Lineage-resolved analysis of embryonic gene expression evolution in C. elegans and C. briggsae

We measured the spatiotemporal divergence of gene expression across embryogenesis by collecting, annotating and comparing the transcriptomes of homologous embryonic cell types, using a dataset comprising >200,000 C. elegans cells and >175,000 C. briggsae cells. What constraints govern the evolution of gene expression patterns across development remains a fundamental question. Single-cell RNA-sequencing can learn these constraints by systematically profiling homologous cells. The conserved invariant embryonic lineage of C. elegans and C. briggsae makes them ideal for comparing cell type gene expression across evolution. Measuring the spatiotemporal divergence of gene expression across embryogenesis, we find a high level of similarity in gene expression programs between species despite tens of millions of years of evolutionary divergence. Nonetheless, thousands of genes show divergence in their cell-type specific expression patterns, with enrichment for functions in environmental response and behavior. Neuronal cell types show higher divergence than others such as the intestine and germline. This work identifies likely constraints on the evolution of developmental gene expression. Highly synchronized L1 larvae were obtained by repeated cycles of bleaching young adults to obtain eggs, which were then hatched in the absence of food to arrest development in the L1 stage. 50,000 of such highly synchronized L1 stage worms were placed on a large peptone rich plate seeded with OP50 bacteria. The L1 worms were allowed to grow at 20 degrees C for around 48 hours to reach the young adult stage, with most adults holding just 1-4 eggs. To obtain a broader range of developmental stages, in some experiments the adults were incubated for additional time before harvesting. The worms were washed off the plate with M9, gently centrifuged and washed again with M9 to remove bacteria. Embryos were released from adult worms with a 1.2 percent bleach solution in 0.5 N KOH. The bleach/adult worm suspension was gently agitated in the tube and checked under the microscope to closely monitor the worm disintegration. Once most of the worms had disintegrated (approximately 5-6 minutes), the tube containing dissociated embryos was vortexed for about 15 seconds and immediately centrifuged. The supernatant was discarded, and the embryos were washed three times with M9 buffer. After the final wash, the embryos were resuspended in M9 and filtered through a 40-micron nylon mesh cell strainer to eliminate remaining worm carcasses. To obtain embryos of different ages, the eggs were incubated on a petri dish with shaking at 20 for the desired time before single cell dissociation. For samples the C. elegans samples wt_600, ceh9_300, ceh9_600, mec3_300, mec3_600, M03D4.4_300 and M03D4.4_500, the adults were harvested as soon as they showed eggs, and the eggs were allowed to develop for the time (minutes) indicated. Similarly, for the C. briggsae samples, Cb_EMB_300, Cb_EMB_400 and Cb_EMB_500, the adults were harvested as soon as they showed eggs and the eggs were allowed to develop for the time indicated before the cells were dispersed. For two samples, Cb_EMB_120 and Cb_ EMB_240, the adults were allowed to develop for 410 minutes after the eggs appeared and the eggs were allowed to develop 120 or 240 minutes. For two samples, Cb_EMB_180 and Cb_EMB_360, the adults were allowed to develop 180 minutes after the first eggs appeared and the eggs dispersed either immediately after capture or after an additional 180 minutes. Embryos were concentrated by centrifugation for single-cell dissociation. To dissociate embryos into single cells, 1U/ml chitinase was added to the embryos at a ratio of 1 ml chitinase to 0.5 ml embryo suspension. The embryo chitinase suspension was transferred to a 30mm petri dish. Degradation of eggshell was continuously monitored for approximately 20-30 min. When the eggshell was degraded, egg buffer was added to the petri dishes. Then 100 microliters of 15 mg/ml pronase solution was added to the sample. The dissociation of embryos into single cells was carried out by passing embryos repeatedly through a 21gauge needle (approx 20 times) into a 1.5ml Eppendorf tube. The single-cell suspension was confirmed under a microscope. Single-cell concentration was determined using a cell counter and subjected to the 10X Chromium GEM version 3.0 single-cell capture procedure immediately. RNA library preparation followed the 10X Chromium procedure. Samples were sequenced with Illumina NextSeq 550 instruments using NextSeq Hi 75 cycles. These sequence files as well as the seven sequence files from the seven samples from GEO Series GSE126954 were processed as described in the data processing step descriptions. *************************************************************** The table below lists GEO accessions reused/reanalyzed for this study. ***************************************************************