Elongation of Caudalized Human Organoids Mimics Neural Tube Development

AbstractAxial elongation of the neural tube is critical during mammalian embryogenesis to establish the anterior-posterior body axis1, but this process is difficult to interrogate directly because it occurs post-implantation2,3. Here we report an organoid model of neural tube extension by caudalized human pluripotent stem cell (hPSC) aggregates that recapitulates the morphologic and temporal gene expression patterns of neural tube development. Axially extending organoids consisting largely of longitudinally elongated neuroepithelial compartments also contained TBXT(+)SOX2(+) neuromesodermal progenitors, PAX6(+)nestin(+) neural progenitor populations, and MEOX1(+) paraxial mesoderm populations. Wnt agonism stimulated singular axial extensions in a dose-dependent manner, and elongated organoids displayed regionalized rostral-caudal HOX gene expression, with spatially distinct hindbrain (HOXB1) expression from brachial (HOXC6) and thoracic (HOXB9) regions. CRISPR-interference-mediated silencing of the TBXT, a downstream Wnt target, increased neuroepithelial compartmentalization and resulted in multiple extensions per aggregate. Further, knock-down of BMP inhibitors, Noggin and Chordin, induced elongation phenotypes that mimicked murine knockout models. These results indicate the potent morphogenic capacity of caudalized hPSC organoids to undergo axial elongation in a manner that can be used to dissect the cellular organization and patterning decisions that dictate early nervous system development in humans.

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Axial elongation of caudalized human organoids mimics aspects of neural tube development

Development ◽

10.1242/dev.198275 ◽

2021 ◽

Vol 148 (12) ◽

Author(s):

Ashley R. G. Libby ◽

David A. Joy ◽

Nicholas H. Elder ◽

Emily A. Bulger ◽

Martina Z. Krakora ◽

...

Keyword(s):

Gene Expression ◽

Neural Tube ◽

System Development ◽

Expression Patterns ◽

Nervous System Development ◽

Critical Threshold ◽

Axial Elongation ◽

Spinal Cord Development ◽

Temporal Gene Expression ◽

Neural Tube Development

ABSTRACT Axial elongation of the neural tube is crucial during mammalian embryogenesis for anterior-posterior body axis establishment and subsequent spinal cord development, but these processes cannot be interrogated directly in humans as they occur post-implantation. Here, we report an organoid model of neural tube extension derived from human pluripotent stem cell (hPSC) aggregates that have been caudalized with Wnt agonism, enabling them to recapitulate aspects of the morphological and temporal gene expression patterns of neural tube development. Elongating organoids consist largely of neuroepithelial compartments and contain TBXT+SOX2+ neuro-mesodermal progenitors in addition to PAX6+NES+ neural progenitors. A critical threshold of Wnt agonism stimulated singular axial extensions while maintaining multiple cell lineages, such that organoids displayed regionalized anterior-to-posterior HOX gene expression with hindbrain (HOXB1) regions spatially distinct from brachial (HOXC6) and thoracic (HOXB9) regions. CRISPR interference-mediated silencing of TBXT, a Wnt pathway target, increased neuroepithelial compartmentalization, abrogated HOX expression and disrupted uniaxial elongation. Together, these results demonstrate the potent capacity of caudalized hPSC organoids to undergo axial elongation in a manner that can be used to dissect the cellular organization and patterning decisions that dictate early human nervous system development.

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The Genome-Wide Impact of Nipblb Loss-of-Function on Zebrafish Gene Expression

International Journal of Molecular Sciences ◽

10.3390/ijms21249719 ◽

2020 ◽

Vol 21 (24) ◽

pp. 9719

Author(s):

Marco Spreafico ◽

Eleonora Mangano ◽

Mara Mazzola ◽

Clarissa Consolandi ◽

Roberta Bordoni ◽

...

Keyword(s):

Gene Expression ◽

Developmental Stages ◽

System Development ◽

Expression Patterns ◽

Nervous System Development ◽

Zebrafish Embryos ◽

Loss Of Function ◽

Zebrafish Model ◽

Genome Wide ◽

Transcriptional Effect

Transcriptional changes normally occur during development but also underlie differences between healthy and pathological conditions. Transcription factors or chromatin modifiers are involved in orchestrating gene activity, such as the cohesin genes and their regulator NIPBL. In our previous studies, using a zebrafish model for nipblb knockdown, we described the effect of nipblb loss-of-function in specific contexts, such as central nervous system development and hematopoiesis. However, the genome-wide transcriptional impact of nipblb loss-of-function in zebrafish embryos at diverse developmental stages remains under investigation. By RNA-seq analyses in zebrafish embryos at 24 h post-fertilization, we examined genome-wide effects of nipblb knockdown on transcriptional programs. Differential gene expression analysis revealed that nipblb loss-of-function has an impact on gene expression at 24 h post fertilization, mainly resulting in gene inactivation. A similar transcriptional effect has also been reported in other organisms, supporting the use of zebrafish as a model to understand the role of Nipbl in gene regulation during early vertebrate development. Moreover, we unraveled a connection between nipblb-dependent differential expression and gene expression patterns of hematological cell populations and AML subtypes, enforcing our previous evidence on the involvement of NIPBL-related transcriptional dysregulation in hematological malignancies.

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Histone methylations in the developing central nervous system and in neural tube defects

Neuroforum ◽

10.1515/nf-2018-a001 ◽

2018 ◽

Vol 24 (2) ◽

pp. A85-A94

Author(s):

Alejandro Villarreal ◽

Henriette Franz ◽

Tanja Vogel

Keyword(s):

Gene Expression ◽

Central Nervous System ◽

Nervous System ◽

Cell Differentiation ◽

Neural Tube ◽

Neural Tube Defects ◽

System Development ◽

Epigenetic Modification ◽

Nervous System Development ◽

Central Nervous System Development

Abstract Understanding central nervous system genesis is of crucial relevance to decode different human diseases such as microcephaly or neural tube defects, which arise from incorrect developmental processes. Epigenetic mechanisms regulate gene expression in a spatio-temporal manner and are implicated in diverse cellular actions one of which is cell differentiation. Therefore, the study of these mechanisms is of great relevance in the context of development and disease. In this article, we will review histone methylations as epigenetic modification and how they impact on gene expression and cell differentiation in central nervous system development and neural differentiation. Further, we will discuss an emerging link between histone methylation in the etiology of neural tube defects. We will specifically highlight the role of the disruptor of telomeric silencing like 1 (DOT1L) and histone H3 lysine 79 methylation (H3K79me), which is an unusual histone modification with implication for proper central nervous system development.

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Large-scale temporal gene expression mapping of central nervous system development

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.95.1.334 ◽

1998 ◽

Vol 95 (1) ◽

pp. 334-339 ◽

Cited By ~ 388

Author(s):

X. Wen ◽

S. Fuhrman ◽

G. S. Michaels ◽

D. B. Carr ◽

S. Smith ◽

...

Keyword(s):

Gene Expression ◽

Central Nervous System ◽

Nervous System ◽

Large Scale ◽

System Development ◽

Nervous System Development ◽

Central Nervous System Development ◽

Temporal Gene Expression ◽

Gene Expression Mapping

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Transcriptional profiling of iPSC-derived neurons with reciprocal monogenic CNV in 3p26.3 region

Nauchno-prakticheskii zhurnal «Medicinskaia genetika» ◽

10.25557/2073-7998.2020.03.10-11 ◽

2020 ◽

pp. 10-11

Author(s):

М.Е. Лопаткина ◽

В.С. Фишман ◽

М.М. Гридина ◽

Н.А. Скрябин ◽

Т.В. Никитина ◽

...

Keyword(s):

Gene Expression ◽

Copy Number ◽

System Development ◽

Transcriptional Profiling ◽

Global Gene Expression ◽

Nervous System Development ◽

Number Variation ◽

The Central Nervous System ◽

Cntn6 Gene ◽

Copy Number Changes

Проведен анализ генной экспрессии в нейронах, дифференцированных из индуцированных плюрипотентных стволовых клеток пациентов с идиопатическими интеллектуальными нарушениями и реципрокными хромосомными мутациями в регионе 3p26.3, затрагивающими единственный ген CNTN6. Для нейронов с различным типом хромосомных аберраций была показана глобальная дисрегуляция генной экспрессии. В нейронах с вариациями числа копий гена CNTN6 была снижена экспрессия генов, продукты которых вовлечены в процессы развития центральной нервной системы. The gene expression analysis of iPSC-derived neurons, obtained from patients with idiopathic intellectual disability and reciprocal microdeletion and microduplication in 3p26.3 region affecting the single CNTN6 gene was performed. The global gene expression dysregulation was demonstrated for cells with CNTN6 copy number variation. Gene expression in neurons with CNTN6 copy number changes was downregulated for genes, whose products are involved in the central nervous system development.

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Microarray Analyses of Gene Expression during Chondrocyte Differentiation Identifies Novel Regulators of Hypertrophy

Molecular Biology of the Cell ◽

10.1091/mbc.e05-01-0084 ◽

2005 ◽

Vol 16 (11) ◽

pp. 5316-5333 ◽

Cited By ~ 88

Author(s):

Claudine G. James ◽

C. Thomas G. Appleton ◽

Veronica Ulici ◽

T. Michael Underhill ◽

Frank Beier

Keyword(s):

Gene Expression ◽

Bone Development ◽

Skeletal Development ◽

Expression Patterns ◽

Chondrocyte Differentiation ◽

Endochondral Bone ◽

Temporal Gene Expression ◽

Affymetrix Microarrays ◽

Time Period ◽

And Cluster Analysis

Ordered chondrocyte differentiation and maturation is required for normal skeletal development, but the intracellular pathways regulating this process remain largely unclear. We used Affymetrix microarrays to examine temporal gene expression patterns during chondrogenic differentiation in a mouse micromass culture system. Robust normalization of the data identified 3300 differentially expressed probe sets, which corresponds to 1772, 481, and 249 probe sets exhibiting minimum 2-, 5-, and 10-fold changes over the time period, respectively. GeneOntology annotations for molecular function show changes in the expression of molecules involved in transcriptional regulation and signal transduction among others. The expression of identified markers was confirmed by RT-PCR, and cluster analysis revealed groups of coexpressed transcripts. One gene that was up-regulated at later stages of chondrocyte differentiation was Rgs2. Overexpression of Rgs2 in the chondrogenic cell line ATDC5 resulted in accelerated hypertrophic differentiation, thus providing functional validation of microarray data. Collectively, these analyses provide novel information on the temporal expression of molecules regulating endochondral bone development.

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Maximal STAT5-Induced Proliferation and Self-Renewal at Intermediate STAT5 Activity Levels

Molecular and Cellular Biology ◽

10.1128/mcb.01025-08 ◽

2008 ◽

Vol 28 (21) ◽

pp. 6668-6680 ◽

Cited By ~ 61

Author(s):

Albertus T. J. Wierenga ◽

Edo Vellenga ◽

Jan Jacob Schuringa

Keyword(s):

Gene Expression ◽

Cell Fate ◽

Target Genes ◽

Expression Patterns ◽

Activity Levels ◽

Dependent Manner ◽

Self Renewal ◽

Hematopoietic Stem ◽

Cell Fate Decisions

ABSTRACT The level of transcription factor activity critically regulates cell fate decisions, such as hematopoietic stem cell (HSC) self-renewal and differentiation. We introduced STAT5A transcriptional activity into human HSCs/progenitor cells in a dose-dependent manner by overexpression of a tamoxifen-inducible STAT5A(1*6)-estrogen receptor fusion protein. Induction of STAT5A activity in CD34+ cells resulted in impaired myelopoiesis and induction of erythropoiesis, which was most pronounced at the highest STAT5A transactivation levels. In contrast, intermediate STAT5A activity levels resulted in the most pronounced proliferative advantage of CD34+ cells. This coincided with increased cobblestone area-forming cell and long-term-culture-initiating cell frequencies, which were predominantly elevated at intermediate STAT5A activity levels but not at high STAT5A levels. Self-renewal of progenitors was addressed by serial replating of CFU, and only progenitors containing intermediate STAT5A activity levels contained self-renewal capacity. By extensive gene expression profiling we could identify gene expression patterns of STAT5 target genes that predominantly associated with a self-renewal and long-term expansion phenotype versus those that identified a predominant differentiation phenotype.

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Differential and Overlapping Effects of 20,23(OH)2D3 and 1,25(OH)2D3 on Gene Expression in Human Epidermal Keratinocytes: Identification of AhR as an Alternative Receptor for 20,23(OH)2D3

International Journal of Molecular Sciences ◽

10.3390/ijms19103072 ◽

2018 ◽

Vol 19 (10) ◽

pp. 3072 ◽

Cited By ~ 36

Author(s):

Andrzej Slominski ◽

Tae-Kang Kim ◽

Zorica Janjetovic ◽

Anna Brożyna ◽

Michal Żmijewski ◽

...

Keyword(s):

Gene Expression ◽

Molecular Modeling ◽

Vitamin D3 ◽

Expression Profiles ◽

Expression Patterns ◽

Canonical Pathway ◽

Epidermal Keratinocytes ◽

Hydroxyl Groups ◽

Dependent Manner ◽

Human Epidermal Keratinocytes

A novel pathway of vitamin D activation by CYP11A has previously been elucidated. To define the mechanism of action of its major dihydroxy-products, we tested the divergence and overlap between the gene expression profiles of human epidermal keratinocytes treated with either CYP11A1-derived 20,23(OH)2D3 or classical 1,25(OH)2D3. Both secosteroids have significant chemical similarity with the only differences being the positions of the hydroxyl groups. mRNA was isolated and examined by microarray analysis using Illumina’s HumanWG-6 chip/arrays and subsequent bioinformatics analyses. Marked differences in the up- and downregulated genes were observed between 1,25(OH)2D3- and 20,23(OH)2D3-treated cells. Hierarchical clustering identified both distinct, opposite and common (overlapping) gene expression patterns. CYP24A1 was a common gene strongly activated by both compounds, a finding confirmed by qPCR. Ingenuity pathway analysis identified VDR/RXR signaling as the top canonical pathway induced by 1,25(OH)2D3. In contrast, the top canonical pathway induced by 20,23(OH)2D3 was AhR, with VDR/RXR being the second nuclear receptor signaling pathway identified. QPCR analyses validated the former finding by revealing that 20,23(OH)2D3 stimulated CYP1A1 and CYP1B1 gene expression, effects located downstream of AhR. Similar stimulation was observed with 20(OH)D3, the precursor to 20,23(OH)2D3, as well as with its downstream metabolite, 17,20,23(OH)3D3. Using a Human AhR Reporter Assay System we showed marked activation of AhR activity by 20,23(OH)2D3, with weaker stimulation by 20(OH)D3. Finally, molecular modeling using an AhR LBD model predicted vitamin D3 hydroxyderivatives to be good ligands for this receptor. Thus, our microarray, qPCR, functional studies and molecular modeling indicate that AhR is the major receptor target for 20,23(OH)2D3, opening an exciting area of investigation on the interaction of different vitamin D3-hydroxyderivatives with AhR and the subsequent downstream activation of signal transduction pathways in a cell-type-dependent manner.

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Harnessing PTEN’s Growth Potential in Neuronal Development and Disease

Neuroscience Insights ◽

10.1177/2633105520959056 ◽

2020 ◽

Vol 15 ◽

pp. 263310552095905

Author(s):

Joachim Fuchs ◽

Britta J. Eickholt ◽

George Leondaritis

Keyword(s):

Neurodevelopmental Disorders ◽

Neuronal Development ◽

System Development ◽

Transmembrane Protein ◽

Nervous System Development ◽

Growth Potential ◽

Dependent Manner ◽

Axon Extension ◽

Growth Promoting ◽

Spatiotemporal Control

PTEN is a powerful regulator of neuronal growth. It globally suppresses axon extension and branching during both nervous system development and regeneration, by antagonizing growth-promoting PI3K/PI(3,4,5)P3 signaling. We recently identified that the transmembrane protein PRG2/LPPR3 functions as a modulator of PTEN function during axon morphogenesis. Our work demonstrates that through inhibition of PTEN activity, PRG2 stabilizes membrane PI(3,4,5)P3. In turn, PRG2 deficiency attenuates the formation of branches in a PTEN-dependent manner, albeit without affecting the overall growth capacity of extending axons. Thus, PRG2 is poised to temporally and locally relieve growth suppression mediated by PTEN in neurons and, in effect, to redirect growth specifically to axonal branches. In this commentary, we discuss potential implications and unresolved questions regarding the regulation of axonal PTEN in neurons. Given their widespread implication during neuronal development and regeneration, identification of mechanisms that confer spatiotemporal control of PTEN may unveil new approaches to reprogram PI3K signaling in neurodevelopmental disorders and regeneration research.

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