Biology subjects

Park, T. J.

Publications and source records attributed to Park, T. J..

4 recordsLinked to original sources

bioRxiv2023

Highly effective gene inactivation in tetraploid Xenopus laevis with low-temperature-active engineered Cas12a

Gene knockout using the CRISPR/Cas (clustered regulatory interspaced short palindromic repeats/CRISPR-associated protein) system revolutionized reverse genetic studies in model and non-model organisms, as almost all genetic elements can be targeted with few limitations. Although the CRISPR/Cas system with SpCas9 (Cas9 derived from Streptococcus pyogenes) remains the most popular for genome editing, another CRISPR/Cas system with Cas12a (Cpf1) has expanded its application. However, Cas12a is challenging to use in some aquatic model organisms, such as Xenopus laevis, because of its low activity at the temperature at which X. laevis embryos are usually raised (lower than 25{degrees}C). Recently, an engineered Cas12a called Cas12a-Ultra was developed, which has improved in vivo endonuclease activity with reduced temperature dependency. Here, we evaluated the performance of these engineered Cas12a enzymes in X. laevis embryos. We first confirmed that they were more active than SpCas9 at the low temperature at which X. laevis embryos are mostly raised (20-22{degrees}C), based on in vitro digestion experiments. Then, we evaluated in vivo activities of Cas12a-Ultra by disrupting several genes in X. laevis whose phenotypic consequences are previously reported. LbCpf1 (Cas12a derived from Lachnospiraceae bacterium)-Ultra outperformed the other enzymes, producing more than 80% of embryos with severely defective phenotypes even in low-temperature conditions. In addition, duplicated copies of two paralogous lysine demethylases (kdm5b and kdm5c) were successfully disrupted, which recapitulated the previously reported phenotypes observed upon morpholino-mediated knockdown. This study demonstrated that this engineered Cas12a is valuable for gene function studies in Xenopus and other model organisms with low growth temperatures.

bioengineering↗

bioRxiv2023

Phylogenetic modeling of enhancer shifts in African mole-rats reveals regulatory changes associated with tissue-specific traits

Changes in gene regulation have long been thought to underlie most phenotypic differences between species. Subterranean rodents, and in particular the naked mole-rat, have attracted substantial attention due to their proposed phenotypic adaptations, which include hypoxia tolerance, metabolic changes and cancer resistance. However, it is largely unknown what regulatory changes may associate with these phenotypic traits, and whether these are unique to the naked mole-rat, the mole-rat clade or also present in other mammals. Here, we investigate regulatory evolution in heart and liver from two African mole-rat species and two rodent outgroups using genome-wide epigenomic profiling. First, we adapted and applied a phylogenetic modeling approach to quantitatively compare epigenomic signals at orthologous regulatory elements, and identified thousands of promoter and enhancer regions with differential epigenomic activity in mole-rats. These elements associate with known mole-rat adaptation in metabolic and functional pathways, and suggest candidate genetic loci that may underlie mole-rat innovations. Second, we evaluated ancestral and species-specific regulatory changes in the study phylogeny, and report several candidate pathways experiencing stepwise remodeling during the evolution of mole-rats - such as the insulin and hypoxia response pathways. Third, we report non-orthologous regulatory elements overlap with lineage-specific repetitive elements and appear to modify metabolic pathways by rewiring of HNF4 and RAR/RXR transcription factor binding sites in mole-rats. These comparative analyses reveal how mole-rat regulatory evolution informs previously reported phenotypic adaptations. Moreover, the phylogenetic modeling framework we propose here improves upon the state-of-the-art by addressing known limitations of inter-species comparisons of epigenomic profiles, and has broad implications in the field of comparative functional genomics.

genomics↗

bioRxiv2023

A single-cell, time-resolved profiling of Xenopus mucociliary epithelium reveals non-hierarchical model of development

The specialized cell-types of the mucociliary epithelium (MCE) lining the respiratory tract enable continuous airway clearing, with its defects leading to chronic respiratory diseases. The molecular mechanisms driving cell-fate acquisition and temporal specialization during mucociliary epithelial development remain largely unknown. Here, we profile the developing Xenopus MCE from pluripotent to mature stages by single-cell transcriptomics, identifying novel, multipotent early epithelial progenitors that execute multi-lineage cues before specialising into late-stage ionocytes, goblet and basal cells. Combining in silico lineage inference, in situ hybridization and single-cell multiplexed RNA imaging, we capture the initial bifurcation into early epithelial and multiciliated progenitors, chart cell- type emergence and fate progression into specialized cell-types. Comparative analysis of nine airway atlases reveals an evolutionary conserved transcriptional module in ciliated cells, whereas secretory and basal types execute distinct function-specific programmes across vertebrates. We uncover a continuous non-hierarchical model of MCE development alongside a significant data resource for understanding respiratory biology.

developmental biology↗

bioRxiv2020

GJA1 Depletion Causes Ciliary Defects by Affecting Rab11 Trafficking to the Ciliary Base

The gap junction complex functions as a transport channel across the membrane. Among gap junction subunits, gap junction protein 1 (GJA1) is the most commonly expressed subunit. A recent study showed that GJA1 is necessary for the maintenance of motile cilia; however, the molecular mechanism and function of GJA1 in ciliogenesis remain unknown. Here, we examined the functions of GJA1 during ciliogenesis in human retinal pigment epithelium-1 and Xenopus laevis embryonic multiciliated-cells. GJA1 localizes to the motile ciliary axonemes or pericentriolar regions beneath the primary cilium. GJA1 depletion caused malformation of both the primary cilium and motile cilia. Further study revealed that GJA1 depletion affected several ciliary proteins such as BBS4, CP110, and Rab11 in the pericentriolar region and basal body. Interestingly, CP110 removal from the mother centriole was significantly reduced by GJA1 depletion. Importantly, Rab11, a key regulator during ciliogenesis, was immunoprecipitated with GJA1, and GJA1 knockdown caused the mislocalization of Rab11. These findings suggest that GJA1 regulates ciliogenesis by interacting with the Rab11-Rab8 ciliary trafficking pathway.

cell biology↗