Cell Cycle Length Research Articles

Decoupled transcript and protein concentrations ensure histone homeostasis in different nutrients

To maintain protein homeostasis in changing nutrient environments, cells must precisely control the amount of their proteins, despite the accompanying changes in cell growth and biosynthetic capacity. As nutrients are major regulators of cell cycle length and progression, a particular challenge arises for the nutrient-dependent regulation of ‘cell cycle genes’, which are periodically expressed during the cell cycle. One important example are histones, which are needed at a constant histone-to-DNA stoichiometry. Here we show that budding yeast achieves histone homeostasis in different nutrients through a decoupling of transcript and protein abundance. We find that cells downregulate histone transcripts in poor nutrients to avoid toxic histone overexpression, but produce constant amounts of histone proteins through nutrient-specific regulation of translation efficiency. Our findings suggest that this allows cells to balance the need for rapid histone production under fast growth conditions with the tight regulation required to avoid toxic overexpression in poor nutrients.

G1 length dictates H3K27me3 landscapes.

Stem cells have lower facultative heterochromatin as defined by trimethylation of histone H3 lysine 27 (H3K27me3) compared to differentiated cells. However, the mechanisms underlying these differential H3K27me3 levels remain elusive. Because H3K27me3 levels are diluted two-fold in every round of replication and then restored through the rest of the cell cycle, we reasoned that the cell cycle length could be a key regulator of total H3K27me3 levels. Here, we propose that a fast cell cycle restricts H3K27me3 levels in stem cells. To test this model, we determined changes to H3K27me3 levels in mESCs globally and at specific loci upon G1 phase lengthening - accomplished by thymidine block or growth in the absence of serum (with the "2i medium"). H3K27me3 levels in mESC increase with G1 arrest when grown in serum and in 2i medium. Additionally, we observed via CUT&RUN and ChIP-seq that regions that gain H3K27me3 in G1 arrest and 2i media overlap, supporting our model of cell cycle length as a critical regulator of the stem cell epigenome and cellular identity. Furthermore, we demonstrate the inverse effect - that G1 shortening in differentiated cells results in a loss of H3K27me3 levels. Finally, in tumor cells with extreme H3K27me3 loss, lengthening of the G1 phase leads to H3K27me3 recovery despite the presence of the dominant negative, sub-stoichiometric H3.1K27M mutation. Our results indicate that G1 length is an essential determinant of H3K27me3 landscapes across diverse cell types.

Cell cycle length governs heterochromatin reprogramming during early development in non-mammalian vertebrates

Heterochromatin marks such as H3K9me3 undergo global erasure and re-establishment after fertilization, and the proper reprogramming of H3K9me3 is essential for early development. Despite the widely conserved dynamics of heterochromatin reprogramming in invertebrates and non-mammalian vertebrates, previous studies have shown that the underlying mechanisms may differ between species. Here, we investigate the molecular mechanism of H3K9me3 dynamics in medaka (Japanese killifish, Oryzias latipes) as a non-mammalian vertebrate model, and show that rapid cell cycle during cleavage stages causes DNA replication-dependent passive erasure of H3K9me3. We also find that cell cycle slowing, toward the mid-blastula transition, permits increasing nuclear accumulation of H3K9me3 histone methyltransferase Setdb1, leading to the onset of H3K9me3 re-accumulation. We further demonstrate that cell cycle length in early development also governs H3K9me3 reprogramming in zebrafish and Xenopus laevis. Together with the previous studies in invertebrates, we propose that a cell cycle length-dependent mechanism for both global erasure and re-accumulation of H3K9me3 is conserved among rapid-cleavage species of non-mammalian vertebrates and invertebrates such as Drosophila, C. elegans, Xenopus and teleost fish.

Atg7 autophagy-independent role on governing neural stem cell fate could be potentially applied for regenerative medicine.

A literature review showed that Atg7 biological role was associated with the development and pathogenesis of nervous system, but very few reports focused on Atg7 role on neurogenesis at the region of spinal cord, so that we are committed to explore the subject. Atg7 expression in neural tube is incrementally increased during neurogenesis. Atg7 neural-specific knockout mice demonstrated the impaired motor function and imbalance of neuronal and glial cell differentiation during neurogenesis, which was similarly confirmed in primary neurosphere culture and reversely verified by Atg7 overexpression in unilateral neural tubes of gastrula chicken embryos. Furthermore, activating autophagy in neural stem cells (NSCs) of neurospheres did not rescue Atg7 deficiency-suppressed neuronal differentiation, but Atg7 overexpression on the basis of autophagy inhibition could reverse Atg7 deficiency-suppressed neuronal differentiation, which provides evidence for the existence of Atg7 role of autophagy-independent function. The underlying mechanism is that Atg7 deficiency directly caused the alteration of cell cycle length of NSCs, which is controlled by Atg7 through specifically binding Mdm2, thereby affecting neuronal differentiation during neurogenesis. Eventually, the effect of overexpressing Atg7-promoting neuronal differentiation was proved in spinal cord injury model as well. Taken together, this study revealed that Atg7 was involved in regulating neurogenesis by a non-autophagic signaling process, and this finding also shed light on the potential application in regenerative medicine.

Nucleotide depletion promotes cell fate transitions by inducing DNA replication stress

Control of cellular identity requires coordination of developmental programs with environmental factors such as nutrient availability, suggesting that perturbing metabolism can alter cell state. Here, we find that nucleotide depletion and DNA replication stress drive differentiation in human and murine normal and transformed hematopoietic systems, including patient-derived acute myeloid leukemia (AML) xenografts. These cell state transitions begin during S phase and are independent of ATR/ATM checkpoint signaling, double-stranded DNA break formation, and changes in cell cycle length. In systems where differentiation is blocked by oncogenic transcription factor expression, replication stress activates primed regulatory loci and induces lineage-appropriate maturation genes despite the persistence of progenitor programs. Altering the baseline cell state by manipulating transcription factor expression causes replication stress to induce genes specific for alternative lineages. The ability of replication stress to selectively activate primed maturation programs across different contexts suggests a general mechanism by which changes in metabolism can promote lineage-appropriate cell state transitions.

UHRF2 accumulates in early G1-phase after serum stimulation or mitotic exit to extend G1 and total cell cycle length

ABSTRACT Ubiquitin like with PHD and ring finger domains 2 (UHRF2) regulates the cell cycle and epigenetics as a multi-domain protein sharing homology with UHRF1. UHRF1 functions with DNMT1 to coordinate daughter strand methylation during DNA replication, but UHRF2 can’t perform this function, and its roles during cell cycle progression are not well defined. UHRF2 role as an oncogene vs. tumor suppressor differs in distinct cell types. UHRF2 interacts with E2F1 to control Cyclin E1 (CCNE1) transcription. UHRF2 also functions in a reciprocal loop with Cyclin E/CDK2 during G1, first as a direct target of CDK2 phosphorylation, but also as an E3-ligase with direct activity toward both Cyclin E and Cyclin D. In this study, we demonstrate that UHRF2 is expressed in early G1 following either serum stimulation out of quiescence or in cells transiting directly out of M-phase, where UHRF2 protein is lost. Further, UHRF2 depletion in G2/M is reversed with a CDK1 specific inhibitor. UHRF2 controls expression levels of cyclins and CDK inhibitors and controls its own transcription in a negative-feedback loop. Deletion of UHRF2 using CRISPR/Cas9 caused a delay in passage through each cell cycle phase. UHRF2 loss culminated in elevated levels of cyclins but also the CDK inhibitor p27KIP1, which regulates G1 passage, to reduce retinoblastoma phosphorylation and increase the amount of time required to reach G1/S passage. Our data indicate that UHRF2 is a central regulator of cell-cycle pacing through its complex regulation of cell cycle gene expression and protein stability.

An Astyanax mexicanus mao knockout line uncovers the developmental roles of monoamine homeostasis in fish brain.

Monoaminergic systems are conserved in vertebrates, yet they present variations in neuroanatomy, genetic components and functions across species. MonoAmine Oxidase, or MAO, is the enzyme responsible for monoamine degradation. While mammals possess two genes, MAO-A and MAO-B, fish possess one single mao gene. To study the function of MAO and monoamine homeostasis on fish brain development and physiology, here we have generated a mao knockout line in Astyanax mexicanus (surface fish), by CRISPR/Cas9 technology. Homozygote mao knockout larvae died at 13 days post-fertilization. Through a time-course analysis, we report that hypothalamic serotonergic neurons undergo fine and dynamic regulation of serotonin level upon loss of mao function, in contrast to those in the raphe, which showed continuously increased serotonin levels - as expected. Dopaminergic neurons were not affected by mao loss-of-function. At behavioral level, knockout fry showed a transient decrease in locomotion that followed the variations in the hypothalamus serotonin neuronal levels. Finally, we discovered a drastic effect of mao knockout on brain progenitors proliferation in the telencephalon and hypothalamus, including a reduction in the number of proliferative cells and an increase of the cell cycle length. Altogether, our results show that MAO has multiple and varied effects on Astyanax mexicanus brain development. Mostly, they bring novel support to the idea that serotonergic neurons in the hypothalamus and raphe of the fish brain are different in nature and identity, and they unravel a link between monoaminergic homeostasis and brain growth.

Linking cell cycle to hematopoietic stem cell fate decisions.

Hematopoietic stem cells (HSCs) have the properties to self-renew and/or differentiate into any blood cell lineages. In order to balance the maintenance of the stem cell pool with supporting mature blood cell production, the fate decisions to self-renew or to commit to differentiation must be tightly controlled, as dysregulation of this process can lead to bone marrow failure or leukemogenesis. The contribution of the cell cycle to cell fate decisions has been well established in numerous types of stem cells, including pluripotent stem cells. Cell cycle length is an integral component of hematopoietic stem cell fate. Hematopoietic stem cells must remain quiescent to prevent premature replicative exhaustion. Yet, hematopoietic stem cells must be activated into cycle in order to produce daughter cells that will either retain stem cell properties or commit to differentiation. How the cell cycle contributes to hematopoietic stem cell fate decisions is emerging from recent studies. Hematopoietic stem cell functions can be stratified based on cell cycle kinetics and divisional history, suggesting a link between Hematopoietic stem cells activity and cell cycle length. Hematopoietic stem cell fate decisions are also regulated by asymmetric cell divisions and recent studies have implicated metabolic and organelle activity in regulating hematopoietic stem cell fate. In this review, we discuss the current understanding of the mechanisms underlying hematopoietic stem cell fate decisions and how they are linked to the cell cycle.

Mettl14-mediated m6A modification ensures the cell-cycle progression of late-born retinal progenitor cells

Neural progenitor cells lengthen their cell cycle to prime themselves for differentiation as development proceeds. It is currently not clear how they counter this lengthening and avoid being halted in the cell cycle. We show that N6-methyladenosine (m6A) methylation of cell-cycle-related mRNAs ensures the proper cell-cycle progression of late-born retinal progenitor cells (RPCs), which are born toward the end of retinogenesis and have long cell-cycle length. Conditional deletion of Mettl14, which is required for depositing m6A, led to delayed cell-cycle exit of late-born RPCs but has no effect on retinal development prior to birth. m6A sequencing and single-cell transcriptomics revealed that mRNAs involved in elongating the cell cycle were highly enriched for m6A, which could target them for degradation and guarantee proper cell-cycle progression. In addition, we identified Zfp292 as a target of m6A and potent inhibitor of RPC cell-cycle progression.

16p11.2 deletion accelerates subpallial maturation and increases variability in human iPSC-derived ventral telencephalic organoids.

Inhibitory interneurons regulate cortical circuit activity, and their dysfunction has been implicated in autism spectrum disorder (ASD). 16p11.2 microdeletions are genetically linked to 1% of ASD cases. However, few studies investigate the effects of this microdeletion on interneuron development. Using ventral telencephalic organoids derived from human induced pluripotent stem cells, we have investigated the effect of this microdeletion on organoid size, progenitor proliferation and organisation into neural rosettes, ganglionic eminence marker expression at early developmental timepoints, and expression of the neuronal marker NEUN at later stages. At early stages, deletion organoids exhibited greater variations in size with concomitant increases in relative neural rosette area and the expression of the ventral telencephalic marker COUPTFII, with increased variability in these properties. Cell cycle analysis revealed an increase in total cell cycle length caused primarily by an elongated G1 phase, the duration of which also varied more than normal. At later stages, deletion organoids increased their NEUN expression. We propose that 16p11.2 microdeletions increase developmental variability and may contribute to ASD aetiology by lengthening the cell cycle of ventral progenitors, promoting premature differentiation into interneurons.

Maternal Immune Activation by Polyinosinic-Polycytidylic Acid Exposure Causes Cerebral Cortical Dysgenesis through Dysregulated Cell Cycle Kinetics of Neural Stem/Progenitor Cells

Maternal immune activation reportedly causes dysregulation of the cell cycle in stem cells and impairment of higher cortical function in rodents. Furthermore, in humans, maternal immune activation during the first to second trimester of pregnancy is strongly correlated with increased incidence of autism spectrum disorder in the offspring. Here, we show that in utero exposure to polyinosinic-polycytidylic acid (poly (I:C)) in mice during the early phase of neuronogenesis increases the probability of differentiation (quiescent fraction [Q fraction]) of neural stem/progenitor cells (NSPCs) without change in the length of cell cycle. This abnormal increase in the Q fraction is assumed to reduce the peak population size of NSPCs, resulting in the thinning of the neocortex in offspring because of the reduced production of neurons. Furthermore, the neocortex of poly (I:C)-exposed mice does not exhibit a layer-specific reduction in radial thickness, possibly because of increased apoptosis caused by poly (I:C) exposure during all stages of cortical development. These results suggest that maternal immune activation by poly (I:C) exposure may affect neocortical histogenesis by altering the cell cycle kinetics of NSPCs. In addition, the timing and amount of poly (I:C) exposure during pregnancy may have profound effects on cerebral cortical histogenesis.

Control of G2 Phase Duration by CDC25B Modulates the Switch from Direct to Indirect Neurogenesis in the Neocortex.

During development, cortical neurons are produced in a temporally regulated sequence from apical progenitors, directly or indirectly, through the production of intermediate basal progenitors. The balance between these major progenitor types is critical for the production of the proper number and types of neurons, and it is thus important to decipher the cellular and molecular cues controlling this equilibrium. Here we address the role of a cell cycle regulator, the CDC25B phosphatase, in this process. We show that, in the developing mouse neocortex of both sex, deleting CDC25B in apical progenitors leads to a transient increase in the production of TBR1+ neurons at the expense of TBR2+ basal progenitors. This phenotype is associated with lengthening of the G2 phase of the cell cycle, the total cell cycle length being unaffected. Using in utero electroporation and cortical slice cultures, we demonstrate that the defect in TBR2+ basal progenitor production requires interaction with CDK1 and is because of the G2 phase lengthening in CDC25B mutants. Together, this study identifies a new role for CDC25B and G2 phase length in direct versus indirect neurogenesis at early stages of cortical development.SIGNIFICANCE STATEMENT This study is the first analysis of the function of CDC25B, a G2/M regulator, in the developing neocortex. We show that removing CDC25B function leads to a transient increase in neuronal differentiation at early stages, occurring simultaneously with a decrease in basal intermediate progenitors (bIPs). Conversely, a CDC25B gain of function promotes production of bIPs, and this is directly related to CDC25B's ability to regulate CDK1 activity. This imbalance of neuron/progenitor production is linked to a G2 phase lengthening in apical progenitors; and using pharmacological treatments on cortical slice cultures, we show that shortening the G2 phase is sufficient to enhance bIP production. Our results reveal the importance of G2 phase length regulation for neural progenitor fate determination.

Initial characterization of gap phase introduction in every cell cycle of C. elegans embryogenesis.

Early embryonic cell cycles usually alternate between S and M phases without any gap phase. When the gap phases are developmentally introduced in various cell types remains poorly defined especially during embryogenesis. To establish the cell-specific introduction of gap phases in embryo, we generate multiple fluorescence ubiquitin cell cycle indicators (FUCCI) in C. elegans. Time-lapse 3D imaging followed by lineal expression profiling reveals sharp and differential accumulation of the FUCCI reporters, allowing the systematic demarcation of cell cycle phases throughout embryogenesis. Accumulation of the reporters reliably identifies both G1 and G2 phases only in two embryonic cells with an extended cell cycle length, suggesting that the remaining cells divide either without a G1 phase, or with a brief G1 phase that is too short to be picked up by our reporters. In summary, we provide an initial picture of gap phase introduction in a metazoan embryo. The newly developed FUCCI reporters pave the way for further characterization of developmental control of cell cycle progression.

CycleFlow simultaneously quantifies cell-cycle phase lengths and quiescence in vivo

SummaryPopulations of stem, progenitor, or cancer cells show proliferative heterogeneity in vivo, comprising proliferating and quiescent cells. Consistent quantification of the quiescent subpopulation and progression of the proliferating cells through the individual phases of the cell cycle has not been achieved. Here, we describe CycleFlow, a method that robustly infers this comprehensive information from standard pulse-chase experiments with thymidine analogs. Inference is based on a mathematical model of the cell cycle, with realistic waiting time distributions for the G1, S, and G2/M phases and a long-term quiescent G0 state. We validate CycleFlow with an exponentially growing cancer cell line in vitro. Applying it to T cell progenitors in steady state in vivo, we uncover strong proliferative heterogeneity, with a minority of CD4+CD8+ T cell progenitors cycling very rapidly and then entering quiescence. CycleFlow is suitable as a routine method for quantitative cell-cycle analysis.

Bilateral Feedback in Oscillator Model Is Required to Explain the Coupling Dynamics of Hes1 with the Cell Cycle

Biological processes are governed by the expression of proteins, and for some proteins, their level of expression can fluctuate periodically over time (i.e., they oscillate). Many oscillatory proteins (e.g., cell cycle proteins and those from the HES family of transcription factors) are connected in complex ways, often within large networks. This complexity can be elucidated by developing intuitive mathematical models that describe the underlying critical aspects of the relationships between these processes. Here, we provide a mathematical explanation of a recently discovered biological phenomenon: the phasic position of the gene Hes1’s oscillatory expression at the beginning of the cell cycle of an individual human breast cancer stem cell can have a predictive value on how long that cell will take to complete a cell cycle. We use a two-component model of coupled oscillators to represent Hes1 and the cell cycle in the same cell with minimal assumptions. Inputting only the initial phase angles, we show that this model is capable of predicting the dynamic mitosis to mitosis behaviour of Hes1 and predicting cell cycle length patterns as found in real-world experimental data. Moreover, we discover that bidirectional coupling between Hes1 and the cell cycle is critical within the system for the data to be reproduced and that nonfixed asymmetry in the interactions between the oscillators is required. The phase dynamics we present here capture the complex interplay between Hes1 and the cell cycle, helping to explain nongenetic cell cycle variability, which has critical implications in cancer treatment contexts.

P-136 Cell exclusion during human embryo development results in altered morphokinetic patterns up to morula formation

Abstract Study question Do embryos that present with cell exclusion show dysregulated cleavage divisions and deviating reproductive success, compared to normally dividing embryos? Summary answer Exclusion embryos have lower reproductive success, showing patterns of delayed and rapid cleavage until morulation, suggesting potential compensatory mechanisms and cellular checkpoints in post-compaction stages What is known already Within assisted reproductive technology, visual observation of embryo development constitutes a major contribution to assessments of embryo competence. Time-lapse technology has allowed for detailed identification and measurement of dynamic events that impact embryo development and reproductive outcome. Two recent studies have identified an anomalous pattern of cell division occurring during early embryo development, termed cell exclusion. These cells seem to arrest during early cleavage stages and fail to participate in downstream compaction, thereby resulting in lower reproductive success. Previous research suggests aberrant chromosomal re-arrangements or irregular cellular content may contribute to negatively associated reproductive outcomes. Study design, size, duration This study is based on a single-clinic retrospective analysis of 515 time-lapse videos collected from 2013-2019. Embryos were cultured in EmbryoScope incubators for 4-5 days post-insemination by conventional in vitro fertilization or intracytoplasmic sperm injection, followed by fresh transfer. Supervised annotations were made for cell divisions and morphological quality by three embryologists. An in-depth morphokinetic analysis was conducted using student’s t-test and analysis of variance for continuous variables. Participants/materials, setting, methods A total of 515 embryo videos and associated annotations were obtained from 502 patients. All videos were individually assessed and grouped according to the presence (n = 122) or absence (n = 393) of an excluded cell. Cell division annotations were collected from the 2-cell stage to blastocyst stage for each video. Time annotations and time intervals between subsequent cell divisions were analyzed to determine differences, if any, between control and exclusion embryos. Main results and the role of chance Following transfer, exclusion embryos resulted in significantly lower rates of fetal heartbeat (28% vs 48%; p < 0.05) and live birth (20% vs 40%; p < 0.05), compared to control embryos. A strong negative association was found between cell exclusion and reproductive success (OR = 0.41; 95% CI = 0.27-0.64; p < 0.0001). Time annotations for 2-cell, 4-cell, and 6-cell to 9-cell stages in the exclusion group showed delayed cell divisions by an average of 3.16 hours up to morulation, compared to control embryos (p < 0.05). However, times to 3-cell and 5-cell stages were significantly earlier than control embryos (p = 0.03 and p = 0.0002, respectively). Exclusion embryos seemed to spend more time in “intermediate” cell stages such as the 3-cell, 5-cell and 6-cell stages (p < 0.01), compared to control embryos. Conversely, they spent less time in commonly known “checkpoint” stages such as the 2-cell, 4-cell, and 8-cell stage (p < 0.001). Moreover, exclusion embryos were found to be more asynchronous than control embryos (p < 0.0001) despite similar cell cycle lengths. No significant morphokinetic differences were found from the start of blastulation to full expansion before zona thinning, between experimental groups. Subgroup analysis revealed no significant trends for known confounding factors pertaining to cell division such as maternal age, paternal age, maternal body mass index and infertility diagnosis. Limitations, reasons for caution Aside from this study being retrospective, there may be unknown confounders that could impact this event. Moreover, identification of cell exclusion was conducted in a single data set generated by one clinic and was only performed by one person, thus limiting the generalizability of the findings. Wider implications of the findings Our results indicate that cell exclusion has negative associations with reproductive outcome. Moreover, morphokinetic analysis has been able to identify aspects of asynchronous and dysregulated cell division that are associated with cell exclusion. Further analysis combining morphokinetics with genetic testing and adhesion protein investigation may improve current embryo assessments. Trial registration number N/A

DNA replication dynamics during erythrocytic schizogony in the malaria parasites Plasmodium falciparum and Plasmodium knowlesi.

Malaria parasites are unusual, early-diverging protozoans with non-canonical cell cycles. They do not undergo binary fission, but divide primarily by schizogony. This involves the asynchronous production of multiple nuclei within the same cytoplasm, culminating in a single mass cytokinesis event. The rate and efficiency of parasite reproduction is fundamentally important to malarial disease, which tends to be severe in hosts with high parasite loads. Here, we have studied for the first time the dynamics of schizogony in two human malaria parasite species, Plasmodium falciparum and Plasmodium knowlesi. These differ in their cell-cycle length, the number of progeny produced and the genome composition, among other factors. Comparing them could therefore yield new information about the parameters and limitations of schizogony. We report that the dynamics of schizogony differ significantly between these two species, most strikingly in the gap phases between successive nuclear multiplications, which are longer in P. falciparum and shorter, but more heterogenous, in P. knowlesi. In both species, gaps become longer as schizogony progresses, whereas each period of active DNA replication grows shorter. In both species there is also extreme variability between individual cells, with some schizonts producing many more nuclei than others, and some individual nuclei arresting their DNA replication for many hours while adjacent nuclei continue to replicate. The efficiency of schizogony is probably influenced by a complex set of factors in both the parasite and its host cell.

Single-cell imaging of the cell cycle reveals CDC25B-induced heterogeneity of G1 phase length in neural progenitor cells.

Although lengthening of the cell cycle and G1 phase is a generic feature of tissue maturation during development, the underlying mechanism remains poorly understood. Here, we develop a time-lapse imaging strategy to measure the four cell cycle phases in single chick neural progenitor cells in their endogenous environment. We show that neural progenitors are widely heterogeneous with respect to cell cycle length. This variability in duration is distributed over all phases of the cell cycle, with the G1 phase contributing the most. Within one cell cycle, each phase duration appears stochastic and independent except for a correlation between S and M phase duration. Lineage analysis indicates that the majority of daughter cells may have a longer G1 phase than mother cells, suggesting that, at each cell cycle, a mechanism lengthens the G1 phase. We identify that the CDC25B phosphatase known to regulate the G2/M transition indirectly increases the duration of the G1 phase, partly through delaying passage through the restriction point. We propose that CDC25B increases the heterogeneity of G1 phase length, revealing a previously undescribed mechanism of G1 lengthening that is associated with tissue development.

Generation of a mouse model of the neurodevelopmental disorder with dysmorphic facies and distal limb anomalies syndrome.

Heterozygous variants in bromodomain and plant homeodomain containing transcription factor (BPTF) cause the neurodevelopmental disorder with dysmorphic facies and distal limb anomalies (NEDDFL) syndrome (MIM#617755) characterized by intellectual disability, speech delay and postnatal microcephaly. BPTF functions within nucleosome and remodeling factor (NURF), a complex comprising sucrose non-fermenting like (SNF2L), an Imitation SWItching (ISWI) chromatin remodeling protein encoded by the SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 1 (SMARCA1) gene. Surprisingly, ablation of Smarca1 resulted in mice with enlarged brains, a direct contrast to the phenotype of NEDDFL patients. To model the NEDDFL syndrome, we generated forebrain-specific Bptf knockout (Bptf conditional Knockout (cKO)) mice. The Bptf cKO mice were born in normal Mendelian ratios, survived to adulthood but were smaller in size with severe cortical hypoplasia. Prolonged progenitor cell cycle length and a high incidence of cell death reduced the neuronal output. Cortical lamination was also disrupted with reduced proportions of deep layer neurons, and neuronal maturation defects that impaired the acquisition of distinct cell fates (e.g. COUP-TF-interacting protein 2 (Ctip2)+ neurons). RNAseq and pathway analysis identified altered expression of fate-determining transcription factors and the biological pathways involved in neural development, apoptotic signaling and amino acid biosynthesis. Dysregulated genes were enriched for MYC Proto-Oncogene, BHLH Transcription Factor (Myc)-binding sites, a known BPTF transcriptional cofactor. We propose the Bptf cKO mouse as a valuable model for further study of the NEDDFL syndrome.

The extracellular matrix molecule tenascin-C modulates cell cycle progression and motility of adult neural stem/progenitor cells from the subependymal zone

Adult neurogenesis has been described in two canonical regions of the adult central nervous system (CNS) of rodents, the subgranular zone (SGZ) of the hippocampus and the subependymal zone (SEZ) of the lateral ventricles. The stem cell niche of the SEZ provides a privileged environment composed of a specialized extracellular matrix (ECM) that comprises the glycoproteins tenascin-C (Tnc) and laminin-1 (LN1). In the present study, we investigated the function of these ECM glycoproteins in the adult stem cell niche. Adult neural stem/progenitor cells (aNSPCs) of the SEZ were prepared from wild type (Tnc+/+) and Tnc knockout (Tnc−/−) mice and analyzed using molecular and cell biological approaches. A delayed maturation of aNSPCs in Tnc−/− tissue was reflected by a reduced capacity to form neurospheres in response to epidermal growth factor (EGF). To examine a potential influence of the ECM on cell proliferation, aNSPCs of both genotypes were studied by cell tracking using digital video microscopy. aNSPCs were cultivated on three different substrates, namely, poly-d-lysine (PDL) and PDL replenished with either LN1 or Tnc for up to 6 days in vitro. On each of the three substrates aNSPCs displayed lineage trees that could be investigated with regard to cell cycle length. The latter appeared reduced in Tnc−/− aNSPCs on PDL and LN1 substrates, less so on Tnc that seemed to compensate the absence of the ECM compound to some extent. Close inspection of the lineage trees revealed a subpopulation of late dividing aNSPCslate that engaged into cycling after a notable delay. aNSPCslate exhibited a clearly different morphology, with a larger cell body and conspicuous processes. aNSPCslate reiterated the reduction in cell cycle length on all substrates tested, which was not rescued on Tnc substrates. When the migratory activity of aNSPC-derived progeny was determined, Tnc−/− neuroblasts displayed significantly longer migration tracks. This was traced to an increased rate of migration episodes compared to the wild-type cells that rested for longer time periods. We conclude that Tnc intervenes in the proliferation of aNSPCs and modulates the motility of neuroblasts in the niche of the SEZ.