To explore the origins and consequences of tetraploidy in the African clawed frog, we sequenced the Xenopus laevis genome and compared it to the related diploid X. tropicalis genome. We demonstrate the allotetraploid origin of X. laevis by partitioning its genome into two homeologous subgenomes, marked by distinct families of “fossil” transposable elements. Based on the activity of these elements and the age of hundreds of unitary pseudogenes, we estimate that the two diploid progenitor species diverged ~34 million years ago (Mya) and combined to form an allotetraploid ~17–18 Mya. 56% of all genes are retained in two homeologous copies. Protein function, gene expression, and the amount of flanking conserved sequence all correlate with retention rates. The subgenomes have evolved asymmetrically, with one chromosome set more often preserving the ancestral state and the other experiencing more gene loss, deletion, rearrangement, and reduced gene expression.

Multiciliate cells employ hundreds of motile cilia to produce fluid flow, which they nucleate and extend by first assembling hundreds of centrioles. In most cells, entry into the cell cycle allows centrioles to undergo a single round of duplication, but in differentiating multiciliate cells, massive centriole assembly occurs in G0 by a process initiated by a small coiled-coil protein, Multicilin. Here we show that Multicilin acts by forming a ternary complex with E2f4 or E2f5 and Dp1 that binds and activates most of the genes required for centriole biogenesis, while other cell cycle genes remain off. This complex also promotes the deuterosome pathway of centriole biogenesis by activating the expression of deup1 but not its paralog, cep63. Finally, we show that this complex is disabled by mutations in human Multicilin that cause a severe congenital mucociliary clearance disorder due to reduced generation of multiple cilia. By coopting the E2f regulation of cell cycle genes, Multicilin drives massive centriole assembly in epithelial progenitors in a manner required for multiciliate cell differentiation.

The developmental bases for species differences in adult phenotypes remain largely unknown. An emerging system for studying such variation is the adult pigment pattern expressed by Danio fishes. These patterns result from several classes of pigment cells including black melanophores and yellow xanthophores, which differentiate during metamorphosis from latent stem cells of presumptive neural crest origin. In the zebrafish D. rerio,alternating light and dark horizontal stripes develop, in part, owing to interactions between melanophores and cells of the xanthophore lineage that depend on the fms receptor tyrosine kinase; zebrafish fmsmutants lack xanthophores and have disrupted melanophore stripes. By contrast,the closely related species D. albolineatus exhibits a uniform pattern of melanophores, and previous interspecific complementation tests identified fms as a potential contributor to this difference between species. Here, we survey additional species and demonstrate marked variation in the fms-dependence of hybrid pigment patterns, suggesting interspecific variation in the fms pathway or fmsrequirements during pigment pattern formation. We next examine the cellular bases for the evolutionary loss of stripes in D. albolineatus and test the simplest model to explain this transformation, a loss of fmsactivity in D. albolineatus relative to D. rerio. Within D. albolineatus, we demonstrate increased rates of melanophore death and decreased melanophore migration, different from wild-type D. rerio but similar to fms mutant D. rerio. Yet, we also find persistent fms expression in D. albolineatus and enhanced xanthophore development compared with wild-type D. rerio,and in stark contrast to fms mutant D. rerio. These findings exclude the simplest model in which stripe loss in D. albolineatusresults from a loss of fms-dependent xanthophores and their interactions with melanophores. Rather, our results suggest an alternative model in which evolutionary changes in pigment cell interactions themselves have contributed to stripe loss, and we test this model by manipulating melanophore numbers in interspecific hybrids. Together, these data suggest evolutionary changes in the fms pathway or fms requirements,and identify changes in cellular interactions as a likely mechanism of evolutionary change in Danio pigment patterns.

SUMMARYSpecialized epithelial cells in the amphibian skin play important roles in ion transport, but how they arise developmentally is largely unknown. Here we show that proton-secreting cells (PSCs) differentiate in the X. laevis larval skin soon after gastrulation, based on the expression of a 'kidney-specific' form of the H + v-ATPase that localizes to the plasma membrane, orthologs of the Cl -/HCO 3 -antiporters ae1 and pendrin, and two isoforms of carbonic anhydrase. Like PSCs in other species, we show that the expression of these genes is likely to be driven by an ortholog of foxi1, which is also sufficient to promote the formation of PSC precursors. Strikingly, the PSCs form in the skin as two distinct subtypes that resemble the alpha-and beta-intercalated cells of the kidney. The alpha-subtype expresses ae1 and localizes H + v-ATPases to the apical plasma membrane, whereas the beta-subtype expresses pendrin and localizes the H + v-ATPase cytosolically or basolaterally. These two subtypes are specified during early PSC differentiation by a binary switch that can be regulated by Notch signaling and by the expression of ubp1, a transcription factor of the grainyhead family. These results have implications for how PSCs are specified in vertebrates and become functionally heterogeneous.

Latent precursors or stem cells of neural crest origin are present in a variety of post-embryonic tissues. Although these cells are of biomedical interest for roles in human health and disease, their potential evolutionary significance has been underappreciated. As a first step towards elucidating the contributions of such cells to the evolution of vertebrate form, we investigated the relative roles of neural crest cells and post-embryonic latent precursors during the evolutionary diversification of adult pigment patterns in Danio fishes. These pigment patterns result from the numbers and arrangements of embryonic melanophores that are derived from embryonic neural crest cells, as well as from post-embryonic metamorphic melanophores that are derived from latent precursors of presumptive neural crest origin. In the zebrafish D. rerio, a pattern of melanophore stripes arises during the larval-to-adult transformation by the recruitment of metamorphic melanophores from latent precursors. Using a comparative approach in the context of new phylogenetic data, we show that adult pigment patterns in five additional species also arise from metamorphic melanophores, identifying this as an ancestral mode of adult pigment pattern development. By contrast, superficially similar adult stripes of D. nigrofasciatus (a sister species to D. rerio) arise by the reorganization of melanophores that differentiated at embryonic stages, with a diminished contribution from metamorphic melanophores. Genetic mosaic and molecular marker analyses reveal evolutionary changes that are extrinsic to D. nigrofasciatus melanophore lineages, including a dramatic reduction of metamorphic melanophore precursors. Finally, interspecific complementation tests identify a candidate genetic pathway for contributing to the evolutionary reduction in metamorphic melanophores and the increased contribution of early larval melanophores to D. nigrofasciatus adult pigment pattern development. These results demonstrate an important role for latent precursors in the diversification of pigment patterns across danios. More generally, differences in the deployment of post-embryonic neural crest-derived stem cells or their specified progeny may contribute substantially to the evolutionary diversification of adult form in vertebrates, particularly in species that undergo a metamorphosis.