Homeobox transcription factor MNX1 is crucial for restraining the expression of pan-neuronal genes in motor neurons

Motor neurons (MNs) control muscle movement and are essential for breathing, walking and fine motor skills. Motor Neuron and Pancreas Homeobox 1 (MNX1) has long been recognized as a key marker of the MN lineage. Deficiency of the Mnx1 gene in mice results in early postnatal lethality - likely by causing abnormal MN development and respiratory malfunction. However, the genome-wide targets and exact regulatory function of Mnx1 in MNs remains unresolved. Using an in vitro model for efficient MN induction from mouse embryonic stem cells, we identified about six thousand MNX1-bound loci, of which half are conserved enhancers co-bound by the core MN-inducing factors ISL1 and LHX3, while the other half are promoters for housekeeping-like genes. Despite its widespread binding, disruption of Mnx1 affects the activity of only a few dozen MNX1-bound loci, and causes mis-regulation of about one hundred genes, the majority of which are up-regulated pan-neuronal genes with relatively higher expression in the brain compared to MNs. Integration of genome-wide binding, transcriptomic and epigenomic data in the wild-type and Mnx1-disrupted MNs predicts that Pbx3 and Pou6f2 are two putative direct targets of MNX1, and both are homeobox transcription factors highly expressed in the central nervous system. Our results suggest that MNX1 is crucial for restraining the expression of many pan-neuronal genes in MNs, likely in an indirect fashion. Further, the rarity of direct targets in contrast to the widespread binding of MNX1 reflects a distinctive mode of transcriptional regulation by homeobox transcriptional factors.

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Construction of a mammalian embryo model from stem cells organized by a morphogen signalling centre

Nature Communications ◽

10.1038/s41467-021-23653-4 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Peng-Fei Xu ◽

Ricardo Moraes Borges ◽

Jonathan Fillatre ◽

Maraysa de Oliveira-Melo ◽

Tao Cheng ◽

...

Keyword(s):

Stem Cells ◽

Cell Tracking ◽

Cardiac Tissue ◽

Embryonic Stem ◽

Mammalian Embryo ◽

Wide Range ◽

Vitro Model ◽

Neurula Stage

AbstractGenerating properly differentiated embryonic structures in vitro from pluripotent stem cells remains a challenge. Here we show that instruction of aggregates of mouse embryonic stem cells with an experimentally engineered morphogen signalling centre, that functions as an organizer, results in the development of embryo-like entities (embryoids). In situ hybridization, immunolabelling, cell tracking and transcriptomic analyses show that these embryoids form the three germ layers through a gastrulation process and that they exhibit a wide range of developmental structures, highly similar to neurula-stage mouse embryos. Embryoids are organized around an axial chordamesoderm, with a dorsal neural plate that displays histological properties similar to the murine embryo neuroepithelium and that folds into a neural tube patterned antero-posteriorly from the posterior midbrain to the tip of the tail. Lateral to the chordamesoderm, embryoids display somitic and intermediate mesoderm, with beating cardiac tissue anteriorly and formation of a vasculature network. Ventrally, embryoids differentiate a primitive gut tube, which is patterned both antero-posteriorly and dorso-ventrally. Altogether, embryoids provide an in vitro model of mammalian embryo that displays extensive development of germ layer derivatives and that promises to be a powerful tool for in vitro studies and disease modelling.

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Genome-Wide Binding Analyses of HOXB1 Revealed a Novel DNA Binding Motif Associated with Gene Repression

Journal of Developmental Biology ◽

10.3390/jdb9010006 ◽

2021 ◽

Vol 9 (1) ◽

pp. 6

Author(s):

Narendra Pratap Singh ◽

Bony De Kumar ◽

Ariel Paulson ◽

Mark E. Parrish ◽

Carrie Scott ◽

...

Keyword(s):

Dna Binding ◽

Target Genes ◽

Embryonic Stem ◽

Binding Motif ◽

Binding Assays ◽

Histone Marks ◽

Genome Wide ◽

Hox Cofactors

Knowledge of the diverse DNA binding specificities of transcription factors is important for understanding their specific regulatory functions in animal development and evolution. We have examined the genome-wide binding properties of the mouse HOXB1 protein in embryonic stem cells differentiated into neural fates. Unexpectedly, only a small number of HOXB1 bound regions (7%) correlate with binding of the known HOX cofactors PBX and MEIS. In contrast, 22% of the HOXB1 binding peaks display co-occupancy with the transcriptional repressor REST. Analyses revealed that co-binding of HOXB1 with PBX correlates with active histone marks and high levels of expression, while co-occupancy with REST correlates with repressive histone marks and repression of the target genes. Analysis of HOXB1 bound regions uncovered enrichment of a novel 15 base pair HOXB1 binding motif HB1RE (HOXB1 response element). In vitro template binding assays showed that HOXB1, PBX1, and MEIS can bind to this motif. In vivo, this motif is sufficient for direct expression of a reporter gene and over-expression of HOXB1 selectively represses this activity. Our analyses suggest that HOXB1 has evolved an association with REST in gene regulation and the novel HB1RE motif contributes to HOXB1 function in part through a repressive role in gene expression.

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Oxygen tension modulates the mitochondrial genetic bottleneck and influences the segregation of a heteroplasmic mtDNA variant in vitro

Communications Biology ◽

10.1038/s42003-021-02069-2 ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Mikael G. Pezet ◽

Aurora Gomez-Duran ◽

Florian Klimm ◽

Juvid Aryaman ◽

Stephen Burr ◽

...

Keyword(s):

Germ Cell ◽

Oxygen Tension ◽

Germ Line ◽

Embryonic Stem ◽

Mitochondrial Diseases ◽

Genetic Bottleneck ◽

Cellular Mechanisms ◽

Germ Cell Specification ◽

Vitro Model

AbstractMost humans carry a mixed population of mitochondrial DNA (mtDNA heteroplasmy) affecting ~1–2% of molecules, but rapid percentage shifts occur over one generation leading to severe mitochondrial diseases. A decrease in the amount of mtDNA within the developing female germ line appears to play a role, but other sub-cellular mechanisms have been implicated. Establishing an in vitro model of early mammalian germ cell development from embryonic stem cells, here we show that the reduction of mtDNA content is modulated by oxygen and reaches a nadir immediately before germ cell specification. The observed genetic bottleneck was accompanied by a decrease in mtDNA replicating foci and the segregation of heteroplasmy, which were both abolished at higher oxygen levels. Thus, differences in oxygen tension occurring during early development likely modulate the amount of mtDNA, facilitating mtDNA segregation and contributing to tissue-specific mutation loads.

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Genome-wide analysis of microrna and mRNA expression in Anti-N-methyl-D-aspartate receptor encephalitis in vitro model

Journal of the Neurological Sciences ◽

10.1016/j.jns.2017.08.1812 ◽

2017 ◽

Vol 381 ◽

pp. 643-644 ◽

Cited By ~ 1

Author(s):

J.S. Jun ◽

J. Moon ◽

T.J. Kim ◽

S.T. Lee ◽

K.H. Jung ◽

...

Keyword(s):

Mrna Expression ◽

In Vitro Model ◽

Genome Wide Analysis ◽

Aspartate Receptor ◽

Genome Wide ◽

Vitro Model

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SOX9 is required for kidney fibrosis and activates NAV3 to drive renal myofibroblast function

Science Signaling ◽

10.1126/scisignal.abb4282 ◽

2021 ◽

Vol 14 (672) ◽

pp. eabb4282 ◽

Cited By ~ 1

Author(s):

Sayyid Raza ◽

Elliot Jokl ◽

James Pritchett ◽

Katherine Martin ◽

Kim Su ◽

...

Keyword(s):

Transcription Factor ◽

Actin Polymerization ◽

Kidney Diseases ◽

Kidney Injury ◽

Pharmacological Intervention ◽

Kidney Fibrosis ◽

Genome Wide ◽

Vitro Model ◽

Sustained Activation

Renal fibrosis is a common end point for kidney injury and many chronic kidney diseases. Fibrogenesis depends on the sustained activation of myofibroblasts, which deposit the extracellular matrix that causes progressive scarring and organ failure. Here, we showed that the transcription factor SOX9 was associated with kidney fibrosis in humans and required for experimentally induced kidney fibrosis in mice. From genome-wide analysis, we identified Neuron navigator 3 (NAV3) as acting downstream of SOX9 in kidney fibrosis. NAV3 increased in abundance and colocalized with SOX9 after renal injury in mice, and both SOX9 and NAV3 were present in diseased human kidneys. In an in vitro model of renal pericyte transdifferentiation into myofibroblasts, we demonstrated that NAV3 was required for multiple aspects of fibrogenesis, including actin polymerization linked to cell migration and sustained activation of the mechanosensitive transcription factor YAP1. In summary, our work identifies a SOX9-NAV3-YAP1 axis involved in the progression of kidney fibrosis and points to NAV3 as a potential target for pharmacological intervention.

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Induction of Recurrent Break Cluster Genes in Neural Progenitor Cells Differentiated from Embryonic Stem Cells In Culture

10.1101/2019.12.30.891168 ◽

2019 ◽

Author(s):

Aseda Tena ◽

Yuxiang Zhang ◽

Nia Kyritsis ◽

Anne Devorak ◽

Jeffrey Zurita ◽

...

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Progenitor Cells ◽

Es Cells ◽

Embryonic Stem ◽

Neural Progenitors ◽

Neural Genes ◽

Genome Wide ◽

Primary Mouse

ABSTRACTMild replication stress enhances appearance of dozens of robust recurrent genomic break clusters, termed RDCs, in cultured primary mouse neural stem and progenitor cells (NSPCs). Robust RDCs occur within genes (“RDC-genes”) that are long and have roles in neural cell communications and/or have been implicated in neuropsychiatric diseases or cancer. We sought to develop an in vitro approach to determine whether specific RDC formation is associated with neural development. For this purpose, we adapted a system to induce neural progenitor cell (NPC) development from mouse embryonic stem cell (ESC) lines deficient for XRCC4 plus p53, a genotype that enhances DNA double-strand break (DSB) persistence to enhance detection. We tested for RDCs by our genome wide DSB identification approach that captures DSBs genome-wide via their ability to join to specific genomic Cas9/sgRNA-generated bait DSBs. In XRCC4/p53-deficient ES cells, we detected 7 RDCs, which were in genes, with two RDCs being robust. In contrast, in NPCs derived from these ES cell lines, we detected 29 RDCs, a large fraction of which were robust and associated with long, transcribed neural genes that were also robust RDC-genes in primary NSPCs. These studies suggest that many RDCs present in NSPCs are developmentally influenced to occur in this cell type and indicate that induced development of NPCs from ES cells provides an approach to rapidly elucidate mechanistic aspects of NPC RDC formation.SIGNIFICANCE STATEMENTWe previously discovered a set of long neural genes susceptible to frequent DNA breaks in primary mouse brain progenitor cells. We termed these genes RDC-genes. RDC-gene breakage during brain development might alter neural gene function and contribute to neurological diseases and brain cancer. To provide an approach to characterize the unknown mechanism of neural RDC-gene breakage, we asked whether RDC-genes appear in neural progenitors differentiated from embryonic stem cells in culture. Indeed, robust RDC-genes appeared in neural progenitors differentiated in culture and many overlapped with robust RDC-genes in primary brain progenitors. These studies indicate that in vitro development of neural progenitors provides a model system for elucidating how RDC-genes are formed.

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