Lexogen and Bluebee co-hosted a webinar last week to introduce the latest updates in the Lexogen-Bluebee sample-to-data mRNA analysis platform, including the new QuantSeq UMI data analysis pipeline. As a part of the webinar the invited speaker Dr. Subham Dasgupta from the David Volz Lab at the University of California, Riverside has presented application of Lexogen’s QuantSeq 3′ mRNA-Seq Library Prep Kit and the integrated data analysis pipeline from Bluebee to unravel the impact of toxicity-induced changes in embryo development.

Tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) is a high-production volume organophosphate flame retardant that is used worldwide and detected within human populations, including children. Within zebrafish, we previously showed that initiation of TDCIPP exposure at 0.75 h post-fertilization (hpf) results in epiboly disruption at 6 hpf, and 76% of epiboly-arrested embryos are strongly dorsalized by 24 hpf – a phenotype that mimics the effects of dorsomorphin (DMP), a bone morphogenetic protein (BMP) antagonist that dorsalizes embryos in the absence of epiboly defects. Therefore, the primary objective of this study was to investigate the potential role of aberrant BMP signaling in TDCIPP-induced toxicity during early embryogenesis. Using epiboly and dorsalization as read-outs, we found that embryos were more susceptible to TDCIPP when exposures were initiated by 2-3 hpf, a maternal-to-zygotic transition characterized by genome activation and initiation of cell motility. We also found that 4’-hydroxychalcone (a BMP agonist) significantly mitigated TDCIPP-induced epiboly arrest as well as TDCIPP- and DMP-induced dorsalization. However, phospho-Smad1/5/9 detection in situ revealed that DMP – but not TDCIPP – blocked the normal progression of BMP signaling gradients at 8 hpf. Therefore, we relied on mRNA-sequencing to identify other signaling pathways that may be altered by TDCIPP during the first 24 h of development. We initiated treatments at 0.75 hpf and collected embryos at 3, 4, 5, 6, 8, 10, 12 and 24 hpf, totaling 90 RNA samples. Libraries were then prepared using the Lexogen Quantseq FWD library prep kit and their qualities were checked using our Agilent Bioanalyzer and quantified using a Qubit fluorometer. The libraries were then sequenced across 8 Illumina High Output (1X75 cycle) flow cells using our Illumina Miniseq and the resulting fastq files were processed and data analyzed using Bluebee’s Quantseq analysis platform. These data revealed that, despite phenotypic similarities starting at 10 hpf, there was minimal overlap in DMP- and TDCIPP-induced effects on the transcriptome, a finding supported by the absence of TDCIPP-induced effects on BMP signaling. However, unlike DMP, TDCIPP exposure resulted in a decrease in transcripts that regulate mesoderm differentiation (tbx16, tbx6, tbx6l, msgn1) at the beginning of segmentation (10-12 hpf), as well as hematopoiesis-specific transcripts (hbae1.3, hbae3, hbbe1.2, hbbe3) at the beginning of pharyngula (24 hpf). Therefore, as red blood cells are derived from the mesoderm during hematopoiesis, we exposed embryos to TDCIPP from 0.75 to 8 hpf and, using o-dianisidine staining, revealed that embryonic hemoglobin levels were significantly decreased at 72 hpf. Overall, our results suggest that initiation of TDCIPP exposure during early-blastula (2-3 hpf) alters the normal trajectory of epiboly, dorsoventral patterning, and hematopoiesis.

Reference: Dasgupta S, Cheng V, Vliet SMF, Mitchell CA, Volz DC. 2018. TDCIPP exposure during early-blastula alters the normal trajectory of zebrafish development. Environmental Science and Technology. 52 (18), pp 10820–10828.

Dr. Dasgupta’s Biography:

Dr-DasguptaDr. Dasgupta is a Postdoctoral Scholar specializing in environmental toxicology in Dr. David Volz’s laboratory at University of California, Riverside. He completed his BS in Human Physiology (2008) and MS in Environmental Sciences (2010) from University of Calcutta in India. In 2011, he joined the PhD program in Marine and Atmospheric Sciences at the Stony Brook University in New York, working with Dr. Anne McElroy on investigating mechanisms of toxicity of crude oil and dispersants used during the Deepwater Horizon oil spill on developing fish embryos and larvae. After graduating in 2016, Dr. Dasgupta joined Dr. Volz’s laboratory, where he is currently using next-generation sequencing, reverse genetics and other molecular and immunological tools to study the mechanisms of toxicity of flame retardants on early development of zebrafish embryos.

Watch the recording:

Learn more about QuantSeq 3’ mRNA-Seq Library Prep Kit here.
Learn more about the Bluebee Genomics Analysis Platform here.