Col11a1a Expression Is Required for Zebrafish Development

The autosomal dominant chondrodystrophies, the Stickler type 2 and Marshall syndromes, are characterized by facial abnormalities, vision deficits, hearing loss, and articular joint issues resulting from mutations in COL11A1. Zebrafish carry two copies of the Col11a1 gene, designated Col11a1a and Col11a1b. Col11a1a is located on zebrafish chromosome 24 and Col11a1b is located on zebrafish chromosome 2. Expression patterns are distinct for Col11a1a and Col11a1b and Col11a1a is most similar to COL11A1 that is responsible for human autosomal chondrodystrophies and the gene responsible for changes in the chondrodystrophic mouse model cho/cho. We investigated the function of Col11a1a in craniofacial and axial skeletal development in zebrafish using a knockdown approach. Knockdown revealed abnormalities in Meckel’s cartilage, the otoliths, and overall body length. Similar phenotypes were observed using a CRISPR/Cas9 gene-editing approach, although the CRISPR/Cas9 effect was more severe compared to the transient effect of the antisense morpholino oligonucleotide treatment. The results of this study provide evidence that the zebrafish gene for Col11a1a is required for normal development and has similar functions to the mammalian COL11A1 gene. Due to its transparency, external fertilization, the Col11a1a knockdown, and knockout zebrafish model systems can, therefore, contribute to filling the gap in knowledge about early events during vertebrate skeletal development that are not as tenable in mammalian model systems and help us understand Col11a1-related early developmental events.

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Differential Requirement of Gata2a and Gata2b for Primitive and Definitive Myeloid Development in Zebrafish

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.708113 ◽

2021 ◽

Vol 9 ◽

Author(s):

Oscar A. Peña ◽

Alexandra Lubin ◽

Jasmine Rowell ◽

Yvette Hoade ◽

Noreen Khokhar ◽

...

Keyword(s):

Progenitor Cells ◽

Expression Patterns ◽

Zebrafish Model ◽

Hematopoietic Stem ◽

Myeloid Development ◽

Stem And Progenitor Cells ◽

Potential Structure ◽

Gata2 Deficiency ◽

Early Developmental

Germline loss or mutation of one copy of the transcription factor GATA2 in humans leads to a range of clinical phenotypes affecting hematopoietic, lymphatic and vascular systems. GATA2 heterozygous mice show only a limited repertoire of the features observed in humans. Zebrafish have two copies of the Gata2 gene as a result of an additional round of ancestral whole genome duplication. These genes, Gata2a and Gata2b, show distinct but overlapping expression patterns, and between them, highlight a significantly broader range of the phenotypes observed in GATA2 deficient syndromes, than each one alone. In this manuscript, we use mutants for Gata2a and Gata2b to interrogate the effects on hematopoiesis of these two ohnologs, alone and in combination, during development in order to further define the role of GATA2 in developmental hematopoiesis. We define unique roles for each ohnolog at different stages of developmental myelopoiesis and for the emergence of hematopoietic stem and progenitor cells. These effects are not additive in the haploinsufficient state suggesting a redundancy between these two genes in hematopoietic stem and progenitor cells. Rescue studies additionally support that Gata2b can compensate for the effects of Gata2a loss. Finally we show that adults with loss of combined heterozygosity show defects in the myeloid compartment consistent with GATA2 loss in humans. These results build on existing knowledge from other models of GATA2 deficiency and refine our understanding of the early developmental effects of GATA2. In addition, these studies shed light on the complexity and potential structure-function relationships as well as sub-functionalization of Gata2 genes in the zebrafish model.

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Loss of Wnt16 Leads to Skeletal Deformities and Downregulation of Bone Developmental Pathway in Zebrafish

International Journal of Molecular Sciences ◽

10.3390/ijms22136673 ◽

2021 ◽

Vol 22 (13) ◽

pp. 6673

Author(s):

Xiaochao Qu ◽

Mei Liao ◽

Weiwei Liu ◽

Yisheng Cai ◽

Qiaorong Yi ◽

...

Keyword(s):

Gene Knockout ◽

Integration Site ◽

Skeletal Development ◽

Rna Seq ◽

Developmental Pathway ◽

Mineral Density ◽

Zebrafish Model ◽

Protein Protein Interaction ◽

Ct Analysis

Wingless-type MMTV integration site family, member 16 (wnt16), is a wnt ligand that participates in the regulation of vertebrate skeletal development. Studies have shown that wnt16 can regulate bone metabolism, but its molecular mechanism remains largely undefined. We obtained the wnt16-/- zebrafish model using the CRISPR-Cas9-mediated gene knockout screen with 11 bp deletion in wnt16, which led to the premature termination of amino acid translation and significantly reduced wnt16 expression, thus obtaining the wnt16-/- zebrafish model. The expression of wnt16 in bone-related parts was detected via in situ hybridization. The head, spine, and tail exhibited significant deformities, and the bone mineral density and trabecular bone decreased in wnt16-/- using light microscopy and micro-CT analysis. RNA sequencing was performed to explore the differentially expressed genes (DEGs). Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis found that the down-regulated DEGs are mainly concentrated in mTOR, FoxO, and VEGF pathways. Protein–protein interaction (PPI) network analysis was performed with the detected DEGs. Eight down-regulated DEGs including akt1, bnip4, ptena, vegfaa, twsg1b, prkab1a, prkab1b, and pla2g4f.2 were validated by qRT-PCR and the results were consistent with the RNA-seq data. Overall, our work provides key insights into the influence of wnt16 gene on skeletal 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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AnIn VivoZebrafish Model for Interrogating ROS-Mediated Pancreaticβ-Cell Injury, Response, and Prevention

Oxidative Medicine and Cellular Longevity ◽

10.1155/2018/1324739 ◽

2018 ◽

Vol 2018 ◽

pp. 1-8 ◽

Cited By ~ 7

Author(s):

Abhishek A. Kulkarni ◽

Abass M. Conteh ◽

Cody A. Sorrell ◽

Anjali Mirmira ◽

Sarah A. Tersey ◽

...

Keyword(s):

Cell Death ◽

Caspase 3 ◽

Cell Injury ◽

Zebrafish Model ◽

Chemical Genetic ◽

Regulated Cell Death ◽

Specific Manner ◽

High Doses

It is well known that a chronic state of elevated reactive oxygen species (ROS) in pancreaticβ-cells impairs their ability to release insulin in response to elevated plasma glucose. Moreover, at its extreme, unmitigated ROS drives regulated cell death. This dysfunctional state of ROS buildup can result both from genetic predisposition and environmental factors such as obesity and overnutrition. Importantly, excessive ROS buildup may underlie metabolic pathologies such as type 2 diabetes mellitus. The ability to monitor ROS dynamics inβ-cells in situ and to manipulate it via genetic, pharmacological, and environmental means would accelerate the development of novel therapeutics that could abate this pathology. Currently, there is a lack of models with these attributes that are available to the field. In this study, we use a zebrafish model to demonstrate that ROS can be generated in aβ-cell-specific manner using a hybrid chemical genetic approach. Using a transgenic nitroreductase-expressing zebrafish line,Tg(ins:Flag-NTR)s950, treated with the prodrug metronidazole (MTZ), we found that ROS is rapidly and explicitly generated inβ-cells. Furthermore, the level of ROS generated was proportional to the dosage of prodrug added to the system. At high doses of MTZ, caspase 3 was rapidly cleaved,β-cells underwent regulated cell death, and macrophages were recruited to the islet to phagocytose the debris. Based on our findings, we propose a model for the mechanism of NTR/MTZ action in transgenic eukaryotic cells and demonstrate the robust utility of this system to model ROS-related disease pathology.

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Mouse Wnt genes exhibit discrete domains of expression in the early embryonic CNS and limb buds

Development ◽

10.1242/dev.119.1.247 ◽

1993 ◽

Vol 119 (1) ◽

pp. 247-261 ◽

Cited By ~ 72

Author(s):

B.A. Parr ◽

M.J. Shea ◽

G. Vassileva ◽

A.P. McMahon

Keyword(s):

Limb Development ◽

Expression Patterns ◽

Limb Buds ◽

Expression Studies ◽

The Family ◽

The Central Nervous System ◽

Discrete Domains ◽

Early Developmental ◽

The Brain

Mutation and expression studies have implicated the Wnt gene family in early developmental decision making in vertebrates and flies. In a detailed comparative analysis, we have used in situ hybridization of 8.0- to 9.5-day mouse embryos to characterize expression of all ten published Wnt genes in the central nervous system (CNS) and limb buds. Seven of the family members show restricted expression patterns in the brain. At least three genes (Wnt-3, Wnt-3a, and Wnt-7b) exhibit sharp boundaries of expression in the forebrain that may predict subdivisions of the region later in development. In the spinal cord, Wnt-1, Wnt-3, and Wnt-3a are expressed dorsally, Wnt-5a, Wnt-7a, and Wnt-7b more ventrally, and Wnt-4 both dorsally and in the floor plate. In the forelimb primordia, Wnt-3, Wnt-4, Wnt-6 and Wnt-7b are expressed fairly uniformly throughout the limb ectoderm. Wnt-5a RNA is distributed in a proximal to distal gradient through the limb mesenchyme and ectoderm. Along the limb's dorsal-ventral axis, Wnt-5a is expressed in the ventral ectoderm and Wnt-7a in the dorsal ectoderm. We discuss the significance of these patterns of restricted and partially overlapping domains of expression with respect to the putative function of Wnt signalling in early CNS and limb development.

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APOL1 polymorphisms and kidney disease: loss-of-function or gain-of-function?

AJP Renal Physiology ◽

10.1152/ajprenal.00426.2018 ◽

2019 ◽

Vol 316 (1) ◽

pp. F1-F8 ◽

Cited By ~ 6

Author(s):

Leslie A. Bruggeman ◽

John F. O’Toole ◽

John R. Sedor

Keyword(s):

Kidney Disease ◽

Human Genetics ◽

Disease Risk ◽

Kidney Diseases ◽

Expression Patterns ◽

Model Systems ◽

Recessive Trait ◽

Loss Of Function ◽

Allele Expression ◽

Basic Biology

The mechanism that explains the association of APOL1 variants with nondiabetic kidney diseases in African Americans remains unclear. Kidney disease risk is inherited as a recessive trait, and many studies investigating the intracellular function of APOL1 have indicated the APOL1 variants G1 and G2 are associated with cytotoxicity. Whether cytotoxicity results from the absence of a protective effect conferred by the G0 allele or is induced by a deleterious effect of variant allele expression has not be conclusively established. A central issue hampering basic biology studies is the lack of model systems that authentically replicate APOL1 expression patterns. APOL1 is present in humans and a few other primates and appears to have important functions in the kidney, as the kidney is the primary target for disease associated with the genetic variance. There have been no studies to date assessing the function of untagged APOL1 protein under native expression in human or primate kidney cells, and no studies have examined the heterozygous state, a disease-free condition in humans. A second major issue is the chronic kidney disease (CKD)-associated APOL1 variants are conditional mutations, where the disease-inducing function is only evident under the appropriate environmental stimulus. In addition, it is possible there may be more than one mechanism of pathogenesis that is dependent on the nature of the stressor or other genetic variabilities. Studies addressing the function of APOL1 and how the CKD-associated APOL1 variants cause kidney disease are challenging and remain to be fully investigated under conditions that faithfully model known human genetics and physiology.

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1450-1545 Young Investigator Awards & Presentations Basic/Translational Exploring cellular subpopulations in glioblastoma and matched organoids using single-cell RNA-seq 52

Canadian Journal of Neurological Sciences / Journal Canadien des Sciences Neurologiques ◽

10.1017/cjn.2018.297 ◽

2018 ◽

Vol 45 (S3) ◽

pp. S13-S14

Author(s):

VG LeBlanc ◽

D Trinh ◽

M Hughes ◽

I Luthra ◽

D Livingstone ◽

...

Keyword(s):

Gene Expression ◽

Single Cell ◽

Tissue Sample ◽

Expression Patterns ◽

Model Systems ◽

Sequencing Data ◽

Tissue Samples ◽

Original Tumour ◽

Current Therapies ◽

Intratumoural Heterogeneity

Glioblastomas (GBMs) account for nearly half of all primary malignant brain tumours, and current therapies are often only marginally effective. Our understanding of the underlying biology of these tumours and the development of new therapies have been complicated in part by widespread inter- and intratumoural heterogeneity. To characterize this heterogeneity, we performed regional subsampling of primary glioblastomas and derived organoids from these tissue samples. We then performed single-cell RNA-sequencing (scRNA-seq) on these primary regional subsamples and 1-3 matched organoids per sample. We have profiled samples from six tumour sets to date and have obtained sequencing data for 21,234 primary tissue cells and 14,742 organoid cells. While the most apparent differences in gene expression appear to be between individual tumours, we were also able to identify similar cellular subpopulations across tissue samples and across organoids. Importantly, organoids derived from the same tissue sample appeared to be composed of similar cellular subpopulations and were highly comparable to each other, indicating that replicate organoids faithfully represent the original tumour tissue. Overall, our scRNA-seq approach will help evaluate the utility of tumour-derived organoids as model systems for GBM and will aid in identifying cellular subpopulations defined by gene expression patterns, both in primary GBM regional subsamples and their associated organoids. These analyses will allow for the characterization of clonal or subclonal populations that are likely to respond to different therapeutic approaches and may also uncover novel therapeutic targets previously unrevealed through bulk analyses.

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Effect of excessive iodine exposure on the placental deiodinase activities and Hoxc8 expression during mouse embryogenesis

British Journal Of Nutrition ◽

10.1017/s0007114507691909 ◽

2007 ◽

Vol 98 (1) ◽

pp. 116-122 ◽

Cited By ~ 8

Author(s):

Xue F. Yang ◽

Jian Xu ◽

Huai L. Guo ◽

Xiao H. Hou ◽

Li P. Hao ◽

...

Keyword(s):

Thyroid Hormone ◽

Thyroid Hormone Receptor ◽

Skeletal Development ◽

Gene Expression Pattern ◽

Skeletal Malformation ◽

Thyroid Hormone Metabolism ◽

Deiodinase Activity ◽

Excessive Iodine ◽

Total Thyroxine

Excessive iodine induces thyroid dysfunction. However, the effect of excessive iodine exposure on maternal–fetal thyroid hormone metabolism and on the expression of genes involved in differentiation, growth and development is poorly understood. Since a thyroid hormone receptor response element was found in the Hoxc8 promoter region, Hoxc8 expression possibly regulated by excessive iodine exposure was firstly investigated. In the present study, Balb/C mice were given different doses of iodine in the form of potassium iodate (KIO3) at the levels of 0 (sterile water), 1·5, 3·0, 6·0, 12·0 and 24·0 μg/ml in drinking water for 4 months, then were mated. On 12·5 d postcoitum, placental type 2 and type 3 deiodinase activities and fetal Hoxc8 expression were determined. The results showed that excessive iodine exposure above 1·5 μg/ml resulted in an increase of total thyroxine and a decrease of total triiodothyronine in the serum of maternal mice, which was mainly associated with the inhibition of type 1 deiodinase activity in liver and kidney. Placental type 2 deiodinase activity decreased, showing an inverse relationship with maternal thyroxine level. Hoxc8 mRNA and protein expression at 12·5 d postcoitum embryos were down regulated. Because Hoxc8 plays an important role in normal skeletal development, this finding provides a possible explanation for the skeletal malformation induced by excessive iodine exposure and also provides a new clue to study the relationship between iodine or thyroid hormones and Hox gene expression pattern.

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Diabetes and Exposure to Environmental Lead (Pb)

Toxics ◽

10.3390/toxics6030054 ◽

2018 ◽

Vol 6 (3) ◽

pp. 54 ◽

Cited By ~ 18

Author(s):

Todd Leff ◽

Paul Stemmer ◽

Jannifer Tyrrell ◽

Ruta Jog

Keyword(s):

Type 2 Diabetes ◽

Lead Exposure ◽

Metabolic Diseases ◽

Disease Incidence ◽

Metabolic Health ◽

Model Systems ◽

Environmental Toxicants ◽

Environmental Lead ◽

Exposure Levels

Although the increased incidence of type 2 diabetes since the 1950s is thought to be primarily due to coincident alterations in lifestyle factors, another potential contributing factor in industrialized countries is exposure of the population to environmental pollutants and industrial chemicals. Exposure levels of many environmental toxicants have risen in the same time-frame as the disease incidence. Of particular interest in this regard is the metal lead. Although overall lead exposure levels have diminished in recent decades, there is an under-recognized but persistent occurrence of lead exposure in poor underserved urban populations. Although the neural developmental pathologies induced by lead exposures have been well documented, very little is known about the effect of lead exposure on the incidence of chronic metabolic diseases such as type 2 diabetes. Although our understanding of the metabolic health effects of lead exposure is incomplete, there are studies in model systems and a small amount of epidemiological data that together suggest a deleterious effect of environmental lead exposure on metabolic health. This article reviews the human, animal and in vitro studies that have examined the effects of lead exposure on the development of diabetes and related metabolic conditions.

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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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