Individual Progenitor Research Articles

The genetics of vertebrate myogenesis

The molecular, genetic and cellular bases for skeletal muscle growth and regeneration have been recently documented in a number of vertebrate species. These studies highlight the role of transient subcompartments of the early somite as a source of distinct waves of myogenic precursors. Individual myogenic progenitor populations undergo a complex series of cell rearrangements and specification events in different regions of the body, all of which are controlled by distinct gene regulatory networks. Collectively, these studies have opened a window into the morphogenetic and molecular bases of the different phases of vertebrate myogenesis, from embryo to adult.

FOXG1 Is Responsible for the Congenital Variant of Rett Syndrome

Rett syndrome is a severe neurodevelopmental disease caused by mutations in the X-linked gene encoding for the methyl-CpG-binding protein MeCP2. Here, we report the identification of FOXG1-truncating mutations in two patients affected by the congenital variant of Rett syndrome. FOXG1 encodes a brain-specific transcriptional repressor that is essential for early development of the telencephalon. Molecular analysis revealed that Foxg1 might also share common molecular mechanisms with MeCP2 during neuronal development, exhibiting partially overlapping expression domain in postnatal cortex and neuronal subnuclear localization.

Temporal Transcription Factors and Their Targets Schedule the End of Neural Proliferation in Drosophila

The timing mechanisms responsible for terminating cell proliferation toward the end of development remain unclear. In the Drosophila CNS, individual progenitors called neuroblasts are known to express a series of transcription factors endowing daughter neurons with different temporal identities. Here we show that Castor and Seven-Up, members of this temporal series, regulate key events in many different neuroblast lineages during late neurogenesis. First, they schedule a switch in the cell size and identity of neurons involving the targets Chinmo and Broad Complex. Second, they regulate the time at which neuroblasts undergo Prospero-dependent cell-cycle exit or Reaper/Hid/Grim-dependent apoptosis. Both types of progenitor termination require the combined action of a late phase of the temporal series and indirect feedforward via Castor targets such as Grainyhead and Dichaete. These studies identify the timing mechanism ending CNS proliferation and reveal how aging progenitors transduce bursts of transcription factors into long-lasting changes in cell proliferation and cell identity.

Individual Retinal Progenitor Cells Display Extensive Heterogeneity of Gene Expression

The development of complex tissues requires that mitotic progenitor cells integrate information from the environment. The highly varied outcomes of such integration processes undoubtedly depend at least in part upon variations among the gene expression programs of individual progenitor cells. To date, there has not been a comprehensive examination of these differences among progenitor cells of a particular tissue. Here, we used comprehensive gene expression profiling to define these differences among individual progenitor cells of the vertebrate retina. Retinal progenitor cells (RPCs) have been shown by lineage analysis to be multipotent throughout development and to produce distinct types of daughter cells in a temporal, conserved order. A total of 42 single RPCs were profiled on Affymetrix arrays. In situ hybridizations performed on both retinal sections and dissociated retinal cells were used to validate the results of the microarrays. An extensive amount of heterogeneity in gene expression among RPCs, even among cells isolated from the same developmental time point, was observed. While many classes of genes displayed heterogeneity of gene expression, the expression of transcription factors constituted a significant amount of the observed heterogeneity. In contrast to previous findings, individual RPCs were found to express multiple bHLH transcription factors, suggesting alternative models to those previously developed concerning how these factors may be coordinated. Additionally, the expression of cell cycle related transcripts showed differences among those associated with G2 and M, versus G1 and S phase, suggesting different levels of regulation for these genes. These data provide insights into the types of processes and genes that are fundamental to cell fate choices, proliferation decisions, and, for cells of the central nervous system, the underpinnings of the formation of complex circuitry.

A Novel Function of DELTA-NOTCH Signalling Mediates the Transition from Proliferation to Neurogenesis in Neural Progenitor Cells

A complete account of the whole developmental process of neurogenesis involves understanding a number of complex underlying molecular processes. Among them, those that govern the crucial transition from proliferative (self-replicating) to neurogenic neural progenitor (NP) cells remain largely unknown. Due to its sequential rostro-caudal gradients of proliferation and neurogenesis, the prospective spinal cord of the chick embryo is a good experimental system to study this issue. We report that the NOTCH ligand DELTA-1 is expressed in scattered cycling NP cells in the prospective chick spinal cord preceding the onset of neurogenesis. These Delta-1-expressing progenitors are placed in between the proliferating caudal neural plate (stem zone) and the rostral neurogenic zone (NZ) where neurons are born. Thus, these Delta-1-expressing progenitors define a proliferation to neurogenesis transition zone (PNTZ). Gain and loss of function experiments carried by electroporation demonstrate that the expression of Delta-1 in individual progenitors of the PNTZ is necessary and sufficient to induce neuronal generation. The activation of NOTCH signalling by DELTA-1 in the adjacent progenitors inhibits neurogenesis and is required to maintain proliferation. However, rather than inducing cell cycle exit and neuronal differentiation by a typical lateral inhibition mechanism as in the NZ, DELTA-1/NOTCH signalling functions in a distinct manner in the PNTZ. Thus, the inhibition of NOTCH signalling arrests proliferation but it is not sufficient to elicit neuronal differentiation. Moreover, after the expression of Delta-1 PNTZ NP continue cycling and induce the expression of Tis21, a gene that is upregulated in neurogenic progenitors, before generating neurons. Together, these experiments unravel a novel function of DELTA–NOTCH signalling that regulates the transition from proliferation to neurogenesis in NP cells. We hypothesize that this novel function is evolutionary conserved.

Spatial specificity of mesodermal even-skipped expression relies on multiple repressor sites

Individual cardiac progenitors emerge at defined positions within each segment in the trunk mesoderm. Their specification depends on segmental information from the pre-patterned ectoderm, which provides positional information to the underlying cardiac mesoderm via inductive signals. This pattern is further reinforced by repressive interactions between transcription factors that are expressed in neighboring sets of cardiac progenitors. For example, even-skipped ( eve) and ladybird early ( lbe) gene products mark adjacent cardiac cell clusters within a segment, and their antagonistic interaction results in mutually exclusive expression domains. Lbe acts directly on the eve mesodermal enhancer (eme) to participate in restricting its expression anteriorly. We hypothesized that additional repressive activities must regulate the precise pattern of eve expression in the cardiac mesoderm via this enhancer. In this study, we identified two additional repressor motifs: 4 copies of an “AT”-rich motif (M1a-d) and 2 copies of an “GC”-rich motif (M2a,b), which when mutated cause expansion of eme-dependent reporter gene expression. We have also examined potential negative regulators of eve and found that their overexpression is sufficient to repress eve as well as the eme enhancer via these sites. Our data suggest that a combination of factors is likely to interact with multiple essential repressor sites to confer precise spatial specificity of eve expression in the cardiac mesoderm.

Statistical analysis of data from retroviral clonal experiments in the developing retina

Retroviral lineage studies have been widely used over the past decade to study retinal development in vivo and in explant culture [Donovan S.L., Dyer, M.A., 2006. Preparation and Square Wave Electroporation of Retinal Explant Cultures, Nature Protocols 1, 2710–2718; Donovan, S.L., Schweers, B., Martins, R., Johnson D., Dyer, M.A., 2001. Compensation by tumor suppressor genes during retinal development in mice and humans, BMC Biol 4 , 14; Dyer M.A., Cepko, C.L., 2001. p27Kip1 and p57Kip2 regulate proliferation in distinct retinal progenitor cell populations, J. of Neurosci 21, 4259–4271; Dyer M.A., Cepko, C.L., 2000. p57(Kip2) regulates progenitor cell proliferation and amacrine interneuron development in the mouse retina, Development 127, 3593–3605; Dyer, M.A., Livesey, F.J., Cepko C.L., Oliver, G., 2003. Prox1 function controls progenitor cell proliferation and horizontal cell genesis in the mammalian retina, Nat Genet 34, 53–58]. These approaches can provide important data on the proliferation, cell fate specification, differentiation and survival of individual neurons and glia derived from single infected retinal progenitor cells. In some experiments, these parameters are compared in retinae from animals with different targeted deletions or transgenes. Alternatively, the effect of ectopic expression of virally encoded transgenes may be studied at the level of individual retinal progenitor cells in vivo and in explant culture. One of the challenges with interpreting retroviral lineage studies is determining the statistical significance of differences in the proliferation, cell fate specification, differentiation of survival of retinal progenitor cells between experimental and control samples. In this study, we provide a clear step-by-step guide to the application of statistical methods to retroviral lineage analyses actual data sets. We anticipate that this will serve as a guide for future statistical analyses of retroviral lineage studies and will help to provide a uniform standard in the field.

In Utero Intraventricular Injection and Electroporation of E15 Mouse Embryos

In-utero in-vivo injection and electroporation of the embryonic mouse neocortex provides a powerful tool for the manipulation of individual progenitors lining the walls of the lateral ventricle. This technique is now widely used to study the processes involved in corticogenesis by over-expressing or knocking down genes and observing the effects on cellular proliferation, migration, and differentiation. In comparison to traditional knockout strategies, in-utero electroporation provides a rapid means to manipulate a population of cells during a specific temporal window. In this video protocol we outline the experimental methodology for preparing mice for surgery, exposing the uterine horns through laporatomy, injecting DNA into the lateral ventricles of the developing embryo, electroporating DNA into the progenitors lining the lateral wall, and caring for animals post-surgery. Our laboratory uses this protocol for surgeries on E13-E16 mice, however it is most commonly performed at E15 as shown in this video.

In Utero Intraventricular Injection and Electroporation of E16 Rat Embryos

In-utero in-vivo injection and electroporation of the embryonic rat neocortex provides a powerful tool for the manipulation of individual progenitors lining the walls of the lateral ventricle. This technique is now widely used to study the processes involved in corticogenesis by over-expressing or knocking down genes and observing the effects on cellular proliferation, migration, and differentiation. In comparison to traditional knockout strategies, in-utero electroporation provides a rapid means to manipulate a population of cells during a specific temporal window. In this video protocol, we outline the experimental methodology for preparing rats for surgery, exposing the uterine horns through laporatomy, injecting DNA into the lateral ventricles of the developing embryo, electroporating DNA into the progenitors lining the lateral wall, and caring for animals post-surgery. Our laboratory uses this protocol for surgeries on E15-E21 rats, however it is most commonly performed at E16 as shown in this video.

In Utero Intraventricular Injection and Electroporation of E16 Rat Embryos

In-utero in-vivo injection and electroporation of the embryonic rat neocortex provides a powerful tool for the manipulation of individual progenitors lining the walls of the lateral ventricle. This technique is now widely used to study the processes involved in corticogenesis by over-expressing or knocking down genes and observing the effects on cellular proliferation, migration, and differentiation. In comparison to traditional knockout strategies, in-utero electroporation provides a rapid means to manipulate a population of cells during a specific temporal window. In this video protocol we outline the experimental methodology for preparing rats for surgery, exposing the uterine horns through laporatomy, injecting DNA into the lateral ventricles of the developing embryo, electroporating DNA into the progenitors lining the lateral wall, and caring for animals post-surgery. Our laboratory uses this protocol for surgeries on E15-E21 rats, however it is most commonly performed at E16 as shown in this video.

No Evidence of Clonal Dominance in Primates up to 4 Years Following Transplantation of Multidrug Resistance 1 Retrovirally Transduced Long-Term Repopulating Cells

Previous murine studies have suggested that retroviral multidrug resistance 1 (MDR1) gene transfer may be associated with a myeloproliferative disorder. Analyses at a clonal level and prolonged long-term follow-up in a model with more direct relevance to human biology were lacking. In this study, we analyzed the contribution of individual CD34-selected peripheral blood progenitor cells to long-term rhesus macaque hematopoiesis after transduction with a retroviral vector either expressing the multidrug resistance 1 gene (HaMDR1 vector) or expressing the neomycin resistance (NeoR) gene (G1Na vector). We found a total of 122 contributing clones from 8 weeks up to 4 years after transplantation. One hundred two clones contained the G1Na vector, whereas only 20 clones contained the HaMDR1 vector. Here, we show for the first time real-time polymerase chain reaction based quantification of individual transduced cell clones constituting 0.0008% +/- 0.0003% to 0.0041% +/- 0.00032% of primate peripheral blood cells. No clonal dominance was observed. Disclosure of potential conflicts of interest is found at the end of this article.

Toll-Like Receptor Ligands and CD154 Stimulate Microglia to Produce a Factor(s) That Promotes Excess Cholinergic Differentiation in the Developing Rat Basal Forebrain: Implications for Neurodevelopmental Disorders

Maternal inflammation plays a role in the etiology of certain neurodevelopmental disorders including autism and schizophrenia. Because maternal inflammation can lead to activation of fetal microglia, we have examined effects of inflamed microglia on cultured neural progenitors from rat embryonic septal region and basal forebrain. These cells give rise to cholinergic neurons projecting to cortex and hippocampus. Microglia stimulated with lipopolysaccharide (LPS), peptidoglycan, Poly I:C and CD154 produce conditioned media (CM) that promotes excessive numbers of cholinergic neurons and levels of choline acetyltransferase (ChAT) activity 6-8 times that of untreated cultures. Expression of the neural-specific transcription factor MATH1 increases substantially within 1 h of plating in LPS-CM. Untreated cultures do not attain equivalent levels until 6 h. By contrast, expression of glial-related transcription factors in LPS-CM-treated cultures never attains the elevated levels of untreated cultures. LPS-CM-treated clones derived from individual progenitors labeled with a LacZ-expressing retrovirus showed >2.5-fold increase in the percentage of cholinergic cells compared with untreated clones. Thus, CM from activated microglia prompts excess cholinergic differentiation from undifferentiated progenitors suggesting that microglial inflammation during critical stages can lead to aberrant brain development.

Sequential Analysis of α‐ and β‐Globin Gene Expression During Erythropoietic Differentiation from Primate Embryonic Stem Cells

The temporal pattern of embryonic, fetal, and adult globin expression in the alpha (zeta --> alpha) and beta (epsilon --> gamma and gamma --> beta) clusters were quantitatively analyzed at the transcriptional and translational levels in erythrocytes induced from primate embryonic stem cells in vitro. When vascular endothelial growth factor receptor-2(high) CD34(+) cells were harvested and reseeded onto OP9 stromal cells, two-wave erythropoiesis occurred sequentially. Immunostaining and real-time reverse transcription-polymerase chain reaction analyses of floating mature erythrocytes revealed that globin switches occurred in parallel with the erythropoietic transition. Colony-forming assays showed replacement of primitive clonogenic progenitor cells with definitive cells during culturing. A decline in embryonic zeta- and epsilon-globin expression at the translational level occurred in individual definitive erythroid progenitors. Expression of beta-globin in individual definitive erythroid progenitors was upregulated in the presence of OP9 stromal cells. Thus, this system reproduces early hematopoietic development in vitro and can serve as a model for analyzing the mechanisms of the globin switch in humans.

Transcription factor profiling in individual hematopoietic progenitors by digital RT-PCR

We report here a systematic, quantitative population analysis of transcription factor expression within developmental progenitors, made possible by a microfluidic chip-based "digital RT-PCR" assay that can count template molecules in cDNA samples prepared from single cells. In a survey encompassing five classes of early hematopoietic precursor, we found markedly heterogeneous expression of the transcription factor PU.1 in hematopoietic stem cells and divergent patterns of PU.1 expression within flk2- and flk2+ common myeloid progenitors. The survey also revealed significant differences in the level of the housekeeping transcript GAPDH across the surveyed populations, which demonstrates caveats of normalizing expression data to endogenous controls and underscores the need to put gene measurement on an absolute, copy-per-cell basis.

Multipotent Flk-1 + Cardiovascular Progenitor Cells Give Rise to the Cardiomyocyte, Endothelial, and Vascular Smooth Muscle Lineages

Cell-tracing studies in the mouse indicate that the cardiac lineage arises from a population that expresses the vascular endothelial growth factor receptor 2 (VEGFR2, Flk-1), suggesting that it may develop from a progenitor with vascular potential. Using the embryonic stem (ES) cell differentiation model, we have identified a cardiovascular progenitor based on the temporal expression of the primitive streak (PS) marker brachyury and Flk-1. Comparable progenitors could also be isolated from head-fold stage embryos. When cultured with cytokines known to function during cardiogenesis, individual cardiovascular progenitors generated colonies that displayed cardiomyocyte, endothelial, and vascular smooth muscle (VSM) potential. Isolation and characterization of this previously unidentified population suggests that the mammalian cardiovascular system develops from multipotential progenitors.

Clonal Analysis of Mouse Development Reveals a Polyclonal Origin for Yolk Sac Blood Islands

Direct clonal analysis of tissue and organ maturation in vivo is a critical step in the interpretation of in vitro cell precursor-progeny relationships. We have developed a method to analyze clonal progenitor contributions in vivo using ES cells stably expressing separate fluorescent proteins and placed into normal blastocysts to form tetrachimeras. Here we applied this method to the analysis of embryonic yolk sac blood islands. In most vertebrates, yolk sac blood islands are the initial sites of appearance of hematopoietic and endothelial cells. It has been proposed that these lineages arise from a common clonal progenitor, the hemangioblast, but this hypothesis has not been tested directly in physiological development in vivo. Our analysis shows that each island has contributions from multiple progenitors. Moreover, contribution by individual hemangioblast progenitors to both endothelial and hematopoietic lineages within an island, if it happens at all, is an infrequent event.

Multilineage Transcriptional Priming and Determination of Alternate Hematopoietic Cell Fates

Hematopoietic stem cells and their progenitors exhibit multilineage patterns of gene expression. Molecular mechanisms underlying the generation and refinement of these patterns during cell fate determination remain unexplored because of the absence of suitable experimental systems. Using PU.1(-/-) progenitors, we demonstrate that at subthreshold levels, this Ets transcription factor regulates a mixed pattern (macrophage/neutrophil) of gene expression within individual myeloid progenitors. Increased PU.1 levels refine the pattern and promote macrophage differentiation by modulating a novel regulatory circuit comprised of counter antagonistic repressors, Egr-1,2/Nab-2 and Gfi-1. Egr-1 and Egr-2 function redundantly to activate macrophage genes and to repress the neutrophil program. These results are used to assemble and mathematically model a gene regulatory network that exhibits both graded and bistable behaviors and accounts for the onset and resolution of mixed lineage patterns during cell fate determination.

Glutamate regulates retinal progenitors cells proliferation during development

The precise coordination of cell cycle exit and cell fate specification is essential for generating the correct proportion of retinal cell types during development. The decision to exit the cell cycle is regulated by intrinsic and extrinsic cues. There is growing evidence that neurotransmitters can regulate cell proliferation and cell fate specification during the early stages of CNS development prior to the formation of synaptic connections. We found that the excitatory neurotransmitter glutamate regulates retinal progenitor cell proliferation during embryonic development of the mouse. AMPA/kainate and N-methyl-d-aspartate receptors are expressed in embryonic retinal progenitor cells. Addition of exogenous glutamate leads to a dose-dependent decrease in cell proliferation without inducing cell death or activating the p53 pathway. Activation of AMPA/kainate receptors induced retinal progenitor cells to prematurely exit the cell cycle. Using a replication-incompetent retrovirus to follow the clonal expansion of individual retinal progenitor cells, it was observed that blockade of AMPA/kainate receptors increased the proportion of large clones, showing that modulation of endogenous glutamatergic activity can have long-term consequences on retinal cell proliferation. Real time reverse transcriptase-polymerase chain reaction and immunoblot analyses demonstrated that glutamate does not alter the levels of the mRNA and proteins that regulate the G1/S-phase transition. Instead, the activity of the Cdk2 kinase is reduced in the presence of glutamate. These data indicate that glutamate regulates retinal progenitor cell proliferation by post-translational modulation of cyclin/Cdk2 kinase activity.

Chalk one up for 'nature' during neocortical neurogenesis

During development, neurons destined for different neocortical layers are sequentially generated. Shen et al. report that this timing is programmed within individual progenitor cells and depends mainly on cell-intrinsic mechanisms.

The timing of cortical neurogenesis is encoded within lineages of individual progenitor cells

In the developing cerebral cortex, neurons are born on a predictable schedule. Here we show in mice that the essential timing mechanism is programmed within individual progenitor cells, and its expression depends solely on cell-intrinsic and environmental factors generated within the clonal lineage. Multipotent progenitor cells undergo repeated asymmetric divisions, sequentially generating neurons in their normal in vivo order: first preplate cells, including Cajal-Retzius neurons, then deep and finally superficial cortical plate neurons. As each cortical layer arises, stem cells and neuroblasts become restricted from generating earlier-born neuron types. Growth as neurospheres or in co-culture with younger cells did not restore their plasticity. Using short-hairpin RNA (shRNA) to reduce Foxg1 expression reset the timing of mid- but not late-gestation progenitors, allowing them to remake preplate neurons and then cortical-plate neurons. Our data demonstrate that neural stem cells change neuropotency during development and have a window of plasticity when restrictions can be reversed.