Browsing by Author "Hockman, Dorit"

Now showing 1 - 8 of 8
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    Deciphering the regulatory code driving neural crest evolution and development
    (2022) Prag, Mayur; Hockman, Dorit
    Neural crest cells are a unique feature of vertebrates. This embryonic cell population is multipotent, giving rise to many structures including peripheral neurons. The sea lamprey, Petromyzon marinus, is at the base of the vertebrate lineage and offers an ideal model for the ancestral neural crest. Comparisons to the modern neural crest gene regulatory network (GRN) such as that of the chicken can elucidate essential conserved regions of the GRN. Previous studies in P. marinus revealed a neural crest-specific enhancer for the neural crest specification gene, SoxE1, which showed conserved activity in chicken and zebrafish neural crest. Here, the SoxE1 enhancer was subdivided to find the core active region, using enhancer-reporter assays in chicken and lamprey. Additionally, the segments were analysed for putative transcription factor binding sites, which were mutated. The central 610 bp of the SoxE1 enhancer retained its activity in lamprey and chicken neural crest. Mutation of a putative Sox10 and Tfap2 binding sites within the core enhancer did not result in complete loss of enhancer activity in the chicken or lamprey, however the number of positive embryos was reduced in the lamprey. Further subdivision of the SoxE1 enhancer core region revealed the 3' half drives expression in the branchial arches of the chicken embryo. Neural crest specific-reporter activity was confirmed by immunological staining embryo sections with the reporter gene overlapping with neuronal and endogenous Sox10 expression. The identified core region of the SoxE1 enhancer shows a conserved regulatory mechanism in vertebrates. Future work will interrogate how this enhancer region interacts directly with neural crest GRN members. In addition, preliminary single cell RNA-seq analysis of dissected dorsal neural tube tissue from the lamprey embryos revealed a neural crest specific cell population that expressed key neural crest specification marker genes. This data can reveal previously unknown genes involved in the neural crest GRN as well as identifying novel cell types during development.
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    Digit formation during embryonic development of bats and mice
    (2018) Parker, Ash; Illing, Nicola; Hockman, Dorit
    The evolution of a strikingly elongated and webbed FL in bats, which contrasts with a small, free-toed HL, has seen extensive research into bat wing development in an effort to determine the molecular mechanism driving limb development. A recent RNA-seq and ChIP-seq study carried out on M. natalensis showed differences in FL and HL activity for several genetic pathways known to be involved in bone formation during key bat development stages CS15-CS17. In this project the prediction made from the literature and the RNA-seq results was that the observed decreased Wnt/β-catenin signalling and increased BMP signalling in the bat FL may lead to elevated levels of Sox9 expression, and larger fields of mesenchymal condensations. This was tested by annotating Sox9 in the M. natalensis genome to further analyse the expression levels and associated ChIP-seq data. In addition the behaviour of condensing mesenchymal cells during bat and mouse limb development was observed by visualising the various stages of chondrogenesis, using H&E and PNA stains. In addition the RNA-seq study found 3000 genes to be differentially expressed. Thus, the project also set out to create an immortalised bat autopod cell line to facilitate future testing and predictions. The Sox9 gene was successfully annotated and revealed to not be differentially expressed between FL and HL as predicted. However downstream targets of Sox9 were further inspected as potential ideas for further investigation. The histological stains provided a set of data characterising mesenchymal condensation in both mouse and bat stages, revealing many interesting features such as the non-specific binding behaviours of PNA prior to digit formation. In addition, quantitative results demonstrated the bat FL digits are already longer than the HL digits at CS16. Cell line work established a working protocol for the storage, dissociation and plating of bat primary cells that retain their bat limb expression identity. Mouse cells were successfully immortalised and a cell line was established from a HL digit cell. This project has facilitated further studies in understanding extreme digit elongation in the bat FL autopod during development.
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    The Emergence of Embryonic Myosin Heavy Chain during Branchiomeric Muscle Development
    (2022-05-25) Yahya, Imadeldin; Böing, Marion; Hockman, Dorit; Brand-Saberi, Beate; Morosan-Puopolo, Gabriela
    A prerequisite for discovering the properties and therapeutic potential of branchiomeric muscles is an understanding of their fate determination, pattering and differentiation. Although the expression of differentiation markers such as myosin heavy chain (MyHC) during trunk myogenesis has been more intensively studied, little is known about its expression in the developing branchiomeric muscle anlagen. To shed light on this, we traced the onset of MyHC expression in the facial and neck muscle anlagen by using the whole-mount in situ hybridization between embryonic days E9.5 and E15.5 in the mouse. Unlike trunk muscle, the facial and neck muscle anlagen express MyHC at late stages. Within the branchiomeric muscles, our results showed variation in the emergence of MyHC expression. MyHC was first detected in the first arch-derived muscle anlagen, while its expression in the second arch-derived muscle and non-somitic neck muscle began at a later time point. Additionally, we show that non-ectomesenchymal neural crest invasion of the second branchial arch is delayed compared with that of the first brachial arch in chicken embryos. Thus, our findings reflect the timing underlying branchiomeric muscle differentiation.
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    Exploring the gene regulatory dynamics of the maturing human brain
    (2022) Fillmore, Stephanie; Hockman, Dorit
    The human brain develops gradually overtime where distinct molecular profiles are established in the embryo. These molecular profiles continue to change through aging and in response to environmental factors. The complexity and dynamics of gene expression and regulation at the cell type-specific level are still poorly understood, especially during the process of brain maturation. The overall aim of this project was to obtain a better understanding of how the brain cell atlas changes over time by contributing to the current brain cell atlas with pediatric single cell data. Bio-banked pediatric and adult brain tissue samples, obtained during surgery to treat epilepsy, were used to optimise and generate a nuclei isolation protocol. Single nuclei RNA-seq (snRNA-seq) libraries were generated using the 10x Genomics Platform. snRNA-seq datasets were then sequenced and analysed using bioinformatics tools, including Cell Ranger and Seurat. The major cell types in the pediatric brain were identified, including the genes being expressed by these cell types. In addition, a pilot differential expression analysis study was conducted between snRNA-seq libraries from the temporal and frontal lobes. Furthermore, Assays for Transposase Accessible Chromatin (ATAC-seq) was performed on pediatric and adult tissue and bulk ATAC-seq libraries were successfully generated. A consensus list of putative enhancers and promoters was generated after testing several bioinformatic pipelines. Differential accessibility analysis was performed on the bulk ATAC-seq datasets and the promoters or enhancers that are being dynamically used over the course of brain development, were also identified. Ultimately, with these findings and with the generation of optimised protocols, this study has contributed to our understanding of gene expression and gene regulation of brain maturation.
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    Limbs gone batty : the role of the anterior-posterior patterning signal, Sonic Hedgehog, in the development of the unique bat limb
    (2007) Hockman, Dorit; Illing, Nicola; Jacobs, David S
    The unique skeletal structure of the bat forelimb and hindlimb provides a new and exciting model for the field of evolutionary developmental biology, which seeks to reveal the molecular mechanisms behind vertebrate limb diversity. The digits of the bat forelimb, excluding the thumb, are considerably elongated and webbed. The hindlimb digits are free of webbing and are of uniform length, lacking the asymmetrical patterning of the forelimb. In this study, gene expression analysis has revealed that changes in the spatial and temporal expression patterns of the anteriorposterior patterning signal, Sonic hedgehog (Shh), and its downstream target, Patched 1 (PtcJ), have contributed to the development of the unique bat limb. The embryonic development of Miniopterus natalensis (Miniopteridae) is described for the first time and the expression patterns of Shh and PtcJ in the developing limbs of this species are compared to those in Carollia perspicillata (Phyllostomidae) and the mouse. Early in bat limb development (stage 14), Shh expression in the ZPA appears to be anteriorly expanded when compared to the mouse. This observation is in line with the reported expansion of Fgf8 expression in the AER (Cretekos et al. 2007) and reveals that an enhancement of the Shh-Fgf positive feedback loop may be responsible for the initial posterior expansion of the bat forelimb. Later in development (stage 16) Shh and PtcJ acquire a novel domain of expression within the interdigital tissue of both the bat forelimb and hindlimb. These expression patterns parallel the reported up-regulation of Fgf8, Gremlin and Bmp2 in the interdigital tissue of C. perspicillata (Weatherbee et al. 2006) and support the hypothesis that the Shh-Fgfpositive feedback loop is re-initiated in the interdigital tissue of the bat limbs. The cell survival and proliferation signals provided by the Shh-Fgf signalling loop most likely contribute to the lengthening of the posterior forelimb digits, the survival of the tissue between the forelimb digits and the extension of digits 1 and 5 of the hindlimb to the same length of the remaining digits. The novel Shh and PtcJ expression patterns were observed in both M natalensis and C. perspicillata, supporting the monophyly of the chiropteran sub-order, Verspertilioniformes.
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    New Insights into the Diversity of Branchiomeric Muscle Development: Genetic Programs and Differentiation
    (Multidisciplinary Digital Publishing Institute, 2022-08-22) Yahya, Imadeldin; Hockman, Dorit; Brand-Saberi, Beate; Morosan-Puopolo, Gabriela
    Branchiomeric skeletal muscles are a subset of head muscles originating from skeletal muscle progenitor cells in the mesodermal core of pharyngeal arches. These muscles are involved in facial expression, mastication, and function of the larynx and pharynx. Branchiomeric muscles have been the focus of many studies over the years due to their distinct developmental programs and common origin with the heart muscle. A prerequisite for investigating these muscles’ properties and therapeutic potential is understanding their genetic program and differentiation. In contrast to our understanding of how branchiomeric muscles are formed, less is known about their differentiation. This review focuses on the differentiation of branchiomeric muscles in mouse embryos. Furthermore, the relationship between branchiomeric muscle progenitor and neural crest cells in the pharyngeal arches of chicken embryos is also discussed. Additionally, we summarize recent studies into the genetic networks that distinguish between first arch-derived muscles and other pharyngeal arch muscles.
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    Profiling the dynamics of the active transcriptome in the juvenile and adult brain using spatial transcriptomics
    (2023) Mishi, Ruvimbo; Hockman, Dorit
    The human brain is made up of a collection of distinct cell types that play specialized roles in maintaining proper brain function. The process of maturation of this complex organ can be assessed by examining the dynamics of gene expression within the various cell types. Previous studies have used single-cell/single nucleus RNA-sequencing (sc/snRNA-seq) to provide vital information on gene expression at a cell-specific level. These studies, which focused on either the pre-natal or adult brain, lacked comprehensive information about the spatial dynamics of cell type-specific gene expression over the course of brain maturation. This information is important for understanding the dynamics of gene expression in the context of changing tissue architecture over the course of maturation. This study aims to contribute to our understanding of the maturing human brain by obtaining the spatial gene expression information of the antemortem brain as it matures from postnatal to adult state. Samples from the human temporal cortex (4-, 15- (x2), and 31-year-old) were obtained during elective surgeries to treat epilepsy. Tissue samples were freshly frozen in OCT. H&E (hematoxylin and eosin) staining was employed for the initial screening of the tissue samples. The 10x Genomics Visium Spatial gene expression system was used to obtain genome-wide spatial gene expression patterns. The Visium FASTQ files were aligned to the human transcriptome using 10X Genomics SpaceRanger. To spatially map the brain cell types, the Visium data was integrated with snRNAseq data using the cell2location method. In situ hybridization chain reaction was used to validate the identified gene expression patterns. Fifty-four cell types, annotated using the current published human temporal lobe cell atlas, were spatially mapped by integrating Visium spatial gene expression and existing snRNA-seq datasets from the same samples. Moreover, this integrative analysis revealed potential changes in the distribution of cell types during brain maturation, highlighting the importance of studying the spatiotemporal dynamics of cell types to better understand brain development and function. Previously identified layer-enriched genes were confirmed to be present in all the samples. Furthermore, layer-enriched genes that showed an increase in expression during brain maturation were identified. The findings of this study contribute to the human brain cell atlas through the provision of spatial gene expression information in the maturing temporal cortex.
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    Towards a complete human cell atlas: a single-nucleus RNA sequencing study of the paediatric and adult human brain
    (2023) Steyn, Christina; Hockman, Dorit
    Postnatal human brain maturation from birth to early adulthood represents a period of susceptibility for neuropsychiatric risk. While temporal gene expression dynamics over this period have been studied extensively, there are no studies exploring the paediatric brain at single cell resolution. To address this, we present the first paediatric brain cell atlas comprising of 6 single nucleus RNA sequencing (snRNA-seq) datasets generated from antemortem human brain tissue samples obtained during elective surgeries to treat epilepsy. To complement these, we included 6 snRNA-seq datasets from adult brain tissue. The 12 samples are all of temporal cortex origin and were produced using the 10X Genomics Single Cell 3' gene expression analysis kits. The datasets were processed using an optimised pipeline and the nuclei were annotated into various cell types using the Allen Institute's middle temporal gyrus dataset as a reference. A novel machine learning method was applied to the annotated datasets to identify combinations of marker genes capable of distinguishing each cell type. Based on this, several minimal marker genes were identified which were shared between paediatric samples and not adults or vice versa. Three different tools were used to identify genes changing in their level of expression with age within each cell type. This revealed hundreds of differentially expressed genes (DEGs), with numerous DEGs being unique to specific cell types and subtypes. From these analyses, two long noncoding RNAs of interest were selected for further in silico characterization which revealed putative functions for these genes. Overall, we have provided a resource which can be interrogated further to explore differences between paediatric and adult samples at the gene expression and cell level. This may promote an expansion in our understanding of brain maturation and brain diseases.