Primate cell fusion disentangles gene regulatory divergence in neurodevelopment.

Among primates, humans display a unique trajectory of development that is responsible for the many traits specific to our species. However, the inaccessibility of primary human and chimpanzee tissues has limited our ability to study human evolution. Comparative in vitro approaches using primate-derived induced pluripotent stem cells have begun to reveal species differences on the cellular and molecular levels1,2. In particular, brain organoids have emerged as a promising platform to study primate neural development in vitro3-5, although cross-species comparisons of organoids are complicated by differences in developmental timing and variability of differentiation6,7. Here we develop a new platform to address these limitations by fusing human and chimpanzee induced pluripotent stem cells to generate a panel of tetraploid hybrid stem cells. We applied this approach to study species divergence in cerebral cortical development by differentiating these cells into neural organoids. We found that hybrid organoids provide a controlled system for disentangling cis- and trans-acting gene-expression divergence across cell types and developmental stages, revealing a signature of selection on astrocyte-related genes. In addition, we identified an upregulation of the human somatostatin receptor 2 gene (SSTR2), which regulates neuronal calcium signalling and is associated with neuropsychiatric disorders8,9. We reveal a human-specific response to modulation of SSTR2 function in cortical neurons, underscoring the potential of this platform for elucidating the molecular basis of human evolution.

Human-chimpanzee fused cells reveal cis-regulatory divergence underlying skeletal evolution.

Gene regulatory divergence is thought to play a central role in determining human-specific traits. However, our ability to link divergent regulation to divergent phenotypes is limited. Here, we utilized human-chimpanzee hybrid induced pluripotent stem cells to study gene expression separating these species. The tetraploid hybrid cells allowed us to separate cis- from trans-regulatory effects, and to control for nongenetic confounding factors. We differentiated these cells into cranial neural crest cells, the primary cell type giving rise to the face. We discovered evidence of lineage-specific selection on the hedgehog signaling pathway, including a human-specific sixfold down-regulation of EVC2 (LIMBIN), a key hedgehog gene. Inducing a similar down-regulation of EVC2 substantially reduced hedgehog signaling output. Mice and humans lacking functional EVC2 show striking phenotypic parallels to human-chimpanzee craniofacial differences, suggesting that the regulatory divergence of hedgehog signaling may have contributed to the unique craniofacial morphology of humans.

An intronic enhancer of Bmp6 underlies evolved tooth gain in sticklebacks.

Threespine stickleback fish offer a powerful system to dissect the genetic basis of morphological evolution in nature. Marine sticklebacks have repeatedly invaded and adapted to numerous freshwater environments throughout the Northern hemisphere. In response to new diets in freshwater habitats, changes in craniofacial morphology, including heritable increases in tooth number, have evolved in derived freshwater populations. Using a combination of quantitative genetics and genome resequencing, here we fine-mapped a quantitative trait locus (QTL) regulating evolved tooth gain to a cluster of ten QTL-associated single nucleotide variants, all within intron four of Bone Morphogenetic Protein 6 (Bmp6). Transgenic reporter assays revealed this intronic region contains a tooth enhancer. We induced mutations in Bmp6, revealing required roles for survival, growth, and tooth patterning. Transcriptional profiling of Bmp6 mutant dental tissues identified significant downregulation of a set of genes whose orthologs were previously shown to be expressed in quiescent mouse hair stem cells. Collectively these data support a model where mutations within a Bmp6 intronic tooth enhancer contribute to evolved tooth gain, and suggest that ancient shared genetic circuitry regulates the regeneration of diverse vertebrate epithelial appendages including mammalian hair and fish teeth.

Partially repeatable genetic basis of benthic adaptation in threespine sticklebacks.

The extent to which convergent adaptation to similar ecological niches occurs by a predictable genetic basis remains a fundamental question in biology. Threespine stickleback fish have undergone an adaptive radiation in which ancestral oceanic populations repeatedly colonized and adapted to freshwater habitats. In multiple lakes in British Columbia, two different freshwater ecotypes have evolved: a deep-bodied benthic form adapted to forage near the lake substrate, and a narrow-bodied limnetic form adapted to forage in open water. Here, we use genome-wide linkage mapping in marine × benthic F2 genetic crosses to test the extent of shared genomic regions underlying benthic adaptation in three benthic populations. We identify at least 100 Quantitative Trait Loci (QTL) harboring genes influencing skeletal morphology. The majority of QTL (57%) are unique to one cross. However, four genomic regions affecting eight craniofacial and armor phenotypes are found in all three benthic populations. We find that QTL are clustered in the genome and overlapping QTL regions are enriched for genomic signatures of natural selection. These findings suggest that benthic adaptation has occurred via both parallel and nonparallel genetic changes.

Disentangling Sources of Selection on Exonic Transcriptional Enhancers.

In addition to coding for proteins, exons can also impact transcription by encoding regulatory elements such as enhancers. It has been debated whether such features confer heightened selective constraint, or evolve neutrally. We have addressed this question by developing a new approach to disentangle the sources of selection acting on exonic enhancers, in which we model the evolutionary rates of every possible substitution as a function of their effects on both protein sequence and enhancer activity. In three exonic enhancers, we found no significant association between evolutionary rates and effects on enhancer activity. This suggests that despite having biochemical activity, these exonic enhancers have no detectable selective constraint, and thus are unlikely to play a major role in protein evolution.

Distinct developmental genetic mechanisms underlie convergently evolved tooth gain in sticklebacks.

Teeth are a classic model system of organogenesis, as repeated and reciprocal epithelial and mesenchymal interactions pattern placode formation and outgrowth. Less is known about the developmental and genetic bases of tooth formation and replacement in polyphyodonts, which are vertebrates with continual tooth replacement. Here, we leverage natural variation in the threespine stickleback fish Gasterosteus aculeatus to investigate the genetic basis of tooth development and replacement. We find that two derived freshwater stickleback populations have both convergently evolved more ventral pharyngeal teeth through heritable genetic changes. In both populations, evolved tooth gain manifests late in development. Using pulse-chase vital dye labeling to mark newly forming teeth in adult fish, we find that both high-toothed freshwater populations have accelerated tooth replacement rates relative to low-toothed ancestral marine fish. Despite the similar evolved phenotype of more teeth and an accelerated adult replacement rate, the timing of tooth number divergence and the spatial patterns of newly formed adult teeth are different in the two populations, suggesting distinct developmental mechanisms. Using genome-wide linkage mapping in marine-freshwater F2 genetic crosses, we find that the genetic basis of evolved tooth gain in the two freshwater populations is largely distinct. Together, our results support a model whereby increased tooth number and an accelerated tooth replacement rate have evolved convergently in two independently derived freshwater stickleback populations using largely distinct developmental and genetic mechanisms.