Oct4 redox sensitivity potentiates reprogramming and differentiation.

The transcription factor Oct4/Pou5f1 is a component of the regulatory circuitry governing pluripotency and is widely used to induce pluripotency from somatic cells. Here we used domain swapping and mutagenesis to study Oct4's reprogramming ability, identifying a redox-sensitive DNA binding domain, cysteine residue (Cys48), as a key determinant of reprogramming and differentiation. Oct4 Cys48 sensitizes the protein to oxidative inhibition of DNA binding activity and promotes oxidation-mediated protein ubiquitylation. Pou5f1 C48S point mutation has little effect on undifferentiated embryonic stem cells (ESCs) but upon retinoic acid (RA) treatment causes retention of Oct4 expression, deregulated gene expression, and aberrant differentiation. Pou5f1 C48S ESCs also form less differentiated teratomas and contribute poorly to adult somatic tissues. Finally, we describe Pou5f1 C48S (Janky) mice, which in the homozygous condition are severely developmentally restricted after E4.5. Rare animals bypassing this restriction appear normal at birth but are sterile. Collectively, these findings uncover a novel Oct4 redox mechanism involved in both entry into and exit from pluripotency.

A function of spalt major as a sequence-specific DNA binding transcription factor mediates repression of knirps in the Drosophila wing imaginal disc.

The Spalt transcriptional regulators participate in a variety of cell fate decisions during multicellular development. Vertebrate Spalt proteins have been mostly associated to the organization of heterochromatic regions, but they also contribute regulatory functions through binding to A/T rich motives present in their target genes. The developmental processes in which the Drosophila spalt genes participate are well known through genetic analysis, but the mechanism by which the Spalt proteins regulate transcription are still unknown. Furthermore, despite the prominent changes in gene expression associated to mutations in the spalt genes, the specific DNA sequences they bind are unknow. Here, we analyze a DNA fragment present in the regulatory region of the knirps gene. Spalt proteins are candidate repressors of knirps expression during the formation of the venation pattern in the wing disc, and we identified a minimal conserved 30bp sequence that binds to Spalt major both in vivo and in vitro. This sequence mediates transcriptional repression in the central region of the wing blade, constituting the first confirmed case of a direct regulatory interaction between Spalt major and its target DNA in Drosophila. Interestingly, we also find similar sequences in a set of eight novel candidate Spalt target genes, pointing to a common mechanism of transcriptional repression mediated by Spalt proteins.

Enhancer-promoter interactions become more instructive in the transition from cell-fate specification to tissue differentiation.

To regulate expression, enhancers must come in proximity to their target gene. However, the relationship between the timing of enhancer-promoter (E-P) proximity and activity remains unclear, with examples of uncoupled, anticorrelated and correlated interactions. To assess this, we selected 600 characterized enhancers or promoters with tissue-specific activity in Drosophila embryos and performed Capture-C in FACS-purified myogenic or neurogenic cells during specification and tissue differentiation. This enabled direct comparison between E-P proximity and activity transitioning from OFF-to-ON and ON-to-OFF states across developmental conditions. This showed remarkably similar E-P topologies between specified muscle and neuronal cells, which are uncoupled from activity. During tissue differentiation, many new distal interactions emerge where changes in E-P proximity reflect changes in activity. The mode of E-P regulation therefore appears to change as embryogenesis proceeds, from largely permissive topologies during cell-fate specification to more instructive regulation during terminal tissue differentiation, when E-P proximity is coupled to activation.

The homeobox transcription factor DUXBL controls exit from totipotency.

In mice, exit from the totipotent two-cell (2C) stage embryo requires silencing of the 2C-associated transcriptional program. However, the molecular mechanisms involved in this process remain poorly understood. Here we demonstrate that the 2C-specific transcription factor double homeobox protein (DUX) mediates an essential negative feedback loop by inducing the expression of DUXBL to promote this silencing. We show that DUXBL gains accessibility to DUX-bound regions specifically upon DUX expression. Furthermore, we determine that DUXBL interacts with TRIM24 and TRIM33, members of the TRIM superfamily involved in gene silencing, and colocalizes with them in nuclear foci upon DUX expression. Importantly, DUXBL overexpression impairs 2C-associated transcription, whereas Duxbl inactivation in mouse embryonic stem cells increases DUX-dependent induction of the 2C-transcriptional program. Consequently, DUXBL deficiency in embryos results in sustained expression of 2C-associated transcripts leading to early developmental arrest. Our study identifies DUXBL as an essential regulator of totipotency exit enabling the first divergence of cell fates.

Epigenetic reprogramming in the transition from pluripotency to totipotency.

Mammalian development commences with the zygote, which can differentiate into both embryonic and extraembryonic tissues, a capability known as totipotency. Only the zygote and embryos around zygotic genome activation (ZGA) (two-cell embryo stage in mice and eight-cell embryo in humans) are totipotent cells. Epigenetic modifications undergo extremely extensive changes during the acquisition of totipotency and subsequent development of differentiation. However, the underlying molecular mechanisms remain elusive. Recently, the discovery of mouse two-cell embryo-like cells, human eight-cell embryo-like cells, extended pluripotent stem cells and totipotent-like stem cells with extra-embryonic developmental potential has greatly expanded our understanding of totipotency. Experiments with these in vitro models have led to insights into epigenetic changes in the reprogramming of pluri-to-totipotency, which have informed the exploration of preimplantation development. In this review, we highlight the recent findings in understanding the mechanisms of epigenetic remodeling during totipotency capture, including RNA splicing, DNA methylation, chromatin configuration, histone modifications, and nuclear organization.

scRNA-seq analysis of cells comprising the amphioxus notochord.

Cephalochordates occupy a key phylogenetic position for deciphering the origin and evolution of chordates, since they diverged earlier than urochordates and vertebrates. The notochord is the most prominent feature of chordates. The amphioxus notochord features coin-shaped cells bearing myofibrils. Notochord-derived hedgehog signaling contributes to patterning of the dorsal nerve cord, as in vertebrates. However, properties of constituent notochord cells remain unknown at the single-cell level. We examined these properties using Iso-seq analysis, single-cell RNA-seq analysis, and in situ hybridization (ISH). Gene expression profiles broadly categorize notochordal cells into myofibrillar cells and non-myofibrillar cells. Myofibrillar cells occupy most of the central portion of the notochord, and some cells extend the notochordal horn to both sides of the ventral nerve cord. Some notochord myofibrillar genes are not expressed in myotomes, suggesting an occurrence of myofibrillar genes that are preferentially expressed in notochord. On the other hand, non-myofibrillar cells contain dorsal, lateral, and ventral Müller cells, and all three express both hedgehog and Brachyury. This was confirmed by ISH, although expression of hedgehog in ventral Müller cells was minimal. In addition, dorsal Müller cells express neural transmission-related genes, suggesting an interaction with nerve cord. Lateral Müller cells express hedgehog and other signaling-related genes, suggesting an interaction with myotomes positioned lateral to the notochord. Ventral Müller cells also expressed genes for FGF- and EGF-related signaling, which may be associated with development of endoderm, ventral to the notochord. Lateral Müller cells were intermediate between dorsal/ventral Müller cells. Since vertebrate notochord contributes to patterning and differentiation of ectoderm (nerve cord), mesoderm (somite), and endoderm, this investigation provides evidence that an ancestral or original form of vertebrate notochord is present in extant cephalochordates.

ISL1 and POU4F1 Directly Interact to Regulate the Differentiation and Survival of Inner Ear Sensory Neurons.

The inner ear sensory neurons play a pivotal role in auditory processing and balance control. Though significant progresses have been made, the underlying mechanisms controlling the differentiation and survival of the inner ear sensory neurons remain largely unknown. During development, ISL1 and POU4F transcription factors are co-expressed and are required for terminal differentiation, pathfinding, axon outgrowth and the survival of neurons in the central and peripheral nervous systems. However, little is understood about their functional relationship and regulatory mechanism in neural development. Here, we have knocked out Isl1 or Pou4f1 or both in mice of both sexes. In the absence of Isl1, the differentiation of cochleovestibular ganglion (CVG) neurons is disturbed and with that Isl1-deficient CVG neurons display defects in migration and axon pathfinding. Compound deletion of Isl1 and Pou4f1 causes a delay in CVG differentiation and results in a more severe CVG defect with a loss of nearly all of spiral ganglion neurons (SGNs). Moreover, ISL1 and POU4F1 interact directly in developing CVG neurons and act cooperatively as well as independently in regulating the expression of unique sets of CVG-specific genes crucial for CVG development and survival by binding to the cis-regulatory elements including the promoters of Fgf10, Pou4f2, and Epha5 and enhancers of Eya1 and Ntng2 These findings demonstrate that Isl1 and Pou4f1 are indispensable for CVG development and maintenance by acting epistatically to regulate genes essential for CVG development.

Degradation and translation of maternal mRNA for embryogenesis.

Maternal mRNAs accumulate during egg growth and must be judiciously degraded or translated to ensure successful development of mammalian embryos. In this review we integrate recent investigations into pathways controlling rapid degradation of maternal mRNAs during the maternal-to-zygotic transition. Degradation is not indiscriminate, and some mRNAs are selectively protected and rapidly translated after fertilization for reprogramming the zygotic genome during early embryogenesis. Oocyte specific cofactors and pathways have been illustrated to control different futures of maternal mRNAs. We discuss mechanisms that control the fate of maternal mRNAs during late oogenesis and after fertilization. Issues to be resolved in current maternal mRNA research are described, and future research directions are proposed.

microRNA-1 regulates sea urchin skeletogenesis by directly targeting skeletogenic genes and modulating components of signaling pathways.

microRNAs are evolutionarily conserved non-coding RNAs that direct post-transcriptional regulation of target transcripts. In vertebrates, microRNA-1 (miR-1) is expressed in muscle and has been found to play critical regulatory roles in vertebrate angiogenesis, a process that has been proposed to be analogous to sea urchin skeletogenesis. Results indicate that both miR-1 inhibitor and miR-1 mimic-injected larvae have significantly less F-actin enriched circumpharyngeal muscle fibers and fewer gut contractions. In addition, miR-1 regulates the positioning of skeletogenic primary mesenchyme cells (PMCs) and skeletogenesis of the sea urchin embryo. Interestingly, the gain-of-function of miR-1 leads to more severe PMC patterning and skeletal branching defects than its loss-of-function. The results suggest that miR-1 directly suppresses Ets1/2, Tbr, and VegfR7 of the skeletogenic gene regulatory network, and Nodal, and Wnt1 signaling components. This study identifies potential targets of miR-1 that impacts skeletogenesis and muscle formation and contributes to a deeper understanding of miR-1's function during development.

Timing Drosophila development through steroid hormone action.

Specifically timed pulses of the moulting hormone ecdysone are necessary for developmental progression in insects, guiding development through important milestones such as larval moults, pupation and metamorphosis. It also coordinates the acquisition of cell identities, known as cell patterning, and growth in a tissue-specific manner. In the absence of ecdysone, the ecdysone receptor heterodimer Ecdysone Receptor and Ultraspiracle represses expression of target primary response genes, which become de-repressed as the ecdysone titre rises. However, ecdysone signalling elicits both repressive and activating responses in a temporal and tissue-specific manner. To understand how ecdysone achieves such specificity, this review explores the layers of gene regulation involved in stage-appropriate ecdysone responses in Drosophila fruit flies.

NOTCH2 promotes osteoclast maturation and metabolism and modulates the transcriptome profile during osteoclastogenesis.

Notch signaling plays a key regulatory role in bone remodeling and NOTCH2 enhances osteoclastogenesis, an effect that is mostly mediated by its target gene Hes1. In the present study, we explored mechanisms responsible for the enhanced osteoclastogenesis in bone marrow-derived macrophages (BMM) from Notch2tm1.1Ecan, harboring a NOTCH2 gain-of-function mutation, and control mice. Notch2tm1.1Ecan mice are osteopenic and have enhanced osteoclastogenesis. Bulk RNA-Seq and gene set enrichment analysis of Notch2tm1.1Ecan BMMs cultured in the presence of macrophage colony stimulating factor (M-CSF) and receptor activator of NF-κB ligand revealed enrichment of genes associated with enhanced cell metabolism, aerobic respiration, and mitochondrial function, all associated with osteoclastogenesis. These pathways were not enhanced in the context of a Hes1 inactivation. Analysis of single cell RNA-Seq data of pooled control and Notch2tm1.1Ecan BMMs treated with M-CSF or M-CSF and receptor activator of NF-κB ligand for 3 days identified 11 well-defined cellular clusters. Pseudotime trajectory analysis indicated a trajectory of clusters expressing genes associated with osteoclast progenitors, osteoclast precursors, and mature cells. There were an increased number of cells expressing gene markers associated with the osteoclast and with an unknown, albeit related, cluster in Notch2tm1.1Ecan than in control BMMs as well as enhanced expression of genes associated with osteoclast progenitors and precursors in Notch2tm1.1Ecan cells. In conclusion, BMM cultures display cellular heterogeneity, and NOTCH2 enhances osteoclastogenesis, increases mitochondrial and metabolic activity of osteoclasts, and affects cell cluster allocation in BMMs.

Functional analysis of the Drosophila eve locus in response to non-canonical combinations of gap gene expression levels.

Transcription factor combinations play a key role in shaping cellular identity. However, the precise relationship between specific combinations and downstream effects remains elusive. Here, we investigate this relationship within the context of the Drosophila eve locus, which is controlled by gap genes. We measure spatiotemporal levels of four gap genes in heterozygous and homozygous gap mutant embryos and correlate them with the striped eve activity pattern. Although changes in gap gene expression extend beyond the manipulated gene, the spatial patterns of Eve expression closely mirror canonical activation levels in wild type. Interestingly, some combinations deviate from the wild-type repertoire but still drive eve activation. Although in homozygous mutants some Eve stripes exhibit partial penetrance, stripes consistently emerge at reproducible positions, even with varying gap gene levels. Our findings suggest a robust molecular canalization of cell fates in gap mutants and provide insights into the regulatory constraints governing multi-enhancer gene loci.

Genome-wide identification of notochord enhancers comprising the regulatory landscape of the brachyury locus in mouse.

The node and notochord are important signaling centers organizing the dorso-ventral patterning of cells arising from neuro-mesodermal progenitors forming the embryonic body anlage. Owing to the scarcity of notochord progenitors and notochord cells, a comprehensive identification of regulatory elements driving notochord-specific gene expression has been lacking. Here, we have used ATAC-seq analysis of FACS-purified notochord cells from Theiler stage 12-13 mouse embryos to identify 8921 putative notochord enhancers. In addition, we established a new model for generating notochord-like cells in culture, and found 3728 of these enhancers occupied by the essential notochord control factors brachyury (T) and/or Foxa2. We describe the regulatory landscape of the T locus, comprising ten putative enhancers occupied by these factors, and confirmed the regulatory activity of three of these elements. Moreover, we characterized seven new elements by knockout analysis in embryos and identified one new notochord enhancer, termed TNE2. TNE2 cooperates with TNE in the trunk notochord, and is essential for notochord differentiation in the tail. Our data reveal an essential role of Foxa2 in directing T-expressing cells towards the notochord lineage.

Evolution of the expression and regulation of the nuclear hormone receptor ERR gene family in the chordate lineage.

The Estrogen Related Receptor (ERR) nuclear hormone receptor genes have a wide diversity of roles in vertebrate development. In embryos, ERR genes are expressed in several tissues, including the central and peripheral nervous systems. Here we seek to establish the evolutionary history of chordate ERR genes, their expression and their regulation. We examine ERR expression in mollusc, amphioxus and sea squirt embryos, finding the single ERR orthologue is expressed in the nervous system in all three, with muscle expression also found in the two chordates. We show that most jawed vertebrates and lampreys have four ERR paralogues, and that vertebrate ERR genes were ancestrally linked to Estrogen Receptor genes. One of the lamprey paralogues shares conserved expression domains with jawed vertebrate ERRγ in the embryonic vestibuloacoustic ganglion, eye, brain and spinal cord. Hypothesising that conserved expression derives from conserved regulation, we identify a suite of pan-vertebrate conserved non-coding sequences in ERR introns. We use transgenesis in lamprey and chicken embryos to show that these sequences are regulatory and drive reporter gene expression in the nervous system. Our data suggest an ancient association between ERR and the nervous system, including expression in cells associated with photosensation and mechanosensation. This includes the origin in the vertebrate common ancestor of a suite of regulatory elements in the 3' introns that drove nervous system expression and have been conserved from this point onwards.

Spatiotemporal transcriptomic maps of whole mouse embryos at the onset of organogenesis.

Spatiotemporal orchestration of gene expression is required for proper embryonic development. The use of single-cell technologies has begun to provide improved resolution of early regulatory dynamics, including detailed molecular definitions of most cell states during mouse embryogenesis. Here we used Slide-seq to build spatial transcriptomic maps of complete embryonic day (E) 8.5 and E9.0, and partial E9.5 embryos. To support their utility, we developed sc3D, a tool for reconstructing and exploring three-dimensional 'virtual embryos', which enables the quantitative investigation of regionalized gene expression patterns. Our measurements along the main embryonic axes of the developing neural tube revealed several previously unannotated genes with distinct spatial patterns. We also characterized the conflicting transcriptional identity of 'ectopic' neural tubes that emerge in Tbx6 mutant embryos. Taken together, we present an experimental and computational framework for the spatiotemporal investigation of whole embryonic structures and mutant phenotypes.

Transmembrane protein 150b attenuates BMP signaling in the Xenopus organizer.

The vertebrate organizer is a specified embryonic tissue that regulates dorsoventral patterning and axis formation. Although numerous cellular signaling pathways have been identified as regulators of the organizer's dynamic functions, the process remains incompletely understood, and as-yet unknown pathways remain to be explored for sophisticated mechanistic understanding of the vertebrate organizer. To identify new potential key factors of the organizer, we performed complementary DNA (cDNA) microarray screening using organizer-mimicking Xenopus laevis tissue. This analysis yielded a list of prospective organizer genes, and we determined the role of six-transmembrane domain containing transmembrane protein 150b (Tmem150b) in organizer function. Tmem150b was expressed in the organizer region and induced by Activin/Nodal signaling. In X. laevis, Tmem150b knockdown resulted in head defects and a shortened body axis. Moreover, Tmem150b negatively regulated bone morphogenetic protein (BMP) signaling, likely via physical interaction with activin receptor-like kinase 2 (ALK2). These findings demonstrated that Tmem150b functions as a novel membrane regulatory factor of BMP signaling with antagonistic effects, contributing to the understanding of regulatory molecular mechanisms of organizer axis function. Investigation of additional candidate genes identified in the cDNA microarray analysis could further delineate the genetic networks of the organizer during vertebrate embryogenesis.

Transcriptional network governing extraembryonic endoderm cell fate choice.

The mechanism by which transcription factor (TF) network instructs cell-type-specific transcriptional programs to drive primitive endoderm (PrE) progenitors to commit to parietal endoderm (PE) versus visceral endoderm (VE) cell fates remains poorly understood. To address the question, we analyzed the single-cell transcriptional signatures defining PrE, PE, and VE cell states during the onset of the PE-VE lineage bifurcation. By coupling with the epigenomic comparison of active enhancers unique to PE and VE cells, we identified GATA6, SOX17, and FOXA2 as central regulators for the lineage divergence. Transcriptomic analysis of cXEN cells, an in vitro model for PE cells, after the acute depletion of GATA6 or SOX17 demonstrated that these factors induce Mycn, imparting the self-renewal properties of PE cells. Concurrently, they suppress the VE gene program, including key genes like Hnf4a and Ttr, among others. We proceeded with RNA-seq analysis on cXEN cells with FOXA2 knockout, in conjunction with GATA6 or SOX17 depletion. We found FOXA2 acts as a potent suppressor of Mycn while simultaneously activating the VE gene program. The antagonistic gene regulatory activities of GATA6/SOX17 and FOXA2 in promoting alternative cell fates, and their physical co-bindings at the enhancers provide molecular insights to the plasticity of the PrE lineage. Finally, we show that the external cue, BMP signaling, promotes the VE cell fate by activation of VE TFs and repression of PE TFs including GATA6 and SOX17. These data reveal a putative core gene regulatory module that underpins PE and VE cell fate choice.

Molecular mechanisms controlling vertebrate retinal patterning, neurogenesis, and cell fate specification.

This review covers recent advances in understanding the molecular mechanisms controlling neurogenesis and specification of the developing retina, with a focus on insights obtained from comparative single cell multiomic analysis. We discuss recent advances in understanding the mechanisms by which extrinsic factors trigger transcriptional changes that spatially pattern the optic cup (OC) and control the initiation and progression of retinal neurogenesis. We also discuss progress in unraveling the core evolutionarily conserved gene regulatory networks (GRNs) that specify early- and late-state retinal progenitor cells (RPCs) and neurogenic progenitors and that control the final steps in determining cell identity. Finally, we discuss findings that provide insight into regulation of species-specific aspects of retinal patterning and neurogenesis, including consideration of key outstanding questions in the field.

Live-cell imaging reveals redox metabolic reprogramming during zygotic genome activation.

Metabolic programming is deeply intertwined with early embryonic development including zygotic genome activation (ZGA), the polarization of zygotic cells, and cell fate commitment. It is crucial to establish a noninvasive imaging technology that spatiotemporally illuminates the cellular metabolism pathways in embryos to track developmental metabolism in situ. In this study, we used two high-quality genetically encoded fluorescent biosensors, SoNar for NADH/NAD+ and iNap1 for NADPH, to characterize the dynamic regulation of energy metabolism and redox homeostasis during early zygotic cleavage. Our imaging results showed that NADH/NAD+ levels decreased from the early to the late two-cell stage, whereas the levels of the reducing equivalent NADPH increased. Mechanistically, transcriptome profiling suggested that during the two-cell stage, zygotic cells downregulated the expression of genes involved in glucose uptake and glycolysis, and upregulated the expression of genes for pyruvate metabolism in mitochondria and oxidative phosphorylation, with a decline in the expression of two peroxiredoxin genes, Prdx1 and Prdx2. Collectively, with the establishment of in situ metabolic monitoring technology, our study revealed the programming of redox metabolism during ZGA.