R-Spondin 3 Regulates Mammalian Dental and Craniofacial Development.

Development of the teeth requires complex signaling interactions between the mesenchyme and the epithelium mediated by multiple pathways. For example, canonical WNT signaling is essential to many aspects of odontogenesis, and inhibiting this pathway blocks tooth development at an early stage. R-spondins (RSPOs) are secreted proteins, and they mostly augment WNT signaling. Although RSPOs have been shown to play important roles in the development of many organs, their role in tooth development is unclear. A previous study reported that mutating Rspo2 in mice led to supernumerary lower molars, while teeth forming at the normal positions showed no significant anomalies. Because multiple Rspo genes are expressed in the orofacial region, it is possible that the relatively mild phenotype of Rspo2 mutants is due to functional compensation by other RSPO proteins. We found that inactivating Rspo3 in the craniofacial mesenchyme caused the loss of lower incisors, which did not progress beyond the bud stage. A simultaneous deletion of Rspo2 and Rspo3 caused severe disruption of craniofacial development from early stages, which was accompanied with impaired development of all teeth. Together, these results indicate that Rspo3 is an important regulator of mammalian dental and craniofacial development.

Anti-osteogenic function of a LIM-homeodomain transcription factor LMX1B is essential to early patterning of the calvaria.

The calvaria (upper part of the skull) is made of plates of bone and fibrous joints (sutures and fontanelles), and the proper balance and organization of these components are crucial to normal development of the calvaria. In a mouse embryo, the calvaria develops from a layer of head mesenchyme that surrounds the brain from shortly after mid-gestation. The mesenchyme just above the eye (supra-orbital mesenchyme, SOM) generates ossification centers for the bones, which then grow toward the apex gradually. In contrast, the mesenchyme apical to SOM (early migrating mesenchyme, EMM), including the area at the vertex, does not generate an ossification center. As a result, the dorsal midline of the head is occupied by sutures and fontanelles at birth. To date, the molecular basis for this regional difference in developmental programs is unknown. The current study provides vital insights into the genetic regulation of calvarial patterning. First, we showed that osteogenic signals were active in both EMM and SOM during normal development, which suggested the presence of an anti-osteogenic factor in EMM to counter the effect of these signals. Subsequently, we identified Lmx1b as an anti-osteogenic gene that was expressed in EMM but not in SOM. Furthermore, head mesenchyme-specific deletion of Lmx1b resulted in heterotopic ossification from EMM at the vertex, and craniosynostosis affecting multiple sutures. Conversely, forced expression of Lmx1b in SOM was sufficient to inhibit osteogenic specification. Therefore, we conclude that Lmx1b plays a key role as an anti-osteogenic factor in patterning the head mesenchyme into areas with different osteogenic competence. In turn, this patterning event is crucial to generating the proper organization of the bones and soft tissue joints of the calvaria.

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

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.

Spindle Assembly and Chromosome Segregation Requires Central Spindle Proteins in Drosophila Oocytes.

Oocytes segregate chromosomes in the absence of centrosomes. In this situation, the chromosomes direct spindle assembly. It is still unclear in this system which factors are required for homologous chromosome bi-orientation and spindle assembly. The Drosophila kinesin-6 protein Subito, although nonessential for mitotic spindle assembly, is required to organize a bipolar meiotic spindle and chromosome bi-orientation in oocytes. Along with the chromosomal passenger complex (CPC), Subito is an important part of the metaphase I central spindle. In this study we have conducted genetic screens to identify genes that interact with subito or the CPC component Incenp. In addition, the meiotic mutant phenotype for some of the genes identified in these screens were characterized. We show, in part through the use of a heat-shock-inducible system, that the Centralspindlin component RacGAP50C and downstream regulators of cytokinesis Rho1, Sticky, and RhoGEF2 are required for homologous chromosome bi-orientation in metaphase I oocytes. This suggests a novel function for proteins normally involved in mitotic cell division in the regulation of microtubule-chromosome interactions. We also show that the kinetochore protein, Polo kinase, is required for maintaining chromosome alignment and spindle organization in metaphase I oocytes. In combination our results support a model where the meiotic central spindle and associated proteins are essential for acentrosomal chromosome segregation.

Lhx6 and Lhx8 promote palate development through negative regulation of a cell cycle inhibitor gene, p57Kip2.

Cleft palate is a common birth defect in humans. Therefore, understanding the molecular genetics of palate development is important from both scientific and medical perspectives. Lhx6 and Lhx8 encode LIM homeodomain transcription factors, and inactivation of both genes in mice resulted in profound craniofacial defects including cleft secondary palate. The initial outgrowth of the palate was severely impaired in the mutant embryos, due to decreased cell proliferation. Through genome-wide transcriptional profiling, we discovered that p57(Kip2) (Cdkn1c), encoding a cell cycle inhibitor, was up-regulated in the prospective palate of Lhx6(-/-);Lhx8(-/-) mutants. p57(Kip2) has been linked to Beckwith-Wiedemann syndrome and IMAGe syndrome in humans, which are developmental disorders with increased incidents of palate defects among the patients. To determine the molecular mechanism underlying the regulation of p57(Kip2) by the Lhx genes, we combined chromatin immunoprecipitation, in silico search for transcription factor-binding motifs, and in vitro reporter assays with putative cis-regulatory elements. The results of these experiments indicated that LHX6 and LHX8 regulated p57(Kip2) via both direct and indirect mechanisms, with the latter mediated by Forkhead box (FOX) family transcription factors. Together, our findings uncovered a novel connection between the initiation of palate development and a cell cycle inhibitor via LHX. We propose a model in which Lhx6 and Lhx8 negatively regulate p57(Kip2) expression in the prospective palate area to allow adequate levels of cell proliferation and thereby promote normal palate development. This is the first report elucidating a molecular genetic pathway downstream of Lhx in palate development.

Identification of a face enhancer reveals direct regulation of LIM homeobox 8 (Lhx8) by wingless-int (WNT)/ß-catenin signaling.

Development of the mammalian face requires a large number of genes that are expressed with spatio-temporal specificity, and transcriptional regulation mediated by enhancers plays a key role in the precise control of gene expression. Using chromatin immunoprecipitation for a histone marker of active enhancers, we generated a genome-wide map of candidate enhancers from the maxillary arch (primordium for the upper jaw) of mouse embryos. Furthermore, we confirmed multiple novel craniofacial enhancers near the genes implicated in human palate defects through functional assays. We characterized in detail one of the enhancers (Lhx8_enh1) located upstream of Lhx8, a key regulatory gene for craniofacial development. Lhx8_enh1 contained an evolutionarily conserved binding site for lymphoid enhancer factor/T-cell factor family proteins, which mediate the transcriptional regulation by the WNT/ß-catenin signaling pathway. We demonstrated in vitro that WNT/ß-catenin signaling was indeed essential for the expression of Lhx8 in the maxillary arch cells and that Lhx8_enh1 was a direct target of the WNT/ß-catenin pathway. Together, we uncovered a molecular mechanism for the regulation of Lhx8, and we provided valuable resources for further investigation into the gene regulatory network of craniofacial development.