Three Dimensional Multi-gene Expression Maps Reveal Cell Fate Changes Associated with Laterality Reversal of Zebrafish Habenula

ABSTRACTThe conserved bilateral habenular nuclei (HA) in vertebrate diencephalon develop into compartmentalized structures containing neurons derived from different cell lineages. Despite extensive studies demonstrated that zebrafish larval HA display distinct left-right (L-R) asymmetry in gene expression and connectivity, the spatial gene expression domains were mainly obtained from two-dimensional (2D) snapshots of colorimetric RNA in situ hybridization staining which could not properly reflect different HA neuronal lineages constructed in three-dimension (3D). Combing the tyramide-based fluorescent mRNA in situ hybridization, confocal microscopy and customized imaging processing procedures, we have created spatial distribution maps of four genes for 4 day old zebrafish and in sibling fish whose L-R asymmetry was spontaneously reversed. 3D volumetric analyses showed that ratios of cpd2, lov, ron and nrp1a expression in L-R reversed HA were reversed according to the parapineal positions. However, the quantitative changes of gene expression in reversed larval brains do not mirror the gene expression level in the obverse larval brains. There were a total 87.78% increase of lov+nrp1a+ and a total 12.45% decrease of lov+ron+ double-positive neurons when the L-R asymmetry of HA was reversed. Thus, our volumetric analyses of the 3D maps indicate that changes of HA neuronal cell fates are associated with the reversal of HA laterality. These changes likely account for the behavior changes associated with HA laterality alterations.

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Three‐dimensional multi‐gene expression maps reveal cell fate changes associated with laterality reversal of zebrafish habenula

Journal of Neuroscience Research ◽

10.1002/jnr.24806 ◽

2021 ◽

Author(s):

Guo‐Tzau Wang ◽

He‐Yen Pan ◽

Wei‐Han Lang ◽

Yuan‐Ding Yu ◽

Chang‐Huain Hsieh ◽

...

Keyword(s):

Gene Expression ◽

Cell Fate ◽

Three Dimensional

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Sex and death: from cell fate specification to dynamic control of X-chromosome structure and gene expression

Molecular Biology of the Cell ◽

10.1091/mbc.e18-06-0397 ◽

2018 ◽

Vol 29 (22) ◽

pp. 2616-2621 ◽

Cited By ~ 4

Author(s):

Barbara J. Meyer

Keyword(s):

Gene Expression ◽

Cell Fate ◽

X Chromosome ◽

Chromosome Structure ◽

Dynamic Control ◽

Three Dimensional ◽

X Chromosomes ◽

Meiotic Chromosomes ◽

The Difference ◽

Developmental Decision

Determining sex is a binary developmental decision that most metazoans must make. Like many organisms, Caenorhabditis elegans specifies sex (XO male or XX hermaphrodite) by tallying X-chromosome number. We dissected this precise counting mechanism to determine how tiny differences in concentrations of signals are translated into dramatically different developmental fates. Determining sex by counting chromosomes solved one problem but created another—an imbalance in X gene products. We found that nematodes compensate for the difference in X-chromosome dose between sexes by reducing transcription from both hermaphrodite X chromosomes. In a surprising feat of evolution, X-chromosome regulation is functionally related to a structural problem of all mitotic and meiotic chromosomes: achieving ordered compaction of chromosomes before segregation. We showed the dosage compensation complex is a condensin complex that imposes a specific three-dimensional architecture onto hermaphrodite X chromosomes. It also triggers enrichment of histone modification H4K20me1. We discovered the machinery and mechanism underlying H4K20me1 enrichment and demonstrated its pivotal role in regulating higher-order X-chromosome structure and gene expression.

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Distinct contracted conformations of the Tcra/Tcrd locus during Tcra and Tcrd recombination

Journal of Experimental Medicine ◽

10.1084/jem.20100772 ◽

2010 ◽

Vol 207 (9) ◽

pp. 1835-1841 ◽

Cited By ~ 36

Author(s):

Han-Yu Shih ◽

Michael S. Krangel

Keyword(s):

In Situ Hybridization ◽

Fluorescence In Situ Hybridization ◽

Three Dimensional ◽

Double Negative ◽

Thymocyte Development ◽

Double Positive ◽

Long Distance ◽

Large Pool ◽

Antigen Receptor Loci

Studies have suggested that antigen receptor loci adopt contracted conformations to promote long-distance interactions between gene segments during V(D)J recombination. The Tcra/Tcrd locus is unique because it undergoes highly divergent Tcrd and Tcra recombination programs in CD4−CD8− double negative (DN) and CD4+CD8+ double positive (DP) thymocytes, respectively. Using three-dimensional fluorescence in situ hybridization, we asked whether these divergent recombination programs are supported by distinct conformational states of the Tcra/Tcrd locus. We found that the 3′ portion of the locus is contracted in DN and DP thymocytes but not in B cells. Remarkably, the 5′ portion of the locus is contracted in DN thymocytes but is decontracted in DP thymocytes. We propose that the fully contracted conformation in DN thymocytes allows Tcrd rearrangements involving Vδ gene segments distributed over 1 Mb, whereas the unique 3′-contracted, 5′-decontracted conformation in DP thymocytes biases initial Tcra rearrangements to the most 3′ of the available Vα gene segments. This would maintain a large pool of distal 5′ Vα gene segments for subsequent rounds of recombination. Thus, distinct contracted conformations of the Tcra/Tcrd locus may facilitate a transition from a Tcrd to a Tcra mode of recombination during thymocyte development.

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Serrate and Notch specify cell fates in the heart field by suppressing cardiomyogenesis

Development ◽

10.1242/dev.127.17.3865 ◽

2000 ◽

Vol 127 (17) ◽

pp. 3865-3876

Author(s):

M.S. Rones ◽

K.A. McLaughlin ◽

M. Raffin ◽

M. Mercola

Keyword(s):

Gene Expression ◽

Notch Signaling ◽

Cell Fate ◽

Notch Pathway ◽

Cell Types ◽

Myocardial Cell ◽

Dominant Negative ◽

Cell Fates ◽

Cell Fate Decisions ◽

Heart Field

Notch signaling mediates numerous developmental cell fate decisions in organisms ranging from flies to humans, resulting in the generation of multiple cell types from equipotential precursors. In this paper, we present evidence that activation of Notch by its ligand Serrate apportions myogenic and non-myogenic cell fates within the early Xenopus heart field. The crescent-shaped field of heart mesoderm is specified initially as cardiomyogenic. While the ventral region of the field forms the myocardial tube, the dorsolateral portions lose myogenic potency and form the dorsal mesocardium and pericardial roof (Raffin, M., Leong, L. M., Rones, M. S., Sparrow, D., Mohun, T. and Mercola, M. (2000) Dev. Biol., 218, 326–340). The local interactions that establish or maintain the distinct myocardial and non-myocardial domains have never been described. Here we show that Xenopus Notch1 (Xotch) and Serrate1 are expressed in overlapping patterns in the early heart field. Conditional activation or inhibition of the Notch pathway with inducible dominant negative or active forms of the RBP-J/Suppressor of Hairless [Su(H)] transcription factor indicated that activation of Notch feeds back on Serrate1 gene expression to localize transcripts more dorsolaterally than those of Notch1, with overlap in the region of the developing mesocardium. Moreover, Notch pathway activation decreased myocardial gene expression and increased expression of a marker of the mesocardium and pericardial roof, whereas inhibition of Notch signaling had the opposite effect. Activation or inhibition of Notch also regulated contribution of individual cells to the myocardium. Importantly, expression of Nkx2. 5 and Gata4 remained largely unaffected, indicating that Notch signaling functions downstream of heart field specification. We conclude that Notch signaling through Su(H) suppresses cardiomyogenesis and that this activity is essential for the correct specification of myocardial and non-myocardial cell fates.

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Spatial- and temporal-restricted pattern for amelogenin gene expression during mouse molar tooth organogenesis

Development ◽

10.1242/dev.104.1.77 ◽

1988 ◽

Vol 104 (1) ◽

pp. 77-85 ◽

Cited By ~ 2

Author(s):

M.L. Snead ◽

W. Luo ◽

E.C. Lau ◽

H.C. Slavkin

Keyword(s):

Gene Expression ◽

In Situ Hybridization ◽

Gene Transcription ◽

Developmental Stages ◽

Three Dimensional ◽

Molar Tooth ◽

Specific Expression ◽

Amelogenin Gene ◽

Stage Of Development

Position- and time-restricted amelogenin gene transcription was analysed in developing tooth organs using in situ hybridization with asymmetric complementary RNA probes produced from a cDNA specific to the mouse 26 × 10(3) Mr amelogenin. In situ analysis was performed on developmentally staged fetal and neonatal mouse mandibular first (M1) and maxillary first (M1) molar tooth organs using serial sections and three-dimensional reconstruction. Amelogenin mRNA was first detected in a cluster of ameloblasts along one cusp of the M1 molar at the newborn stage of development. In subsequent developmental stages, amelogenin transcripts were detected within foci of ameloblasts lining each of the five cusps comprising the molar crown form. The number of amelogenin transcripts appeared to be position-dependent, being more abundant on one cusp surface while reduced along the opposite surface. Amelogenin gene transcription was found to be bilaterally symmetric between the developing right and left M1 molars, and complementary between the M1 and M1 developing molars; indicating position-restricted gene expression resulting in organ stereoisomerism. The application of in situ hybridization to forming tooth organ geometry provides a novel strategy to define epithelial-mesenchymal signal(s) which are believed to be responsible for organ morphogenesis, as well as for temporal- and spatial-restricted tissue-specific expression of enamel extracellular matrix.

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Modeling binary and graded cone cell fate patterning in the mouse retina

10.1101/650606 ◽

2019 ◽

Author(s):

Kiara C. Eldred ◽

Cameron Avelis ◽

Robert J. Johnston ◽

Elijah Roberts

Keyword(s):

Gene Expression ◽

Thyroid Hormone ◽

Cell Fate ◽

Regulatory Network ◽

Photoreceptor Cells ◽

Mouse Retina ◽

Hormone Signaling ◽

Cell Fates ◽

Opsin Expression ◽

A Cell

AbstractNervous systems are incredibly diverse, with myriad neuronal subtypes defined by gene expression. How binary and graded fate characteristics are patterned across tissues is poorly understood. Expression of opsin photopigments in the cone photoreceptors of the mouse retina provides an excellent model to address this question. Individual cones express S-opsin only, M-opsin, or both S-opsin and M-opsin. These cell populations are patterned along the dorsal-ventral axis, with greater M-opsin expression in the dorsal region and greater S-opsin expression in the ventral region. Thyroid hormone signaling plays a critical role in activating M-opsin and repressing S-opsin. Here, we developed an image analysis approach to identify individual cone cells and evaluate their opsin expression from immunofluorescence imaging tiles spanning roughly 6 mm along the D-V axis of the mouse retina. From analyzing the opsin expression of ∼250,000 cells, we found that cones make a binary decision between S-opsin only and co-expression competent fates. Co-expression competent cells express graded levels of S- and M-opsins, depending nonlinearly on their position in the dorsal-ventral axis. M- and S-opsin expression display differential, inverse patterns. Using these single-cell data we developed a quantitative, stochastic model of cone cell decisions in the retinal tissue based on thyroid hormone signaling activity. The model recovers the probability distribution for cone fate patterning in the mouse retina and describes a minimal set of interactions that are necessary to reproduce the observed cell fates. Our study provides a paradigm describing how differential responses to regulatory inputs generate complex patterns of binary and graded cell fates.Author SummaryThe development of a cell in a mammalian tissue is governed by a complex regulatory network that responds to many input signals to give the cell a distinct identity, a process referred to as cell-fate specification. Some of these cell fates have binary on-or-off gene expression patterns, while others have graded gene expression that changes across the tissue. Differentiation of the photoreceptor cells that sense light in the mouse retina provides a good example of this process. Here, we explore how complex patterns of cell fates are specified in the mouse retina by building a computational model based on analysis of a large number of photoreceptor cells from microscopy images of whole retinas. We use the data and the model to study what exactly it means for a cell to have a binary or graded cell fate and how these cell fates can be distinguished from each other. Our study shows how tens-of-thousands of individual photoreceptor cells can be patterned across a complex tissue by a regulatory network, creating a different outcome depending upon the received inputs.

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In situelectro-sequencing in three-dimensional tissues

10.1101/2021.04.22.440941 ◽

2021 ◽

Author(s):

Qiang Li ◽

Zuwan Lin ◽

Ren Liu ◽

Xin Tang ◽

Jiahao Huang ◽

...

Keyword(s):

Gene Expression ◽

Single Cell ◽

Three Dimensional ◽

Cell Types ◽

Developmental Trajectory ◽

Single Cell Level ◽

Cell Level ◽

Cell Gene Expression ◽

Cell Gene

AbstractPairwise mapping of single-cell gene expression and electrophysiology in intact three-dimensional (3D) tissues is crucial for studying electrogenic organs (e.g., brain and heart)1–5. Here, we introducein situelectro-sequencing (electro-seq), combining soft bioelectronics within situRNA sequencing to stably map millisecond-timescale cellular electrophysiology and simultaneously profile a large number of genes at single-cell level across 3D tissues. We appliedin situelectro-seq to 3D human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) patches, precisely registering the CM gene expression with electrophysiology at single-cell level, enabling multimodalin situanalysis. Such multimodal data integration substantially improved the dissection of cell types and the reconstruction of developmental trajectory from spatially heterogeneous tissues. Using machine learning (ML)-based cross-modal analysis,in situelectro-seq identified the gene-to-electrophysiology relationship over the time course of cardiac maturation. Further leveraging such a relationship to train a coupled autoencoder, we demonstrated the prediction of single-cell gene expression profile evolution using long-term electrical measurement from the same cardiac patch or 3D millimeter-scale cardiac organoids. As exemplified by cardiac tissue maturation,in situelectro-seq will be broadly applicable to create spatiotemporal multimodal maps and predictive models in electrogenic organs, allowing discovery of cell types and gene programs responsible for electrophysiological function and dysfunction.

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The prospero transcription factor is asymmetrically localized to the cell cortex during neuroblast mitosis in Drosophila

Development ◽

10.1242/dev.121.10.3187 ◽

1995 ◽

Vol 121 (10) ◽

pp. 3187-3195 ◽

Cited By ~ 53

Author(s):

E.P. Spana ◽

C.Q. Doe

Keyword(s):

Gene Expression ◽

Transcription Factor ◽

Cell Fate ◽

Daughter Cell ◽

Cell Cortex ◽

Cell Fates ◽

Extrinsic Factors ◽

Cortical Localization ◽

Ganglion Mother Cell ◽

Intrinsic And Extrinsic Factors

Both intrinsic and extrinsic factors are known to regulate sibling cell fate. Here we describe a novel mechanism for the asymmetric localization of a transcription factor to one daughter cell at mitosis. The Drosophila CNS develops from asymmetrically dividing neuroblasts, which give rise to a large neuroblast and a smaller ganglion mother cell (GMC). The prospero gene encodes a transcription factor necessary for proper GMC gene expression. We show that the prospero protein is synthesized in the neuroblast where it is localized to the F-actin cell cortex. At mitosis, prospero is asymmetrically localized to the budding GMC and excluded from the neuroblast. After cytokinesis, prospero is translocated from the GMC cortex into the nucleus. Asymmetric cortical localization of prospero in neuroblasts requires entry into mitosis; it does not depend on numb function. prospero is also observed in cortical crescents in dividing precursors of the peripheral nervous system and adult midgut. The asymmetric cortical localization of prospero at mitosis is a mechanism for rapidly establishing distinct sibling cell fates in the CNS and possibly other tissues.

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H4K20me3 methyltransferase SUV420H2 shapes the chromatin landscape of pluripotent embryonic stem cells

Development ◽

10.1242/dev.188516 ◽

2020 ◽

Vol 147 (23) ◽

pp. dev188516

Author(s):

Jiji T. Kurup ◽

Zhijun Han ◽

Wenfei Jin ◽

Benjamin L. Kidder

Keyword(s):

Gene Expression ◽

Cell Fate ◽

Es Cells ◽

Three Dimensional ◽

Embryonic Stem ◽

Pericentric Heterochromatin ◽

Es Cell ◽

Chromatin Interactions ◽

Repressive Histone Modification ◽

Dna Elements

ABSTRACTHeterochromatin, a densely packed chromatin state that is transcriptionally silent, is a critical regulator of gene expression. However, it is unclear how the repressive histone modification H4K20me3 or the histone methyltransferase SUV420H2 regulates embryonic stem (ES) cell fate by patterning the epigenetic landscape. Here, we report that depletion of SUV420H2 leads to a near-complete loss of H4K20me3 genome wide, dysregulated gene expression and delayed ES cell differentiation. SUV420H2-bound regions are enriched with repetitive DNA elements, which are de-repressed in SUV420H2 knockout ES cells. Moreover, SUV420H2 regulation of H4K20me3-marked heterochromatin controls chromatin architecture, including fine-scale chromatin interactions in pluripotent ES cells. Our results indicate that SUV420H2 plays a crucial role in stabilizing the three-dimensional chromatin landscape of ES cells, as loss of SUV420H2 resulted in A/B compartment switching, perturbed chromatin insulation, and altered chromatin interactions of pericentric heterochromatin and surrounding regions, indicative of localized decondensation. In addition, depletion of SUV420H2 resulted in compromised interactions between H4K20me3 and gene-regulatory regions. Together, these findings describe a new role for SUV420H2 in regulating the chromatin landscape of ES cells.

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ClusterMap: multi-scale clustering analysis of spatial gene expression

10.1101/2021.02.18.431337 ◽

2021 ◽

Cited By ~ 1

Author(s):

Yichun He ◽

Xin Tang ◽

Jiahao Huang ◽

Haowen Zhou ◽

Kevin Chen ◽

...

Keyword(s):

Gene Expression ◽

Dimensional Space ◽

Expression Patterns ◽

Three Dimensional ◽

Point Pattern Analysis ◽

Point Pattern ◽

Computational Framework ◽

Spatially Resolved ◽

Tissue Organization

AbstractQuantifying RNAs in their spatial context is crucial to understanding gene expression and regulation in complex tissues. In situ transcriptomic methods generate spatially resolved RNA profiles in intact tissues. However, there is a lack of a unified computational framework for integrative analysis of in situ transcriptomic data. Here, we present an unsupervised and annotation-free framework, termed ClusterMap, which incorporates physical proximity and gene identity of RNAs, formulates the task as a point pattern analysis problem, and thus defines biologically meaningful structures and groups. Specifically, ClusterMap precisely clusters RNAs into subcellular structures, cell bodies, and tissue regions in both two- and three-dimensional space, and consistently performs on diverse tissue types, including mouse brain, placenta, gut, and human cardiac organoids. We demonstrate ClusterMap to be broadly applicable to various in situ transcriptomic measurements to uncover gene expression patterns, cell-cell interactions, and tissue organization principles from high-dimensional transcriptomic images.

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