Regulation of proliferation in developing human tooth germs by MSX homeodomain proteins and cyclin-dependent kinase inhibitor p19INK4d

Before the secretion of hard dental tissues, tooth germs undergo several distinctive stages of development (dental lamina, bud, cap and bell). Every stage is characterized by specific proliferation patterns, which is regulated by various morphogens, growth factors and homeodomain proteins. The role of MSX homeodomain proteins in odontogenesis is rather complex. Expression domains of genes encoding for murine Msx1/2 during development are observed in tissues containing highly proliferative progenitor cells. Arrest of tooth development in Msx knockout mice can be attributed to impaired proliferation of progenitor cells. In Msx1 knockout mice, these progenitor cells start to differentiate prematurely as they strongly express cyclin-dependent kinase inhibitor p19^INK4d. p19^INK4d induces terminal differentiation of cells by blocking the cell cycle in mitogen-responsive G1 phase. Direct suppression of p19^INK4d by Msx1 protein is, therefore, important for maintaining proliferation of progenitor cells at levels required for the normal progression of tooth development. In this study, we examined the expression patterns of MSX1, MSX2 and p19^INK4d in human incisor tooth germs during the bud, cap and early bell stages of development. The distribution of expression domains of p19^INK4d throughout the investigated period indicates that p19^INK4d plays active role during human tooth development. Furthermore, comparison of expression domains of p19^INK4d with those of MSX1, MSX2 and proliferation markers Ki67, Cyclin A2 and pRb, indicates that MSX-mediated regulation of proliferation in human tooth germs might not be executed by the mechanism similar to one described in developing tooth germs of wild-type mouse.     

Single-cell gene profiling defines differential progenitor subclasses in mammalian neurogenesis

Cellular diversity of the brain is largely attributed to the spatial and temporal heterogeneity of progenitor cells. In mammalian cerebral development, it has been difficult to determine how heterogeneous the neural progenitor cells are, owing to dynamic changes in their nuclear position and gene expression. To address this issue, we systematically analyzed the cDNA profiles of a large number of single progenitor cells at the mid-embryonic stage in mouse. By cluster analysis and in situ hybridization, we have identified a set of genes that distinguishes between the apical and basal progenitors. Despite their relatively homogeneous global gene expression profiles, the apical progenitors exhibit highly variable expression patterns of Notch signaling components, raising the possibility that this causes the heterogeneous division patterns of these cells. Furthermore, we successfully captured the nascent state of basal progenitor cells. These cells are generated shortly after birth from the division of the apical progenitors, and show strong expression of the major Notch ligand delta-like 1, which soon fades away as the cells migrate in the ventricular zone. We also demonstrated that attenuation of Notch signals immediately induces differentiation of apical progenitors into nascent basal progenitors. Thus, a Notch-dependent feedback loop is likely to be in operation to maintain both progenitor populations.     

Secreted frizzled related protein 1 is a target to improve fracture healing

Genetic studies have identified a high bone mass of phenotype in both human and mouse when canonical Wnt signaling is increased. Secreted frizzled related protein 1 (sFRP1) is one of several Wnt antagonists and among the loss‐of‐function mouse models in which 32‐week‐old mice exhibit a high bone mass phenotype. Here we show that impact fracture healing is enhanced in this mouse model of increased Wnt signaling at a physiologic level in young (8 weeks) sFRP1^?/? mice which do not yet exhibit significant increases in BMD. In vivo deletion of sFRP1 function improves fracture repair by promoting early bone union without adverse effects on the quality of bone tissue reflected by increased mechanical strength. We observe a dramatic reduction of the cartilage callous, increased intramembranous bone formation with bone bridging by 14 days, and early bone remodeling during the 28‐day fracture repair process in the sFRP1^?/? mice. Our molecular analyses of gene markers indicate that the effect of sFRP1 loss‐of‐function during fracture repair is to accelerate bone healing after formation of the initial hematoma by directing mesenchymal stem cells into the osteoblast lineage via the canonical pathway. Further evidence to support this conclusion is the observation of maximal sFRP1 levels in the cartilaginous callus of a WT mouse. Hence sFRP1^?/? mouse progenitor cells are shifted directly into the osteoblast lineage. Thus, developing an antagonist to specifically inhibit sFRP1 represents a safe target for stimulating fracture repair and bone formation in metabolic bone disorders, osteoporosis and aging. J. Cell. Physiol. 220: 174–181, 2009. © 2009 Wiley‐Liss, Inc.     

Stage-specific gene expression in erythroid progenitor cells (CFU-E)

In erythropoietic differentiation, mature red blood cells are generated from specific progenitor cells through the action of specific growth regulatory molecules. To know the mechanism of differentiation, it is important to examine the control of gene expression in these progenitor cells in combination with growth regulatory molecules. We have cloned two genes expressing at a maximal level in the CFU-E (colony forming unit-erythroid), one of the erythroid progenitor cells from novel murine erythroleukemia (MEL) cell line (TSA8) which can be induced to CFU-E in vitro. The expression of these genes is well correlated with the appearance of CFU-E during induction of TSA8 cells, and is higher in the CFU-E-cells enriched from mouse fetal livers than in the more differentiated erythroid cells. Combining these with our previous results, it is suggested that in the erythropoiesis the progenitor cells have distinct patterns of gene expression. This expression is replaced through each progenitor cell rather than by the continuous increase in the expression of a set of genes specific to the mature erythroid cell following the commitment process.     

Induction and enhancement of cardiac cell differentiation from mouse and human induced pluripotent stem cells with cyclosporin-A

Induced pluripotent stem cells (iPSCs) are novel stem cells derived from adult mouse and human tissues by reprogramming. Elucidation of mechanisms and exploration of efficient methods for their differentiation to functional cardiomyocytes are essential for developing cardiac cell models and future regenerative therapies. We previously established a novel mouse embryonic stem cell (ESC) and iPSC differentiation system in which cardiovascular cells can be systematically induced from Flk1(+) common progenitor cells, and identified highly cardiogenic progenitors as Flk1(+)/CXCR4(+)/VE-cadherin(-) (FCV) cells. We have also reported that cyclosporin-A (CSA) drastically increases FCV progenitor and cardiomyocyte induction from mouse ESCs. Here, we combined these technologies and extended them to mouse and human iPSCs. Co-culture of purified mouse iPSC-derived Flk1(+) cells with OP9 stroma cells induced cardiomyocyte differentiation whilst addition of CSA to Flk1(+) cells dramatically increased both cardiomyocyte and FCV progenitor cell differentiation. Spontaneously beating colonies were obtained from human iPSCs by co-culture with END-2 visceral endoderm-like cells. Appearance of beating colonies from human iPSCs was increased approximately 4.3 times by addition of CSA at mesoderm stage. CSA-expanded human iPSC-derived cardiomyocytes showed various cardiac marker expressions, synchronized calcium transients, cardiomyocyte-like action potentials, pharmacological reactions, and ultra-structural features as cardiomyocytes. These results provide a technological basis to obtain functional cardiomyocytes from iPSCs.     

Conditional expression of the TVA receptor allows clonal analysis of descendents from Cre-expressing progenitor cells

An understanding of the number and types of progeny produced by progenitor cells during development provides a foundation for studies of when and where cell fate determination takes place. Lineal relationships can be revealed by the identification of descendents of cells that express a recombinase, such as Cre or Flp. This method provides data concerning gene expression history, but does not provide clonal resolution among the descendents. An alternative method employs retroviral labeling, which permits the identification of clones, but does not allow for the tracking of gene expression history. Here we report a combination of these methods to circumvent each method's limitations. By employing the specificity of Cre expression, and by selecting only a subset of cells with a Cre history for retroviral infection, clones with a gene expression history can be labeled. The method utilizes a conditional allele of the avian tumor virus receptor A (TVA), which allows infection of mouse cells following Cre activity, with mammalian retroviral vectors pseudotyped with the ASLV-A envelope glycoprotein (EnvA). We quantified the efficiency and specificity of this system in vivo and in vitro. We also generated a series of retroviral vectors encoding a variety of histochemical and fluorescent reporter genes that enable the tracking of mixtures of clones, thus enabling better resolution of clonal boundaries. This method and new vectors can be used to further our understanding of the gene expression patterns of progenitor cells that make particular daughter cells, as well as provide a platform for manipulating identified subsets of developing cells.     

The pattern of gene expression in mouse Gr-1(+) myeloid progenitor cells

To understand the pattern of gene expression in mouse myeloid progenitor cells, we carried out a genome-wide analysis of gene expression in mouse bone marrow Gr-1(+) cells using SAGE and GLGI techniques. We identified 22,033 unique SAGE tags with quantitative information from 73,869 collected SAGE tags. Among these unique tags, 64% match known sequences, including many genes important for myeloid differentiation, and 36% have no matches to known sequences and are likely to represent novel genes. We compared the expression of mouse Gr-1(+) and human CD15(+) myeloid progenitor cells and showed that the pattern of gene expression of these two cell populations had some similarities. We also compared the expression of mouse Gr-1(+) myeloid progenitor cells with that of mouse brain tissue and found a highly tissue-specific manner of gene expression in these two samples. Our data provide a basis for studying altered gene expression in myeloid disorders using mouse models.     

Bromodomain‐dependent stage‐specific male genome programming by Brdt

Male germ cell differentiation is a highly regulated multistep process initiated by the commitment of progenitor cells into meiosis and characterized by major chromatin reorganizations in haploid spermatids. We report here that a single member of the double bromodomain BET factors, Brdt, is a master regulator of both meiotic divisions and post‐meiotic genome repackaging. Upon its activation at the onset of meiosis, Brdt drives and determines the developmental timing of a testis‐specific gene expression program. In meiotic and post‐meiotic cells, Brdt initiates a genuine histone acetylation‐guided programming of the genome by activating essential genes and repressing a ‘progenitor cells’ gene expression program. At post‐meiotic stages, a global chromatin hyperacetylation gives the signal for Brdt's first bromodomain to direct the genome‐wide replacement of histones by transition proteins. Brdt is therefore a unique and essential regulator of male germ cell differentiation, which, by using various domains in a developmentally controlled manner, first drives a specific spermatogenic gene expression program, and later controls the tight packaging of the male genome.     

Grhl2 is required in nonneural tissues for neural progenitor survival and forebrain development

Grainyhead‐like genes are part of a highly conserved gene family that play a number of roles in ectoderm development and maintenance in mammals. Here we identify a novel allele of Grhl2, cleft‐face 3 (clft3), in a mouse line recovered from an ENU mutagenesis screen for organogenesis defects. Homozygous clft3 mutants have a number of phenotypes in common with other alleles of Grhl2. We note a significant effect of genetic background on the clft3 phenotype. One of these is a reduction in size of the telencephalon where we find abnormal patterns of neural progenitor mitosis and apoptosis in mutant brains. Interestingly, Grhl2 is not expressed in the developing forebrain, suggesting this is a survival factor for neural progenitors exerting a paracrine effect on the neural tissue from the overlying ectoderm where Grhl2 is highly expressed. genesis 53:573–582, 2015. © 2015 Wiley Periodicals, Inc.