Cerebral cortex size differs dramatically between reptiles, birds, and mammals, owing to developmental differences in neuron production. In mammals, signaling pathways regulating neurogenesis have been identified, but genetic differences behind their evolution across amniotes remain unknown. We show that direct neurogenesis from radial glia cells, with limited neuron production, dominates the avian, reptilian, and mammalian paleocortex, whereas in the evolutionarily recent mammalian neocortex, most neurogenesis is indirect via basal progenitors. Gain- and loss-of-function experiments in mouse, chick, and snake embryos and in human cerebral organoids demonstrate that high Slit/Robo and low Dll1 signaling, via Jag1 and Jag2, are necessary and sufficient to drive direct neurogenesis. Attenuating Robo signaling and enhancing Dll1 in snakes and birds recapitulates the formation of basal progenitors and promotes indirect neurogenesis. Our study identifies modulation in activity levels of conserved signaling pathways as a primary mechanism driving the expansion and increased complexity of the mammalian neocortex during amniote evolution.

Feathers are arranged in a precise pattern in avian skin. They first arise during development in a row along the dorsal midline, with rows of new feather buds added sequentially in a spreading wave. We show that the patterning of feathers relies on coupled fibroblast growth factor (FGF) and bone morphogenetic protein (BMP) signalling together with mesenchymal cell movement, acting in a coordinated reaction-diffusion-taxis system. This periodic patterning system is partly mechanochemical, with mechanical-chemical integration occurring through a positive feedback loop centred on FGF20, which induces cell aggregation, mechanically compressing the epidermis to rapidly intensify FGF20 expression. The travelling wave of feather formation is imposed by expanding expression of Ectodysplasin A (EDA), which initiates the expression of FGF20. The EDA wave spreads across a mesenchymal cell density gradient, triggering pattern formation by lowering the threshold of mesenchymal cells required to begin to form a feather bud. These waves, and the precise arrangement of feather primordia, are lost in the flightless emu and ostrich, though via different developmental routes. The ostrich retains the tract arrangement characteristic of birds in general but lays down feather primordia without a wave, akin to the process of hair follicle formation in mammalian embryos. The embryonic emu skin lacks sufficient cells to enact feather formation, causing failure of tract formation, and instead the entire skin gains feather primordia through a later process. This work shows that a reaction-diffusion-taxis system, integrated with mechanical processes, generates the feather array. In flighted birds, the key role of the EDA/Ectodysplasin A receptor (EDAR) pathway in vertebrate skin patterning has been recast to activate this process in a quasi-1-dimensional manner, imposing highly ordered pattern formation. Feathers are a defining feature of birds. In flighted birds, the feathers are arranged in a highly ordered pattern that arises during embryonic development. Feather rudiments are produced in rows, with these rows appearing one after another in a spreading wave. We show that the formation of highly regular feather patterns depends on this wave of formation, with the row-by-row progression triggered by a spreading wave of the signal Ectodysplasin A (EDA). The EDA signal cooperates with a spreading wave of cellular density to spark the formation of regularly spaced cell aggregates, each of which serves as the precursor to a feather. The EDA wave and the cell density wave are themselves independent of feather formation, as they can be detected travelling across the embryonic skin even when feather formation is impaired. In the flightless birds examined these waves are missing and a highly ordered feather pattern is not produced. However, the manner of wave loss differs in different species, with ostrich embryos lacking the wave of EDA, whereas the emu lacks sufficient skin cells at the appropriate developmental stage to make precisely organised feather rudiments.

Vertebrate skin is characterized by its patterned array of appendages, whether feathers, hairs, or scales. In avian skin the distribution of feathers occurs on two distinct spatial levels. Grouping of feathers within discrete tracts, with bare skin lying between the tracts, is termed the macropattern, while the smaller scale periodic spacing between individual feathers is referred to as the micropattern. The degree of integration between the patterning mechanisms that operate on these two scales during development and the mechanisms underlying the remarkable evolvability of skin macropatterns are unknown. A striking example of macropattern variation is the convergent loss of neck feathering in multiple species, a trait associated with heat tolerance in both wild and domestic birds. In chicken, a mutation called Naked neck is characterized by a reduction of body feathering and completely bare neck. Here we perform genetic fine mapping of the causative region and identify a large insertion associated with the Naked neck trait. A strong candidate gene in the critical interval, BMP12/GDF7, displays markedly elevated expression in Naked neck embryonic skin due to a cis-regulatory effect of the causative mutation. BMP family members inhibit embryonic feather formation by acting in a reaction-diffusion mechanism, and we find that selective production of retinoic acid by neck skin potentiates BMP signaling, making neck skin more sensitive than body skin to suppression of feather development. This selective production of retinoic acid by neck skin constitutes a cryptic pattern as its effects on feathering are not revealed until gross BMP levels are altered. This developmental modularity of neck and body skin allows simple quantitative changes in BMP levels to produce a sparsely feathered or bare neck while maintaining robust feather patterning on the body. The distribution of hairs or feathers across the body is not homogeneous, and many animals have characteristic regions of their skin with either profuse or reduced coverage. These features, such as manes, crests, or bald patches, are seen in diverse species, suggesting that they can be selectively advantageous and also that the mechanisms by which the skin develops somehow enables such features to appear repeatedly in the course of evolution. In this study we explore the basis of loss of neck feathering, a feature associated with heat tolerance that has arisen independently several times during bird evolution. We find that in chickens a bare neck is caused by increased production of BMPs, factors previously implicated in defining the size of the gaps between neighboring feathers. Selective production of retinoic acid by embryonic neck skin enhances BMP signaling, thereby bringing this skin region close to the threshold of BMP action required to completely suppress feather development. This usually innocuous distinction between neck and body skin enables mutations that increase BMP action to render the neck completely bare while permitting normal feathering on the body. Thus an underlying map within the skin provides a one-step route to a bare neck.

Hox genes are required for the development of the intestinal cecum, a major organ of plant-eating species. We have analyzed the transcriptional regulation of Hoxd genes in cecal buds and show that they are controlled by a series of enhancers located in a gene desert flanking the HoxD cluster. The start site of two opposite long noncoding RNAs (lncRNAs), Hotdog and Twin of Hotdog, selectively contacts the expressed Hoxd genes in the framework of a topological domain, coinciding with robust transcription of these genes during cecum budding. Both lncRNAs are specifically transcribed in the cecum, albeit bearing no detectable function in trans. Hedgehogs have kept this regulatory potential despite the absence of the cecum, suggesting that these mechanisms are used in other developmental situations. In this context, we discuss the implementation of a common "budding toolkit" between the cecum and the limbs.

Significance: Syncytins are genes of retroviral origin that have been captured by their host as symbionts for a function in placentation. They can mediate cell–cell fusion, consistent with their ancestral retroviral envelope gene status, and are involved in fusion of mononucleate trophoblast cells to form the syncytial layer—the syncytiotrophoblast—of the feto–maternal interface. We proposed that such genes have been pivotal for the emergence of placental mammals from egg-laying animals and should be present all along the Placentalia radiation. We searched for syncytins in a superorder of eutherian mammals that emerged ancestrally during the Cretaceous terrestrial revolution and identified syncytin-Ten1 , conserved over millions years of evolution of the Afrotherian tenrecs, regarded as among the most primitive of living mammals.