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. 2022 Sep 22;11:e79247. doi: 10.7554/eLife.79247

Figure 4. Wild-type gene expression studies reveal mef2 paralog expression dynamics, strain-specific expression levels, and standing paralog expression variation.

(A) Wild-type head expression of all mef2 paralogs was quantified by RT-quantitative PCR (qPCR) at 1 hr intervals from 20 to 30 hpf. Expression of each paralog was normalized to rps18. Error bars are SD. (B) Expression of each paralog in cranial neural crest cells at 24 hpf was determined by single cell RNA-sequencing on sorted cells. Seurat-based clustering subdivided the cells into four populations as described (Mitchell et al., 2021): anterior arches (aa), frontonasal (fn), posterior arches (pa), and a satellite population containing melanocyte lineage cells (m). (C) Wild-type head expression of all mef2 paralogs was quantified by RT-qPCR at 24 hpf to compare paralog expression levels between the low- and high-penetrance strains. Expression of each paralog was normalized to rps18. Asterisks indicate significant difference (*≤0.05 and **≤0.01). Error bars are SD. (D) Wild-type head expression of all mef2 paralogs was quantified by RT-qPCR at 24 hpf to compare paralog expression levels between two families from the unselected AB strain. Expression of each paralog was normalized to rps18. Asterisks indicate significant difference (*≤0.05, ***≤0.001, and ****≤0.0001). Error bars are SEM.

Figure 4—source data 1. qPCR raw values.
Figure 4—source data 2. qPCR raw values.
Figure 4—source data 3. qPCR raw values.
Figure 4—source data 4. qPCR raw values.

Figure 4.

Figure 4—figure supplement 1. Overall transcription is not increased in the low-penetrance strain.

Figure 4—figure supplement 1.

We observed increased expression of many mef2 paralogs in wild types from the low-penetrance strain compared with high-penetrance strain wild types. To determine if overall gene expression is generally upregulated in the low-penetrance strain, we quantified two housekeeping genes by RT-quantitative PCR (qPCR) at 24 hpf to compare expression levels between strains. Expression of each gene was normalized to rps18, the same normalization gene we used for comparing paralogs. In contrast to the mef2 paralogs, neither housekeeping gene is significantly upregulated in the low-penetrance strain. In fact, expression of these housekeeping genes was higher in the high- versus low-penetrance strain wild types. Error bars are SD, p-values (**≤0.01 and *≤0.05).