Automated profiling of gene function during embryonic development.

Systematic functional profiling of the gene set that directs embryonic development is an important challenge. To tackle this challenge, we used 4D imaging of C. elegans embryogenesis to capture the effects of 500 gene knockdowns and developed an automated approach to compare developmental phenotypes. The automated approach quantifies features-including germ layer cell numbers, tissue position, and tissue shape-to generate temporal curves whose parameterization yields numerical phenotypic signatures. In conjunction with a new similarity metric that operates across phenotypic space, these signatures enabled the generation of ranked lists of genes predicted to have similar functions, accessible in the PhenoBank web portal, for ∼25% of essential development genes. The approach identified new gene and pathway relationships in cell fate specification and morphogenesis and highlighted the utilization of specialized energy generation pathways during embryogenesis. Collectively, the effort establishes the foundation for comprehensive analysis of the gene set that builds a multicellular organism.

Transcriptional priming and chromatin regulation during stochastic cell fate specification.

Stochastic cell fate specification, in which a cell chooses between two or more fates with a set probability, diversifies cell subtypes in development. Although this is a vital process across species, a common mechanism for these cell fate decisions remains elusive. This review examines two well-characterized stochastic cell fate decisions to identify commonalities between their developmental programmes. In the fly eye, two subtypes of R7 photoreceptors are specified by the stochastic ON/OFF expression of a transcription factor, spineless. In the mouse olfactory system, olfactory sensory neurons (OSNs) randomly select to express one copy of an olfactory receptor (OR) gene out of a pool of 2800 alleles. Despite the differences in these sensory systems, both stochastic fate choices rely on the dynamic interplay between transcriptional priming, chromatin regulation and terminal gene expression. The coupling of transcription and chromatin modifications primes gene loci in undifferentiated neurons, enabling later expression during terminal differentiation. Here, we compare these mechanisms, examine broader implications for gene regulation during development and posit key challenges moving forward. This article is part of a discussion meeting issue 'Causes and consequences of stochastic processes in development and disease'.

Mechanisms of lineage specification in Caenorhabditis elegans.

The studies of cell fate and lineage specification are fundamental to our understanding of the development of multicellular organisms. Caenorhabditis elegans has been one of the premiere systems for studying cell fate specification mechanisms at single cell resolution, due to its transparent nature, the invariant cell lineage, and fixed number of somatic cells. We discuss the general themes and regulatory mechanisms that have emerged from these studies, with a focus on somatic lineages and cell fates. We next review the key factors and pathways that regulate the specification of discrete cells and lineages during embryogenesis and postembryonic development; we focus on transcription factors and include numerous lineage diagrams that depict the expression of key factors that specify embryonic founder cells and postembryonic blast cells, and the diverse somatic cell fates they generate. We end by discussing some future perspectives in cell and lineage specification.

Low Levels of Amyloid Precursor Protein (APP) Promote Neurogenesis and Decrease Gliogenesis in Human Neural Stem Cells.

Amyloid precursor protein (APP) has been widely studied due to its association with Alzheimer's disease (AD). However, the physiological functions of APP are still largely unexplored. APP is a transmembrane glycoprotein whose expression in humans is abundant in the central nervous system. Specifically, several studies have revealed the high expression of APP during brain development. Previous studies in our laboratory revealed that a transient increase in APP expression induces early cell cycle exit of human neural stem cells (hNSCs) and directs their differentiation towards glial cells (gliogenesis) while decreasing their differentiation towards neurons (neurogenesis). In the present study, we have evaluated the intrinsic cellular effects of APP down-expression (using siRNA) on cell death, cell proliferation, and cell fate specification of hNSCs. Our data indicate that APP silencing causes cellular effects opposite to those obtained in previous APP overexpression assays, inducing cell proliferation in hNS1 cells (a model line of hNSCs) and favoring neurogenesis instead of gliogenesis in these cells. In addition, we have analyzed the gene and protein expression levels of ß-Catenin as a possible molecule involved in these cellular effects. These data could help to understand the biological role of APP, which is necessary to deepen the knowledge of AD.

Lmx1a is a master regulator of the cortical hem.

Development of the nervous system depends on signaling centers - specialized cellular populations that produce secreted molecules to regulate neurogenesis in the neighboring neuroepithelium. In some cases, signaling center cells also differentiate to produce key types of neurons. The formation of a signaling center involves its induction, the maintenance of expression of its secreted molecules, and cell differentiation and migration events. How these distinct processes are coordinated during signaling center development remains unknown. By performing studies in mice, we show that Lmx1a acts as a master regulator to orchestrate the formation and function of the cortical hem (CH), a critical signaling center that controls hippocampus development. Lmx1a co-regulates CH induction, its Wnt signaling, and the differentiation and migration of CH-derived Cajal-Retzius neurons. Combining RNAseq, genetic, and rescue experiments, we identified major downstream genes that mediate distinct Lmx1a-dependent processes. Our work revealed that signaling centers in the mammalian brain employ master regulatory genes and established a framework for analyzing signaling center development.

An expandable FLP-ON::TIR1 system for precise spatiotemporal protein degradation in Caenorhabditis elegans.

The auxin-inducible degradation system has been widely adopted in the Caenorhabditis elegans research community for its ability to empirically control the spatiotemporal expression of target proteins. This system can efficiently degrade auxin-inducible degron (AID)-tagged proteins via the expression of a ligand-activatable AtTIR1 protein derived from A. thaliana that adapts target proteins to the endogenous C. elegans proteasome. While broad expression of AtTIR1 using strong, ubiquitous promoters can lead to rapid degradation of AID-tagged proteins, cell type-specific expression of AtTIR1 using spatially restricted promoters often results in less efficient target protein degradation. To circumvent this limitation, we have developed an FLP/FRT3-based system that functions to reanimate a dormant, high-powered promoter that can drive sufficient AtTIR1 expression in a cell type-specific manner. We benchmark the utility of this system by generating a number of tissue-specific FLP-ON::TIR1 drivers to reveal genetically separable cell type-specific phenotypes for several target proteins. We also demonstrate that the FLP-ON::TIR1 system is compatible with enhanced degron epitopes. Finally, we provide an expandable toolkit utilizing the basic FLP-ON::TIR1 system that can be adapted to drive optimized AtTIR1 expression in any tissue or cell type of interest.

Excitotoxic glutamate levels drive spinal cord ependymal stem cell proliferation and fate specification through CP-AMPAR signaling.

The adult spinal cord contains a population of ependymal-derived neural stem/progenitor cells (epNSPCs) that are normally quiescent, but are activated to proliferate, differentiate, and migrate after spinal cord injury. The mechanisms that regulate their response to injury cues, however, remain unknown. Here, we demonstrate that excitotoxic levels of glutamate promote the proliferation and astrocytic fate specification of adult spinal cord epNSPCs. We show that glutamate-mediated calcium influx through calcium-permeable alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors (CP-AMPARs) in concert with Notch signaling increases the proliferation of epNSPCs via pCREB, and induces astrocytic differentiation through Hes1 upregulation. Furthermore, the in vivo targeting of this pathway via positive modulation of AMPARs after spinal cord injury enhances epNSPC proliferation, astrogliogenesis, neurotrophic factor production and increases neuronal survival. Our study uncovers an important mechanism by which CP-AMPARs regulate the growth and phenotype of epNSPCs, which can be targeted therapeutically to harness the regenerative potential of these cells after injury.

The making of the Drosophila mushroom body.

The mushroom body (MB) is a computational center in the Drosophila brain. The intricate neural circuits of the mushroom body enable it to store associative memories and process sensory and internal state information. The mushroom body is composed of diverse types of neurons that are precisely assembled during development. Tremendous efforts have been made to unravel the molecular and cellular mechanisms that build the mushroom body. However, we are still at the beginning of this challenging quest, with many key aspects of mushroom body assembly remaining unexplored. In this review, I provide an in-depth overview of our current understanding of mushroom body development and pertinent knowledge gaps.

Setting the clock of neural progenitor cells during mammalian corticogenesis.

Radial glial cells (RGCs) as primary neural stem cells in the developing mammalian cortex give rise to diverse types of neurons and glial cells according to sophisticated developmental programs with remarkable spatiotemporal precision. Recent studies suggest that regulation of the temporal competence of RGCs is a key mechanism for the highly conserved and predictable development of the cerebral cortex. Various types of epigenetic regulations, such as DNA methylation, histone modifications, and 3D chromatin architecture, play a key role in shaping the gene expression pattern of RGCs. In addition, epitranscriptomic modifications regulate temporal pre-patterning of RGCs by affecting the turnover rate and function of cell-type-specific transcripts. In this review, we summarize epigenetic and epitranscriptomic regulatory mechanisms that control the temporal competence of RGCs during mammalian corticogenesis. Furthermore, we discuss various developmental elements that also dynamically regulate the temporal competence of RGCs, including biochemical reaction speed, local environmental changes, and subcellular organelle remodeling. Finally, we discuss the underlying mechanisms that regulate the interspecies developmental tempo contributing to human-specific features of brain development.

Enhanced Transcriptional Strength of HIV-1 Subtype C Minimizes Gene Expression Noise and Confers Stability to the Viral Latent State.

Stochastic fluctuations in gene expression emanating from the HIV-1 long terminal repeat (LTR), amplified by the Tat positive feedback circuit, determine the choice between viral infection fates: active transcription (ON) or transcriptional silence (OFF). The emergence of several transcription factor binding site (TFBS) variant strains in HIV-1 subtype C (HIV-1C), especially those containing the duplication of the NF-κB motif, mandates the evaluation of the effect of enhanced transcriptional strength on gene expression noise and its influence on viral fate selection switch. Using a panel of subgenomic LTR-variant strains containing different copy numbers of the NF-κB motif (ranging from 0 to 4), we used flow cytometry, mRNA quantification, and pharmacological perturbations to demonstrate an inverse correlation between promoter strength and gene expression noise in Jurkat T cells and primary CD4+ T cells. The inverse correlation is consistent in clonal cell populations at constant intracellular concentrations of Tat and when NF-κB levels were regulated pharmacologically. Further, we show that strong LTRs containing at least two copies of the NF-κB motif in the enhancer establish a more stable latent state and demonstrate more rapid latency reversal than weak LTRs containing fewer motifs. We also demonstrate a cooperative binding of NF-κB to the motif cluster in HIV-1C LTRs containing two, three, or four NF-κB motifs (Hill coefficient [H] = 2.61, 3.56, and 3.75, respectively). The present work alludes to a possible evolution of the HIV-1C LTR toward gaining transcriptional strength associated with attenuated gene expression noise with implications for viral latency. IMPORTANCE Over the past two consecutive decades, HIV-1 subtype C (HIV-1C) has been undergoing directional evolution toward augmenting the transcriptional strength of the long terminal repeat (LTR) by adding more copies of the existing transcription factor binding site (TFBS) by sequence duplication. Additionally, the duplicated elements are genetically diverse, suggesting broader-range signal receptivity by variant LTRs. The HIV-1 promoter is inherently noisy, and the stochastic fluctuations in gene expression of variant LTRs may influence the active transcription (ON)/transcriptional silence (OFF) latency decisions. The evolving NF-κB motif variations of HIV-1C offer a powerful opportunity to examine how the transcriptional strength of the LTR might influence gene expression noise. Our work here shows that the augmented transcriptional strength of the HIV-1C LTR leads to concomitantly reduced gene expression noise, consequently leading to stabler latency maintenance and rapid latency reversal. The present work offers a novel lead toward appreciating the molecular mechanisms governing HIV-1 latency.

Paracrine interactions through FGFR1 and FGFR2 receptors regulate the development of preimplantation mouse chimaeric embryo.

The preimplantation mammalian embryo has the potential to self-organize, allowing the formation of a correctly patterned embryo despite experimental perturbation. To better understand the mechanisms controlling the developmental plasticity of the early mouse embryo, we used chimaeras composed of an embryonic day (E)3.5 or E4.5 inner cell mass (ICM) and cleaving 8-cell embryo. We revealed that the restricted potential of the ICM can be compensated for by uncommitted 8-cell embryo-derived blastomeres, thus leading to the formation of a normal chimaeric blastocyst that can undergo full development. However, whether such chimaeras maintain developmental competence depends on the presence or specific orientation of the polarized primitive endoderm layer in the ICM component. We also demonstrated that downregulated FGFR1 and FGFR2 expression in 8-cell embryos disturbs intercellular interactions between both components and results in an inverse proportion of primitive endoderm and epiblast within the resulting ICM and abnormal embryo development. This finding suggests that FGF signalling is a key part of the regulatory mechanism that assigns cells to a given lineage and ensures the proper composition of the blastocyst, which is a prerequisite for its successful implantation in the uterus and for further development.

Fmrp regulates neuronal balance in embryonic motor circuit formation.

Motor behavior requires the balanced production and integration of a variety of neural cell types. Motor neurons are positioned in discrete locations in the spinal cord, targeting specific muscles to drive locomotive contractions. Specialized spinal interneurons modulate and synchronize motor neuron activity to achieve coordinated motor output. Changes in the ratios and connectivity of spinal interneurons could drastically alter motor output by tipping the balance of inhibition and excitation onto target motor neurons. Importantly, individuals with Fragile X syndrome (FXS) and associated autism spectrum disorders often have significant motor challenges, including repetitive behaviors and epilepsy. FXS stems from the transcriptional silencing of the gene Fragile X Messenger Ribonucleoprotein 1 (FMR1), which encodes an RNA binding protein that is implicated in a multitude of crucial neurodevelopmental processes, including cell specification. Our work shows that Fmrp regulates the formation of specific interneurons and motor neurons that comprise early embryonic motor circuits. We find that zebrafish fmr1 mutants generate surplus ventral lateral descending (VeLD) interneurons, an early-born cell derived from the motor neuron progenitor domain (pMN). As VeLD interneurons are hypothesized to act as central pattern generators driving the earliest spontaneous movements, this imbalance could influence the formation and long-term function of motor circuits driving locomotion. fmr1 embryos also show reduced expression of proteins associated with inhibitory synapses, including the presynaptic transporter vGAT and the postsynaptic scaffold Gephyrin. Taken together, we show changes in embryonic motor circuit formation in fmr1 mutants that could underlie persistent hyperexcitability.

Cell Fate Decisions Within the Vascular Cambium-Initiating Wood and Bast Formation.

Precise coordination of cell fate decisions is a hallmark of multicellular organisms. Especially in tissues with non-stereotypic anatomies, dynamic communication between developing cells is vital for ensuring functional tissue organization. Radial plant growth is driven by a plant stem cell niche known as vascular cambium, usually strictly producing secondary xylem (wood) inward and secondary phloem (bast) outward, two important structures serving as much-needed CO2 depositories and building materials. Because of its bidirectional nature and its developmental plasticity, the vascular cambium serves as an instructive paradigm for investigating principles of tissue patterning. Although genes and hormones involved in xylem and phloem formation have been identified, we have a yet incomplete picture of the initial steps of cell fate transitions of stem cell daughters into xylem and phloem progenitors. In this mini-review perspective, we describe two possible scenarios of cell fate decisions based on the current knowledge about gene regulatory networks and how cellular environments are established. In addition, we point out further possible research directions.

Identification of the Shared Gene Signatures and Biological Mechanism in Type 2 Diabetes and Pancreatic Cancer.

Background: The relationship between pancreatic cancer (PC) and type 2 diabetes mellitus (T2DM) has long been widely recognized, but the interaction mechanisms are still unknown. This study was aimed to investigate the shared gene signatures and molecular processes between PC and T2DM. Methods: The Gene Expression Omnibus (GEO) database was used to retrieve the RNA sequence and patient information of PC and T2DM. Weighted gene co-expression network analysis (WGCNA) was performed to discover a co-expression network associated with PC and T2DM. Enrichment analysis of shared genes present in PC and T2DM was performed by ClueGO software. These results were validated in the other four cohorts based on differential gene analysis. The predictive significance of S100A6 in PC was evaluated using univariate and multivariate Cox analyses, as well as Kaplan-Meier plots. The biological process of S100A6 enrichment in PC was detected using Gene Set Enrichment Analysis (GSEA). The involvement of S100A6 in the tumor immune microenvironment (TIME) was assessed by CIBERSORT. In vitro assays were used to further confirm the function of S100A6 in PC. Results: WGCNA recognized three major modules for T2DM and two major modules for PC. There were 44 shared genes identified for PC and T2DM, and Gene Ontology (GO) analysis showed that regulation of endodermal cell fate specification was primarily enriched. In addition, a key shared gene S100A6 was derived in the validation tests. S100A6 was shown to be highly expressed in PC compared to non-tumor tissues. PC patients with high S100A6 expression had worse overall survival (OS) than those with low expression. GSEA revealed that S100A6 is involved in cancer-related pathways and glycometabolism-related pathways. There is a strong relationship between S100A6 and TIME. In vitro functional assays showed that S100A6 helped to induce the PC cells' proliferation and migration. We also proposed a diagram of common mechanisms of PC and T2DM. Conclusions: This study firstly revealed that the regulation of endodermal cell fate specification may be common pathogenesis of PC and T2DM and identified S100A6 as a possible biomarker and therapeutic target for PC and T2DM patients.

ECM-integrin signalling instructs cellular position sensing to pattern the early mouse embryo.

Development entails patterned emergence of diverse cell types within the embryo. In mammals, cells positioned inside the embryo give rise to the inner cell mass (ICM), which eventually forms the embryo itself. Yet, the molecular basis of how these cells recognise their 'inside' position to instruct their fate is unknown. Here, we show that provision of extracellular matrix (ECM) to isolated embryonic cells induces ICM specification and alters the subsequent spatial arrangement between epiblast (EPI) and primitive endoderm (PrE) cells that emerge within the ICM. Notably, this effect is dependent on integrin ß1 activity and involves apical-to-basal conversion of cell polarity. We demonstrate that ECM-integrin activity is sufficient for 'inside' positional signalling and is required for correct EPI/PrE patterning. Thus, our findings highlight the significance of ECM-integrin adhesion in enabling position sensing by cells to achieve tissue patterning.

An expansion of the non-coding genome and its regulatory potential underlies vertebrate neuronal diversity.

Proper assembly and function of the nervous system requires the generation of a uniquely diverse population of neurons expressing a cell-type-specific combination of effector genes that collectively define neuronal morphology, connectivity, and function. How countless partially overlapping but cell-type-specific patterns of gene expression are controlled at the genomic level remains poorly understood. Here we show that neuronal genes are associated with highly complex gene regulatory systems composed of independent cell-type- and cell-stage-specific regulatory elements that reside in expanded non-coding genomic domains. Mapping enhancer-promoter interactions revealed that motor neuron enhancers are broadly distributed across the large chromatin domains. This distributed regulatory architecture is not a unique property of motor neurons but is employed throughout the nervous system. The number of regulatory elements increased dramatically during the transition from invertebrates to vertebrates, suggesting that acquisition of new enhancers might be a fundamental process underlying the evolutionary increase in cellular complexity.

hlh-12, a gene that is necessary and sufficient to promote migration of gonadal regulatory cells in Caenorhabditis elegans, evolved within the Caenorhabditis clade.

Specialized cells of the somatic gonad primordium of nematodes play important roles in the final form and function of the mature gonad. Caenorhabditis elegans hermaphrodites are somatic females that have a two-armed, U-shaped gonad that connects to the vulva at the midbody. The outgrowth of each gonad arm from the somatic gonad primordium is led by two female distal tip cells (fDTCs), while the anchor cell (AC) remains stationary and central to coordinate uterine and vulval development. The bHLH protein HLH-2 and its dimerization partners LIN-32 and HLH-12 had previously been shown to be required for fDTC specification. Here, we show that ectopic expression of both HLH-12 and LIN-32 in cells with AC potential transiently transforms them into fDTC-like cells. Furthermore, hlh-12 was known to be required for the fDTCs to sustain gonad arm outgrowth. Here, we show that ectopic expression of HLH-12 in the normally stationary AC causes displacement from its normal position and that displacement likely results from activation of the leader program of fDTCs because it requires genes necessary for gonad arm outgrowth. Thus, HLH-12 is both necessary and sufficient to promote gonadal regulatory cell migration. As differences in female gonadal morphology of different nematode species reflect differences in the fate or migratory properties of the fDTCs or of the AC, we hypothesized that evolutionary changes in the expression of hlh-12 may underlie the evolution of such morphological diversity. However, we were unable to identify an hlh-12 ortholog outside of Caenorhabditis. Instead, by performing a comprehensive phylogenetic analysis of all Class II bHLH proteins in multiple nematode species, we found that hlh-12 evolved within the Caenorhabditis clade, possibly by duplicative transposition of hlh-10. Our analysis suggests that control of gene regulatory hierarchies for gonadogenesis can be remarkably plastic during evolution without adverse phenotypic consequence.

Getting in touch with your senses: Mechanisms specifying sensory interneurons in the dorsal spinal cord.

The spinal cord is functionally and anatomically divided into ventrally derived motor circuits and dorsally derived somatosensory circuits. Sensory stimuli originating either at the periphery of the body, or internally, are relayed to the dorsal spinal cord where they are processed by distinct classes of sensory dorsal interneurons (dIs). dIs convey sensory information, such as pain, heat or itch, either to the brain, and/or to the motor circuits to initiate the appropriate response. They also regulate the intensity of sensory information and are the major target for the opioid analgesics. While the developmental mechanisms directing ventral and dorsal cell fates have been hypothesized to be similar, more recent research has suggested that dI fates are specified by novel mechanisms. In this review, we will discuss the molecular events that specify dorsal neuronal patterning in the spinal cord, thereby generating diverse dI identities. We will then discuss how this molecular understanding has led to the development of robust stem cell methods to derive multiple spinal cell types, including the dIs, and the implication of these studies for treating spinal cord injuries and neurodegenerative diseases. This article is categorized under: Neurological Diseases > Stem Cells and Development.

Notch signaling mediates olfactory multiciliated cell specification.

Epithelial multiciliated cells (MCCs) use motile cilia to direct external fluid flow, the disruption of which is associated with human diseases in a broad array of organs such as those in the respiratory, reproductive, and renal systems. While many of the signaling pathways that regulate MCC formation in these organ systems have been identified, similar characterization of MCC differentiation in the developing olfactory system has been lacking. Here, using live cell tracking, targeted cell ablation, and temporally-specific inhibition of the Notch signaling pathway, we identify the earliest time window of zebrafish olfactory MCC (OMCC) differentiation and demonstrate these cells' derivation from peridermal cells. We also describe regionally segregated Notch signaling across time points of rapid OMCC differentiation and show that Notch signaling downregulation yields an increase in OMCCs, suggesting that OMCC fate is normally repressed in a region-specific manner during olfactory development. Finally, we describe Notch signaling's regulation of the differentiation/ciliogenesis-associated genes foxj1a and foxj1b. Taken together, these findings provide new insights into the origins and developmental programming of OMCCs in vivo.

DDX21 is a p38-MAPK-sensitive nucleolar protein necessary for mouse preimplantation embryo development and cell-fate specification.

Successful navigation of the mouse preimplantation stages of development, during which three distinct blastocyst lineages are derived, represents a prerequisite for continued development. We previously identified a role for p38-mitogen-activated kinases (p38-MAPK) regulating blastocyst inner cell mass (ICM) cell fate, specifically primitive endoderm (PrE) differentiation, that is intimately linked to rRNA precursor processing, polysome formation and protein translation regulation. Here, we develop this work by assaying the role of DEAD-box RNA helicase 21 (DDX21), a known regulator of rRNA processing, in the context of p38-MAPK regulation of preimplantation mouse embryo development. We show nuclear DDX21 protein is robustly expressed from the 16-cell stage, becoming exclusively nucleolar during blastocyst maturation, a localization dependent on active p38-MAPK. siRNA-mediated clonal Ddx21 knockdown within developing embryos is associated with profound cell-autonomous and non-autonomous proliferation defects and reduced blastocyst volume, by the equivalent peri-implantation blastocyst stage. Moreover, ICM residing Ddx21 knockdown clones express the EPI marker NANOG but rarely express the PrE differentiation marker GATA4. These data contribute further significance to the emerging importance of lineage-specific translation regulation, as identified for p38-MAPK, during mouse preimplantation development.