes that allow colocalization of an immunostained section with an endogenous GFP and a strong lacZ signal emanating from a β-gal marker gene. We present an approach for recording multiple images from the same histological section that allows colocalization of a GFP signal with subsequent stains and procedures that destroy GFP. Examples that illustrate the flexibility for dual imaging of various fluorescent signals are described in this study. The same imaging approach can serve as a vehicle for archiving, retrieving, and sharing histological images among research groups.

Region- and stage-specific effects of FGFs and BMPs in chick mandibular morphogenesis

The mandibular processes are specified as at least two independent functional regions: two large lateral regions where morphogenesis is dependent on fibroblast growth factor (FGF)‐8 signaling, and a small medial region where morphogenesis is independent of FGF‐8 signaling. To gain insight into signaling pathways that may be involved in morphogenesis of the medial region, we have examined the roles of pathways regulated by FGFs and bone morphogenetic proteins (BMPs) in morphogenesis of the medial and lateral regions of the developing chick mandible. Our results show that, unlike in the lateral region, the proliferation and growth of the mesenchyme in the medial region is dependent on signals derived from the overlying epithelium. We also show that medial and lateral mandibular mesenchyme respond differently to exogenous FGFs and BMPs. FGF‐2 and FGF‐4 can mimic many of the effects of mandibular epithelium from the medial region, including supporting the expression of Msx genes, outgrowth of the mandibular processes and elongation of Meckels cartilage. On the other hand, laterally placed FGF beads did not induce ectopic expression of Msx genes and did not affect the growth of the mandibular processes. These functional studies, together with our tissue distribution studies, suggest that FGF‐mediated signaling (other than FGF‐8), through interactions with FGF receptor‐2 and downstream target genes including Msx genes, is part of the signaling pathway that mediates the growth‐promoting interactions in the medial region of the developing mandible. Our observations also suggest that BMPs play multiple stage‐ and region‐specific roles in mandibular morphogenesis. In this study, we show that exogenous BMP‐7 applied to the lateral region at early stages of development (stage 20) caused apoptosis, ectopic expression of Msx genes, and inhibited outgrowth of the mandibular processes and the formation of Meckels cartilage. Our additional experiments suggest that the differences between the effects of BMP‐7 on lateral mandibular mesenchyme at stage 20 and previously reported results at stage 23 (Wang et al., [1999] Dev. Dyn. 216:320–335) are related to differences in stages of differentiation in that BMP‐7 promotes apoptosis in undifferentiated lateral mandibular mesenchyme, whereas it promotes chondrogenesis at later stages of development. We also showed that, unlike mandibular epithelium and medially placed FGF beads, medially placed BMP‐7 did not support outgrowth of the isolated mesenchyme and at stage 20 induced the formation of a duplicated rod of cartilage extending from the body of Meckels cartilage. These observations suggest that BMPs do not play essential roles in growth‐promoting interactions in the medial region of the developing mandible. However, BMP‐mediated signaling is a part of the signaling pathways regulating chondrogenesis of the mandibular mesenchyme.

Expression and Function of Dlx Genes in the Osteoblast Lineage

Our laboratory and others have shown that overexpression of Dlx5 stimulates osteoblast differentiation. Dlx5(-/-)/Dlx6(-/-) mice have more severe craniofacial and limb defects than Dlx5(-/-), some of which are potentially due to defects in osteoblast maturation. We wished to investigate the degree to which other Dlx genes compensate for the lack of Dlx5, thus allowing normal development of the majority of skeletal elements in Dlx5(-/-) mice. Dlx gene expression in cells from different stages of the osteoblast lineage isolated by FACS sorting showed that Dlx2, Dlx5 and Dlx6 are expressed most strongly in less mature osteoblasts, whereas Dlx3 is very highly expressed in differentiated osteoblasts and osteocytes. In situ hybridization and Northern blot analysis demonstrated the presence of endogenous Dlx3 mRNA within osteoblasts and osteocytes. Dlx3 strongly upregulates osteoblastic markers with a potency comparable to Dlx5. Cloned chick or mouse Dlx6 showed stimulatory effects on osteoblast differentiation. Our results suggest that Dlx2 and Dlx6 have the potential to stimulate osteoblastic differentiation and may compensate for the absence of Dlx5 to produce relatively normal osteoblastic differentiation in Dlx5 knockout mice, while Dlx3 may play a distinct role in late stage osteoblast differentiation and osteocyte function.

Overexpression of Dlx5 in chicken calvarial cells accelerates osteoblastic differentiation

Our laboratory and others have shown that a homeodomain protein binding site plays an important role in transcription of the Col1a1 gene in osteoblasts. This suggests that homeodomain proteins have an important role in osteoblast differentiation. We have investigated the role of Dlx5 in osteoblastic differentiation. In situ hybridization studies indicated that Dlx5 is expressed in chick calvarial osteoblasts (cCOB) in vivo. Northern blot analysis indicated that Dlx5 expression in cultured cCOBs is induced concurrently with osteoblastic markers. To study the effect of overexpression of Dlx5 on osteoblast differentiation, we infected primary osteoblast cultures from 15‐day‐old embryonal chicken calvaria with replication competent retroviral vectors [RCASBP(A)] expressing Dlx5 or control replication competent avian splice acceptor brianhightiter polymerase subtype A [RCASBP(A)]. Expression of Col1a1, osteopontin, alkaline phosphatase, and osteocalcin messenger RNA (mRNA) occurred sooner and at higher levels in cultures infected with RCASBP(A)DLX5 than in RCASBP(A)‐infected cultures. Mineralization of Dlx5‐expressing cultures was evident by days 12‐14, and RCAS‐infected control osteoblasts did not begin to mineralize until day 17. Dlx5 also stimulated osteoblastic differentiation of calvarial cells that do not normally undergo osteoblastic differentiation in vitro. Our results suggest that Dlx5 plays an important role in inducing calvarial osteoblast differentiation.

Regulation of Mandibular Growth and Morphogenesis

The development of the vertebrate face is a dynamic process that starts with the formation of facial processes/prominences. Facial processes are small buds made up of mesenchymal masses enclosed by an epithelial layer that surround the primitive mouth. The 2 maxillary processes, the 2 lateral nasal processes, and the frontonasal processes form the upper jaw. The lower jaw is formed by the 2 mandibular processes. Although the question of the embryonic origin of facial structures has received considerable attention, the mechanisms that control differential growth of the facial processes and patterning of skeletal tissues within these structures have been difficult to study and still are not well-understood. This has been partially due to the lack of readily identifiable morphologically discrete regions in the developing face that regulate patterning of the face. Nonetheless, in recent years there has been significant progress in the understanding of the signaling network controlling the patterning and development of the face (for review, see Richman et al., 1991; Francis-West et al., 1998). This review focuses on current understanding of the processes and signaling molecules that are involved in the formation of the mandibular arch.

Effects of BMP-7 on mouse tooth mesenchyme and chick mandibular mesenchyme

BMP‐7 is a member of the BMP family of signaling molecules that are thought to play key roles in mediating inductive events during embryogenesis. In the present study the possible roles of BMP‐7 in mediating inductive events during the initiation phase of odontogenesis and mandibular morphogenesis were investigated. To do so, we have examined the effects of agarose beads soaked in recombinant BMP‐7 on E11 mouse molar‐forming mesenchyme and stage 23 chick mandibular mesenchyme, and analyzed the patterns of expression of Bmp‐7 in developing mouse and chick first branchial arches. Beads releasing BMP‐7 induced a translucent zone, cellular proliferation, and expression of Msx‐1, Msx‐2, and Bmp‐4 in molar‐forming mesenchyme after 24 hr. The effects of BMP‐7 on molar‐forming mesenchyme are similar to the effects of BMP‐4 and are consistent with their overlapping patterns of expression in the thickened epithelium of the early developing tooth buds, which is suggestive of cooperative and/or redundant roles of BMPs in mediating the inductive interactions during the early stages of odontogenesis. Our studies in the developing chick mandible showed that Bmp‐7 is expressed in the mandibular epithelium. In the absence of mandibular epithelium, BMP‐7 beads maintained cell proliferation and Msx expression in the medial mandibular mesenchyme and were able to induce cell proliferation, cell death, and Msx expression in the lateral chick mandibular mesenchyme. The effects of BMP‐7 on the expression of Msx genes in lateral chick mandibular mesenchyme, although different from the effects of lateral mandibular epithelium, are similar to the effects of epithelium from the medial region where multiple Bmps are expressed. We also showed that laterally placed BMP‐7 beads induced ectopic expression of Msx genes and changes in the development of posterior skeletal elements in the maxillary and mandibular arches. However, despite its proliferative effects on mandibular mesenchyme, BMP‐7 did not support the directional outgrowth of the mandible. These observations suggest that epithelial–mesenchymal interactions in the medial region of the mandibular arch regulating directional outgrowth of the mandibular mesenchyme are mediated by cooperative interactions between BMPs and other growth factors. Our observations also indicated that EGF, another growth factor implicated in mediating epithelial–mesenchymal interactions in the initiation phase of odontogenesis and morphogenesis of the developing mandible, induces an extensive translucent zone and cellular proliferation in the E11 mouse molar‐forming mesenchyme and stage 23 chick mandibular mesenchyme. However, in contrast to BMPs, EGF did not induce Msx‐1, Msx‐2, and Bmp‐4, but modulated the effects of BMPs on the expression of Msx‐1 and Msx‐2 in these mesenchymes. Our combined data suggest that BMP‐7 is a component of the signaling network mediating epithelial–mesenchymal interactions during the initiation phase of odontogenesis and morphogenesis of the mandibular arch. Dev Dyn 1999;216:320–335. ©1999 Wiley‐Liss, Inc.

Odontogenic epithelium induces similar molecular responses in chick and mouse mandibular mesenchyme

Previous observations have shown that, during the initiation phase of odontogenesis, signals from mouse odontogenic epithelium can elicit teeth in non‐odontogenic but neural crest–derived mesenchyme isolated from ectopic sites including chick mandibular mesenchyme. In the present study the formation of ectopic tooth buds and dental mesenchyme in chick mandibular mesenchyme was examined using heterospecific recombinations between E11 mouse odontogenic epithelium and stage 23 chick lateral mandibular mesenchyme. Both morphological criteria and chick‐specific probes for Msx‐1, Msx‐2, and Bmp‐4 mRNAs were used as markers for early dental mesenchyme. Our results demonstrated that interactions of mouse odontogenic epithelium with chick mandibular mesenchyme induce early changes in the chick mandibular mesenchyme including the appearance of a translucent zone, cell proliferation, and induction of expression of Msx‐1, Msx‐2, and Bmp‐4, which have been shown to be associated with the formation of dental mesenchyme. In addition, tooth bud–like structures that resemble E13 tooth buds in vivo both morphologically and in their patterns of gene expression formed after 6 days in the heterospecific recombinations. The tooth bud–like structures consist of invaginated mouse mandibular epithelium and condensed chick mandibular mesenchyme expressing high levels of Msx‐1 and Bmp‐4, but undetectable levels of Msx‐2. Unlike the induction of Msx‐1, Msx‐2, and Bmp‐4 in the underlying mesenchyme, which is specific for signals derived from odontogenic epithelium, the induction of a translucent zone and cellular proliferation in the underlying mesenchyme may be related to the growth‐promoting potential of embryonic epithelia and not be specific to signals derived from the odontogenic epithelium. Similar to mouse odontogenic epithelium, agarose beads soaked in recombinant BMP‐4 induced a translucent zone, cellular proliferation, and expression of Msx‐1, Msx‐2, and Bmp‐4 in chick mandibular mesenchyme after 24 hours. These observations together showed that avian mandibular mesenchyme has odontogenic potential that is expressed upon interactions with inductive signals from mouse odontogenic epithelium. Similar to odontogenesis in vivo, formation of dental mesenchyme in chick mandibular mesenchyme is mediated by the activation of Msx‐1, Msx‐2, and Bmp‐4. Dev. Dyn. 1998;213:386–397.