-
Development (Cambridge, England) May 2024Collective migration of caudal visceral mesoderm (CVM) cells in Drosophila embryos helps form the longitudinal muscles of the larval gut. In their study, Angelike...
Collective migration of caudal visceral mesoderm (CVM) cells in Drosophila embryos helps form the longitudinal muscles of the larval gut. In their study, Angelike Stathopoulos and colleagues reveal that cell division coordinates two gene expression programmes in migrating CVM cells. To know more about their work, we spoke to the first author, Jingjing Sun, and the corresponding author, Angelike Stathopoulos, Professor in the Division of Biology at the California Institute of Technology, USA.
Topics: Animals; Developmental Biology; History, 20th Century; History, 21st Century; Mesoderm; Drosophila; Cell Movement; Humans
PubMed: 38757779
DOI: 10.1242/dev.203022 -
Neuroradiology May 2024Neurofibromatosis type 1 (NF1) is a multisystem neurocutaneous disorder. Scoliosis and dural ectasia are features of the associated mesodermal dysplasia. Lateral...
Neurofibromatosis type 1 (NF1) is a multisystem neurocutaneous disorder. Scoliosis and dural ectasia are features of the associated mesodermal dysplasia. Lateral thoracic meningoceles can develop in NF1 and progressively enlarge due to cerebrospinal fluid (CSF) pulsations. Large meningoceles can cause compressive symptoms in the thorax. We are reporting a case of a NF1 presenting with acute onset respiratory distress, who also had chronic orthostatic headaches. CT chest showed unruptured enlarging bilateral lateral thoracic meningoceles causing lung compression. MRI of the brain and spine showed features of CSF hypotension, explaining the headaches. CSF hypotension with unruptured meningoceles is extremely rare. Management of the condition is challenging since surgical removal is prone to complications due to underlying mesodermal abnormalities. Cystoperitoneal shunting to relieve lung compression may worsen CSF hypotension. A shunt with a programmable valve allowed controlled drainage and successfully relieved lung compression without worsening of orthostatic headaches in our case.
PubMed: 38755334
DOI: 10.1007/s00234-024-03381-4 -
Nature Communications May 2024Angiogenesis, the growth of new blood vessels from pre-existing vasculature, is essential for the development of new organ systems, but transcriptional control of...
Angiogenesis, the growth of new blood vessels from pre-existing vasculature, is essential for the development of new organ systems, but transcriptional control of angiogenesis remains incompletely understood. Here we show that FOXC1 is essential for retinal angiogenesis. Endothelial cell (EC)-specific loss of Foxc1 impairs retinal vascular growth and expression of Slc3a2 and Slc7a5, which encode the heterodimeric CD98 (LAT1/4F2hc) amino acid transporter and regulate the intracellular transport of essential amino acids and activation of the mammalian target of rapamycin (mTOR). EC-Foxc1 deficiency diminishes mTOR activity, while administration of the mTOR agonist MHY-1485 rescues perturbed retinal angiogenesis. EC-Foxc1 expression is required for retinal revascularization and resolution of neovascular tufts in a model of oxygen-induced retinopathy. Foxc1 is also indispensable for pericytes, a critical component of the blood-retina barrier during retinal angiogenesis. Our findings establish FOXC1 as a crucial regulator of retinal vessels and identify therapeutic targets for treating retinal vascular disease.
Topics: Animals; Forkhead Transcription Factors; Retinal Neovascularization; Mice; Endothelial Cells; Blood-Retinal Barrier; TOR Serine-Threonine Kinases; Pericytes; Fusion Regulatory Protein 1, Heavy Chain; Retinal Vessels; Humans; Large Neutral Amino Acid-Transporter 1; Mice, Knockout; Mice, Inbred C57BL; Retina; Male; Angiogenesis
PubMed: 38755144
DOI: 10.1038/s41467-024-48134-2 -
Development (Cambridge, England) May 2024The patterning of somites is coordinated by presomitic mesoderm cells through synchronised oscillations of Notch signalling, creating sequential waves of gene expression...
The patterning of somites is coordinated by presomitic mesoderm cells through synchronised oscillations of Notch signalling, creating sequential waves of gene expression that propagate from the posterior to the anterior end of the tissue. In a new study, Klepstad and Marcon propose a new theoretical framework that recapitulates the dynamics of mouse somitogenesis observed in vivo and in vitro. To learn more about the story behind the paper, we caught up with first author Julie Klepstad and corresponding author Luciano Marcon, Principal Investigator at the Andalusian Center for Developmental Biology.
Topics: Animals; Developmental Biology; Mice; Somites; History, 21st Century; Humans; Body Patterning; History, 20th Century; Receptors, Notch
PubMed: 38752392
DOI: 10.1242/dev.203021 -
Circulation May 2024Cardiomyocyte differentiation involves a stepwise clearance of repressors and fate-restricting regulators through the modulation of BMP (bone morphogenic...
BACKGROUND
Cardiomyocyte differentiation involves a stepwise clearance of repressors and fate-restricting regulators through the modulation of BMP (bone morphogenic protein)/Wnt-signaling pathways. However, the mechanisms and how regulatory roadblocks are removed with specific developmental signaling pathways remain unclear.
METHODS
We conducted a genome-wide CRISPR screen to uncover essential regulators of cardiomyocyte specification in human embryonic stem cells using a myosin heavy chain 6 ()-GFP (green fluorescence protein) reporter system. After an independent secondary sgRNA validation of 25 candidates, we identified NF2 (neurofibromin 2), a moesin-ezrin-radixin like (MERLIN) tumor suppressor, as an upstream driver of early cardiomyocyte lineage specification. Independent monoclonal knockouts were generated using CRISPR-Cas9, and cell states were inferred through bulk RNA sequencing and protein expression analysis across differentiation time points. Terminal lineage differentiation was assessed by using an in vitro 2-dimensional-micropatterned gastruloid model, trilineage differentiation, and cardiomyocyte differentiation. Protein interaction and post-translation modification of NF2 with its interacting partners were assessed using site-directed mutagenesis, coimmunoprecipitation, and proximity ligation assays.
RESULTS
Transcriptional regulation and trajectory inference from -null cells reveal the loss of cardiomyocyte identity and the acquisition of nonmesodermal identity. Sustained elevation of early mesoderm lineage repressor and upregulation of late anticardiac regulators and in knockout cells reflect a necessary role for in removing regulatory roadblocks. Furthermore, we found that NF2 and AMOT (angiomotin) cooperatively bind to YAP (yes-associated protein) during mesendoderm formation, thereby preventing YAP activation, independent of canonical MST (mammalian sterile 20-like serine-threonine protein kinase)-LATS (large tumor suppressor serine-threonine protein kinase) signaling. Mechanistically, cardiomyocyte lineage identity was rescued by wild-type and NF2 serine-518 phosphomutants, but not NF2 FERM (ezrin-radixin-meosin homology protein) domain blue-box mutants, demonstrating that the critical FERM domain-dependent formation of the AMOT-NF2-YAP scaffold complex at the adherens junction is required for early cardiomyocyte lineage differentiation.
CONCLUSIONS
These results provide mechanistic insight into the essential role of NF2 during early epithelial-mesenchymal transition by sequestering the repressive effect of YAP and relieving regulatory roadblocks en route to cardiomyocytes.
PubMed: 38752370
DOI: 10.1161/CIRCULATIONAHA.122.061335 -
Frontiers in Cell and Developmental... 2024Hematopoiesis continues throughout life to produce all types of blood cells from hematopoietic stem cells (HSCs). Metabolic state is a known regulator of HSC...
Hematopoiesis continues throughout life to produce all types of blood cells from hematopoietic stem cells (HSCs). Metabolic state is a known regulator of HSC self-renewal and differentiation, but whether and how metabolic sensor -GlcNAcylation, which can be modulated via an inhibition of its cycling enzymes -GlcNAcase (OGA) and -GlcNAc transferase (OGT), contributes to hematopoiesis remains largely unknown. Herein, isogenic, single-cell clones of -depleted (OGAi) and -depleted (OGTi) human induced pluripotent stem cells (hiPSCs) were successfully generated from the master hiPSC line MUSIi012-A, which were reprogrammed from CD34 hematopoietic stem/progenitor cells (HSPCs) containing epigenetic memory. The established OGAi and OGTi hiPSCs exhibiting an increase or decrease in cellular -GlcNAcylation concomitant with their loss of OGA and OGT, respectively, appeared normal in phenotype and karyotype, and retained pluripotency, although they may favor differentiation toward certain germ lineages. Upon hematopoietic differentiation through mesoderm induction and endothelial-to-hematopoietic transition, we found that OGA inhibition accelerates hiPSC commitment toward HSPCs and that disruption of -GlcNAc homeostasis affects their commitment toward erythroid lineage. The differentiated HSPCs from all groups were capable of giving rise to all hematopoietic progenitors, thus confirming their functional characteristics. Altogether, the established single-cell clones of OGTi and OGAi hiPSCs represent a valuable platform for further dissecting the roles of -GlcNAcylation in blood cell development at various stages and lineages of blood cells. The incomplete knockout of and in these hiPSCs makes them susceptible to additional manipulation, i.e., by small molecules, allowing the molecular dynamics studies of -GlcNAcylation.
PubMed: 38752196
DOI: 10.3389/fcell.2024.1361943 -
Journal of Medical Virology May 2024The coronavirus disease of 2019 (COVID-19) pandemic has led to more than 700 million confirmed cases and nearly 7 million deaths. Although severe acute respiratory...
The coronavirus disease of 2019 (COVID-19) pandemic has led to more than 700 million confirmed cases and nearly 7 million deaths. Although severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) virus mainly infects the respiratory system, neurological complications are widely reported in both acute infection and long-COVID cases. Despite the success of vaccines and antiviral treatments, neuroinvasiveness of SARS-CoV-2 remains an important question, which is also centered on the mystery of whether the virus is capable of breaching the barriers into the central nervous system. By studying the K18-hACE2 infection model, we observed clear evidence of microvascular damage and breakdown of the blood-brain barrier (BBB). Mechanistically, SARS-CoV-2 infection caused pericyte damage, tight junction loss, endothelial activation and vascular inflammation, which together drive microvascular injury and BBB impairment. In addition, the blood-cerebrospinal fluid barrier at the choroid plexus was also impaired after infection. Therefore, cerebrovascular and choroid plexus dysfunctions are important aspects of COVID-19 and may contribute to neurological complications both acutely and in long COVID.
Topics: Blood-Brain Barrier; Animals; Choroid Plexus; COVID-19; Mice; SARS-CoV-2; Tight Junctions; Disease Models, Animal; Angiotensin-Converting Enzyme 2; Inflammation; Humans; Pericytes
PubMed: 38747003
DOI: 10.1002/jmv.29671 -
BioRxiv : the Preprint Server For... May 2024Over the last decade, single-cell approaches have become the gold standard for studying gene expression dynamics, cell heterogeneity, and cell states within samples....
UNLABELLED
Over the last decade, single-cell approaches have become the gold standard for studying gene expression dynamics, cell heterogeneity, and cell states within samples. Before single-cell advances, the feasibility of capturing the dynamic cellular landscape and rapid cell transitions during early development was limited. In this paper, we designed a robust pipeline to perform single-cell and nuclei analysis on mouse embryos from E6.5 to E8, corresponding to the onset and completion of gastrulation. Gastrulation is a fundamental process during development that establishes the three germinal layers: mesoderm, ectoderm, and endoderm, which are essential for organogenesis. Extensive literature is available on single-cell omics applied to WT perigastrulating embryos. However, single-cell analysis of mutant embryos is still scarce and often limited to FACS-sorted populations. This is partially due to the technical constraints associated with the need for genotyping, timed pregnancies, the count of embryos with desired genotypes per pregnancy, and the number of cells per embryo at these stages. Here, we present a methodology designed to overcome these limitations. This method establishes breeding and timed pregnancy guidelines to achieve a higher chance of synchronized pregnancies with desired genotypes. Optimization steps in the embryo isolation process coupled with FAST genotyping protocol (3 hours) allow for microdroplet-based single-cell to be performed on the same day, ensuring the high viability of cells and robust results. We also include guidelines for optimal nuclei isolations from embryos. Thus, these approaches increase the feasibility of single-cell approaches of mutant embryos at the gastrulation stage. We anticipate this method will facilitate the analysis of how mutations shape the cellular landscape of the gastrula.
SUMMARY
We establish a pipeline for high-quality single-cell and nuclei suspensions of gastrulating mouse embryos for sequencing of single cells and nuclei.
PubMed: 38746120
DOI: 10.1101/2024.04.29.591777 -
Methods in Molecular Biology (Clifton,... 2024The blood-brain barrier (BBB) is one of several barriers between the brain and the peripheral blood system to maintain homeostasis. Understanding the interactions...
The blood-brain barrier (BBB) is one of several barriers between the brain and the peripheral blood system to maintain homeostasis. Understanding the interactions between infectious agents such as human immunodeficiency virus type 1 (HIV-1), which are capable of traversing the BBB and causing neuroinflammation requires modeling an authentic BBB in vitro. Such an in vitro BBB model also helps develop means of targeting viruses that reside in the brain via natural immune effectors such as antibodies. The BBB consists of human brain microvascular endothelial cells (HBMECs), astrocytes, and pericytes. Here we report in vitro methods to establish a dual-cell BBB model consisting of primary HBMECs and primary astrocytes to measure the integrity of the BBB and antibody penetration of the BBB, as well as a method to establish a single cell BBB model to study the impact of HIV-1 infected medium on the integrity of such a BBB.
Topics: Blood-Brain Barrier; Humans; Astrocytes; Endothelial Cells; HIV-1; HIV Infections; Pericytes; Neuroinflammatory Diseases; Coculture Techniques; Cells, Cultured; Brain
PubMed: 38743235
DOI: 10.1007/978-1-0716-3862-0_19 -
Human Cell May 2024Mesenchymal stem/stromal cells (MSCs), originating from the mesoderm, represent a multifunctional stem cell population capable of differentiating into diverse cell types... (Review)
Review
Mesenchymal stem/stromal cells (MSCs), originating from the mesoderm, represent a multifunctional stem cell population capable of differentiating into diverse cell types and exhibiting a wide range of biological functions. Despite more than half a century of research, MSCs continue to be among the most extensively studied cell types in clinical research projects globally. However, their significant heterogeneity and phenotypic instability have significantly hindered their exploration and application. Single-cell sequencing technology emerges as a powerful tool to address these challenges, offering precise dissection of complex cellular samples. It uncovers the genetic structure and gene expression status of individual contained cells on a massive scale and reveals the heterogeneity among these cells. It links the molecular characteristics of MSCs with their clinical applications, contributing to the advancement of regenerative medicine. With the development and cost reduction of single-cell analysis techniques, sequencing technology is now widely applied in fundamental research and clinical trials. This study aimed to review the application of single-cell sequencing in MSC research and assess its prospects.
PubMed: 38743204
DOI: 10.1007/s13577-024-01076-9