Embryonic stem (ES) cells, derived from pre-implantation embryo and embryonic germ (EG) cells, derived from embryonic precursors of gametes, primordial germ cells (PGCs), can differentiate into any cell type in the body. Moreover, ES cells have a capacity to differentiate into PGCs in vitro. In the present study we have shown the differentiation capacity of six EG cell lines to form PGCs in vitro, in comparison to ES cells. Cell lines were differentiated via embryoid body (EB) formation using the co-expression of mouse vasa homologue (Mvh) and Oct-4 to identify newly formed PGCs in vitro. We found an increase of PGC numbers in almost all analysed cell lines in 5 days old EBs thus suggesting that EG and ES cells have similar efficiency to generate PGCs. The addition of retinoic acid confirmed that the cultures had attained a PGC like identity and continued to proliferate. Furthermore we have shown that the expression pattern of Prmt5 and H3K27me3 in newly formed PGCs is similar to that observed at embryonic day E11.5 PGCs in vivo. By co-culturing EBs with CHO cells some of the PGCs entered into meiosis, as judged by Scp3 expression. The derivation of germ cells from pluripotent stem cells in vitro could provide an invaluable model system to study both the genetic and epigenetic programming of germ cell development in vivo.
Pluripotent stem cell; EG cells; ES cell; Differentiation; Embryoid bodies; PGCs
Multipotent P19CL6 cells differentiate into cardiac myocytes or neural lineages when stimulated with dimethyl sulfoxide (DMSO) or retinoic acid (RA), respectively. Expression of the transcription factor Tbx6 was found to increase during cardiac myocyte differentiation and to decrease during neural differentiation. Overexpression of Tbx6 was not sufficient to drive P19CL6 cells to a cardiac myocyte fate or to accelerate DMSO-induced differentiation. In contrast, knockdown of Tbx6 dramatically inhibited DMSO-induced differentiation of P19CL6 cells to cardiac myocytes, as evidenced by the loss of striated muscle-specific markers and spontaneous beating. Tbx6 knockdown was also accompanied by almost complete loss of Nkx2.5, a transcription factor involved in the specification of the cardiac myocyte lineage, indicating that Nkx2.5 is downstream of Tbx6. In distinction to its positive role in cardiac myocyte differentiation, Tbx6 knockdown augmented RA-induced differentiation of P19CL6 cells to both neurons and glia, and accelerated the rate of neurite formation. Conversely, Tbx6 overexpression attenuated differentiation to neural lineages. Thus, in the P19CL6 model, Tbx6 is required for cardiac myocyte differentiation and represses neural differentiation. We propose a model in which Tbx6 is a part of a molecular switch that modulates divergent differentiation programs within a single progenitor cell.
Tbx6; Cardiac myocyte; Neuron/glial cells; Differentiation; P19CL6 cells
The heterogeneous nature of stem cells is an important issue in both research and therapeutic use in terms of directing cell lineage differentiation pathways, as well as self-renewal properties. Using flow cytometry we have identified two distinct subpopulations by size, large and small, within cultures of human embryonic stem (hES) cell lines. These two cell populations respond differentially to retinoic acid (RA) differentiation and several endocrine disruptor compounds (EDC). The large cell population responds to retinoic acid differentiation with a greater than a 50% reduction in cell number and loss of Oct-4 expression, whereas the number of the small cell population does not change and Oct-4 protein expression is maintained. In addition, four estrogenic compounds altered SSEA-3 expression differentially between the two cell subpopulations changing their ratios relative to each other. Both populations express stem cell markers Oct-4, Nanog, Tra-1-60, Tra-1-80 and SSEA-4, but express low levels of differentiation markers common to the three germ layers. Cloning studies indicate that both populations can revive the parental population. Furthermore, whole genome microarray identified approximately 400 genes with significantly different expression between the two populations (p<0.01). We propose the differential response to RA in these populations is due to differential gene expression of Notch signaling members, CoupTF1 and CoupTF2, chromatin remodeling and histone modifying genes that render the small population resistant to RA differentiation. The findings that hES cells exist as heterogeneous populations with distinct responses to differentiation signals and environmental stimuli will be relevant for their use for drug discovery and disease therapy.
Human embryonic stem cell; Heterogeneous population; Retinoic acid differentiation; Endocrine disruptor compounds; Notch signaling; Chromatin remodeling
Exposure to exogenous hormones during development can result in permanent health problems. In utero exposure to diethylstilbestrol (DES) is probably the most well documented case in human history. DES, an orally active synthetic estrogen, was believed to prevent adverse pregnancy outcome and thus was routinely given to selected pregnant women from the 1940s to the 1960s. It has been estimated that 5 million pregnant women worldwide were prescribed with DES during this period. In the early 1970s, vaginal clear cell adenocarcinomas (CCACs) were diagnosed in daughters whose mother took DES during pregnancy (known as DES daughters). Follow up studies demonstrated that exposure to DES in utero causes a spectrum of congenital anomalies in female reproductive tracts and CCACs. Among those, cervical and vaginal adenoses are most commonly found, which are believed to be the precursors of CCACs. Transformation related protein 63 (TRP63/p63) marks the cell fate decision of Müllerian duct epithelium (MDE) to become squamous epithelium in the cervix and vagina. DES disrupts the TRP63 expression in mice and induces adenosis lesions in the cervix and vagina. This review describes mouse models can be used to study the development of DES-induced anomalies, focusing on cervical and vaginal adenoses, and discusses its molecular pathogenesis.
diethylstilbestrol; vaginal adenocarcinoma; adenosis; TRP63; p63
The purpose of this study was to validate a combined in situ hybridization (ISH)/immunohistochemistry (IHC) staining method for visualizing and quantifying mouse prostatic buds. To refine animal usage in prostate development studies, we also determined whether a comparable number of prostatic buds were formed in male and female mouse urogenital sinus (UGS) explants grown in vitro in the presence of androgen. We used IHC to label UGS epithelium and ISH to label prostatic buds with one of three different prostatic bud marking riboprobes: a previously identified prostatic bud marker, NK-3 transcription factor, locus 1 (Nkx3-1), and two newly identified prostatic bud markers, wingless-related MMTV integration site 10b (Wnt10b) and ectodysplasin-A receptor (Edar). We calculated total buds formed per UGS and the proportion marked by each mRNA after male UGS development in vivo and male and female UGS development in vitro. Nkx3-1 was first to mark the prostate field during UGS development in vivo but all three mRNAs marked prostatic buds during later developmental stages. The mRNAs localized to different domains: Nkx3-1 was present along about half the prostatic bud length while Edar and Wnt10b were restricted to distal bud tips. None of the mRNAs marked all buds formed in vitro and the proportion marked was developmental stage- and gender-dependent. Nkx3-1 marked the highest proportion of prostatic buds during in vitro UGS development. Together, our results reveal that ISH staining of mouse UGS can be used to quantify prostatic bud number, Nkx3-1 is currently the best suited riboprobe for this method, and female UGSs cannot be used interchangeably with male UGSs when conducting prostate development studies in vitro. We also found that Nkx3-1, Edar, and Wnt10b mark different prostatic bud regions and are likely to be useful in future studies of regional differences in prostatic bud gene expression.
Prostate; UGS; Organ culture; LUT; Explant
External genitalia development occurs through a combination of hormone independent, hormone dependent, and endocrine pathways. Perturbation of these pathways can lead to abnormal external genitalia development. We review human and animal mechanisms of normal and abnormal external genitalia development, and we evaluate abnormal mechanisms that lead to hypospadias. We also discuss recent laboratory findings that further our understanding of animal models of hypospadias.
external genitalia; development; hypospadias
The objective of this study was to perform a comprehensive morphologic analysis of developing mouse external genitalia (ExG) and to determine specific sexual differentiation features that are responsive to androgens or estrogens. To eliminate sex steroid signaling postnatally, male and female mice were gonadectomized on the day of birth, and then injected intraperitoneally every other day with DES (200 ng/g), DHT (1 μg/g), or oil. On day-10 postnatal male and female ExG were dissected, fixed, embedded, serially sectioned and analyzed. We identified 10 sexually dimorphic anatomical features indicative of normal penile and clitoral differentiation in intact mice. Several (but not all) penile features were impaired or abolished as a result of neonatal castration. Those penile features remaining after neonatal castration were completely abolished with attendant clitoral development in androgen receptor (AR) mutant male mice (XTfm/Y and X/Y AR-null) in which AR signaling is absent both pre- and postnatally. Administration of DHT to neonatally castrated males restored development of all 10 masculine features to almost normal levels. Neonatal ovariectomy of female mice had little effect on clitoral development, whereas treatment of ovariectomized female mice with DHT induced partial masculinization of the clitoris. Administration of DES to neonatally gonadectomized male and female mice elicited a spectrum of development abnormalities. These studies demonstrate that the presence or absence of androgen prenatally specifies penile versus clitoral identity. Differentiated penile features emerge postnatally and are sensitive to and dependent upon prenatal or pre- and postnatal androgen. Emergence of differentiated clitoral features occurs postnatally in either intact or ovariectomized females. It is likely that each penile and clitoral feature has a unique time-course of hormonal dependency/sensitivity.
External genitalia; Sex differentiation; Estrogen receptors; Androgen receptor
The severe degeneration of the germinal epithelium and subsequent male sterility observed in mice null for the retinoic acid receptor α (RARα) gene suggested its critical role in spermatogenesis, although the etiology and progression of these abnormalities remain to be determined. Previous studies have revealed that elongated spermatids in RARα−/− testes were improperly aligned at the tubular lumen and did not undergo spermiation at stage VIII*. We now report a distinctive failure of step 8–9 spermatids to orient properly with regard to the basal aspect of Sertoli cells, resulting in stage VIII*–IX* tubules with randomly oriented spermatids. By in situ terminal deoxynucleo-tidyltransferase-mediated deoxy-UTP nick end labeling (TUNEL), we noted that elongating spermatids frequently underwent apoptosis. Immunohistochemical analysis revealed that while activated caspase-3, the primary effector caspase in the apoptotic cell death machinery, was detected in the nuclei of primary spermatocytes in the first wave of spermatogenesis and occasionally in spermatogonia of both normal and mutant testes, it was not involved in the death of elongating spermatids in RARα−/− testes. Thus, sterility in RARα−/− males was associated with specific defects in spermiogenesis, which may correlate with a failure in both spermatid release and spermatid orientation to the basal aspect of Sertoli cells at stage VIII* in young adult RARα−/− testis. Further, the resulting apoptosis in elongating spermatids appears to involve pathways other than that mediated by activated caspase-3.
apoptosis; spermiogenesis; ectoplasmic specializations; acrosome; RARα; caspase-3; retinoid signaling
Mesenchymal stromal cells (MSCs) show promise for treatment of a variety of neurological and other disorders. Cat has a high degree of linkage with the human genome and has been used as a model for analysis of neurological disorders such as stroke, Alzheimer’s disease and motor disorders. The present study was designed to characterize bone marrow-derived MSCs from cats and to investigate the capacity to generate functional peptidergic neurons. MSCs were expanded with cells from the femurs of cats and then characterized by phenotype and function. Phenotypically, feline and human MSCs shared surface markers, and lacked hematopoietic markers, with similar morphology. As compared to a subset of human MSCs, feline MSCs showed no evidence of the major histocompatibility class II. Since the literature suggested Stro-1 as an indicator of pluripotency, we compared early and late passages feline MSCs and found its expression in >90% of the cells. However, the early passage cells showed two distinct populations of Stro-1-expressing cells. At passage 5, the MSCs were more homogeneous with regards to Stro-1 expression. The passage 5 MSCs differentiated to osteogenic and adipogenic cells, and generated neurons with electrophysiological properties. This correlated with the expression of mature neuronal markers with concomitant decrease in stem cell-associated genes. At day 12 induction, the cells were positive for MAP2, Neuronal Nuclei, tubulin βIII, Tau and synaptophysin. This correlated with electrophysiological maturity as presented by excitatory postsynaptic potentials (EPSPs). The findings indicate that the cat may constitute a promising biomedical model for evaluation of novel therapies such as stem cell therapy in such neurological disorders as Alzheimer’s disease and stroke.
cat; mesenchymal stem cells; neuronal differentiation; immunohistochemistry; feline; bone marrow
In the central nervous system (CNS), neural stem cells (NSCs) differentiate into neurons, astrocytes, and oligodendrocytes - these cell lineages are considered unidirectional and irreversible under normal conditions. The introduction of a defined set of transcription factors has been shown to directly convert terminally differentiated cells into pluripotent stem cells, reinforcing the notion that preserving cellular identity is an active process. Indeed, recent studies highlight that tumor suppressor genes (TSGs) such as Ink4a/Arf and p53, control the barrier to efficient reprogramming, leaving open the question whether the same TSGs function to maintain the differentiated state. During malignancy or following brain injury, mature astrocytes have been reported to re-express neuronal genes and re-gain neurogenic potential to a certain degree, yet few studies have addressed the underlying mechanisms due to a limited number of cellular models or tools to probe this process. Here, we use a synthetic small-molecule (isoxazole) to demonstrate that highly malignant EGFRvIII-expressing Ink4a/Arf-/-; Pten-/- astrocytes down-regulated their astrocyte character, re-entered the cell cycle, and upregulated neuronal gene expression. As a collateral discovery, isoxazole small-molecules blocked tumor cell proliferation in vitro, a phenotype likely coupled to activation of neuronal gene expression. Similarly, histone deacetylase inhibitors induced neuronal gene expression and morphologic changes associated with the neuronal phenotype, suggesting the involvement of epigenetic-mediated gene activation. Our study assesses the contribution of specific genetic pathways underlying the de-differentiation potential of astrocytes and uncovers a novel pharmacological tool to explore astrocyte plasticity, which may bring insight to reprogramming and anti-tumor strategies.
astrocyte plasticity; de-differentiation; epigenetics; glioblastoma; cancer stem cell
This review summarizes the concept that the neo-formation of ductal-acinar architecture in the pathogenesis of benign prostatic hyperplasia (BPH) is due to the reactivation of embryonic inductive activity by BPH stroma, an idea enunciated by John McNeal. The concept is the synthesis of McNeal's astute pathological inference based upon developmental biology and supported by the mesenchyme-epithelial interaction studies. In a broader context, McNeal's concept of framing epithelial pathogenesis in terms of developmental biological principals has been extended more recently into the field of carcinogenesis under the umbrella of tumor microenvironment.
Corneal differentiation and maturation are associated with major changes in the expression levels of numerous genes, including those coding for the chromatin-binding high-mobility group (HMG) proteins. Here we report that HMGN1, a nucleosome-binding protein that alters the structure and activity of chromatin, affects the development of the corneal epithelium in mice. The corneal epithelium of Hmgn1−/− mice is thin, has a reduced number of cells, is poorly stratified, is depleted of supra-basal wing cells, and its most superficial cell layer blisters. In mature Hmgn1−/− mice, the basal cells retain the ovoid shape of immature cells, and rest directly on the basal membrane which is disorganized. Gene expression was modified in Hmgn1−/− corneas: glutathione-S-transferase (GST)α 4and GST ω 1, epithelial layer-specific markers, were selectively reduced while E-cadherin and α-, β-, and γ-catenin, components of adherens junctions, were increased. Immunofluorescence analysis reveals a complete co-localization of HMGN1 and p63 in small clusters of basal corneal epithelial cells of wild-type mice, and an absence of p63 expressing cells in the central region of the Hmgn1−/− cornea. We suggest that interaction of HMGN1 with chromatin modulates the fidelity of gene expression and affects corneal development and maturation.
HMG proteins; eye differentiation; corneal epithelium; chromatin
Induction of smooth muscle differentiation from bladder mesenchyme depends on signals that originate from the urothelium. We hypothesize Sonic hedgehog (Shh) is the urothelial signal that promotes bladder mesenchymal proliferation and induces bladder smooth muscle differentiation.
Pregnant FVB mice were euthanized on embryonic day (E) 12.5 and fetal bladders were harvested. Two experimental protocols were utilized:
Bladder mesenchyme (BLM) was isolated by incubating intact bladders (IB) in 0.02 M EDTA and then removing the urothelium by microdissection. IB and BLM were cultured in Shh-deficient media or BLM was cultured in Shh-supplemented (480 nM) media for 72 h.IB were cultured for 72 h in media containing different concentrations of Shh (0, 48, and 480 nM).
Specimens were sized by serial sectioning. Cell counts were performed after trypsin digestion. Immunohistochemistry was performed to detect smooth muscle-specific protein expression. α-Actin expression was quantified using Western blot.
All specimens were viable at 72 h. BLM cultured without Shh survived but did not grow or undergo smooth muscle differentiation. IB cultured without Shh and BLM cultured with Shh grew and expressed smooth muscle proteins at 72 h. IB cultured with Shh were larger and contained more cells than IB cultured without Shh (all p <0.05). Increasing Shh concentration from 48 to 480 nM did not change bladder size, cell counts, or the level of α-actin expression. Prior to culture, IB did not express α-actin. After culture of IB in Shh-deficient media, α-actin was detected throughout the mesenchyme except in the submucosal layer. The IB submucosa was thinner after culture with 48 nM Shh and smooth muscle completely obliterated the submucosa after culture with 480 nM Shh.
In fetal mouse bladders, urothelium-derived Shh is necessary for mesenchymal proliferation and smooth muscle differentiation. Shh concentration affects mesenchymal proliferation and patterning of bladder smooth muscle.
Bladder; Urothelium; Sonic hedgehog; Smooth muscle differentiation
Smooth muscle differentiation and patterning is a fundamental process in urinary bladder development that involves a complex array of local environmental factors, epithelial-mesenchymal interaction, and signaling pathways. An epithelial signal is necessary to induce smooth muscle differentiation in the adjacent bladder mesenchyme. The bladder epithelium (urothelium) also influences the spatial organization of the bladder wall. Sonic hedgehog (Shh), which is expressed by the urothelium, promotes mesenchymal proliferation and induces differentiation of smooth muscle from embryonic bladder mesenchyme. Shh, whose signal is mediated through various transcription factors including Gli2 and BMP4, is likely also important in the patterning of bladder smooth muscle. However, it is not known to what extent early mediators of mesenchymal migration, other Shh-associated transcription factors, and crosstalk between the Shh signaling cascade and other pathways, are involved in the patterning of bladder smooth muscle. Here we review the role of epithelial-mesenchymal interaction and Shh signaling in smooth muscle differentiation and patterning in the bladder. We also discuss emerging signaling molecules, transcription factors, and mesenchyme properties that might be fruitful areas of future research in the process of smooth muscle formation in the bladder.
bladder; smooth muscle; differentiation; patterning; sonic hedgehog
Atrioventricular valve development commences with an EMT event whereby endocardial cells transform into mesenchyme. The molecular events that induce this phenotypic change are well understood and include many growth factors, signaling components, and transcription factors. Besides their clear importance in valve development, the role of these transformed mesenchyme and the function they serve in the developing prevalve leaflets is less understood. Indeed, we know that these cells migrate, but how and why do they migrate? We also know that they undergo a transition to a mature, committed cell, largely defined as an interstitial fibroblast due to their ability to secrete various matrix components including collagen type I. However, we have yet to uncover mechanisms by which the matrix is synthesized, how it is secreted, and how it is organized. As valve disease is largely characterized by altered cell number, cell activation, and matrix disorganization, answering questions of how the valves are built will likely provide us with information of real clinical relevance. Although expression profiling and descriptive or correlative analyses are insightful, to advance the field, we must now move past the simplicity of these assays and ask fundamental, mechanistic based questions aimed at understanding how valves are ‘built”. Herein we review current understandings of atrioventricular valve development and present what is known and what isn’t known. In most cases, basic, biological questions and hypotheses that were presented decades ago on valve development still are yet to be answered but likely hold keys to uncovering new discoveries with relevance to both embryonic development and the developmental basis of adult heart valve diseases. Thus, the goal of this review is to remind us of these questions and provide new perspectives on an old theme of valve development.
Valve Development; EMT; Post-EMT; Valve Disease; cushions; Fibroblasts
Partitioning of the four-chambered heart requires the proper formation, interaction and fusion of several mesenchymal tissues derived from different precursor populations that together form the atrioventricular mesenchymal complex. This includes the major endocardial cushions and the mesenchymal cap of the septum primum, which are of endocardial origin, and the dorsal mesenchymal protrusion (DMP), which is derived from the Second Heart Field. Failure of these structures to develop and/or fully mature results in atrial septal defects (ASDs) and atrioventricular septal defects (AVSD). AVSDs are congenital malformations in which the atria are permitted to communicate due to defective septation between the inferior margin of the septum primum and the atrial surface of the common atrioventricular valve. The clinical presentation of AVSDs is variable and depends on both the size and/or type of defect; less severe defects may be asymptomatic while the most severe defect, if untreated, results in infantile heart failure. For many years, maldevelopment of the endocardial cushions was thought to be the sole etiology of AVSDs. More recent work, however, has demonstrated that perturbation of DMP development also results in AVSD. Here, we discuss in detail the formation of the DMP, its contribution to cardiac septation and describe the morphological features as well as potential etiologies of ASDs and AVSDs.
atrioventricular septal defect; AVSD; atrial septal defect; ASD; dorsal mesenchymal protrusion; DMP; ostium primum defect; ostium secundum defect; second heart field; SHF
The heart is a complex organ that is composed of numerous cell types, which must integrate their programs for proper specification, differentiation and cardiac morphogenesis. During cardiogenesis members of the Twist-family of basic helix-loop-helix (bHLH) transcription factors play distinct roles within cardiac lineages such as the endocardium and extra-cardiac lineages such as the cardiac neural crest (cNCC) and epicardium. While the study of these cell populations is often eclipsed by that of cardiomyocytes, the contributions of non-cardiomyocytes to development and disease are increasingly being appreciated as both dynamic and essential. This review summarizes what is known regarding Twist-family bHLH function in extra-cardiac cell populations and the endocardium, with a focus on regulatory mechanisms, downstream targets, and expression profiles. Improving our understanding of the molecular pathways that Twist-family bHLH factors mediate in these lineages will be necessary to ascertain how their dysfunction leads to congenital disease and adult pathologies such as myocardial infarctions and cardiac fibroblast induced fibrosis. Indeed, this knowledge will prove to be critical to clinicians seeking to improve current treatments.
This review discusses the function of neural crest as they relate to cardiovascular defects. The cardiac neural crest cells are a subpopulation of cranial neural crest discovered nearly 30 years ago by ablation of premigratory neural crest. The cardiac neural crest cells are necessary for normal cardiovascular development. We begin with a description of the crest cells in normal development, including their function in remodeling the pharyngeal arch arteries, outflow tract septation, valvulogenesis, and development of the cardiac conduction system. The cells are also responsible for modulating signaling in the caudal pharynx, including the second heart field. Many of the molecular pathways that are known to influence specification, migration, patterning and final targeting of the cardiac neural crest cells are reviewed. The cardiac neural crest cells play a critical role in the pathogenesis of various human cardiocraniofacial syndromes such as DiGeorge, Velocardiofacial, CHARGE, Fetal Alcohol, Alagille, LEOPARD, and Noonan syndromes, as well as Retinoic Acid Embryopathy. The loss of neural crest cells or their dysfunction may not always directly cause abnormal cardiovascular development, but are involved secondarily because crest cells represent a major component in the complex tissue interactions in the head, pharynx and outflow tract. Thus many of the human syndromes linking defects in the heart, face and brain can be better understood when considered within the context of a single cardiocraniofacial developmental module with the neural crest being a key cell type that interconnects the regions.
cardiac neural crest; aortic arch arteries; persistent truncus arteriosus; congentital heart defects; DiGeorge Syndrome; Retinoic Acid; second heart field
Animal models are critically important for mechanistic understanding of embryonic morphogenesis. For decades, visualizing these rapid and complex multidimensional events has relied on projection images and thin section reconstructions. While much insight has been gained, fixed tissue specimens offer limited information on dynamic processes that are essential for tissue assembly and organ patterning. Quantitative imaging is required to unlock the important basic science and clinically relevant secrets that remain hidden. Recent advances in live imaging technology have enabled quantitative longitudinal analysis of embryonic morphogenesis at multiple length and time scales. Four different imaging modalities are currently being used to monitor embryonic morphogenesis: optical, ultrasound, magnetic resonance imaging (MRI), and micro-computed tomography (micro-CT). Each has its advantages and limitations with respect to spatial resolution, depth of field, scanning speed, and tissue contrast. In addition, new processing tools have been developed to enhance live imaging capabilities. In this review, we analyze each type of imaging source and its use in quantitative study of embryonic morphogenesis in small animal models. We describe the physics behind their function, identify some examples in which the modality has revealed new quantitative insights, and then conclude with a discussion of new research directions with live imaging.
Several tissue-specific regulatory genes have been found to play essential roles in both organogenesis and carcinogenesis. In the prostate, the Nkx3.1 homeobox gene plays an important role in normal differentiation of the prostatic epithelium while its loss of function is an initiating event in prostate carcinogenesis in both mouse models and human patients. Thus, the Nkx3.1 homeobox gene provides a paradigm for understanding the relationship between normal differentiation and cancer, as well as studying the roles of homeobox genes in these processes. Here, we review recent findings concerning the roles of Nkx3.1 in development and discuss how its normal function is disrupted in processes of early prostate carcinogenesis.
prostate; homeobox; nkx3.1
Embryonic stem (ES) cells are pluripotent cells that can differentiate into all three main germ layers: endoderm, mesoderm, and ectoderm. Although a number of methods have been developed to differentiate ES cells into neuronal phenotypes such as sensory and motor neurons, the efficient generation of GABAergic interneurons from ES cells still presents an ongoing challenge. Because the main output of inhibitory GABAergic interneurons is the gamma-aminobutyric-acid (GABA), a neurotransmitter whose controlled homeostasis is required for normal brain function, the efficient generation in culture of functional interneurons may have future implications on the treatment of neurological disorders such as epilepsy, autism, and schizophrenia. The goal of this work was to examine the generation of GABAergic neurons from mouse ES cells by comparing an embryoid body-based methodology versus a hydrogel-based encapsulation protocol that involves the use of all-trans-retinoid acid (RA). We observed that 1) there was a 2-fold increase in neuronal differentiation in encapsulated versus non-encapsulated cells and 2) there was an increase in the specificity for interneuronal differentiation in encapsulated cells, as assessed by mRNA expression and electrophysiology approaches. Furthermore, our results indicate that most of the neurons obtained from encapsulated mouse ES cells are GABA-positive (~87%). Thus, these results suggest that combining encapsulation of ES cells and RA treatment provide a more efficient and scalable differentiation strategy for the generation in culture of functional GABAergic interneurons. This technology may have implications for future cell replacement therapies and the treatment of CNS disorders.
stem cell; neuron; encapsulation; GABAergic; differentiation
Activation of Wnt/β-catenin signaling is crucial for the differentiation of pluripotent stem cells, namely the epiblast, embryonic stem, and embryonal carcinoma cells, into mesendoderm. However, downstream events of Wnt/β-catenin signaling that control the formation of mesendoderm are still unclear. In the present study, we used mouse P19 embryonal carcinoma cells as a model, and identified a homeodomain protein Nkx1-2 as a key regulator of mesendoderm formation. In the mouse embryo, Nkx1-2 was expressed in the primitive streak, in which the nascent mesendoderm emerges. In P19 cells, the expression of Nkx1-2 was activated by Wnt/β-catenin signaling independently of Brachyury, an evolutionary conserved early mesendoderm gene. In contrast, the expression of Nkx1-2 was both necessary and sufficient for the activation of Brachyury. Nkx1-2 acted as a transcriptional repressor to mediate the action of Wnt/β-catenin signaling to activate the Brachyury expression. We found Tcf3 as a potential target of gene repression by Nkx1-2, and the down-regulation of Tcf3 was partly required for effective activation of Brachyury by Wnt/β-catenin signaling. These results suggest that Nkx1-2 is a critical component of the gene regulatory network that operates downstream of Wnt/β-catenin signaling to regulate the formation of mesendoderm.
Mesendoderm; Wnt/β-catenin signaling; P19 embryonal carcinoma cell; Brachyury; Primitive streak; Transcription factor
While the pathologies associated with in utero smoke exposure are well established, their underlying molecular mechanisms are incompletely understood. We differentiated human embryonic stem cells in the presence of physiological concentrations of tobacco smoke and nicotine. Using post hoc microarray analysis, quantitative PCR, and immunoblot analysis, we demonstrated that tobacco smoke has lineage- and stage-specific effects on human embryonic stem cell differentiation, through both nicotine-dependent and -independent pathways. We show that three major stem cell pluripotency/differentiation pathways, Notch, canonical Wnt, and transforming growth factor-β, are affected by smoke exposure, and that Nodal signaling through SMAD2 is specifically impacted by effects on Lefty1, Nodal, and FoxH1. These events are associated with upregulation of microRNA-302a, a post-transcriptional silencer of Lefty1. The described studies provide insight into the mechanisms by which tobacco smoke influences fetal development at the cellular level, and identify specific transcriptional, post-transcriptional, and signaling pathways by which this likely occurs.
human embryonic stem cell; differentiation; mesoderm; microRNA; tobacco; nicotine
Congenital limb reduction defects occurring in isolation of other developmental abnormalities continue to be an important medical problem in which little progress has been made. Herein we generated transgenic mice expressing Dkk1 in an appendicular mesodermal pattern. Prx1-Dkk1 mice recapitulate a full spectrum of human congenital limb reduction defects, without other developmental issues, and have normal life-spans. Importantly, a close examination of the inheritance pattern suggests that there is a significant degree of incomplete penetrance as progeny of phenotypically positive or phenotypically negative, but genotypically positive Prx1-Dkk1 mice, consistently give rise to both phenotypically positive mice and phenotypically normal-appearing mice. Thus, this heterogeneous phenotype is reproducible with each generation regardless of the phenotype of the parents. We further go on to identify that mesenchymal stem cells from Prx1-Dkk1 mice have limited proliferative ability, but normal differentiation potential which may explain the mechanism for the limb reduction defects observed. We believe Prx1-Dkk1 mice may prove useful in the future to study the mechanisms underlying the development of congenital limb reduction defects.
Dkk1; limb reduction; mesenchymal stem cell
Ca2+ plays a complex role in the differentiation of committed pre-adipocytes into mature, fat laden adipocytes. Stim1 is a single pass transmembrane protein that has an essential role in regulating the influx of Ca2+ ions through specific plasma membrane store-operated Ca2+ channels. Stim1 is a sensor of endoplasmic reticulum Ca2+ store content and when these stores are depleted ER-localized Stim1 interacts with molecular components of store-operated Ca2+ channels in the plasma membrane to activate these channels and induce Ca2+ influx. To investigate the potential role of Stim1 in Ca2+-mediated adipogenesis, we investigated the expression of Stim1 during adipocyte differentiation and the effects of altering Stim1 expression on the differentiation process. Western blotting revealed that Stim1 was expressed at low levels in 3T3-L1 pre-adipocytes and was upregulated 4 days following induction of differentiation. However, overexpression of Stim1 potently inhibited their ability to differentiate and accumulate lipid, and reduced the expression of C/EBP alpha and adiponectin. Stim1-mediated differentiation was shown to be dependent on store-operated Ca2+ entry, which was increased upon overexpression of Stim1. Overexpression of Stim1 did not disrupt cell proliferation, mitotic clonal expansion or subsequent growth arrest. siRNA-mediated knockdown of endogenous Stim1 had the opposite effect, with increased 3T3-L1 differentiation and increased expression of C/EBP alpha and adiponectin. We thus demonstrate for the first time the presence of store-operated Ca2+ entry in 3T3-L1 adipocytes, and that Stim1-mediated Ca2+ entry negatively regulates adipocyte differentiation. We suggest that increased expression of Stim1 during 3T3-L1 differentiation may act, through its ability to modify the level of Ca2+ influx through store-operated channels, to balance the level of differentiation in these cells in vitro.
Stim1; 3T3-L1; Adipocyte; Store-operated Ca2+ entry; BTP2; Differentiation