Biology subjects

Gontarz, P.

Publications and source records attributed to Gontarz, P..

10 recordsLinked to original sources

bioRxiv2025

Regulation of the Yolk Microtubule and Actin Cytoskeleton by Dachsous Cadherins during Zebrafish Epiboly

Epiboly is a crucial morphogenetic process during early animal embryogenesis that expands surface area of embryonic tissues while thinning them. During zebrafish development, epiboly spreads the superficial enveloping layer (EVL), germ layers, and yolk syncytial layer to cover the yolk cell. Here we investigated functions of the three zebrafish dchs genes, dchs1a, dchs1b and dchs2 that encode large atypical cadherins and report that they have partially overlapping functions in epiboly progression. We have inserted GFP at the C-terminal Dchs1b intracellular domain of the endogenous dchs1b locus using homologous recombination. We observed the resulting Dchs1b-GFP fusion protein localized in both the cell membrane and the cytoplasm of EVL and embryonic cells during gastrulation. The dynamic microtubule and actin cytoskeleton of the yolk cell are essential for epiboly. Our studies of the yolk microtubule network demonstrate that these microtubules are more bundled and show faster polymerization during epiboly in dchs triple loss-of-function mutant embryos than in wild-type embryos, indicating that dchs genes are required for limiting microtubule polymerization and promoting dynamics during epiboly. The epiboly progression defects of dchs1b deficient mutants were suppressed by mutations in the tetratricopeptide repeat protein 28 (ttc28) gene encoding a cytoplasmic protein previously shown to bind to Dchs1b intracellular domain and alter microtubule dynamics during early cleavages. We further demonstrate that MZdchs1b mutants exhibit abnormal organization and dynamics of yolk cell actin cytoskeleton during epiboly. Together, these lines of evidence as well as our transcriptomic analyses support the notion that like during early embryonic cleavages, Dchs1b plays a major role, while Dchs1a and Dchs2 proteins have supporting roles in regulating microtubule dynamics and organization of both microtubule and actin cytoskeleton to ensure normal epiboly.

developmental biology↗

bioRxiv2024

Autism and Intellectual Disability-Associated MYT1L Mutation Alters Human Cortical Interneuron Differentiation, Maturation, and Physiology

MYT1L is a neuronal transcription factor highly expressed in the developing and adult brain. While pathogenic MYT1L mutation causes neurodevelopmental disorders, these have not been characterized in human models of neurodevelopment. Here, we defined the consequences of pathogenic MYT1L mutation in human pluripotent stem cell-derived cortical interneurons. During differentiation, mutation reduced MYT1L expression and increased progenitor cell cycle exit and neuronal differentiation and synapse-related gene expression, morphological complexity, and synaptic puncta formation. Conversely, interneuron maturation was compromised, while variant neurons exhibited altered sodium and potassium channel activity and reduced function in electrophysiological analyses. CRISPRi-based knockdown similarly impaired interneuron differentiation and maturation, supporting loss of function-based effects. We further defined MYT1L genome-wide occupancy in interneurons and related this to the transcriptomic dysregulation resulting from MYT1L mutation, to identify direct targets that could mediate these phenotypic consequences. Together, this work delineates contributors to the etiology of neurodevelopmental disorders resulting from MYT1L mutation.

developmental biology↗

bioRxiv2024

A conserved regulation of cell expansion underlies notochord mechanics, spine morphogenesis, and endochondral bone lengthening

Cell size is a key contributor to tissue morphogenesis1. As a notable example, growth plate hypertrophic chondrocytes use cellular biogenesis and disproportionate fluid uptake to expand 10-20 times in size to drive lengthening of endochondral bone2,3. Similarly, notochordal cells expand to one of the largest cell types in the developing embryo to drive axial extension4-6. In zebrafish, the notochord vacuolated cells undergo vacuole fusion to form a single large, fluid-filled vacuole that fills the cytoplasmic space and contributes to vacuolated cell expansion7. When this process goes awry, the notochord lacks sufficient hydrostatic pressure to support vertebral bone deposition resulting in adult spines with misshapen vertebral bones and scoliosis8. However, it remains unclear whether endochondral bone and the notochord share common genetic and cellular mechanisms for regulating cell and tissue expansion. Here, we demonstrate that the 5-inositol phosphatase gene, inppl1a, regulates notochord expansion, spine morphogenesis, and endochondral bone lengthening in zebrafish. Furthermore, we show that inppl1a regulates notochord expansion independent of vacuole fusion, thereby genetically decoupling these processes. We demonstrate that inppl1a-dependent notochord expansion is essential to establish normal mechanical properties of the notochord to facilitate the development of a straight spine. Finally, we find that inppl1a is also important for endochondral bone lengthening in fish, as has been shown in the human INPPL1-related endochondral bone disorder, Opsismodysplasia9. Overall, this work reveals a conserved mechanism of cell size regulation that influences disparate tissues critical for skeletal development and short-stature disorders.

developmental biology↗

bioRxiv2021

A genetic screen for regulators of muscle morphogenesis in Drosophila

The mechanisms that determine the final topology of skeletal muscles remain largely unknown. We have been developing Drosophila body wall musculature as a model to identify and characterize the pathways that control muscle size, shape, and orientation during embryogenesis (Johnson et al., 2013; Williams et al., 2015; Yang et al., 2020a; Yang et al., 2020b). Our working model argues muscle morphogenesis is regulated by (1) extracellular guidance cues that direct muscle cells toward muscle attachment sites, and (2) contact dependent interactions between muscles and tendons. While we have identified several pathways that regulate muscle morphogenesis, our understanding is far from complete. Here we report the results of a recent EMS-based forward genetic screen that identified a myriad of loci not previously associated with muscle morphogenesis. We recovered new alleles of known muscle morphogenesis genes, including bsd, kon, ths, and tum, arguing our screening strategy was effective and efficient. We also identified and sequenced new alleles of salm, barr, and ptc that presumably disrupt independent pathways directing muscle morphogenesis. Equally as important, our screen shows that at least 11 morphogenetic loci remain to be identified. This screen has developed exciting new tools to study muscle morphogenesis, which may provide future insights into the mechanisms that determine skeletal muscle topology.

developmental biology↗

bioRxiv2020

Alterations in neuronal physiology, development, and function associated with a common duplication of chromosome 15 involving CHRNA7

BackgroundCopy number variants at chromosome 15q13.3 contribute to liability for multiple intellectual and developmental disabilities including Autism Spectrum Disorder (ASD). Individuals with duplications of this interval, which includes the gene CHRNA7, have multiple psychiatric disorders with widely variable penetrance. However, the basis of such differential affectation remains uncharacterized. MethodsInduced pluripotent stem cell (iPSC) models were generated from two first degree relatives with the same 15q13.3 duplication, a boy with distinct features of autism and emotional dysregulation (the affected proband, AP) and his clinically unaffected mother (the UM). These models were compared to unrelated control subjects lacking this duplication (UC, male and female). iPSC-derived neural progenitors and cortical neuroids consisting of cortical excitatory and inhibitory neurons were used to model potential contributors to neuropsychiatric impairment. ResultsThe AP-derived model uniquely exhibited disruptions of cellular physiology and neurodevelopment not observed in either the UM or the unrelated male and female controls. These included enhanced neural progenitor proliferation but impaired neuronal differentiation, maturation, and migration, and increased endoplasmic reticulum (ER) stress. Both the AP models neuronal migration deficit and elevated ER stress could be selectively rescued by different pharmacologic agents. Neuronal gene expression was also specifically dysregulated in the AP, including reduced expression of genes related to behavior, psychological disorders, neuritogenesis, neuronal migration, and WNT, axonal guidance, and GABA receptor signaling. Interestingly, the UM model exhibited upregulated expression of genes in many of these same pathways, by comparison with both the AP and UC models, suggesting that cell intrinsic molecular compensation could have contributed to the lack of neurodevelopmental phenotypes in the UM model. However, by contrast with the AP-specific neurodevelopmental phenotypes, both the AP- and UM-derived neurons exhibited shared alterations of neuronal function, including increased action potential firing and elevated cholinergic activity, consistent with increased homomeric CHRNA7 channel activity. ConclusionTogether, these data define both affectation-specific phenotypes seen only in the AP, as well as abnormalities observed in both individuals with CHRNA7 duplication, the AP and UM, but not in UC-derived neurons. This is, to our knowledge, the first study to use a human stem cell-based platform to study the basis of variable affectation in cases of 15q13.3 duplication at the cellular, molecular, and functional levels. This work suggests potential approaches for suppressing abnormal neurodevelopment or physiology that may contribute to severity of affectation. Some of these AP-specific neurodevelopmental anomalies, or the functional anomalies observed in both 15q13.3 duplication carriers (the AP and UM), could also contribute to the variable phenotypic penetrance seen in other individuals with 15q13.3 duplication.

developmental biology↗

bioRxiv2020

Geminin is required for Hox gene regulation to pattern the developing limb

Development of the complex structure of the vertebrate limb requires carefully orchestrated interactions between multiple regulatory pathways and proteins. Among these, precise regulation of 5 Hox transcription factor expression is essential for proper limb bud patterning and elaboration of distinct limb skeletal elements. Here, we identified Geminin (Gmnn) as a novel regulator of this process. A conditional model of Gmnn deficiency resulted in loss or severe reduction of forelimb skeletal elements, while both the forelimb autopod and hindlimb were unaffected. 5 Hox gene expression expanded into more proximal and anterior regions of the embryonic forelimb buds in this Gmnn-deficient model. A second conditional model of Gmnn deficiency instead caused a similar but less severe reduction of hindlimb skeletal elements and hindlimb polydactyly, while not affecting the forelimb. An ectopic posterior SHH signaling center was evident in the anterior hindlimb bud of Gmnn-deficient embryos in this model. This center ectopically expressed Hoxd13, the HOXD13 target Shh, and the SHH target Ptch1, while these mutant hindlimb buds also had reduced levels of the cleaved, repressor form of GLI3, a SHH pathway antagonist. Together, this work delineates a new role for Gmnn in modulating Hox expression to pattern the vertebrate limb. SummaryThis work identifies a new role for Geminin in mouse limb development. Geminin is a nuclear protein that regulates gene expression to control several other aspects of vertebrate development.

developmental biology↗

bioRxiv2019

The Reissner Fiber is Highly Dynamic in vivo and Controls Morphogenesis of the Spine

Spine morphogenesis requires the integration of multiple musculoskeletal tissues with the nervous system. Cerebrospinal fluid (CSF) physiology is important for development and homeostasis of the central nervous system and its disruption has been linked to scoliosis in zebrafish [1, 2]. Suspended in the CSF is an enigmatic glycoprotein thread called the Reissner fiber, which is secreted from the subcomissural organ (SCO) in the brain and extends caudally through the central canal to where it terminates at the base of the spinal cord. In zebrafish, scospondin null mutants are unable to assemble the Reissner fiber and fail to extend a straight body axis during embryonic development [3]. Here, we describe zebrafish hypomorphic missense alleles, which assemble the Reissner fiber and straighten the body axis during early embryonic development, yet progressively lose the fiber, concomitant with the emergence of body curvature, alterations in neuronal gene expression, and scoliosis in adults. Using an endogenously tagged scospondin-GFP zebrafish knock-in line, we directly visualized Reissner fiber dynamics during the normal development and during the progression of scoliosis, and demonstrate that the Reissner fiber is critical for the morphogenesis of the spine. Our study establishes a framework for future investigations of mechanistic roles of the Reissner fiber including its dynamic properties, molecular interactions, and how these processes are involved in the regulation of spine morphogenesis and scoliosis. HighlightsO_LIHypomorphic mutations in zebrafish scospondin result in progressive scoliosis C_LIO_LIThe disassembly of the Reissner fiber in scospondin hypomorphic mutants results in the strong upregulation of neuronal receptors and synaptic transport components C_LIO_LIAn endogenous fluorescent knock-in allele of scospondin reveals dynamic properties of the Reissner fiber during zebrafish development C_LIO_LILoss of the Reissner fiber during larval development is a common feature of zebrafish scoliosis models C_LI

genetics↗

bioRxiv2019

Improving ATAC-seq Data Analysis with AIAP, a Quality Control and Integrative Analysis Package

ATAC-seq is a technique widely used to investigate genome-wide chromatin accessibility. The recently published Omni-ATAC-seq protocol substantially improves the signal/noise ratio and reduces the input cell number. High-quality data are critical to ensure accurate analysis. Several tools have been developed for assessing sequencing quality and insertion size distribution for ATAC-seq data; however, key quality control (QC) metrics have not yet been established to accurately determine the quality of ATAC-seq data. Here, we optimized the analysis strategy for ATAC-seq and defined a series of QC metrics, including reads under peak ratio (RUPr), background (BG), promoter enrichment (ProEn), subsampling enrichment (SubEn), and other measurements. We incorporated these QC tests into our recently developed ATAC-seq Integrative Analysis Package (AIAP) to provide a complete ATAC-seq analysis system, including quality assurance, improved peak calling, and downstream differential analysis. We demonstrated a significant improvement of sensitivity (20%~60%) in both peak calling and differential analysis by processing paired-end ATAC-seq datasets using AIAP. AIAP is compiled into Docker/Singularity, and with one command line execution, it generates a comprehensive QC report. We used ENCODE ATAC-seq data to benchmark and generate QC recommendations, and developed qATACViewer for the user-friendly interaction with the QC report.

bioinformatics↗

bioRxiv2019

Cellular and molecular characterization of multiplex autism in human induced pluripotent stem cell-derived neurons

BackgroundAutism spectrum disorder (ASD) is a neurodevelopmental disorder with pronounced heritability in the general population. This is largely attributable to effects of polygenic susceptibility, with inherited liability exhibiting distinct sex differences in phenotypic expression. Attempts to model ASD in human cellular systems have principally involved rare de novo mutations associated with ASD phenocopies. However, by definition, these models are not representative of polygenic liability, which accounts for the vast share of population-attributable risk.\n\nMethodsHere, we performed what is, to our knowledge, the first attempt to model multiplex autism using patient-derived induced pluripotent stem cells (iPSCs) in a family manifesting incremental degrees of phenotypic expression of inherited liability (absent, intermediate, severe). The family members share an inherited variant of unknown significance in GPD2, a gene that was previously associated with developmental disability but here is insufficient by itself to cause ASD. iPSCs from three first-degree relatives and an unrelated control were differentiated into both cortical excitatory (cExN) and cortical inhibitory (cIN) neurons, and cellular phenotyping and transcriptomic analysis were conducted.\n\nResultscExN neurospheres from the two affected individuals were reduced in size, compared to those derived from unaffected related and unrelated individuals. This reduction was, at least in part, due to increased apoptosis of cells from affected individuals upon initiation of cExN neural induction. Likewise, cIN neural progenitor cells from affected individuals exhibited increased apoptosis, compared to both unaffected individuals. Transcriptomic analysis of both cExN and cIN neural progenitor cells revealed distinct molecular signatures associated with affectation, including misregulation of suites of genes associated with neural development, neuronal function, and behavior, as well as altered expression of ASD risk-associated genes.\n\nConclusionsWe have provided evidence of morphological, physiological, and transcriptomic signatures of polygenic liability to ASD from an analysis of cellular models derived from a multiplex autism family. ASD is commonly inherited on the basis of additive genetic liability. Therefore, identifying convergent cellular and molecular phenotypes resulting from polygenic and monogenic susceptibility may provide a critical bridge for determining which of the disparate effects of rare highly deleterious mutations might also apply to common autistic syndromes.

neuroscience↗

bioRxiv2017

The chromatin factor Gon4l regulates embryonic axis extension by promoting mediolateral cell polarity and notochord boundary formation through negative regulation of cell adhesion

Anteroposterior axis extension during vertebrate gastrulation requires cell proliferation, embryonic patterning, and morphogenesis to be spatiotemporally coordinated, but the underlying genetic mechanisms remain poorly understood. Here we define a role for the conserved chromatin factor Gon4l, encoded by ugly duckling (udu), in coordinating tissue patterning and axis extension during zebrafish gastrulation. Although identified as a recessive enhancer of short axis phenotypes in planar cell polarity (PCP) mutants, we found that Gon4l functions in a genetically independent, partially overlapping fashion with PCP signaling to regulate mediolateral cell polarity underlying axis extension in part by promoting notochord boundary formation. We identified direct genomic targets of Gon4l and found that it acts as both a positive and negative regulator of gene expression, including limiting expression of the cell-cell and cell-matrix adhesion molecules EpCAM and Integrin3b. Excess epcam or itga3b in wild-type gastrulae phenocopied notochord boundary defects of udu mutants, while downregulation of itga3b suppressed them. By promoting formation of this anteroposteriorly aligned boundary and associated cell polarity, Gon4l cooperates with PCP signaling to coordinate morphogenesis with the anteroposterior embryonic axis.

developmental biology↗