To connect human biology to fish biomedical models, we sequenced the genome of spotted gar (Lepisosteus oculatus), whose lineage diverged from teleosts before the teleost genome duplication (TGD). The slowly evolving gar genome conserved in content and size many entire chromosomes from bony vertebrate ancestors. Gar bridges teleosts to tetrapods by illuminating the evolution of immunity, mineralization, and development (e.g., Hox, ParaHox, and miRNA genes). Numerous conserved non-coding elements (CNEs, often cis-regulatory) undetectable in direct human-teleost comparisons become apparent using gar: functional studies uncovered conserved roles of such cryptic CNEs, facilitating annotation of sequences identified in human genome-wide association studies. Transcriptomic analyses revealed that the sum of expression domains and levels from duplicated teleost genes often approximate patterns and levels of gar genes, consistent with subfunctionalization. The gar genome provides a resource for understanding evolution after genome duplication, the origin of vertebrate genomes, and the function of human regulatory sequences.

It was a zoological sensation when a living specimen of the coelacanth was first discovered in 1938, as this lineage of lobe-finned fish was thought to have gone extinct 70 million years ago. The modern coelacanth looks remarkably similar to many of its ancient relatives, and its evolutionary proximity to our own fish ancestors provides a glimpse of the fish that first walked on land. Here we report the genome sequence of the African coelacanth, Latimeria chalumnae. Through a phylogenomic analysis, we conclude that the lungfish, and not the coelacanth, is the closest living relative of tetrapods. Coelacanth protein-coding genes are significantly more slowly evolving than those of tetrapods, unlike other genomic features . Analyses of changes in genes and regulatory elements during the vertebrate adaptation to land highlight genes involved in immunity, nitrogen excretion and the development of fins, tail, ear, eye, brain, and olfaction. Functional assays of enhancers involved in the fin-to-limb transition and in the emergence of extra-embryonic tissues demonstrate the importance of the coelacanth genome as a blueprint for understanding tetrapod evolution.

There is no obvious morphological counterpart of the autopod (wrist/ankle and digits) in living fishes. Comparative molecular data may provide insight into understanding both the homology of elements and the evolutionary developmental mechanisms behind the fin to limb transition. In mouse limbs the autopod is built by a "late" phase of Hoxd and Hoxa gene expression, orchestrated by a set of enhancers located at the 5′ end of each cluster. Despite a detailed mechanistic understanding of mouse limb development, interpretation of Hox expression patterns and their regulation in fish has spawned multiple hypotheses as to the origin and function of "autopod" enhancers throughout evolution. Using phylogenetic footprinting, epigenetic profiling, and transgenic reporters, we have identified and functionally characterized hoxD and hoxA enhancers in the genomes of zebrafish and the spotted gar, Lepisosteus oculatus, a fish lacking the whole genome duplication of teleosts. Gar and zebrafish "autopod" enhancers drive expression in the distal portion of developing zebrafish pectoral fins, and respond to the same functional cues as their murine orthologs. Moreover, gar enhancers drive reporter gene expression in both the wrist and digits of mouse embryos in patterns that are nearly indistinguishable from their murine counterparts. These functional genomic data support the hypothesis that the distal radials of bony fish are homologous to the wrist and/or digits of tetrapods.he origin of novel features is a key question in evolutionary biology, and the autopod-wrists, fingers, ankles, and toesis a hallmark example (1). Although paleontological data, such as that from the Devonian lobe fin Tiktaalik roseae, reveal a sequence of changes in the elaboration of the bony elements of fins into limbs (2), in living taxa there is a lack of obvious homology between the wrist and digits of tetrapod limbs and the pectoral fin skeleton of extant fish (3). Tetrapod forelimbs are generally composed of a series of long bones (upper arm and forearm), followed by small nodular bones (wrist), and ending in another group of long bones (digits). Ray-finned (Actinopterygian) pectoral fins are diverse but are usually composed of a series of long proximal radials, followed by a set of smaller distal radials. The open question remains: do extant fish have the equivalent of wrists or digits?The molecular mechanisms governing the development of mammalian limbs have been approached in mouse models through multiple levels of analysis, from chromatin dynamics, to enhancer sequence, to gene expression patterns (4). Murine limbs display two successive phases of gene expression of the HoxD and HoxA gene clusters. The initial or "early" phase of expression begins with members at the 3′ end of the clusters being expressed broadly, and members at the 5′ end of the cluster being activated in an increasingly restricted number of cells (5). This "early" phase of Hox expression is associated with the development of the upper arm (stylopod) and forearm (zeugopod). The ...

Establishment of the left-right axis is essential for normal organ morphogenesis and function. Ca 2+ signaling and cilia function in the zebrafish Kuppfer's Vesicle (KV) have been implicated in laterality. Here we describe an endogenous Ca 2+ release event in the region of the KV precursors (dorsal forerunner cells, DFCs), prior to KV and cilia formation. Manipulation of Ca 2+ release to disrupt this early flux does not impact early DFC specification, but results in altered DFC migration or cohesion in the tailbud at somite stages. This leads to disruption of KV formation followed by bilateral expression of asymmetrical genes, and randomized organ laterality. We identify ␤-catenin inhibition as a Ca 2+-signaling target and demonstrate that localized loss of Ca 2+ within the DFC region or DFC-specific activation of ␤-catenin is sufficient to alter laterality in zebrafish. We identify a previously unknown DFC-like cell population in Xenopus and demonstrate a similar Ca 2+ -sensitive stage. As in zebrafish, manipulation of Ca 2+ release results in ectopic nuclear ␤-catenin and altered laterality. Overall, our data support a conserved early Ca 2+ requirement in DFC-like cell function in zebrafish and Xenopus.

Salamanders are the only living tetrapods capable of fully regenerating limbs. The discovery of salamander lineage-specific genes (LSGs) expressed during limb regeneration suggests that this capacity is a salamander novelty. Conversely, recent paleontological evidence supports a deeper evolutionary origin, before the occurrence of salamanders in the fossil record. Here we show that lungfishes, the sister group of tetrapods, regenerate their fins through morphological steps equivalent to those seen in salamanders. Lungfish de novo transcriptome assembly and differential gene expression analysis reveal notable parallels between lungfish and salamander appendage regeneration, including strong downregulation of muscle proteins and upregulation of oncogenes, developmental genes and lungfish LSGs. MARCKS-like protein (MLP), recently discovered as a regeneration-initiating molecule in salamander, is likewise upregulated during early stages of lungfish fin regeneration. Taken together, our results lend strong support for the hypothesis that tetrapods inherited a bona fide limb regeneration programme concomitant with the fin-to-limb transition.