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.

Icefishes (suborder Notothenioidei; family Channichthyidae) are the only vertebrates that lack functional haemoglobin genes and red blood cells. Here, we report a high-quality genome assembly and linkage map for the Antarctic blackfin icefish Chaenocephalus aceratus, highlighting evolved genomic features for its unique physiology. Phylogenomic analysis revealed that Antarctic fish of the teleost suborder Notothenioidei, including icefishes, diverged from the stickleback lineage about 77 million years ago and subsequently evolved cold-adapted phenotypes as the Southern Ocean cooled to sub-zero temperatures. Our results show that genes involved in protection from ice damage, including genes encoding antifreeze glycoprotein and zona pellucida proteins, are highly expanded in the icefish genome. Furthermore, genes that encode enzymes that help to control cellular redox state, including members of the sod3 and nqo1 gene families, are expanded, probably as evolutionary adaptations to the relatively high concentration of oxygen dissolved in cold Antarctic waters. In contrast, some crucial regulators of circadian homeostasis (cry and per genes) are absent from the icefish genome, suggesting compromised control of biological rhythms in the polar light environment. The availability of the icefish genome sequence will accelerate our understanding of adaptation to extreme Antarctic environments.

Transmitter-gated cation channels are detectors of excitatory chemical signals at synapses in the nervous system. Here we show that structurally distinct alpha3beta4 nicotinic and P2X2 channels influence each other when co-activated. The activation of one channel type affects distinct kinetic and conductance states of the other, and co-activation results in non-additive responses owing to inhibition of both channel types. State-dependent inhibition of nicotinic channels is revealed most clearly with mutant P2X2 channels, and inhibition is decreased at lower densities of channel expression. In synaptically coupled myenteric neurons, nicotinic fast excitatory postsynaptic currents are occluded during activation of endogenously co-expressed P2X channels. Our data provide a molecular basis and a synaptic context for cross-inhibition between transmitter-gated channels.

MicroRNAs (miRNAs) can have organ-specific expression and functions; they can originate from dedicated miRNA genes, from non-canonical miRNA genes, or from mirror-miRNA genes and can also experience post-transcriptional variation. It remains unclear, however, which mechanisms of miRNA production or modification are organ-specific and the extent of their evolutionary conservation. To address these issues, we developed the software Prost! (PRocessing Of Short Transcripts), which, among other features, helps quantify mature miRNAs, accounts for post-transcriptional processing, such as nucleotide editing, and identifies mirror-miRNAs. Here, we applied Prost! to annotate and analyze miRNAs in three-spined stickleback ( Gasterosteus aculeatus ), a model fish for evolutionary biology reported to have a miRNome larger than most teleost fish. Zebrafish ( Danio rerio ), a distantly related teleost with a well-known miRNome, served as comparator. Our results provided evidence for the existence of 286 miRNA genes and 382 unique mature miRNAs (excluding mir430 gene duplicates and the vaultRNA-derived mir733 ), which doesn’t represent a miRNAome larger than other teleost miRNomes. In addition, small RNA sequencing data from brain, heart, testis, and ovary in both stickleback and zebrafish identified suites of mature miRNAs that display organ-specific enrichment, many of which are evolutionarily-conserved in the brain and heart in both species. These data also supported the hypothesis that evolutionarily-conserved, organ-specific mechanisms may regulate post-transcriptional variations in miRNA sequence. In both stickleback and zebrafish, miR2188-5p was edited frequently with similar nucleotide changes in the seed sequence with organ specific editing rates, highest in the brain. In summary, Prost! is a new tool to identify and understand small RNAs, to help clarify a species’ miRNA biology as shown here for an important model for the evolution of developmental mechanisms, and to provide insight into organ-enriched expression and the evolutionary conservation of miRNA post-transcriptional modifications.

Fetal mammalian testes secrete Anti-Müllerian hormone (Amh), which inhibits female reproductive tract (Müllerian duct) development. Amh also derives from mature mammalian ovarian follicles, which marks oocyte reserve and characterizes polycystic ovarian syndrome. Zebrafish (Danio rerio) lacks Müllerian ducts and the Amh receptor gene amhr2 but, curiously, retains amh. To discover the roles of Amh in the absence of Müllerian ducts and the ancestral receptor gene, we made amh null alleles in zebrafish. Results showed that normal amh prevents female-biased sex ratios. Adult male amh mutants had enormous testes, half of which contained immature oocytes, demonstrating that Amh regulates male germ cell accumulation and inhibits oocyte development or survival. Mutant males formed sperm ducts and some produced a few offspring. Young female mutants laid a few fertile eggs, so they also had functional sex ducts. Older amh mutants accumulated nonvitellogenic follicles in exceedingly large but sterile ovaries, showing that Amh helps control ovarian follicle maturation and proliferation. RNA-sequencing data partitioned juveniles at 21 days postfertilization (dpf) into two groups that each contained mutant and wild-type fish. Group21-1 upregulated ovary genes compared to Group21-2, which were likely developing as males. By 35 dpf, transcriptomes distinguished males from females and, within each sex, mutants from wild types. In adult mutants, ovaries greatly underexpressed granulosa and theca genes, and testes underexpressed Leydig cell genes. These results show that ancestral Amh functions included development of the gonadal soma in ovaries and testes and regulation of gamete proliferation and maturation. A major gap in our understanding is the identity of the gene encoding a zebrafish Amh receptor; we show here that the loss of amhr2 is associated with the breakpoint of a chromosome rearrangement shared among cyprinid fishes. KEYWORDS germ cells; PGC; male fertility; female fertility; gonad development; Genetics of Sex D EVELOPING mammalian embryos form the rudiments of both male and female sex ducts, the Wolffian and Müllerian ducts, respectively. Over 70 years ago, Alfred Jost conducted remarkable experiments to learn if gonads control sex duct development (Jost 1947). He removed undifferentiated gonads from rabbit fetuses and reimplanted them into the uterus of surrogate rabbit hosts. Gonadectomized kits lost male sex ducts but retained female sex ducts. He concluded that developing testes maintain male ducts (epididymis, seminal vesicles, and vas deferens) but destroy female sex duct anlagen (fallopian tubes and uterus). In contrast, developing ovaries neither maintain male ducts nor destroy female ducts. Subsequent experiments showed that one testisderived substance (testosterone) maintains male sex duct rudiments and another [anti-Müllerian hormone (AMH), also called Müllerian Inhibiting Substance (MIS)], inhibits female reproductive duct anlagen (Elger 1966; Josso 1972). Although AMH from testes represses female duct d...