Naïve human pluripotent stem cells (hPSCs) provide a unique experimental platform of cell fate decisions during pre-implantation development, but their lineage potential remains incompletely ...characterized. As naïve hPSCs share transcriptional and epigenomic signatures with trophoblast cells, it has been proposed that the naïve state may have enhanced predisposition for differentiation along this extraembryonic lineage. Here we examined the trophoblast potential of isogenic naïve and primed hPSCs. We found that naïve hPSCs can directly give rise to human trophoblast stem cells (hTSCs) and undergo further differentiation into both extravillous and syncytiotrophoblast. In contrast, primed hPSCs do not support hTSC derivation, but give rise to non-self-renewing cytotrophoblasts in response to BMP4. Global transcriptome and chromatin accessibility analyses indicate that hTSCs derived from naïve hPSCs are similar to blastocyst-derived hTSCs and acquire features of post-implantation trophectoderm. The derivation of hTSCs from naïve hPSCs will enable elucidation of early mechanisms that govern normal human trophoblast development and associated pathologies.
During vertebrate gastrulation, convergence and extension (C and E) of the primary anteroposterior (AP) embryonic axis is driven by polarized mediolateral (ML) cell intercalations and is influenced ...by AP axial patterning. Nodal signaling is essential for patterning of the AP axis while planar cell polarity (PCP) signaling polarizes cells with respect to this axis, but how these two signaling systems interact during C and E is unclear. We find that the neuroectoderm of Nodal-deficient zebrafish gastrulae exhibits reduced C and E cell behaviors, which require Nodal signaling in both cell- and non-autonomous fashions. PCP signaling is partially active in Nodal-deficient embryos and its inhibition exacerbates their C and E defects. Within otherwise naïve zebrafish blastoderm explants, however, Nodal induces C and E in a largely PCP-dependent manner, arguing that Nodal acts both upstream of and in parallel with PCP during gastrulation to regulate embryonic axis extension cooperatively.
Custom-designed nucleases afford a powerful reverse genetic tool for direct gene disruption and genome modification in vivo. Among various applications of the nucleases, homologous recombination ...(HR)-mediated genome editing is particularly useful for inserting heterologous DNA fragments, such as GFP, into a specific genomic locus in a sequence-specific fashion. However, precise HR-mediated genome editing is still technically challenging in zebrafish. Here, we establish a GFP reporter system for measuring the frequency of HR events in live zebrafish embryos. By co-injecting a TALE nuclease and GFP reporter targeting constructs with homology arms of different size, we defined the length of homology arms that increases the recombination efficiency. In addition, we found that the configuration of the targeting construct can be a crucial parameter in determining the efficiency of HR-mediated genome engineering. Implementing these modifications improved the efficiency of zebrafish knock-in generation, with over 10% of the injected F0 animals transmitting gene-targeting events through their germline. We generated two HR-mediated insertion alleles of sox2 and gfap loci that express either superfolder GFP (sfGFP) or tandem dimeric Tomato (tdTomato) in a spatiotemporal pattern that mirrors the endogenous loci. This efficient strategy provides new opportunities not only to monitor expression of endogenous genes and proteins and follow specific cell types in vivo, but it also paves the way for other sophisticated genetic manipulations of the zebrafish genome.
In the mammalian intestine, crypts of Leiberkühn house intestinal epithelial stem/progenitor cells at their base. The mammalian intestine also harbors a diverse array of microbial metabolite ...compounds that potentially modulate stem/progenitor cell activity. Unbiased screening identified butyrate, a prominent bacterial metabolite, as a potent inhibitor of intestinal stem/progenitor proliferation at physiologic concentrations. During homeostasis, differentiated colonocytes metabolized butyrate likely preventing it from reaching proliferating epithelial stem/progenitor cells within the crypt. Exposure of stem/progenitor cells in vivo to butyrate through either mucosal injury or application to a naturally crypt-less host organism led to inhibition of proliferation and delayed wound repair. The mechanism of butyrate action depended on the transcription factor Foxo3. Our findings indicate that mammalian crypt architecture protects stem/progenitor cell proliferation in part through a metabolic barrier formed by differentiated colonocytes that consume butyrate and stimulate future studies on the interplay of host anatomy and microbiome metabolism.
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•Microbial metabolite screening identifies intestinal stem cell effectors•Butyrate suppresses intestinal stem cell proliferation upon exposure•Crypt structure and colonocytes protect stem/progenitor cells
The architecture of intestinal crypts protects the stem cells at their base from a growth-inhibiting metabolite derived from the gut microbiome. Might these findings suggest co-evolution of mammalian anatomy with commensal flora?
Distal arthrogryposis (DA) is group of syndromes characterized by congenital joint contractures. Treatment development is hindered by the lack of vertebrate models. Here, we describe a zebrafish ...model in which a common MYH3 missense mutation (R672H) was introduced into the orthologous zebrafish gene smyhc1 (slow myosin heavy chain 1) (R673H). We simultaneously created a smyhc1 null allele (smyhc1−), which allowed us to compare the effects of both mutant alleles on muscle and bone development, and model the closely related disorder, spondylocarpotarsal synostosis syndrome. Heterozygous smyhc1R673H/+ embryos developed notochord kinks that progressed to scoliosis with vertebral fusions; motor deficits accompanied the disorganized and shortened slow‐twitch skeletal muscle myofibers. Increased dosage of the mutant allele in both homozygous smyhc1R673H/R673H and transheterozygous smyhc1R673H/− embryos exacerbated the notochord and muscle abnormalities, causing early lethality. Treatment of smyhc1R673H/R673H embryos with the myosin ATPase inhibitor, para‐aminoblebbistatin, which decreases actin–myosin affinity, normalized the notochord phenotype. Our zebrafish model of MYH3‐associated DA2A provides insight into pathogenic mechanisms and suggests a beneficial therapeutic role for myosin inhibitors in treating disabling contractures.
Synopsis
Introduction of the common distal arthrogryposis (DA)‐associated missense variant (MYH3R672H) into the analogous zebrafish gene (smyhc1R673H) largely reproduced the human DA phenotype. This model provides key mechanistic insight into the pathogenesis of developmental musculoskeletal disorders as well as exploration of novel therapeutics.
Smyhc1R673H mutant zebrafish exhibit muscle hypercontraction.
Apparent hypercontraction deforms the muscle, shortening muscle fiber length.
The notochord is bent early in development by tension caused by the hypercontracted muscle.
Vertebral fusions and scoliosis develop from kinks in the notochord.
The notochord phenotype is normalized by myosin ATPase inhibitors, and show promise for human therapeutics.
Introduction of the common distal arthrogryposis (DA)‐associated missense variant (MYH3R672H) into the analogous zebrafish gene (smyhc1R673H) largely reproduced the human DA phenotype. This model provides key mechanistic insight into the pathogenesis of developmental musculoskeletal disorders as well as exploration of novel therapeutics.
Morphogen gradients expose cells to different signal concentrations and induce target genes with different ranges of expression. To determine how the Nodal morphogen gradient induces distinct gene ...expression patterns during zebrafish embryogenesis, we measured the activation dynamics of the signal transducer Smad2 and the expression kinetics of long- and short-range target genes. We found that threshold models based on ligand concentration are insufficient to predict the response of target genes. Instead, morphogen interpretation is shaped by the kinetics of target gene induction: the higher the rate of transcription and the earlier the onset of induction, the greater the spatial range of expression. Thus, the timing and magnitude of target gene expression can be used to modulate the range of expression and diversify the response to morphogen gradients.
Chemokines are secreted proteins that regulate a range of processes in eukaryotic organisms. Interestingly, different chemokine receptors control distinct biological processes, and the same receptor ...can direct different cellular responses, but the basis for this phenomenon is not known. To understand this property of chemokine signaling, we examined the function of the chemokine receptors Cxcr4a, Cxcr4b, Ccr7, Ccr9 in the context of diverse processes in embryonic development in zebrafish. Our results reveal that the specific response to chemokine signaling is dictated by cell-type-specific chemokine receptor signal interpretation modules (CRIM) rather than by chemokine-receptor-specific signals. Thus, a generic signal provided by different receptors leads to discrete responses that depend on the specific identity of the cell that receives the signal. We present the implications of employing generic signals in different contexts such as gastrulation, axis specification and single-cell migration.
Vertebrate gastrulation entails massive cell movements that establish and shape the germ layers. During gastrulation, the individual cell behaviors are strictly coordinated in time and space by ...various signaling pathways. These pathways instruct the cells about proliferation, shape, fate and migration into proper location. Convergence and extension (C&E) movements during vertebrate gastrulation play a major role in the shaping of the embryonic body. In vertebrates, the Wnt/Planar Cell Polarity (Wnt/PCP) pathway is a key regulator of C&E movements, essential for several polarized cell behaviors, including directed cell migration, and mediolateral and radial cell intercalation. However, the molecular mechanisms underlying the acquisition of Planar Cell Polarity by highly dynamic mesenchymal cells engaged in C&E are still not well understood. Here we review new evidence implicating the Wnt/PCP pathway in specific cell behaviors required for C&E during zebrafish gastrulation, in comparison to other vertebrates. We also discuss findings on the molecular regulation and the interaction of the Wnt/PCP pathway with other signaling pathways during gastrulation movements.
During vertebrate gastrulation, the evolutionarily conserved morphogenetic movements of epiboly, internalization, convergence and extension cooperate to generate germ layers and to sculpt the body ...plan. In zebrafish, these movements are driven by a variety of cell behaviors, including slow and fast directed migration, radial and mediolateral intercalation, and cell shape changes. Whereas some signaling pathways are required for a subset of these behaviors, other molecules, such as E-cadherin or Gα12 and Gα13 proteins, appear to have a widespread role in different gastrulation cell behaviors.
Cerebrospinal fluid (CSF) physiology is important for the development and homeostasis of the central nervous system, and its disruption has been linked to scoliosis in zebrafish 1, 2. Suspended in ...the CSF is an extracellular structure called the Reissner fiber, which extends from the brain through the central canal of the spinal cord. Zebrafish scospondin-null mutants are unable to assemble a Reissner fiber and fail to form a straight body axis during embryonic development 3. Here, we describe hypomorphic missense mutations of scospondin, which allow Reissner fiber assembly and extension of a straight axis. However, during larval development, these mutants display progressive Reissner fiber disassembly, which is concomitant with the emergence of axial curvatures and scoliosis in adult animals. Using a scospondin-GFP knockin zebrafish line, we demonstrate several dynamic properties of the Reissner fiber in vivo, including embryonic fiber assembly, the continuous rostral to caudal movement of the fiber within the brain and central canal, and subcommissural organ (SCO)-spondin-GFP protein secretion from the floor plate to merge with the fiber. Finally, we show that disassembly of the Reissner fiber is also associated with the progression of axial curvatures in distinct scoliosis mutant zebrafish models. Together, these data demonstrate a critical role for the Reissner fiber for the maintenance of a straight body axis and spine morphogenesis in adult zebrafish. Our study establishes a framework for future investigations to address the cellular effectors responsible for Reissner-fiber-dependent regulation of axial morphology.
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•Hypomorphic scospondin mutants show disassembly of the Reissner fiber and scoliosis•SCO-spondin mislocalization in floor plate cells is associated with fiber loss•A scospondin-GFP knockin strain reveals dynamic properties of Reissner fiber in vivo•Loss of the Reissner fiber is a common feature of scoliosis in zebrafish
Troutwine et al. report a new scospondin-GFP knockin zebrafish strain and demonstrate intriguing dynamic properties of the Reissner fiber in the brain and central canal in vivo. Using forward genetics and cell biological approaches, they demonstrate that Reissner fiber assembly is critical during spine morphogenesis in zebrafish.
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