Systems biology of facial development: contributions of ectoderm and mesenchyme.

The rapid increase in gene-centric biological knowledge coupled with analytic approaches for genomewide data integration provides an opportunity to develop systems-level understanding of facial development. Experimental analyses have demonstrated the importance of signaling between the surface ectoderm and the underlying mesenchyme are coordinating facial patterning. However, current transcriptome data from the developing vertebrate face is dominated by the mesenchymal component, and the contributions of the ectoderm are not easily identified. We have generated transcriptome datasets from critical periods of mouse face formation that enable gene expression to be analyzed with respect to time, prominence, and tissue layer. Notably, by separating the ectoderm and mesenchyme we considerably improved the sensitivity compared to data obtained from whole prominences, with more genes detected over a wider dynamic range. From these data we generated a detailed description of ectoderm-specific developmental programs, including pan-ectodermal programs, prominence- specific programs and their temporal dynamics. The genes and pathways represented in these programs provide mechanistic insights into several aspects of ectodermal development. We also used these data to identify co-expression modules specific to facial development. We then used 14 co-expression modules enriched for genes involved in orofacial clefts to make specific mechanistic predictions about genes involved in tongue specification, in nasal process patterning and in jaw development. Our multidimensional gene expression dataset is a unique resource for systems analysis of the developing face; our co-expression modules are a resource for predicting functions of poorly annotated genes, or for predicting roles for genes that have yet to be studied in the context of facial development; and our analytic approaches provide a paradigm for analysis of other complex developmental programs.

MEMO1 drives cranial endochondral ossification and palatogenesis.

The cranial base is a component of the neurocranium and has a central role in the structural integration of the face, brain and vertebral column. Consequently, alteration in the shape of the human cranial base has been intimately linked with primate evolution and defective development is associated with numerous human facial abnormalities. Here we describe a novel recessive mutant mouse strain that presented with a domed head and fully penetrant cleft secondary palate coupled with defects in the formation of the underlying cranial base. Mapping and non-complementation studies revealed a specific mutation in Memo1 - a gene originally associated with cell migration. Expression analysis of Memo1 identified robust expression in the perichondrium and periosteum of the developing cranial base, but only modest expression in the palatal shelves. Fittingly, although the palatal shelves failed to elevate in Memo1 mutants, expression changes were modest within the shelves themselves. In contrast, the cranial base, which forms via endochondral ossification had major reductions in the expression of genes responsible for bone formation, notably matrix metalloproteinases and markers of the osteoblast lineage, mirrored by an increase in markers of cartilage and extracellular matrix development. Concomitant with these changes, mutant cranial bases showed an increased zone of hypertrophic chondrocytes accompanied by a reduction in both vascular invasion and mineralization. Finally, neural crest cell-specific deletion of Memo1 caused a failure of anterior cranial base ossification indicating a cell autonomous role for MEMO1 in the development of these neural crest cell derived structures. However, palate formation was largely normal in these conditional mutants, suggesting a non-autonomous role for MEMO1 in palatal closure. Overall, these findings assign a new function to MEMO1 in driving endochondral ossification in the cranium, and also link abnormal development of the cranial base with more widespread effects on craniofacial shape relevant to human craniofacial dysmorphology.

Improved efficacy of doxorubicin delivery by a novel dual-ligand-modified liposome in hepatocellular carcinoma.

Liposomes have been widely used as drug carriers in both biomedical research and for clinical applications, allowing the stabilisation of therapeutic compounds and overcoming obstacles to cellular and tissue uptake. However, liposomes still have low targeting efficiency, resulting in insufficient killing of tumour cells and unnecessary damage to normal cells. In this study, glycyrrhetinic acid (GA) and peanut agglutinin (PNA) were used as ligands to prepare dual-ligand-modified doxorubicin-loaded liposomes (DOX-GA/PNA-Lips) to enhance the targeting accuracy and efficacy of drug delivery against malignant liver cancer. PNA and GA modification enhanced the binding ability of liposomes to liver cancer cells, leading to excellent tissue and cell targeting of DOX-GA/PNA-Lips. DOX-GA/PNA-Lips showed an effective anti-tumour effect in vivo and in vitro, with its targeted delivery facilitating attenuation of the toxic side effects of DOX. These results demonstrated that dual-ligand-modified liposomes may provide an effective strategy for the treatment of hepatocellular carcinoma.

RUNX2 contributes to TGF-ß1-induced expression of Wdr72 in ameloblasts during enamel mineralization.

The elaborate modulation of the transforming growth factor ß (TGF-ß) superfamily signaling network plays an essential role in tooth morphogenesis and differentiation. In our previous studies, we have demonstrated that TGF-ß1 promotes enamel mineralization and maturation using TGF-ß1 gene conditional knockout (TGF-ß1-cKO) mice. However, the specific regulatory mechanisms of TGF-ß1 during enamel development remain unclear. Furthermore, we have previously found that the expression of WD repeat-containing protein 72(WDR72)in mouse enamel epithelium is decreased significantly in the absence of TGF-ß1. Therefore, the aim of the present study was to investigate how TGF-ß1 affects amelogenesis by regulating the expression of Wdr72. Histological examination showed that the absence of TGF-ß1 in ameloblastic epithelial cells resulted in a reduction in enamel mineralization and a delay in enamel matrix protein absorption. TGF-ß1, Runt-related transcription factor 2(RUNX2) and WDR72 were revealed to be colocalized in ameloblasts by immunohistochemistry, and it was also found that the expression of Runx2 and Wdr72 was markedly different between TGF-ß1-cKO mice and wild type(TGF-ß1-WT)mice. In addition, the effect of exogenous TGF-ß1 on Wdr72 was more significant when RUNX2 was present than when RUNX2 was absent. Furthermore, we found that there were binding sites for RUNX2 on the promoter of Wdr72 and that Wdr72 expression was regulated by RUNX2. Collectively, our results suggest that TGF-ß1 affects enamel mineralization by modulating RUNX2 and thus affecting the expression of Wdr72.

Gene expression profile data for mouse facial development.

This article contains data related to the research articles "Spatial and Temporal Analysis of Gene Expression during Growth and Fusion of the Mouse Facial Prominences" (Feng et al., 2009) [1] and "Systems Biology of facial development: contributions of ectoderm and mesenchyme" (Hooper et al., 2017 In press) [2]. Embryonic mammalian craniofacial development is a complex process involving the growth, morphogenesis, and fusion of distinct facial prominences into a functional whole. Aberrant gene regulation during this process can lead to severe craniofacial birth defects, including orofacial clefting. As a means to understand the genes involved in facial development, we had previously dissected the embryonic mouse face into distinct prominences: the mandibular, maxillary or nasal between E10.5 and E12.5. The prominences were then processed intact, or separated into ectoderm and mesenchyme layers, prior analysis of RNA expression using microarrays (Feng et al., 2009, Hooper et al., 2017 in press) [1], [2]. Here, individual gene expression profiles have been built from these datasets that illustrate the timing of gene expression in whole prominences or in the separated tissue layers. The data profiles are presented as an indexed and clickable list of the genes each linked to a graphical image of that gene׳s expression profile in the ectoderm, mesenchyme, or intact prominence. These data files will enable investigators to obtain a rapid assessment of the relative expression level of any gene on the array with respect to time, tissue, prominence, and expression trajectory.

Spatial and temporal analysis of gene expression during growth and fusion of the mouse facial prominences.

Orofacial malformations resulting from genetic and/or environmental causes are frequent human birth defects yet their etiology is often unclear because of insufficient information concerning the molecular, cellular and morphogenetic processes responsible for normal facial development. We have, therefore, derived a comprehensive expression dataset for mouse orofacial development, interrogating three distinct regions - the mandibular, maxillary and frontonasal prominences. To capture the dynamic changes in the transcriptome during face formation, we sampled five time points between E10.5-E12.5, spanning the developmental period from establishment of the prominences to their fusion to form the mature facial platform. Seven independent biological replicates were used for each sample ensuring robustness and quality of the dataset. Here, we provide a general overview of the dataset, characterizing aspects of gene expression changes at both the spatial and temporal level. Considerable coordinate regulation occurs across the three prominences during this period of facial growth and morphogenesis, with a switch from expression of genes involved in cell proliferation to those associated with differentiation. An accompanying shift in the expression of polycomb and trithorax genes presumably maintains appropriate patterns of gene expression in precursor or differentiated cells, respectively. Superimposed on the many coordinated changes are prominence-specific differences in the expression of genes encoding transcription factors, extracellular matrix components, and signaling molecules. Thus, the elaboration of each prominence will be driven by particular combinations of transcription factors coupled with specific cell:cell and cell:matrix interactions. The dataset also reveals several prominence-specific genes not previously associated with orofacial development, a subset of which we externally validate. Several of these latter genes are components of bidirectional transcription units that likely share cis-acting sequences with well-characterized genes. Overall, our studies provide a valuable resource for probing orofacial development and a robust dataset for bioinformatic analysis of spatial and temporal gene expression changes during embryogenesis.