Modulation of bioelectric cues in the evolution of flying fishes.

Changes to allometry, or the relative proportions of organs and tissues within organisms, is a common means for adaptive character change in evolution. However, little is understood about how relative size is specified during development and shaped during evolution. Here, through a phylogenomic analysis of genome-wide variation in 35 species of flying fishes and relatives, we identify genetic signatures in both coding and regulatory regions underlying the convergent evolution of increased paired fin size and aerial gliding behaviors. To refine our analysis, we intersected convergent phylogenomic signatures with mutants with altered fin size identified in distantly related zebrafish. Through these paired approaches, we identify a surprising role for an L-type amino acid transporter, lat4a, and the potassium channel, kcnh2a, in the regulation of fin proportion. We show that interaction between these genetic loci in zebrafish closely phenocopies the observed fin proportions of flying fishes. The congruence of experimental and phylogenomic findings point to conserved, non-canonical signaling integrating bioelectric cues and amino acid transport in the establishment of relative size in development and evolution.

Bioelectric-calcineurin signaling module regulates allometric growth and size of the zebrafish fin.

The establishment of relative size of organs and structures is paramount for attaining final form and function of an organism. Importantly, variation in the proportions of structures frequently underlies adaptive change in morphology in evolution and maybe a common mechanism underlying selection. However, the mechanism by which growth is integrated within tissues during development to achieve proper proportionality is poorly understood. We have shown that signaling by potassium channels mediates coordinated size regulation in zebrafish fins. Recently, calcineurin inhibitors were shown to elicit changes in zebrafish fin allometry as well. Here, we identify the potassium channel kcnk5b as a key player in integrating calcineurin's growth effects, in part through regulation of the cytoplasmic C-terminus of the channel. We propose that the interaction between Kcnk5b and calcineurin acts as a signaling node to regulate allometric growth. Importantly, we find that this regulation is epistatic to inherent mechanisms instructing overall size as inhibition of calcineurin is able to bypass genetic instruction of size as seen in sof and wild-type fins, however, it is not sufficient to re-specify positional memory of size of the fin. These findings integrate classic signaling mediators such as calcineurin with ion channel function in the regulation of size and proportion during growth.

Bioelectric signaling regulates size in zebrafish fins.

The scaling relationship between the size of an appendage or organ and that of the body as a whole is tightly regulated during animal development. If a structure grows at a different rate than the rest of the body, this process is termed allometric growth. The zebrafish another longfin (alf) mutant shows allometric growth resulting in proportionally enlarged fins and barbels. We took advantage of this mutant to study the regulation of size in vertebrates. Here, we show that alf mutants carry gain-of-function mutations in kcnk5b, a gene encoding a two-pore domain potassium (K(+)) channel. Electrophysiological analysis in Xenopus oocytes reveals that these mutations cause an increase in K(+) conductance of the channel and lead to hyperpolarization of the cell. Further, somatic transgenesis experiments indicate that kcnk5b acts locally within the mesenchyme of fins and barbels to specify appendage size. Finally, we show that the channel requires the ability to conduct K(+) ions to increase the size of these structures. Our results provide evidence for a role of bioelectric signaling through K(+) channels in the regulation of allometric scaling and coordination of growth in the zebrafish.

Gene expression analysis of zebrafish melanocytes, iridophores, and retinal pigmented epithelium reveals indicators of biological function and developmental origin.

In order to facilitate understanding of pigment cell biology, we developed a method to concomitantly purify melanocytes, iridophores, and retinal pigmented epithelium from zebrafish, and analyzed their transcriptomes. Comparing expression data from these cell types and whole embryos allowed us to reveal gene expression co-enrichment in melanocytes and retinal pigmented epithelium, as well as in melanocytes and iridophores. We found 214 genes co-enriched in melanocytes and retinal pigmented epithelium, indicating the shared functions of melanin-producing cells. We found 62 genes significantly co-enriched in melanocytes and iridophores, illustrative of their shared developmental origins from the neural crest. This is also the first analysis of the iridophore transcriptome. Gene expression analysis for iridophores revealed extensive enrichment of specific enzymes to coordinate production of their guanine-based reflective pigment. We speculate the coordinated upregulation of specific enzymes from several metabolic pathways recycles the rate-limiting substrate for purine synthesis, phosphoribosyl pyrophosphate, thus constituting a guanine cycle. The purification procedure and expression analysis described here, along with the accompanying transcriptome-wide expression data, provide the first mRNA sequencing data for multiple purified zebrafish pigment cell types, and will be a useful resource for further studies of pigment cell biology.

Lineage relationship of direct-developing melanocytes and melanocyte stem cells in the zebrafish.

Previous research in zebrafish has demonstrated that embryonic and larval regeneration melanocytes are derived from separate lineages. The embryonic melanocytes that establish the larval pigment pattern do not require regulative melanocyte stem cell (MSC) precursors, and are termed direct-developing melanocytes. In contrast, the larval regeneration melanocytes that restore the pigment pattern after ablation develop from MSC precursors. Here, we explore whether embryonic melanocytes and MSCs share bipotent progenitors. Furthermore, we explore when fate segregation of embryonic melanocytes and MSCs occurs in zebrafish development. In order to achieve this, we develop and apply a novel lineage tracing method. We first demonstrate that Tol2-mediated genomic integration of reporter constructs from plasmids injected at the 1-2 cell stage occurs most frequently after the midblastula transition but prior to shield stage, between 3 and 6 hours post-fertilization. This previously uncharacterized timing of Tol2-mediated genomic integration establishes Tol2-mediated transposition as a means for conducting lineage tracing in zebrafish. Combining the Tol2-mediated lineage tracing strategy with a melanocyte regeneration assay previously developed in our lab, we find that embryonic melanocytes and larval regeneration melanocytes are derived from progenitors that contribute to both lineages. We estimate 50-60 such bipotent melanogenic progenitors to be present in the shield-stage embryo. Furthermore, our examination of direct-developing and MSC-restricted lineages suggests that these are segregated from bipotent precursors after the shield stage, but prior to the end of convergence and extension. Following this early fate segregation, we estimate approximately 100 embryonic melanocyte and 90 MSC-restricted lineages are generated to establish or regenerate the zebrafish larval pigment pattern, respectively. Thus, the dual strategies of direct-development and MSC-derived development are established in the early gastrula, via fate segregation of the two lineages.