Lindenberg, T. | bioRxiv Science

bioRxiv2023

EZH2 specifically regulates ISL1 during embryonic urinary tract formation

Isl1 has been described as an embryonic master control gene expressed in the pericloacal mesenchyme. Deletion of Isl1 from the genital mesenchyme in mice leads to an ectopic urethral opening and epispadias-like phenotype. Using genome wide association methods, we identified ISL1 as the key susceptibility gene for classic bladder exstrophy (CBE), comprising epispadias and exstrophy of the urinary bladder. The most significant marker (rs6874700) identified in our recent GWAS meta-analysis achieved a p value of 1.48 x 10-24 within the ISL1 region. In silico analysis of rs6874700 and all other genome-wide significant markers in Linkage Disequilibrium (LD) with rs6874700 (D = 1.0; R2 > 0.90) revealed marker rs2303751 (p value 8.12 x 10-20) as the marker with the highest regulatory effect predicted. Here, we describe a novel 1.2 kb intragenic promoter residing between 6.2 and 7.4 kb downstream of the ISL1 transcription starting site, which is located in the reverse DNA strand and harbors a binding side for EZH2 at the exact region of marker rs2303751. We show, that EZH2 silencing in HEK cells reduces ISL1 expression. We show that ezh2-/- ko zebrafish larvae display tissues specificity of ISL1 regulation with reduced expression of Isl1 in the pronephric region of zebrafish larvae. In addition, a shorter and malformed nephric duct is observed in ezh2-/- ko zebrafish Tg(wt1{beta}:eGFP) reporter lines. Our study shows, that Ezh2 is a key regulator of Isl1 during urinary tract formation and suggests tissue specific ISL1 dysregulation as an underlying mechanism for CBE formation.

genetics↗

bioRxiv2023

DanioCTC: Injection of circulating tumor cells from metastatic breast cancer patients in zebrafish xenografts for analysis of metastasis

Circulating tumor cells (CTCs) are considered as metastatic precursor cells, and zebrafish xenografts provide an in vivo model to study cancer cell spread. Currently, the low number of patient-derived CTCs limits their analysis in animal models. We present DanioCTC, a xenograft workflow for injecting CTCs from metastatic breast cancer (MBC) patients into zebrafish embryos to study cell dissemination in vivo. The study successfully adapts existing workflows and combines diagnostic leukapheresis (DLA), the Parsortix microfluidic system, flow cytometry, and the automated cell micromanipulator CellCelector setup to enrich and isolate MBC-derived CTCs and to finally inject them into Zebrafish embryos, where their dissemination was tracked up to 3 days post-injection. MDA-MB-231 cells were used as a standard xenotransplantation control, and these cells were frequently found in the head and blood-forming regions of the tail. Using DLA aliquots spiked with MBA-MB-231 cells, the newly established DanioCTC workflow confirmed the dissemination of MDA-MB-231 cells into these regions. CTCs from an MBC patient were then enriched by DLA, Parsortix, and flow cytometry, isolated with the CellCelectorTM and xenografted into zebrafish embryos. CTCs were mainly detected in the head and trunk, unlike MDA-MB-231 cells, which were present in the head and tail. DanioCTC presents a significant breakthrough in the use of zebrafish embryos as a model to study CTC dissemination in vivo, which can be used for patient-derived CTCs instead of cell culture-derived cancer cells as a crucial step towards understanding the biology of metastatic breast cancer. Statement of significanceDanioCTC is a novel workflow to inject patient-derived CTCs into zebrafish, enabling studies on CTC dissemination and personalized treatment in vivo, therefore advancing our toolkit to fight metastatic cancer.

cancer biology↗