Recapitulating cranial osteogenesis with neural crest cells in 3-D microenvironments.

The experimental systems that recapitulate the complexity of native tissues and enable precise control over the microenvironment are becoming essential for the pre-clinical tests of therapeutics and tissue engineering. Here, we described a strategy to develop an in vitro platform to study the developmental biology of craniofacial osteogenesis. In this study, we directly osteo-differentiated cranial neural crest cells (CNCCs) in a 3-D in vitro bioengineered microenvironment. Cells were encapsulated in the gelatin-based photo-crosslinkable hydrogel and cultured up to three weeks. We demonstrated that this platform allows efficient differentiation of p75 positive CNCCs to cells expressing osteogenic markers corresponding to the sequential developmental phases of intramembranous ossification. During the course of culture, we observed a decrease in the expression of early osteogenic marker Runx2, while the other mature osteoblast and osteocyte markers such as Osterix, Osteocalcin, Osteopontin and Bone sialoprotein increased. We analyzed the ossification of the secreted matrix with alkaline phosphatase and quantified the newly secreted hydroxyapatite. The Field Emission Scanning Electron Microscope (FESEM) images of the bioengineered hydrogel constructs revealed the native-like osteocytes, mature osteoblasts, and cranial bone tissue morphologies with canaliculus-like intercellular connections. This platform provides a broadly applicable model system to potentially study diseases involving primarily embryonic craniofacial bone disorders, where direct diagnosis and adequate animal disease models are limited.

A molecular mechanism for the origin of a key evolutionary innovation, the bird beak and palate, revealed by an integrative approach to major transitions in vertebrate history.

The avian beak is a key evolutionary innovation whose flexibility has permitted birds to diversify into a range of disparate ecological niches. We approached the problem of the mechanism behind this innovation using an approach bridging paleontology, comparative anatomy, and experimental developmental biology. First, we used fossil and extant data to show the beak is distinctive in consisting of fused premaxillae that are geometrically distinct from those of ancestral archosaurs. To elucidate underlying developmental mechanisms, we examined candidate gene expression domains in the embryonic face: the earlier frontonasal ectodermal zone (FEZ) and the later midfacial WNT-responsive region, in birds and several reptiles. This permitted the identification of an autapomorphic median gene expression region in Aves. To test the mechanism, we used inhibitors of both pathways to replicate in chicken the ancestral amniote expression. Altering the FEZ altered later WNT responsiveness to the ancestral pattern. Skeletal phenotypes from both types of experiments had premaxillae that clustered geometrically with ancestral fossil forms instead of beaked birds. The palatal region was also altered to a more ancestral phenotype. This is consistent with the fossil record and with the tight functional association of avian premaxillae and palate in forming a kinetic beak.

Conserved MicroRNA miR-8/miR-200 and its target USH/FOG2 control growth by regulating PI3K.

How body size is determined is a long-standing question in biology, yet its regulatory mechanisms remain largely unknown. Here, we find that a conserved microRNA miR-8 and its target, USH, regulate body size in Drosophila. miR-8 null flies are smaller in size and defective in insulin signaling in fat body that is the fly counterpart of liver and adipose tissue. Fat body-specific expression and clonal analyses reveal that miR-8 activates PI3K, thereby promoting fat cell growth cell-autonomously and enhancing organismal growth non-cell-autonomously. Comparative analyses identify USH and its human homolog, FOG2, as the targets of fly miR-8 and human miR-200, respectively. USH/FOG2 inhibits PI3K activity, suppressing cell growth in both flies and humans. FOG2 directly binds to p85alpha, the regulatory subunit of PI3K, and interferes with the formation of a PI3K complex. Our study identifies two novel regulators of insulin signaling, miR-8/miR-200 and USH/FOG2, and suggests their roles in adolescent growth, aging, and cancer.

Functional links between clustered microRNAs: suppression of cell-cycle inhibitors by microRNA clusters in gastric cancer.

microRNAs (miRNAs) play integral roles in diverse processes including tumorigenesis. miRNA gene loci are often found in close conjunction, and such clustered miRNA genes are transcribed from a common promoter to generate polycistronic primary transcript. The primary transcript (pri-miRNA) is then processed by two RNase III proteins to release the mature miRNAs. Although it has been speculated that the miRNAs in the same cluster may play related biological functions, this has not been experimentally addressed. Here we report that the miRNAs in two clusters (miR-106b approximately 93 approximately 25 and miR-222 approximately 221) suppress the Cip/Kip family members of Cdk inhibitors (p57(Kip2), p21(Cip1) and p27(Kip1)). We show that miR-25 targets p57 through the 3'-UTR. Furthermore, miR-106b and miR-93 control p21 while miR-222 and miR-221 regulate both p27 and p57. Ectopic expression of these miRNAs results in activation of Cdk2 and facilitation of G1/S phase transition. Consistent with these results, both clusters are abnormally upregulated in gastric cancer tissues compared to the corresponding normal tissues. Ectopic expression of miR-222 cluster enhanced tumor growth in the mouse xenograft model. Our study demonstrates the functional associations between clustered miRNAs and further implicates that effective cancer treatment may require a combinatorial approach to target multiple oncogenic miRNA clusters.