Intergeneric hybrids inform reproductive isolating barriers in the Antarctic icefish radiation.

Interspecific hybridization or barriers to hybridization may have contributed to the diversification of Antarctic icefishes (Channichthyidae), but data supporting these hypotheses is scarce. To understand the potential for hybridization and to investigate reproductive isolating mechanisms among icefish species, we performed in vitro fertilization experiments using eggs from a female blackfin icefish Chaenocephalus aceratus and sperm from a male of another genera, the ocellated icefish Chionodraco rastrospinosus. Sequencing of genomic and mitochondrial DNA confirmed the intergeneric hybrid nature of resulting embryos which successfully developed and hatched as active larvae at about four and a half months during the Antarctic winter. This result demonstrates the compatibility of gametes of these two species and the viability of resulting zygotes and larvae. Due to logistic constraints and the slow developmental rate of icefishes, we could not test for long-term hybrid viability, fertility, fitness, or hybrid breakdown. Analysis of our fishing records and available literature, however, suggests that the strongest barriers to hybridization among parapatric icefish species are likely to be behavioral and characterized by assortative mating and species-specific courtship and nesting behaviors. This conclusion suggests that, in long-lived fish species with late sexual maturity and high energetic investment in reproduction like icefishes, pre-mating barriers are energetically more efficient than post-mating barriers to prevent hybridization.

Divergent Hemogen genes of teleosts and mammals share conserved roles in erythropoiesis: analysis using transgenic and mutant zebrafish.

Hemogen is a vertebrate transcription factor that performs important functions in erythropoiesis and testicular development and may contribute to neoplasia. Here we identify zebrafish Hemogen and show that it is considerably smaller (∼22 kDa) than its human ortholog (∼55 kDa), a striking difference that is explained by an underlying modular structure. We demonstrate that Hemogens are largely composed of 21-25 amino acid repeats, some of which may function as transactivation domains (TADs). Hemogen expression in embryonic and adult zebrafish is detected in hematopoietic, renal, neural and gonadal tissues. Using Tol2- and CRISPR/Cas9-generated transgenic zebrafish, we show that Hemogen expression is controlled by two Gata1-dependent regulatory sequences that act alone and together to control spatial and temporal expression during development. Partial depletion of Hemogen in embryos by morpholino knockdown reduces the number of erythrocytes in circulation. CRISPR/Cas9-generated zebrafish lines containing either a frameshift mutation or an in-frame deletion in a putative, C-terminal TAD display anemia and embryonic tail defects. This work expands our understanding of Hemogen and provides mutant zebrafish lines for future study of the mechanism of this important transcription factor.

Assisted protein folding at low temperature: evolutionary adaptation of the Antarctic fish chaperonin CCT and its client proteins.

Eukaryotic ectotherms of the Southern Ocean face energetic challenges to protein folding assisted by the cytosolic chaperonin CCT. We hypothesize that CCT and its client proteins (CPs) have co-evolved molecular adaptations that facilitate CCT-CP interaction and the ATP-driven folding cycle at low temperature. To test this hypothesis, we compared the functional and structural properties of CCT-CP systems from testis tissues of an Antarctic fish, Gobionotothen gibberifrons (Lönnberg) (habitat/body T = -1.9 to +2°C), and of the cow (body T = 37°C). We examined the temperature dependence of the binding of denatured CPs (ß-actin, ß-tubulin) by fish and bovine CCTs, both in homologous and heterologous combinations and at temperatures between -4°C and 20°C, in a buffer conducive to binding of the denatured CP to the open conformation of CCT. In homologous combination, the percentage of G. gibberifrons CCT bound to CP declined linearly with increasing temperature, whereas the converse was true for bovine CCT. Binding of CCT to heterologous CPs was low, irrespective of temperature. When reactions were supplemented with ATP, G. gibberifrons CCT catalyzed the folding and release of actin at 2°C. The ATPase activity of apo-CCT from G. gibberifrons at 4°C was ∼2.5-fold greater than that of apo-bovine CCT, whereas equivalent activities were observed at 20°C. Based on these results, we conclude that the catalytic folding cycle of CCT from Antarctic fishes is partially compensated at their habitat temperature, probably by means of enhanced CP-binding affinity and increased flexibility of the CCT subunits.

Evolution and function of the globin intergenic regulatory regions of the antarctic dragonfishes (Notothenioidei: Bathydraconidae).

As the Southern Ocean cooled to -1.8 °C over the past 40 My, the teleostean clade Notothenioidei diversified and, under reduced selection pressure for an oxygen-transporting apparatus, became less reliant on hemoglobin and red blood cells. At the extreme of this trend, the crown group of Antarctic icefishes (Channichthyidae) lost both components of oxygen transport. Under the decreased selection scenario, we hypothesized that the Antarctic dragonfishes (Bathydraconidae, the red-blooded sister clade to the icefishes) evolved lower blood hemoglobin concentrations because their globin gene complexes (α- and ß-globin gene pairs linked by a regulatory intergene) transcribe globin mRNAs less effectively than those of basal notothenioids (e.g., the Nototheniidae [notothens]). To test our hypothesis, we 1) sequenced the α/ß-intergenes of the adult globin complexes of three notothen and eight dragonfish species and 2) measured globin transcript levels in representative species from each group. The typical nototheniid intergene was ∼3-4 kb in length. The bathydraconid intergenes resolved into three subclasses (long [3.8 kb], intermediate [3.0 kb], and short [1.5-2.3 kb]) that corresponded to the three subclades proposed for the taxon. Although they varied in length due to indels, the three notothen and eight dragonfish intergenes contained a conserved ∼90-nt element that we have previously shown to be required for globin gene transcription. Using the quantitative polymerase chain reaction, we found that globin mRNA levels in red cells from one notothen species and from one species of each dragonfish subclade were equivalent statistically. Thus, our results indicate that the bathydraconids have evolved adult globin loci whose regulatory intergenes tend to be shorter than those of the more basal nototheniids yet are equivalent in transcriptional efficacy. Their low blood hemoglobin concentrations are most likely due to reduction in hematocrit.

A model for cleavage plane determination in early amphibian and fish embryos.

Current models for cleavage plane determination propose that metaphase spindles are positioned and oriented by interactions of their astral microtubules with the cellular cortex, followed by cleavage in the plane of the metaphase plate [1, 2]. We show that in early frog and fish embryos, where cells are unusually large, astral microtubules in metaphase are too short to position and orient the spindle. Rather, the preceding interphase aster centers and orients a pair of centrosomes prior to nuclear envelope breakdown, and the spindle assembles between these prepositioned centrosomes. Interphase asters center and orient centrosomes with dynein-mediated pulling forces. These forces act before astral microtubules contact the cortex; thus, dynein must pull from sites in the cytoplasm, not the cell cortex as is usually proposed for smaller cells. Aster shape is determined by interactions of the expanding periphery with the cell cortex or with an interaction zone that forms between sister-asters in telophase. We propose a model to explain cleavage plane geometry in which the length of astral microtubules is limited by interaction with these boundaries, causing length asymmetries. Dynein anchored in the cytoplasm then generates length-dependent pulling forces, which move and orient centrosomes.

Genome enablement of the notothenioidei: genome size estimates from 11 species and BAC libraries from 2 representative taxa.

The perciform suborder Notothenoidei provides a compelling opportunity to study the adaptive radiation of a marine species flock in the cold Southern Ocean surrounding Antarctica. To enable genome-level studies of these psychrophilic fishes, we estimated the sizes of the genomes of 11 Antarctic species and generated high-quality BAC libraries for 2, the notothen Notothenia coriiceps and the icefish Chaenocephalus aceratus. Our results indicate that evolution of phylogenetically derived notothenioid families, [e.g., the icefishes (Channichthyidae)], was accompanied by genome expansion. Species (n=6) of the basal family Nototheniidae had C values that ranged between 0.98 and 1.20 pg, whereas those of the icefishes, the notothenioid crown group, were 1.66-1.83 pg (n=4 species). The BAC libraries VMRC-19 (N. coriiceps) and VMRC-21 (C. aceratus) comprised 12X and 10X coverage of the respective genomes and had average insert sizes of 138 and 168 kb. Greater than 60% of paired BAC ends sampled from each library ( approximately 0.1% of each genome) contained repetitive sequences, and the repetitive element landscapes of the 2 genomes (13.4% of the N. coriiceps genome and 14.5% for C. aceratus) were similar. The representation and depth of coverage of the libraries were verified by identification of multiple Hox gene contigs: six discrete Hox clusters were found in N. coriiceps and at least five Hox clusters were found in C. aceratus. Given the unusual anatomical and physiological adaptations of the notothenioids, the availability of these BAC libraries sets the stage for expanded analysis of the psychrophilic mode of life.

A genomic fossil reveals key steps in hemoglobin loss by the antarctic icefishes.

Antarctic icefishes are the only vertebrates that do not have hemoglobin and erythrocytes in their blood. These startling phenotypes are associated in several icefish species with deletions of juvenile and adult globin loci, which in red-blooded teleosts are typically composed of tightly linked pairs of alpha- and beta-globin genes. It is unknown if the loss of hemoglobin expression in icefishes was the direct result of such deletions or if other mutational events compromised globin chain synthesis prior to globin gene loss. In this study, we show that 15 of the 16 icefish species have lost the adult beta-globin gene but retain a truncated alpha-globin pseudogene. Surprisingly, a phylogenetically derived icefish species, Neopagetopsis ionah, possesses a complete, but nonfunctional, adult alphabeta-globin complex. This cluster contains 2 distinct beta-globin pseudogenes whose phylogenetic origins span the entire Antarctic notothenioid radiation, consistent with an origin via introgression. Maximum likelihood ancestral state reconstruction supports a scenario of icefish globin gene evolution that involves a single loss of the transcriptionally active adult alphabeta-globin cluster prior to the diversification of the extant species in the clade. Through lineage sorting of ancestral polymorphism, 2 types of alleles became fixed in the clade: 1) the alpha-globin pseudogene of the majority of species and 2) the inactive alphabeta-globin complex of N. ionah. We conclude that the globin pseudogene complex of N. ionah is a "genomic fossil" that reveals key intermediate steps on the pathway to loss of hemoglobin expression by all icefish species.

Characterization of the cytoplasmic chaperonin containing TCP-1 from the Antarctic fish Notothenia coriiceps.

The cytoplasmic chaperonin containing TCP-1 (CCT) plays a critically important role in the folding and biogenesis of many cytoskeletal proteins, including tubulin and actin. For marine ectotherms, the chronically cold Southern Ocean (-2 to +2 degrees C) poses energetic challenges to protein folding, both at the level of substrate proteins and with respect to the chaperonin/chaperone folding system. Here we report the partial functional and structural characterization of CCT from an Antarctic notothenioid fish, Notothenia coriiceps. We find that the mechanism of folding by the Antarctic fish CCT differed from that of mammalian CCT: (1) the former complex was able to bind denatured beta-tubulin but (2) when reconstituted with rabbit Cofactor A, failed to release the protein to yield the tubulin/cofactor intermediate. Moreover, the amino acid sequences of the N. coriiceps CCT beta and theta chains contained residue substitutions in the equatorial, apical, and intermediate domains that would be expected to increase the flexibility of the subunits, thus facilitating function of the chaperonin in an energy poor environment. Our work contributes to the growing realization that protein function in cold-adapted organisms reflects a delicate balance between the necessity of structural flexibility for catalytic activity and the concomitant hazard of cold-induced denaturation.

bloodthirsty, an RBCC/TRIM gene required for erythropoiesis in zebrafish.

The Antarctic icefishes (family Channichthyidae, suborder Notothenioidei) constitute the only vertebrate taxon that fails to produce red blood cells. These fishes can be paired with closely related, but erythrocyte-producing, notothenioids to discover erythropoietic genes via representational difference analysis. Using a B30.2-domain-encoding DNA probe so derived from the hematopoietic kidney (pronephros) of a red-blooded Antarctic rockcod, Notothenia coriiceps, we discovered a related, novel gene, bloodthirsty (bty), that encoded a 547-residue protein that contains sequential RING finger, B Box, coiled-coil, and B30.2 domains. bty mRNA was expressed by the pronephric kidney of N. coriiceps at a steady-state level 10-fold greater than that found in the kidney of the icefish Chaenocephalus aceratus. To test the function of bty, we cloned the orthologous zebrafish gene from a kidney cDNA library. Whole-mount in situ hybridization of zebrafish embryos showed that bty mRNA was present throughout development and, after the mid-blastula transition, was expressed in the head and in or near the site of primitive erythropoiesis in the tail just prior to red cell production. One- to four-cell embryos injected with two distinct antisense morpholino oligonucleotides (MOs) targeted to the 5'-end of the bty mRNA failed to develop red cells, whereas embryos injected with 4- and 5-bp mismatch control MOs produced wild-type quantities of erythrocytes. The morphant phenotype was rescued by co-injection of synthetic bty mRNA containing an artificial 5'-untranslated region (UTR) with the antisense MO that bound the 5'-UTR of the wild-type bty transcript. Furthermore, the expression of genes that mark terminal erythroid differentiation was greatly reduced in the antisense-MO-treated embryos. We conclude that bty is likely to play a role in differentiation of the committed red cell progenitor.

Posttranslational modification of brain tubulins from the Antarctic fish Notothenia coriiceps: reduced C-terminal glutamylation correlates with efficient microtubule assembly at low temperature.

We have shown previously that the tubulins of Antarctic fish assemble into microtubules efficiently at low temperatures (-2 to +2 degrees C) due to adaptations intrinsic to the tubulin subunits. To determine whether changes in posttranslational glutamylation of the fish tubulins may contribute to cold adaptation of microtubule assembly, we have characterized C-terminal peptides from alpha- and beta-tubulin chains from brains of adult specimens of the Antarctic rockcod Notothenia coriiceps by MALDI-TOF mass spectrometry and by Edman degradation amino acid sequencing. Of the four fish beta-tubulin isotypes, nonglutamylated isoforms were more abundant than glutamylated isoforms. In addition, maximal glutamyl side-chain length was shorter than that observed for mammalian brain beta tubulins. For the nine fish alpha-tubulin isotypes, nonglutamylated isoforms were also generally more abundant than glutamylated isoforms. When glutamylated, however, the maximal side-chain lengths of the fish alpha tubulins were generally longer than those of adult rat brain alpha chains. Thus, Antarctic fish adult brain tubulins are glutamylated differently than mammalian brain tubulins, resulting in a more heterogeneous population of alpha isoforms and a reduction in the number of beta isoforms. By contrast, neonatal rat brain tubulin possesses low levels of glutamylation that are similar to that of the adult fish brain tubulins. We suggest that unique residue substitutions in the primary structures of Antarctic fish tubulin isotypes and quantitative changes in isoform glutamylation act synergistically to adapt microtubule assembly to low temperatures.