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. Author manuscript; available in PMC: 2023 Nov 23.
Published in final edited form as: Cell. 2022 Nov 23;185(24):4587–4603.e23. doi: 10.1016/j.cell.2022.10.016

Figure 5: Rapid divergence of hominin-specific neurodevelopmental enhancers near FOXD4 family genes followed multiple segmental duplications.

Figure 5:

(A) Experimental design. We cloned the human or inferred human-chimpanzee ancestral sequence of HAQER0059 into an PGK-EGFP reporter plasmid and delivered plasmids to the developing cerebral cortex via in utero electroporation at E15.5 alongside an mCherry injection reporter. We performed dissection, sectioning, and imaging 24 hours later. (B) Representative images of Hoechst-stained coronal sections imaged for the mCherry injection reporter and EGFP enhancer reporter. Scale bar, 100 μm. (C) Left: Quantification of PGK-EGFP reporter signal normalized to the mCherry injection reporter for HAQER0059. Right: Corresponding in vivo STARR-seq results. (*: p<0.05, ***: p<0.001, FDR t-test. Dotted line: Negative Control Mean + 3SD). (D) Phylogeny of HAQER0059 homologs in humans and other great apes. (E) Genomic context for the paralogous regions near the genes FOXD4L3, FOXD4L1, and FOXD4. We present genomic context for the region near FOXD4, which contains HAQER0059, on the reverse strand. The region near FOXD4L3 does not have a nearby HAQER and shares synteny with the great ape ortholog. (F) A model of recent FOXD4 evolution. The great ape ortholog of the human gene FOXD4L3 generated the paralog FOXD4 in the chromosome 9 subtelomere through paired inversion and duplication. Subsequent duplication produced the paralog FOXD4L1 at the fusion site between the ancestral chromosomes 2a/2b, which formed the modern human chromosome 2. See also Figure S6.