Defects In Cell Differentiation Research Articles

ROW1 maintains quiescent centre identity by confining WOX5 expression to specific cells.

The quiescent centre (QC) in the Arabidopsis root apical meristem is essential for stem cell organization. Here we show that the loss of REPRESSOR OF WUSCHEL1 (ROW1), a PHD domain-containing protein, leads to QC failure, defects in cell differentiation and ectopic expression of WUSCHEL-RELATED HOMEOBOX 5 (WOX5) in cells that normally express ROW1. The wox5-1/row1-3 double mutants show similar phenotypes to wox5-1 indicating that WOX5 is epistatic to ROW1. ROW1 specifically binds trimethylated histone H3 lysine 4 (H3K4me3) in the WOX5 promoter region to repress its transcription. QC expression of ROW1 results in a wox5-1-like phenotype with undetectable WOX5 transcripts. We propose that ROW1 is essential for QC maintenance and for stem cell niche development through the repression of WOX5 in the proximal meristem.

Zebrafish retinal defects induced by ethanol exposure are rescued by retinoic acid and folic acid supplement

Fetal Alcohol Spectrum Disorder (FASD) is caused by prenatal alcohol exposure, producing craniofacial, sensory, motor, and cognitive defects. FASD is highly prevalent in low socioeconomic populations, which are frequently accompanied by malnutrition. FASD-associated ocular pathologies include microphthalmia, optic nerve hypoplasia, and cataracts. The present study characterizes specific retinal tissue defects, identifies ethanol-sensitive stages during retinal development, and dissects the effect of nutrient supplements, such as retinoic acid (RA) and folic acid (FA) on ethanol-induced retinal defects. Exposure to pathophysiological concentrations of ethanol (during midblastula transition through somitogenesis; 2–24 h post fertilization [hpf]) altered critical transcription factor expression involved in retinal cell differentiation, and produced severe retinal ganglion cell, photoreceptor, and Müller glial differentiation defects. Ethanol exposure did not alter retinal cell differentiation induction, but increased retinal cell death and proliferation. RA and FA nutrient co-supplementation rescued retinal photoreceptor and ganglion cell differentiation defects. Ethanol exposure during retinal morphogenesis stages (16–24 hpf) produced retinal defects like those seen with ethanol exposure between 2 and 24 hpf. Significantly, during an ethanol-sensitive time window (16–24 hpf), RA co-supplementation moderately rescued these defects, whereas FA co-supplementation showed significant rescue of optic nerve and photoreceptor differentiation defects. Interestingly, RA, but not FA, supplementation after ethanol exposure could reverse ethanol-induced optic nerve and photoreceptor differentiation defects. Our results indicate that various ethanol-sensitive events underlie FASD-associated retinal defects. Nutrient supplements like retinoids and folate were effective in alleviating ethanol-induced retinal defects.

Abstract 460: Base resolution epigenomic analysis reveals a role for Tet2 in modulating enhancer activity

Abstract Cytosine methylation (5mC) is a well-established epigenetic mechanism essential for genomic imprinting, silencing of retrotransposons, and lineage-specific expression of developmental genes. This epigenetic mark is extensively remodeled during mammalian development through the action of several DNA methyltransferases (DNMT) and the Ten-Eleven-Translocation (TET) family of protein dioxygenases. TET proteins function in DNA de-methylation by catalyzing the oxidation of 5mC to 5-hydroxymethylcytosine (5hmC) and other oxidized intermediates. Recurrent loss of function mutations of Tet2 are found in 27% of acute myeloid leukemia patients. In mouse models, TET2 loss is sufficient to cause myeloid malignancy through defects in hematopoietic stem cell differentiation. However, it remains unclear how TET2-dependent de-methylation regulates differentiation, a process often disrupted in tumorigenesis. Base-resolution methylomes have revealed that 5mC is dynamically regulated in a cell-type specific manner. Notably, DNA methylation is depleted at distal regulatory elements such as enhancers, where reduction of 5mC is correlated with the activity of these sequences. 5hmC is also significantly enriched at distal cis-regulatory sequences, suggesting that dynamic DNA methylation at these regions is likely mediated by interplays between DNMT-mediated methylation and TET-mediated de-methylation. Despite these discoveries, several open questions remain. First, are the different TET proteins functionally redundant? Does Tet2 uniquely modulate the methylome? If so, how? Second, what is the precise role of DNA hypomethylation and hydroxymethylation at enhancers? Do these modifications play active roles in gene regulation? We have employed genome-wide technologies to examine the effects of loss of Tet1 and Tet2 genes on DNA methylation, chromatin modification and gene expression landscapes in mouse embryonic stem cells. We generated base resolution DNA methylation and hydroxymethylation maps in wild type and Tet1 or Tet2 mutant mES cells, and determined the genomic regions where dynamic DNA methylation depends on either Tet1 or Tet2. Loss of Tet2 leads to dramatic reduction of DNA hydroxymethylation genome-wide, and local hypermethylation predominantly at enhancers. Furthermore, we observed reduced activities of re-methylated enhancers in Tet2-/- mES cells, supporting an active role for Tet2 and DNA hypomethylation at enhancers. Together, our results clarify the distinct functions of Tet1 and Tet2 in mammalian cells and highlight an active role of DNA hypomethylation at enhancers. Citation Format: Gary Hon, Chun-Xiao Song, Fulai Jin, Siddarth Selvaraj, Ah Young Lee, Chia-an Yen, Zhen Ye, Shi-Qing Mao, Bang-An Wang, Samantha Kuan, Lee Edsall, Boxuan Simen Zhao, Guo-Liang Xu, Chuan He, Bing Ren. Base resolution epigenomic analysis reveals a role for Tet2 in modulating enhancer activity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 460. doi:10.1158/1538-7445.AM2014-460

Out-of-sequence signal 3 as a mechanism for virus-induced immune suppression of CD8 T cell responses.

Virus infections are known to induce a transient state of immune suppression often associated with an inhibition of T cell proliferation in response to mitogen or cognate-antigen stimulation. Recently, virus-induced immune suppression has been linked to responses to type 1 interferon (IFN), a signal 3 cytokine that normally can augment the proliferation and differentiation of T cells exposed to antigen (signal 1) and co-stimulation (signal 2). However, pre-exposure of CD8 T cells to IFN-inducers such as viruses or poly(I∶C) prior to antigen signaling is inhibitory, indicating that the timing of IFN exposure is of essence. We show here that CD8 T cells pretreated with poly(I∶C) down-regulated the IFN receptor, up-regulated suppressor of cytokine signaling 1 (SOCS1), and were refractory to IFNβ-induced signal transducers and activators of transcription (STAT) phosphorylation. When exposed to a viral infection, these CD8 T cells behaved more like 2-signal than 3-signal T cells, showing defects in short lived effector cell differentiation, reduced effector function, delayed cell division, and reduced levels of survival proteins. This suggests that IFN-pretreated CD8 T cells are unable to receive the positive effects that type 1 IFN provides as a signal 3 cytokine when delivered later in the signaling process. This desensitization mechanism may partially explain why vaccines function poorly in virus-infected individuals.

Molecular Characterization of Gastric Neuroendocrine Carcinoma Based on Extensive Sequence Variation and Genome-Wide Methylation Analysis

ABSTRACT Aim: Neuroendocrine cancer (NEC) of the stomach is a rare type of cancer that is often diagnosed at an advanced stage and with poor prognosis. Its molecular characterization associated with its pathogenesis and therapeutic targets are poorly understood, although chemotherapy based on small cell type of lung cancer is recommended. In this study, we aimed to clarify the molecular characterization of gastric NEC and to find NEC-specific alterations in comparison with adenocarcinoma. Methods: Twenty samples from gastric cancers with advanced stage (11 adenocarcinomas and 9 NECs) and paired non-cancerous mucosa as controls were obtained by endoscopic biopsy. Analysis of sequence variations of 55 cancer-related genes was performed using the Ion AmpliSeq Panel Kit (Life Technologies), and analysis of DNA methylation was performed using the Infinium HumanMethylation 450 BeadChip array, covering 482,421 CpG sites (Illumina). DNA methylation and sequencing variation was confirmed using methylation-specific PCR and dideoxy sequencing. Results: Extensive mutation analysis revealed mutations of tumor suppressor genes (TP53 and CDKN2A) were frequently detected, whereas any oncogene mutation was not detected in gastric NECs. However, ERBB2 and KRAS were amplified and overexpressed, which were therapeutic targets. Comprehensive methylation analysis revealed 842 gene promoters were methylated, and gene silencing associated with neuronal differentiation (p=7.68E-21) and regulation of transcription (P=1.24E-21) were significantly enriched in gastric NECs. Among these genes, 13 genes were identified as NEC-specific methylations in comparison with adenocarcinoma, and TLE1 and HNF1B were known to be cancer-related genes in blood and ovarian cancers. Conclusions: To the best of our knowledge, this is the first study in which both genetic and epigenetic alterations were extensively analyzed in gastric NECs. Gene alteration with therapeutic targets was detected in NEC, and cell differentiation defects and cell cycle progression with DNA methylation are suggested to be involved in carcinogenesis of gastric NECs. Disclosure: All authors have declared no conflicts of interest.

Increased production of interleukin-17 over interleukin-10 by treg cells implicates inducible costimulator molecule in experimental spondyloarthritis.

HLA-B27/human β2 -microglobulin (hβ2 m)-transgenic (B27-transgenic) rats develop an inflammatory disorder resembling spondyloarthritis, with accumulation of proinflammatory Th17 cells. Because Treg cells and Th17 cells have opposing effects in inflammatory disorders, we sought to determine whether biased expansion of Th17 cells could result from altered Treg cell frequency and/or function in B27-transgenic rats. We characterized the phenotype and function of Treg cells from B27-transgenic rats in comparison with those from control rats, by examining their expression of cell surface markers, suppressive activity, cytokine production, and differentiation pattern. In B27-transgenic rats, the preferential accumulation of CD4+ Teff cells over Treg cells was not associated with a defect in Treg cell differentiation or suppressive activity. The expression of Treg cell markers was similar between B27-transgenic and control rats, with the exception of the inducible costimulator (ICOS) molecule, which was overexpressed in B27-transgenic rats. High levels of ICOS are considered to be a hallmark of Treg cells with heightened suppressive activity and interleukin-10 (IL-10) expression. Paradoxically, the production of IL-10 by Treg cells was reduced in B27-transgenic rats, whereas the production of IL-17 was enhanced. Moreover, the addition of anti-ICOS monoclonal antibodies during Treg cell differentiation in the presence of dendritic cells from B27-transgenic rats reversed this cytokine profile, restoring the balance between IL-10 and IL-17 in Treg cells from B27-transgenic rats. We observed dysregulated production of IL-10 and IL-17 by Treg cells from B27-transgenic rats, which may contribute to disease development. Moreover, our data highlight a key role for ICOS signaling in the generation of imbalanced production of IL-10 and IL-17 by Treg cells in this experimental model of spondyloarthritis.

Rictor is required for early B cell development in bone marrow.

The development of early B cells, which are generated from hematopoietic stem cells (HSCs) in a series of well-characterized stages in bone marrow (BM), represents a paradigm for terminal differentiation processes. Akt is primarily regulated by phosphorylation at Thr308 by PDK1 and at Ser473 by mTORC2, and Akt signaling plays a key role in hematopoiesis. However, the role of mTORC2 in the development of early B cells remains poorly understood. In this study, we investigated the functional role of mTORC2 by specifically deleting an integral component, Rictor, in a hematopoietic system. We demonstrated that the deletion of Rictor induced an aberrant increase in the FoxO1 and Rag-1 proteins in BM B cells and that this increase was accompanied by a significant decrease in the abundance of B cells in the peripheral blood (PB) and the spleen, suggesting impaired development of early B cells in adult mouse BM. A BM transplantation assay revealed that the B cell differentiation defect induced by Rictor deletion was not affected by the BM microenvironment, thus indicating a cell-intrinsic mechanism. Furthermore, the knockdown of FoxO1 in Rictor-deleted HSCs and hematopoietic progenitor cells (HPCs) promoted the maturation of B cells in the BM of recipient mice. In addition, we revealed that treatment with rapamycin (an mTORC1 inhibitor) aggravated the deficiency in B cell development in the PB and BM. Taken together, our results provide further evidence that Rictor regulates the development of early B cells in a cell-intrinsic manner by modifying the expression of FoxO1 and Rag-1.

Important aspects of placental-specific gene transfer

The placenta is a unique and highly complex organ that develops only during pregnancy and is essential for growth and survival of the developing fetus. The placenta provides the vital exchange of gases and wastes, the necessary nutrients for fetal development, acts as immune barrier that protects against maternal rejection, and produces numerous hormones and growth factors that promote fetal maturity to regulate pregnancy until parturition. Abnormal placental development is a major underlying cause of pregnancy-associated disorders that often result in preterm birth. Defects in placental stem cell propagation, growth, and differentiation are the major factors that affect embryonic and fetal well-being and dramatically increase the risk of pregnancy complications. Understanding the processes that regulate placentation is important in determining the underlying factors behind abnormal placental development. The ability to manipulate genes in a placenta-specific manner provides a unique tool to analyze development and eliminates potentially confounding results that can occur with traditional gene knockouts. Trophoblast stem cells and mouse embryos are not overly amenable to traditional gene transfer techniques. Most viral vectors, however, have a low infection rate and often lead to mosaic transgenesis. Although the traditional method of embryo transfer is intrauterine surgical implantation, the methodology reported here, combining lentiviral blastocyst infection and nonsurgical embryo transfer, leads to highly efficient and placental-specific gene transfer. Numerous advantages of our optimized procedures include increased investigator safety, a reduction in animal stress, rapid and noninvasive embryo transfer, and higher a rate of pregnancy and live birth.

RNA-seq analysis to identify novel roles of scleraxis during embryonic mouse heart valve remodeling.

Heart valve disease affects up to 30% of the population and has been shown to have origins during embryonic development. Valvulogenesis begins with formation of endocardial cushions in the atrioventricular canal and outflow tract regions. Subsequently, endocardial cushions remodel, elongate and progressively form mature valve structures composed of a highly organized connective tissue that provides the necessary biomechanical function throughout life. While endocardial cushion formation has been well studied, the processes required for valve remodeling are less well understood. The transcription factor Scleraxis (Scx) is detected in mouse valves from E15.5 during initial stages of remodeling, and expression remains high until birth when formation of the highly organized mature structure is complete. Heart valves from Scx-/- mice are abnormally thick and develop fibrotic phenotypes similar to human disease by juvenile stages. These phenotypes begin around E15.5 and are associated with defects in connective tissue organization and valve interstitial cell differentiation. In order to understand the etiology of this phenotype, we analyzed the transcriptome of remodeling valves isolated from E15.5 Scx-/- embryos using RNA-seq. From this, we have identified a profile of protein and non-protein mRNAs that are dependent on Scx function and using bioinformatics we can predict the molecular functions and biological processes affected by these genes. These include processes and functions associated with gene regulation (methyltransferase activity, DNA binding, Notch signaling), vitamin A metabolism (retinoic acid biosynthesis) and cellular development (cell morphology, cell assembly and organization). In addition, several mRNAs are affected by alternative splicing events in the absence of Scx, suggesting additional roles in post-transcriptional modification. In summary, our findings have identified transcriptome profiles from abnormal heart valves isolated from E15.5 Scx-/- embryos that could be used in the future to understand mechanisms of heart valve disease in the human population.

Out-of-sequence signal 3 as a mechanism for virus-induced immune suppression of CD8 T cell proliferation (VIR1P.960)

Abstract Virus infections are known to induce a transient state of immune suppression often associated with an inhibition of T cell proliferation in response to mitogen or cognate-antigen stimulation. Recently, virus-induced immune suppression has been linked to responses to type 1 IFN, a signal 3 cytokine that normally can augment the proliferation and differentiation of T cells exposed to antigen (signal 1) and co-stimulation (signal 2). However, pre-exposure of CD8 T cells to IFN-inducers such as viruses or poly(I:C) prior to antigen signaling is inhibitory, indicating that the timing of IFN exposure is of essence. Studies with T cells lacking IFN receptors implicated a direct role for type 1 IFN in this process. CD8 T cells pretreated with poly(I:C) down-regulated the IFN receptor, up-regulated SOCS1, and were refractory to IFNβ-induced STAT phosphorylation. When exposed to a viral infection these CD8 T cells behaved more like 2-signal than 3-signal T cells, showing defects in short lived effector cell differentiation, reduced effector function, delayed cell division, and reduced levels of survival proteins. This suggests that IFN-pretreated CD8 T cells are unable to receive the positive effects that type 1 IFN provides as a signal 3 cytokine when delivered later in the signaling process. This desensitization mechanism may partially explain why vaccines function poorly in virus-infected individuals.

Prenylation is required for polar cell elongation, cell adhesion, and differentiation in Physcomitrella patens

Protein prenylation is required for a variety of growth and developmental processes in flowering plants. Here we report the consequences of loss of function of all known prenylation subunits in the moss Physcomitrella patens. As in Arabidopsis, protein farnesyltransferase and protein geranylgeranyltransferase type I are not required for viability. However, protein geranylgeranyltransferase type I activity is required for cell adhesion, polar cell elongation, and cell differentiation. Loss of protein geranylgeranyltransferase activity results in colonies of round, single-celled organisms that resemble unicellular algae. The loss of protein farnesylation is not as severe but also results in polar cell elongation and differentiation defects. The complete loss of Rab geranylgeranyltransferase activity appears to be lethal in P.patens. Labeling with antibodies to cell wall components support the lack of polarity establishment and the undifferentiated state of geranylgeranyltransferase type I mutant plants. Our results show that prenylated proteins play key roles in P.patens development and differentiation processes.

Piwi Is Required in Multiple Cell Types to Control Germline Stem Cell Lineage Development in the Drosophila Ovary

The piRNA pathway plays an important role in maintaining genome stability in the germ line by silencing transposable elements (TEs) from fly to mammals. As a highly conserved piRNA pathway component, Piwi is widely expressed in both germ cells and somatic cells in the Drosophila ovary and is required for piRNA production in both cell types. In addition to its known role in somatic cap cells to maintain germline stem cells (GSCs), this study has demonstrated that Piwi has novel functions in somatic cells and germ cells of the Drosophila ovary to promote germ cell differentiation. Piwi knockdown in escort cells causes a reduction in escort cell (EC) number and accumulation of undifferentiated germ cells, some of which show active BMP signaling, indicating that Piwi is required to maintain ECs and promote germ cell differentiation. Simultaneous knockdown of dpp, encoding a BMP, in ECs can partially rescue the germ cell differentiation defect, indicating that Piwi is required in ECs to repress dpp. Consistent with its key role in piRNA production, TE transcripts increase significantly and DNA damage is also elevated in the piwi knockdown somatic cells. Germ cell-specific knockdown of piwi surprisingly causes depletion of germ cells before adulthood, suggesting that Piwi might control primordial germ cell maintenance or GSC establishment. Finally, Piwi inactivation in the germ line of the adult ovary leads to gradual GSC loss and germ cell differentiation defects, indicating the intrinsic role of Piwi in adult GSC maintenance and differentiation. This study has revealed new germline requirement of Piwi in controlling GSC maintenance and lineage differentiation as well as its new somatic function in promoting germ cell differentiation. Therefore, Piwi is required in multiple cell types to control GSC lineage development in the Drosophila ovary.

MAIN‐LIKE1 is a crucial factor for correct cell division and differentiation in Arabidopsis thaliana

Plant development requires accurate coordination of gene expression, both in actively dividing meristematic cells and differentiated cells. Cell fate establishment and maintenance, among others, are mediated by chromatin organization complexes that determine the stable transcriptional states of specific cell types. Here, we focus on MAIN-LIKE1 (MAIL1), one of three homologs of MAINTENANCE OF MERISTEMS (MAIN), which form a plant-specific gene family in Arabidopsis thaliana. We show that MAIL1 encodes a ubiquitously expressed nuclear protein. A mail1 loss-of-function mutant developed short primary roots, in which the meristematic cells accumulated DNA double-strand breaks and underwent massive cell death. In addition, mail1 mutant showed also cell differentiation defects in root and shoot tissues, and developed disorganized callus-like structures. The genetic interaction between main and mail1 mutants suggests that they act in the same pathway, and that both are essential for maintaining correct cell division acitivity in meristematic cells, while MAIL1 has an additional function in differentiating cells.

Transcription Factor STAT3 and Type I Interferons Are Corepressive Insulators for Differentiation of Follicular Helper and T Helper 1 Cells

Follicular helper T (Tfh) cells are required for the establishment of T-dependent B cell memory and high affinity antibody-secreting cells. We have revealed herein opposing roles for signal transducer and activator of transcription 3 (STAT3) and type I interferon (IFN) signaling in the differentiation of Tfh cells following viral infection. STAT3-deficient CD4(+) Tcells had a profound defect in Tfh cell differentiation, accompanied by decreased germinal center (GC) B cells and antigen-specific antibody production during acute infection with lymphocytic choriomeningitis virus. STAT3-deficient Tfh cells had strikingly increased expression of a number of IFN-inducible genes, in addition to enhanced T-bet synthesis, thus adopting a T helper 1 (Th1) cell-like effector phenotype. Conversely, IFN-αβ receptor blockade restored Tfh and GC B cell phenotypes in mice containing STAT3-deficient CD4(+) Tcells. These data suggest mutually repressive roles for STAT3 andtype I IFN signaling pathways in the differentiation of Tfh cells following viral infection.

TGFβ regulates epithelial-mesenchymal interactions through WNT signaling activity to control muscle development in the soft palate

Clefting of the soft palate occurs as a congenital defect in humans and adversely affects the physiological function of the palate. However, the molecular and cellular mechanism of clefting of the soft palate remains unclear because few animal models exhibit an isolated cleft in the soft palate. Using three-dimensional microCT images and histological reconstruction, we found that loss of TGFβ signaling in the palatal epithelium led to soft palate muscle defects in Tgfbr2(fl/fl);K14-Cre mice. Specifically, muscle mass was decreased in the soft palates of Tgfbr2 mutant mice, following defects in cell proliferation and differentiation. Gene expression of Dickkopf (Dkk1 and Dkk4), negative regulators of WNT-β-catenin signaling, is upregulated in the soft palate of Tgfbr2(fl/fl);K14-Cre mice, and WNT-β-catenin signaling is disrupted in the palatal mesenchyme. Importantly, blocking the function of DKK1 and DKK4 rescued the cell proliferation and differentiation defects in the soft palate of Tgfbr2(fl/fl);K14-Cre mice. Thus, our findings indicate that loss of TGFβ signaling in epithelial cells compromises activation of WNT signaling and proper muscle development in the soft palate through tissue-tissue interactions, resulting in a cleft soft palate. This information has important implications for prevention and non-surgical correction of cleft soft palate.

Basal Cell Signaling by p63 Controls Luminal Progenitor Function and Lactation via NRG1

The mammary epithelium is organized as a bilayer ofluminal and basal/myoepithelial cells. During pregnancy, the luminal compartment expands for milk production, while basal cells are thought to provide structural and contractile support. Here, we reveal a pregnancy-specific role of basal epithelia as a central coordinator of lactogenesis. We demonstrate that genetic deletion of the transcription factor p63 (Trp63) gene exclusively within basal cells of the adult gland during pregnancy leads to dramatic defects in luminal cell proliferation and differentiation, resulting in lactation failure. This phenotype is explained by direct transcriptional activation of the epidermal growth factor family ligand gene Nrg1 by p63 selectively in basal cells, which is required for luminal ERBB4/STAT5A activation and consequent luminal progenitor cell maturation. Thus, paracrine basal-to-luminal cell signaling, controlled by p63 via NRG1, orchestrates the entire lactation program. Collectively, these findings redefine the paradigm for cellular interactions specifying the functional maturation of the mammary gland.

Integrins regulate epithelial cell differentiation by modulating Notch activity.

Coordinating exit from the cell cycle with differentiation is crucial for proper development and tissue homeostasis. Failure to do so can lead to aberrant organogenesis and tumorigenesis. However, little is known about the developmental signals that regulate the switch from cell cycle exit to differentiation. Signals downstream of two key developmental pathways, Notch and Salvador-Warts-Hippo (SWH), and signals downstream of myosin activity regulate this switch during the development of the follicle cell epithelium of the Drosophila ovary. Here, we have identified a fourth player, the integrin signaling pathway. Elimination of integrin function blocks the mitosis-to-endocycle switch and differentiation in posterior follicle cells (PFCs), by regulation of the cyclin-dependent kinase inhibitor (CKI) dacapo. In addition, integrin-mutant PFCs show defective Notch signaling and endocytosis. Furthermore, integrins act in PFCs by modulating the activity of the Notch pathway, as reducing the amount of Hairless, the major antagonist of Notch, or misexpressing Notch intracellular domain rescues the cell cycle and differentiation defects. Taken together, our findings reveal a direct involvement of integrin signaling on the spatial and temporal regulation of epithelial cell differentiation during development.

LT-HSC Methylcellulose Assay.

Hematopoietic differentiation is a highly complex process originating from an extraordinary population of cells called long-term repopulating hematopoietic stem cells (LT-HSCs). The unique feature of all stem cells, including HSCs, is their exceptional ability to divide asymmetrically giving rise to two different kinds of offspring. One daughter cell becomes an LT-HSC itself (self-renews) to maintain the LT-HSC pool, whereas the second daughter cell pursues a differentiation fate to ultimately give rise to terminally differentiated mature blood cells (Orkin and Zon, 2008). Quantification of phenotypic LT-HSCs can be performed by multi-color flow cytometry and the gold standard for assessment of LT-HSC self-renewal and function is competitive bone marrow transplantation (Miller et al., 2008). Although these methods are irreplaceable to determine LT-HSC abundance and functionality, they have their disadvantages and limitations. For example, competitive bone marrow transplantation is typically monitored as a function of peripheral blood donor contribution over 12-16 weeks. While reduced peripheral blood donor contribution by itself signifies impairment in the stem/progenitor cells compartment, it cannot unambiguously discriminate between reduced LT-HSC self-renewal, impaired LT-HSC differentiation or compromised progenitor cell differentiation. Here we describe an LT-HSCs methylcellulose colony-forming assay, as a fast complementary in vitro method to directly assess LT-HSC differentiation capacity. As described in Kerenyi et al. (2013), this technique acts as a powerful tool to differentiate between LT-HSC or progenitor cell differentiation defects.

Interaction between Foxc1 and Fgf8 during Mammalian Jaw Patterning and in the Pathogenesis of Syngnathia

Syngnathia (bony fusion of the upper and lower jaw) is a rare human congenital condition, with fewer than sixty cases reported in the literature. Syngnathia typically presents as part of a complex syndrome comprising widespread oral and maxillofacial anomalies, but it can also occur in isolation. Most cartilage, bone, and connective tissue of the head and face is derived from neural crest cells. Hence, congenital craniofacial anomalies are often attributed to defects in neural crest cell formation, survival, migration, or differentiation. The etiology and pathogenesis of syngnathia however remains unknown. Here, we report that Foxc1 null embryos display bony syngnathia together with defects in maxillary and mandibular structures, and agenesis of the temporomandibular joint (TMJ). In the absence of Foxc1, neural crest cell derived osteogenic patterning is affected, as osteoblasts develop ectopically in the maxillary prominence and fuse with the dentary bone. Furthermore, we observed that the craniofacial musculature is also perturbed in Foxc1 null mice, which highlights the complex tissue interactions required for proper jaw development. We present evidence that Foxc1 and Fgf8 genetically interact and that Fgf8 dosage is associated with variation in the syngnathic phenotype. Together our data demonstrates that Foxc1 – Fgf8 signaling regulates mammalian jaw patterning and provides a mechanistic basis for the pathogenesis of syngnathia. Furthermore, our work provides a framework for understanding jaw patterning and the etiology of other congenital craniofacial anomalies, including temporomandibular joint agenesis.

Numb family proteins are essential for cardiac morphogenesis and progenitor differentiation

Numb family proteins (NFPs), including Numb and numb-like (Numbl), are cell fate determinants for multiple progenitor cell types. Their functions in cardiac progenitor differentiation and cardiac morphogenesis are unknown. To avoid early embryonic lethality and study NFP function in later cardiac development, Numb and Numbl were deleted specifically in heart to generate myocardial double-knockout (MDKO) mice. MDKOs were embryonic lethal and displayed a variety of defects in cardiac progenitor differentiation, cardiomyocyte proliferation, outflow tract (OFT) and atrioventricular septation, and OFT alignment. By ablating NFPs in different cardiac populations followed by lineage tracing, we determined that NFPs in the second heart field (SHF) are required for OFT and atrioventricular septation and OFT alignment. MDKOs displayed an SHF progenitor cell differentiation defect, as revealed by a variety of methods including mRNA deep sequencing. Numb regulated cardiac progenitor cell differentiation in an endocytosis-dependent manner. Studies including the use of a transgenic Notch reporter line showed that Notch signaling was upregulated in the MDKO. Suppression of Notch1 signaling in MDKOs rescued defects in p57 expression, proliferation and trabecular thickness. Further studies showed that Numb inhibits Notch1 signaling by promoting the degradation of the Notch1 intracellular domain in cardiomyocytes. This study reveals that NFPs regulate trabecular thickness by inhibiting Notch1 signaling, control cardiac morphogenesis in a Notch1-independent manner, and regulate cardiac progenitor cell differentiation in an endocytosis-dependent manner. The function of NFPs in cardiac progenitor differentiation and cardiac morphogenesis suggests that NFPs might be potential therapeutic candidates for cardiac regeneration and congenital heart diseases.