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During embryogenesis, the processes that control how cells differentiate and interact to form particular tissues and organs with precise timing and shape are of fundamental importance. One prominent example of such processes is vertebrate somitogenesis, which is governed by a molecular oscillator called the segmentation clock. The segmentation clock system is initiated in the presomitic mesoderm in which a set of genes and signaling pathways exhibit coordinated spatiotemporal dynamics to establish regularly spaced boundaries along the body axis; these boundaries provide a blueprint for the development of segment-like structures such as spines and skeletal muscles. The highly complex and dynamic nature of this in vivo event and the design principles and their regulation in both normal and abnormal embryogenesis are not fully understood. Recently, live-imaging has been used to quantitatively analyze the dynamics of selected components of the circuit, particularly in combination with well-designed experiments to perturb the system. Here, we review recent progress from studies using live imaging and manipulation, including attempts to recapitulate the segmentation clock in vitro. In combination with mathematical modeling, these techniques have become essential for disclosing novel aspects of the clock.

Vertebrate cranial mesoderm is a discrete developmental unit compared to the mesoderm below the developing neck. An extraordinary feature of the cranial mesoderm is that it includes a common progenitor pool contributing to the chambered heart and the craniofacial skeletal muscles. This striking developmental potential and the excitement it generated led to advances in our understanding of cranial mesoderm developmental mechanism. Remarkably, recent findings have begun to unravel the origin of its distinct developmental characteristics. Here, we take a detailed view of the ontogenetic trajectory of cranial mesoderm and its regulatory network. Based on the emerging evidence, we propose that cranial and posterior mesoderm diverge at the earliest step of the process that patterns the mesoderm germ layer along the anterior-posterior body axis. Further, we discuss the latest evidence and their impact on our current understanding of the evolutionary origin of cranial mesoderm. Overall, the review highlights the findings from contemporary research, which lays the foundation to probe the molecular basis of unique developmental potential and evolutionary origin of cranial mesoderm.

Hepatic stellate cells (HSCs) play a principal role in Vitamin A metabolism and are considered the major matrix-producing cell type in the diseased liver. Rat HSCs are identified by immunohistochemistry with myogenic or mesenchymal (desmin, vimentin, and alpha-smooth muscle actin) or neural (e.g., GFAP or neuronal cell adhesion molecule) markers. Embryonic origin of rat HSCs was determined using these markers. Nestin, an intermediate filament protein originally identified in neuronal stem or progenitor cells, is widely used as a stem cell marker, including hepatic stem cells in adult rat livers. Additionally, nestin is reportedly expressed in activated HSCs during liver injury and hepatic regeneration. However, little is known about nestin expression in rat fetal liver HSCs. The present study aimed to clarify nestin-positive HSC expression during rat liver development. At embryonic day (ED) 10.5, nestin expression in mesenchymal cells adjacent to the liver bud was detected by immunohistochemistry. At ED 11.5, nestin-positive cells were also detected in desmin-positive cells appearing and increasing in intensity by ED 16.5. However, nestin-positive cells in the parenchyma decreased by ED 20.5 or later. These findings reveal that the nestin-positive HSCs during rat liver development originate from nestin-positive mesenchymal cells in the septum transversum.

Recent advances in understanding the molecular events underlying hypohidrotic ectodermal dysplasia (HED) caused by mutations of the genes encoding proteins of the tumor necrosis factor α (TNFα)-related signaling pathway have been presented. These proteins are involved in signal transduction from ectoderm to mesenchyme during development of the fetus and are indispensable for the differentiation of ectoderm-derived structures such as eccrine sweat glands, teeth, hair, skin, and/or nails. Novel data were reviewed and discussed on the structure and functions of the components of TNFα-related signaling pathway, the consequences of mutations of the genes encoding these proteins, and the prospect for further investigations, which might elucidate the origin of HED.

Staurosporine has been known to induce chondrogenesis in monolayer cultures of mesenchymal cells by dissolving actin stress fibers. The aim of this study was to further elucidate how the alteration of actin filaments by staurosporine induces chondrogenesis. Specifically, we examined whether the transforming growth factor (TGF)-β pathway is implicated. SB505124 strongly suppressed staurosporine-induced chondrogenesis without affecting the drug's action on the actin cytoskeleton. Staurosporine increased the phosphorylation of TGF-β receptor I (TβRI) but had no significant effect on the expression levels of TGF-β1, TGF-β2, TGF-β3, TβRI, TβRII, and TβRIII. Phosphorylation of Smad2 and Smad3 was not increased by staurosporine. However, SB505124 almost completely suppressed the phosphorylation of Smad2 and Smad3. In addition, inhibition of Smad3 blocked staurosporine-induced chondrogenesis. Inhibition of Akt, p38 mitogen-activated protein kinase (MAPK), and c-jun N-terminal kinase (JNK) suppressed chondrogenesis induced by staurosporine. Phosphorylation of Akt, p38 MAPK, and JNK was increased by staurosporine. SB505124 reduced the phosphorylation of Akt and p38 MAPK, while it had no effect on the phosphorylation of JNK. The phosphorylation level of extracellular signal-regulated kinase (ERK) was not significantly affected by staurosporine. In addition, inhibition of ERK with PD98059 alone did not induce chondrogenesis. Taken together, these results suggest that staurosporine induces chondrogenesis through TGF-β pathways including canonical Smads and non-canonical Akt and p38 MAPK signaling.

Hertwig's epithelial root sheath/epithelial rests of Malassez (HERS/ERM) have been suggested to play an important role in tooth root formation, particularly in periodontal development. Epithelial mesenchymal transition (EMT) has been suggested to contribute to root development in tooth. However, the mechanism of interaction between HERS/ERM cells and dental mesenchymal cells has not been fully understood. In this study, we investigated the effect of exogenous transforming growth factor beta 1 (TGF-β1) in human HERS/ERM cells in order to verify the role of granulocyte macrophage colony-stimulating factor (GM-CSF) in EMT process. Antibody array was used to screen secretion factors by exogenous TGF-β1. Secretion of GM-CSF was increased by exogenous TGF-β1. Expression levels of EMT markers, vimentin, ZEB1 (zinc finger E-box binding homeobox 1), and E-cadherin, were confirmed using reverse transcription polymerase chain reaction and immunocytochemistry. Treatment with GM-CSF increased the expression of vimentin and ZEB1, similar to TGF-β1 treatment, and decreased the expression of E-cadherin. Our results suggest that GM-CSF could induce EMT in human HERS/ERM cells.

Odontogenesis is the result of the reciprocal interactions between epithelial-mesenchymal cells leading to terminally differentiated odontoblasts. This process from dental papilla mesenchymal cells to odontoblasts is regulated by a complex signaling pathway. When isolated from the developing tooth germs, odontoblasts quickly lose their potential to maintain the odontoblast-specific phenotype. Therefore, generation of an odontoblast-like cell line would be a good surrogate model for studying the dental mesenchymal cell differentiation into odontoblasts and the molecular events of dentin formation. In this study, immortalized dental papilla mesenchymal cell lines were generated from the first mouse mandibular molars at postnatal day 3 using pSV40. These transformed cells were characterized by RT-PCR, immunohistochemistry, Western blot, and analyzed for alkaline phosphatase activity and mineralization nodule formation. One of these immortalized cell lines, iMDP-3, displayed a high proliferation rate, but retained the genotypic and phenotypic characteristics similar to primary cells as determined by expression of tooth-specific markers and demonstrated the ability to differentiate and form mineralized nodules. Furthermore, iMDP-3 cells had high transfection efficiency as well as were inducible and responded to BMP2 stimulation. We conclude that the establishment of the stable murine dental papilla mesenchymal cell line might be used for studying the mechanisms of dental cell differentiation and dentin formation.

Adipose-derived stem cells are an attractive alternative as a source of stem cells that can easily be extracted from adipose tissue. Isolation, characterization, and multi-lineage differentiation of adipose-derived stem cells have been described for human and a number of other species. Here we aimed to isolate and characterize camel adipose-derived stromal cell frequency and growth characteristics and assess their adipogenic, osteogenic, and chondrogenic differentiation potential. Samples were obtained from five adult dromedary camels. Fat from abdominal deposits were obtained from each camel and adipose-derived stem cells were isolated by enzymatic digestion as previously reported elsewhere for adipose tissue. Cultures were kept until confluency and subsequently were subjected to differentiation protocols to evaluate adipogenic, osteogenic, and chondrogenic potential. The morphology of resultant camel adipose-derived stem cells appeared to be spindle-shaped fibroblastic morphology, and these cells retained their biological properties during in vitro expansion with no sign of abnormality in karyotype. Under inductive conditions, primary adipose-derived stem cells maintained their lineage differentiation potential into adipogenic, osteogenic, and chondrogenic lineages during subsequent passages. Our observation showed that like human lipoaspirate, camel adipose tissue also contain multi-potent cells and may represent an important stem cell source both for veterinary cell therapy and preclinical studies as well.

Fates of digits in amniotes, i.e., free or webbed digits, are determined by the size of programmed interdigital cell death (ICD) area. However, no (or very few) cell death has thus far been observed in developing limb buds of non-amniotic terrestrial vertebrates including other anuran or urodela amphibians. We speculate that the undetectable situation of amphibian ICD is the result of their less frequency due to slow developmental speed characteristic to most amphibian species. Here, we present three strategies for detecting difficult-to-find ICD in the frog, Xenopus laevis. (1) Addition of triiodo-L-thyronine (T(3)) accelerated two to three times the limb development and increased two to four times the appearance frequency of vital dye-stainable cells in limb buds of the accelerated tadpoles (stage 54 to 55). (2) Application of human bone morphogenetic protein-4 to the autopods of tadpoles at stage 53 to 54 enhanced digital cartilage formation and induced vital dye-stainable cells around the enhanced digital cartilages within 2 d. (3) In cell culture, T(3) increased the chondrogenic and cell death activities of limb mesenchymal cells. The augmentation of both activities by T(3) was stronger in the forelimb cells than in the hindlimb cells. This situation is well coincided with the limb fates of non-webbed forelimbs and webbed hindlimbs in X. laevis adulthood. Collectively, all three approaches showed that it become possible to detect X. laevis ICD with appropriate strategies.

The aim of this study was to determine whether passaged rat fetal liver cells are functional hepatoblasts. Hepatocyte/hepatoblast- and liver myofibroblast-gene-expressions were studied in adult and fetal rat liver tissues as well as in primary and passaged cultures of isolated rat fetal liver cells at both the mRNA and protein level. Desmin- and Alpha-Smooth Muscle Actin (SMA)-positive cells were located in the walls of liver vessels, whereas Desmin-positive/SMA-negative cells were distributed within the liver parenchyma. Primary cultures contained Prox1-positive hepatoblasts, Desmin/SMA-positive myofibroblasts and only a few Desmin-positive/SMA-negative cells. Albumin and alpha-fetoprotein (AFP) could be detected in the primary cultures and to a lesser extent after the first passage. The number of Desmin-positive/SMA-negative cells decreased with successive passage, such that after the second passage, only Desmin/SMA-positive cells could be detected. SMA-gene-expression increased during the passages, suggesting that myofibroblasts become the major cell population of fetal liver cell cultures over time. This observation needs to be taken into account, should passaged fetal liver cells be used for liver cell transplantation. Moreover it contradicts the concept of epithelial-mesenchymal transformation and suggests rather that selective overgrowth of mesenchymal cells occurs in culture.