Barsi Lab Internship
Barsi Lab Research
Dr. Julius Barsi’s research takes a systems biology approach to understanding and explaining the mechanisms by which genetic information leads to anatomical structure. By systematically collecting cell specific data on gene expression he is able to catalogue differentially expressed genes and gene regulatory networks throughout purple sea urchin (Strongylocentrotus purpuratus) embryonic development. Once completed, a catalogue such as this may enable the reverse engineering of sea urchin development for the first time.
Dr. Barsi has reached the stage in S. purpuratus development where the Archenteron cells become highly specialised and will soon differentiate into various gut cells. Data has been collected on Archenteron cells as well as a transcriptome of the entire embryo during mid-gastrulation (30 hpf). This data will be analysed during the internship.
to read about the Barsi Lab visit: https://barsi-leidenfrost.org/
Data Collection
Many steps have gone into the data collection by Dr. Barsi at at the Bermuda Institute for Ocean Sciences (the procedure is described by Barsi et al. (2014)). These steps are roughly laid here:
1 Create genetically engineered artificial chromosomes whose expression defines a unique cell type (Fox A in the Archenteron), and inject these into sea urchin embryos.
2 Cultivate ~8000 embryos & disaggregate into single cells at the desired developmental
stage (here 30hpf).
3 Using fluoresence-activated cell sorting to sort the GFP+ (cell specific) from the GFP- (control) cells.
4 Following rolling circle amplification and next generation sequencing library preparation, conduct Illumina RNA sequencing on both populations of cells and calculate relative enrichment.
5 Normalising data across biological replicates and for gene length.
The Purple Sea Urchin
Strongylocentrotus purpuratus
Sea urchins are model organisms commonly used for this type of research for various reasons, hence the purple sea urchins gene regulatory network is one of the best understood at the moment. The main advantage that purple sea urchins provide is their ability to integrate foreign DNA, introduced at the single cell stage, into their own DNA.
The images below illustrate their embryonic development, highlighting the stage and tissue of interest:
Green and Batterman, 2017. Stud. His. Philos. Sci. A. Part C, 61, pp.20-34
Green and Batterman, 2017. Stud. His. Philos. Sci. A. Part C, 61, pp.20-34
Ettensohn, 2020. Mech. Dev., 162, p103599
Signalling Genes
The Internship Focus
Signalling genes are genes that specifically have a role in signalling processes by encoding for the production of receptors and signalling ligands. This allows for communication between cells which is crucial throughout embryonic development as it provides each cell with information on its surroundings and location during this process. Development is controlled by gene regulation of transcription factors and signalling genes (Levine and Davidson, 2005), where gene regulation refers to the process of regulating which subset of genes should be expressed in each cell via transcription factors (Davidson, 2010). The transcription factors present in a cell nucleus determines a cells regulatory state and actively regulates gene expression, and the signalling gene products then allow for communication between cells to ensure each cell is in the correct regulatory state (Davidson, 2010; Cui et al., 2014). Gene regulation occurs as a network, where transcription factors lie upstream of all other genes, meaning that the expression of transcription factors cascades into a network of gene activation and suppression (Davidson, 2010). Signalling genes fit into this network by communicating between the gene regulatory networks of different tissues.
Signalling Types in Sea Urchin Embryonic Development
Signalling can either occur short-range or long-range, and during early embryonic development the short-range signalling types are utilised. These consist of contact-dependant, paracrine, and autocrine signalling. Paracrine signalling, where two nearby cells that are not in direct contact communicate, is most often used in sea urchin development around 30hpf. Once a signal is received in a cell it typically triggers a cascade of reactions, i.e. a signalling pathway, that ultimately achieves the goal of the signal. The main pathways used throughout embryonic development in sea urchins are: hedgehog, Wnt, TGF-beta, FGF, RTK, and Notch which all use paracrine signalling with the exception of Notch signalling which uses contact-dependent signalling (e.g. Basson, 2012; Egana and Ernst, 2004; Lapraz et al., 2006; Sanz-Ezquerro et al., 2017; Walton et al., 2006). There are multiple pathways that fall under each of these main ones. Wnt-signalling pathways for example include the canonical Wnt/B-catenin pathway, and the non-canonical WNT/planar cell polarity (PCP) and WNT/ Ca 2+ pathways (Sanz-Ezquerro et al., 2017).
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