Fig 1.
RNA-seq of hybrid Drosophila embryos reveals extensive spatially patterned allele-specific expression.
A) Each embryo was cryosliced along the anterior-posterior axis in 14μm sections, followed by RNA-seq in each slice. Allele-specific expression (ASE) was called for each gene in each slice by assigning unambiguous reads to the parent of origin; shown here are the reads for the gene Ance, with blue indicating D. simulans reads and red indicating D. melanogaster reads. For each gene, we fit either a step-like or peak-like (shown) function. B-C) Genes with a step-like pattern (B, best fit by a logistic function) or peak-like pattern (C, best fit by a Gaussian function). For each gene, anterior is left and posterior is right. The green line indicates the best fit pattern, with higher indicating D. simulans biased expression, and lower indicating D. melanogaster biased expression. The heat maps are from the first female replicate of each direction of the cross.
Fig 2.
Hybrid embryos show strong melanogaster-specific expression of hunchback in the anterior.
A) Heat map of svASE of hb shows a significant D. melanogaster bias in the anterior tip of the embryo. Each row is a different embryo. Embryos with a melanogaster mother are above the horizontal line. B-C) In situ hybridization for hb in parental embryos at the 26-50% membrane invagination stage from [31] (C) and [13] (D). Images are arranged anterior to the left and dorsal up. D) Computed bias for each nucleus. Nuclei with low expression in both species (less than 20% of the peak expression value) are colored white to reflect no callable bias. E) Overall computed bias for each 4% section of the embryo by x-position. D. melanogaster and D. simulans expression levels are summed for each nucleus in that section of the embryo, then bias computed. Bias is not computed for the middle sections of the embryo where no RNA-seq bias data is available due to low hb expression.
Fig 3.
Cis-regulatory changes in hb regulatory regions could cause the observed svASE.
A) Regulatory elements near the zygotic hunchback-RA transcript. B-C) FIMO binding motifs and inter-specific variants of the anterior activator (B) and shadow CRM from [35] (C). Species-specific predicted binding sites are highlighted with arrows. D) Overview of the logistic expression model. A function is fit for wild-type D. melanogaster, then individual activation/repression coefficients are independently adjusted for each TF. E-J) Predicted ASE from adjusting strength of each TF in the model in order to maximize the variance in the real ASE explained by the predicted ASE. Predicted absolute expression is shown in purple above, ASE per nucleus is shown in the middle panel in opposed red/blue, and predicted ASE in a sliced embryo is shown below. Note that in panel I, although a Bicoid site is gained in D. simulans, the best fit according to the model would be to decrease the coefficient.
Fig 4.
CRISPR-Cas9-mediated editing shows a Bicoid site in D. simulans is responsible for the change in expression pattern.
A) A pair of SNPs in the canonical hb CRM at the indicated coordinates on D. melanogaster chromosome 3R. SNPs between D. melanogaster and D. simulans marked in red. B) The Bicoid binding motif aligned to the site of the binding change. C) The Huckebein binding motif aligned to the site of the binding change. D-E) Staining of hb in the two most closely staged wild-type (D) and CRISPR-edited (E) D. melanogaster embryos. F) We created hybrid embryos with the wild-type alleles on the wild-type chromosomes (orange), or with the D. simulans copy of hb driven by the more D. melanogaster-like CRM and the D. melanogaster copy of hb driven by the more D. simulans-like CRM (purple). G) Allele-specific expression measured by pyrosequencing in slices from 4 wild-type hybrid and 3 allele-swapped hybrid embryos. Error bars indicate standard deviation across 3 SNPs in all slices at the indicated slice. Significance markers indicate results of 2-sample, 2-sided t-tests. As usual, +1 is 100% D. simulans bias, and -1 is 100% D. melanogaster bias.