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PLoS Biology Jun 2024In the highly regulative embryo of the sea urchin Paracentrotus lividus, establishment of the dorsal-ventral (D/V) axis critically depends on the zygotic expression of...
In the highly regulative embryo of the sea urchin Paracentrotus lividus, establishment of the dorsal-ventral (D/V) axis critically depends on the zygotic expression of the TGF-β nodal in the ventral ectoderm. nodal expression is first induced ubiquitously in the 32-cell embryo and becomes progressively restricted to the presumptive ventral ectoderm by the early blastula stage. This early spatial restriction of nodal expression is independent of Lefty, and instead relies on the activity of Panda, a maternally expressed TGF-β ligand related to Lefty and Inhibins, which is required maternally for D/V axis specification. However, the mechanism by which Panda restricts the early nodal expression has remained enigmatic and it is not known if Panda works like a BMP ligand by opposing Nodal and antagonizing Smad2/3 signaling, or if it works like Lefty by sequestering an essential component of the Nodal signaling pathway. In this study, we report that Panda functions as an antagonist of the TGF-β type II receptor ACVRII (Activin receptor type II), which is the only type II receptor for Nodal signaling in the sea urchin and is also a type II receptor for BMP ligands. Inhibiting translation of acvrII mRNA disrupted D/V patterning across all 3 germ layers and caused acvrII morphants to develop with a typical Nodal loss-of-function phenotype. In contrast, embryos overexpressing acvrII displayed strong ectopic Smad1/5/8 signaling at blastula stages and developed as dorsalized larvae, a phenotype very similar to that caused by over activation of BMP signaling. Remarkably, embryos co-injected with acvrII mRNA and panda mRNA did not show ectopic Smad1/5/8 signaling and developed with a largely normal dorsal-ventral polarity. Furthermore, using an axis induction assay, we found that Panda blocks the ability of ACVRII to orient the D/V axis when overexpressed locally. Using co-immunoprecipitation, we showed that Panda physically interacts with ACVRII, as well as with the Nodal co-receptor Cripto, and with TBR3 (Betaglycan), which is a non-signaling receptor for Inhibins in mammals. At the molecular level, we have traced back the antagonistic activity of Panda to the presence of a single proline residue, conserved with all the Lefty factors, in the ACVRII binding motif of Panda, instead of a serine as in most of TGF-β ligands. Conversion of this proline to a serine converted Panda from an antagonist that opposed Nodal signaling and promoted dorsalization to an agonist that promoted Nodal signaling and triggered ventralization when overexpressed. Finally, using phylogenomics, we analyzed the emergence of the agonist and antagonist form of Panda in the course of evolution. Our data are consistent with the idea that the presence of a serine at that position, like in most TGF-β, was the ancestral condition and that the initial function of Panda was possibly in promoting and not in antagonizing Nodal signaling. These results highlight the existence of key functional and structural elements conserved between Panda and Lefty, allow to draw an intriguing parallel between sea urchin Panda and mammalian Inhibin α and raise the unexpected possibility that the original function of Panda may have been in activation of the Nodal pathway rather than in its inhibition.
PubMed: 38913712
DOI: 10.1371/journal.pbio.3002701 -
Stem Cell Research Jun 2024Ten-Eleven Translocation methylcytosine dioxygenase 1 (TET1) is known to play a broad tumor suppressor role through demethylating and activating tumor suppressor genes....
Ten-Eleven Translocation methylcytosine dioxygenase 1 (TET1) is known to play a broad tumor suppressor role through demethylating and activating tumor suppressor genes. TET1 missense mutations are previously reported in many types of leukemia. Here, the human induced pluripotent stem cell line MURAi001-A was generated from skin fibroblasts derived from a 56-year-old female patient carrying the TET1 gene mutation c.4404A > G (p.I1468M), who had a history of ovarian germ cell tumor. The MURAi001-A cell line demonstrated embryonic-like characteristics as it expressed specific stemness markers, differentiated into the three germ layers, and retained normal karyotyping.
PubMed: 38909482
DOI: 10.1016/j.scr.2024.103474 -
Nature Communications Jun 2024To uncover molecular changes underlying blood-brain-barrier dysfunction in Alzheimer's disease, we performed single nucleus RNA sequencing in 24 Alzheimer's disease and...
To uncover molecular changes underlying blood-brain-barrier dysfunction in Alzheimer's disease, we performed single nucleus RNA sequencing in 24 Alzheimer's disease and control brains and focused on vascular and astrocyte clusters as main cell types of blood-brain-barrier gliovascular-unit. The majority of the vascular transcriptional changes were in pericytes. Of the vascular molecular targets predicted to interact with astrocytic ligands, SMAD3, upregulated in Alzheimer's disease pericytes, has the highest number of ligands including VEGFA, downregulated in Alzheimer's disease astrocytes. We validated these findings with external datasets comprising 4,730 pericyte and 150,664 astrocyte nuclei. Blood SMAD3 levels are associated with Alzheimer's disease-related neuroimaging outcomes. We determined inverse relationships between pericytic SMAD3 and astrocytic VEGFA in human iPSC and zebrafish models. Here, we detect vast transcriptome changes in Alzheimer's disease at the gliovascular-unit, prioritize perturbed pericytic SMAD3-astrocytic VEGFA interactions, and validate these in cross-species models to provide a molecular mechanism of blood-brain-barrier disintegrity in Alzheimer's disease.
Topics: Alzheimer Disease; Humans; Blood-Brain Barrier; Smad3 Protein; Zebrafish; Astrocytes; Vascular Endothelial Growth Factor A; Animals; Pericytes; Male; Induced Pluripotent Stem Cells; Female; Aged; Transcriptome; Brain; Aged, 80 and over; Disease Models, Animal
PubMed: 38902234
DOI: 10.1038/s41467-024-48926-6 -
Nature Communications Jun 2024DNA replication in differentiated cells follows a defined program, but when and how it is established during mammalian development is not known. Here we show using...
DNA replication in differentiated cells follows a defined program, but when and how it is established during mammalian development is not known. Here we show using single-cell sequencing, that late replicating regions are established in association with the B compartment and the nuclear lamina from the first cell cycle after fertilization on both maternal and paternal genomes. Late replicating regions contain a relative paucity of active origins and few but long genes and low G/C content. In both bovine and mouse embryos, replication timing patterns are established prior to embryonic genome activation. Chromosome breaks, which form spontaneously in bovine embryos at sites concordant with human embryos, preferentially locate to late replicating regions. In mice, late replicating regions show enhanced fragility due to a sparsity of dormant origins that can be activated under conditions of replication stress. This pattern predisposes regions with long neuronal genes to fragility and genetic change prior to separation of soma and germ cell lineages. Our studies show that the segregation of early and late replicating regions is among the first layers of genome organization established after fertilization.
Topics: Animals; DNA Replication; Mice; Embryo, Mammalian; Cattle; Nuclear Lamina; Female; Male; Humans; Embryonic Development; Genome; Single-Cell Analysis
PubMed: 38898078
DOI: 10.1038/s41467-024-49565-7 -
Nature Communications Jun 2024Mutations in the FOXF1 gene, a key transcriptional regulator of pulmonary vascular development, cause Alveolar Capillary Dysplasia with Misalignment of Pulmonary Veins,...
Mutations in the FOXF1 gene, a key transcriptional regulator of pulmonary vascular development, cause Alveolar Capillary Dysplasia with Misalignment of Pulmonary Veins, a lethal lung disease affecting newborns and infants. Identification of new FOXF1 upstream regulatory elements is critical to explain why frequent non-coding FOXF1 deletions are linked to the disease. Herein, we use multiome single-nuclei RNA and ATAC sequencing of mouse and human patient lungs to identify four conserved endothelial and mesenchymal FOXF1 enhancers. We demonstrate that endothelial FOXF1 enhancers are autoactivated, whereas mesenchymal FOXF1 enhancers are regulated by EBF1 and GLI1. The cell-specificity of FOXF1 enhancers is validated by disrupting these enhancers in mouse embryonic stem cells using CRISPR/Cpf1 genome editing followed by lineage-tracing of mutant embryonic stem cells in mouse embryos using blastocyst complementation. This study resolves an important clinical question why frequent non-coding FOXF1 deletions that interfere with endothelial and mesenchymal enhancers can lead to the disease.
Topics: Forkhead Transcription Factors; Animals; Humans; Persistent Fetal Circulation Syndrome; Mice; Enhancer Elements, Genetic; Mesoderm; Lung; Endothelial Cells; Zinc Finger Protein GLI1; Embryonic Stem Cells; Pulmonary Alveoli
PubMed: 38898031
DOI: 10.1038/s41467-024-49477-6 -
Nature Communications Jun 2024Single-cell RNA sequencing (scRNA-seq) has been widely used to characterize cell types based on their average gene expression profiles. However, most studies do not...
Single-cell RNA sequencing (scRNA-seq) has been widely used to characterize cell types based on their average gene expression profiles. However, most studies do not consider cell type-specific variation across donors. Modelling this cell type-specific inter-individual variation could help elucidate cell type-specific biology and inform genes and cell types underlying complex traits. We therefore develop a new model to detect and quantify cell type-specific variation across individuals called CTMM (Cell Type-specific linear Mixed Model). We use extensive simulations to show that CTMM is powerful and unbiased in realistic settings. We also derive calibrated tests for cell type-specific interindividual variation, which is challenging given the modest sample sizes in scRNA-seq. We apply CTMM to scRNA-seq data from human induced pluripotent stem cells to characterize the transcriptomic variation across donors as cells differentiate into endoderm. We find that almost 100% of transcriptome-wide variability between donors is differentiation stage-specific. CTMM also identifies individual genes with statistically significant stage-specific variability across samples, including 85 genes that do not have significant stage-specific mean expression. Finally, we extend CTMM to partition interindividual covariance between stages, which recapitulates the overall differentiation trajectory. Overall, CTMM is a powerful tool to illuminate cell type-specific biology in scRNA-seq.
Topics: Humans; Single-Cell Analysis; Sequence Analysis, RNA; Induced Pluripotent Stem Cells; Transcriptome; Cell Differentiation; Gene Expression Profiling; RNA-Seq; Endoderm
PubMed: 38898015
DOI: 10.1038/s41467-024-49242-9 -
Stem Cell Research Jun 2024Here we present the generation of HIMRi006-A and HIMRi007-A Pompe disease (PD) patient derived human induced pluripotent stem cell (hiPSC) lines. HIMRi006-A represents...
Here we present the generation of HIMRi006-A and HIMRi007-A Pompe disease (PD) patient derived human induced pluripotent stem cell (hiPSC) lines. HIMRi006-A represents an infantile onset disease (IOPD) phenotype caused by a homozygous c.307 T > G mutation in the GAA gene. HIMRi007-A is characterized by heterozygous mutations c.-32-13 T > G/c.1716C > G and is associated with an adult onset of disease symptoms (LOPD). Both lines are generated via lentiviral expression of OCT4, SOX2, KLF4, and c-MYC. The lines display a typical embryonic stem cell morphology, express pluripotency markers, retain a normal karyotype (46, XX/XY) and have the differentiation capacity in all three germ layers. Altogether, both lines provide a resource tool to the community for future in depth molecular studies of PD pathomechanism.
PubMed: 38896971
DOI: 10.1016/j.scr.2024.103459 -
Stem Cell Research Jun 2024Calcium indicators are sensitive tools to image neural activity. However, their use in human induced pluripotent stem cells (iPSC)-derived neurons is limited by...
Calcium indicators are sensitive tools to image neural activity. However, their use in human induced pluripotent stem cells (iPSC)-derived neurons is limited by silencing of the transgene. We generated the iPSC line MSE2336A carrying heterozygous insertion in the safe-harbor locus AAVS1 of the ultrasensitive protein calcium sensor (GCaMP6) under the control of CAG promoter and UCOE to maintain robust transgene expression in differentiated cells. The iPSC exhibited normal cell morphology, expression of pluripotency markers, genome integrity, and the ability to differentiate into the three primary germ layers. This line provides a powerful model to study activity in human neurons.
PubMed: 38896969
DOI: 10.1016/j.scr.2024.103455 -
Heterochromatin protein ERH represses alternative cell fates during early mammalian differentiation.BioRxiv : the Preprint Server For... Jun 2024During development, H3K9me3 heterochromatin is dynamically rearranged, silencing repeat elements and protein coding genes to restrict cell identity. Enhancer of...
During development, H3K9me3 heterochromatin is dynamically rearranged, silencing repeat elements and protein coding genes to restrict cell identity. Enhancer of Rudimentary Homolog (ERH) is an evolutionarily conserved protein originally characterized in fission yeast and recently shown to be required for H3K9me3 maintenance in human fibroblasts, but its function during development remains unknown. Here, we show that ERH is required for proper segregation of the inner cell mass and trophectoderm cell lineages during mouse development by repressing totipotent and alternative lineage programs. During human embryonic stem cell (hESC) differentiation into germ layer lineages, ERH is crucial for silencing naïve and pluripotency genes, transposable elements, and alternative lineage genes. Strikingly, ERH depletion in somatic cells reverts the H3K9me3 landscape to an hESC state and enables naïve and pluripotency gene and transposable element activation during iPSC reprogramming. Our findings reveal a role for ERH in initiation and maintenance of developmentally established gene repression.
PubMed: 38895478
DOI: 10.1101/2024.06.06.597604 -
International Journal of Molecular... May 2024Derived from axial structures, Sonic Hedgehog (Shh) is secreted into the paraxial mesoderm, where it plays crucial roles in sclerotome induction and myotome...
Derived from axial structures, Sonic Hedgehog (Shh) is secreted into the paraxial mesoderm, where it plays crucial roles in sclerotome induction and myotome differentiation. Through conditional loss-of-function in quail embryos, we investigate the timing and impact of Shh activity during early formation of sclerotome-derived vertebrae and ribs, and of lateral mesoderm-derived sternum. To this end, Hedgehog interacting protein (Hhip) was electroporated at various times between days 2 and 5. While the vertebral body and rib primordium showed consistent size reduction, rib expansion into the somatopleura remained unaffected, and the sternal bud developed normally. Additionally, we compared these effects with those of locally inhibiting BMP activity. Transfection of Noggin in the lateral mesoderm hindered sternal bud formation. Unlike Hhip, BMP inhibition via Noggin or Smad6 induced myogenic differentiation of the lateral dermomyotome lip, while impeding the growth of the myotome/rib complex into the somatic mesoderm, thus affirming the role of the lateral dermomyotome epithelium in rib guidance. Overall, these findings underscore the continuous requirement for opposing gradients of Shh and BMP activity in the morphogenesis of proximal and distal flank skeletal structures, respectively. Future research should address the implications of these early interactions to the later morphogenesis and function of the musculo-skeletal system and of possible associated malformations.
Topics: Animals; Hedgehog Proteins; Ribs; Spine; Gene Expression Regulation, Developmental; Mesoderm; Quail; Somites; Bone Morphogenetic Proteins; Carrier Proteins
PubMed: 38891790
DOI: 10.3390/ijms25115602