Molecular basis of the unique osmoregulatory strategy in the inshore hagfish, Eptatretus burgeri.

Hagfishes are characterized by omo- and iono-conforming nature similar to marine invertebrates. Conventionally, hagfishes had been recognized as the most primitive living vertebrate that retains plesiomorphic features. However, some of the "ancestral" features of hagfishes, such as rudimentary eyes and the lack of vertebrae, have been proven to be deceptive. Similarly, by the principle of maximum parsimony, the unique body fluid regulatory strategy of hagfishes seems to be apomorphic, since the lamprey, another cyclostome, adopts osmo- and iono-regulatory mechanisms as in jawed vertebrates. Although hagfishes are unequivocally important upon discussing the origin and evolution of the vertebrate osmoregulatory system, the molecular basis for the body fluid homeostasis in hagfishes has been poorly understood. In the present study, we explored this matter in the inshore hagfish, Eptatretus burgeri, by analyzing the transcriptomes obtained from the gill, kidney and muscle of the animals acclimated to distinct environmental salinities. Together with the measurement of parameters in the muscular fluid compartment, our data indicate that the hagfish possesses an ability to conduct free amino acids (FAAs)-based osmoregulation at a cellular level, which is in coordination with the renal and branchial FAA absorption. We also revealed that the hagfish does possess the orthologs of the known osmoregulatory genes, and that the transepithelial movement of inorganic ions in the hagfish gill and kidney is more complex than previously thought. These observations pose a challenge to the conventional view that the physiological features of hagfishes have been inherited from the last common ancestor of the extant vertebrates.

Prognostic Impact of the Early Use of Tolvaptan in Elderly Patients with Acute Decompensated Heart Failure.

The number of elderly patients with acute decompensated heart failure (ADHF) is increasing, and it is often difficult to treat. This study aimed to evaluate the efficacy and safety of using tolvaptan early after hospitalization in elderly patients with ADHF and the prognosis one year after hospitalization. This study enrolled 185 patients with ADHF who were admitted for the first time. Tolvaptan was administered within 24 h after admission. These patients were assigned to two groups: over 80 years old (n = 109) and under 80 years old (n = 76). There were no significant differences between the two groups in the occurrence of MACCE within one year (25% vs. 20%, p = 0.59). All-cause mortality was significantly higher in the over-80 group (12% vs. 2%, p = 0.01). There were no significant differences in the incidence of worsening renal failure (11% vs. 7%, p = 0.46) and hypernatremia (5% vs. 9%, p = 1.0), and in the duration of hospitalization (19.2 days vs. 18.8 days, p = 0.8). Tolvaptan might be effective and safe in elderly patients with ADHF, and there was no difference in the incidence of MACCE within one year.

Progesterone initiates tendril formation in the oviducal gland during egg encapsulation in cloudy catshark (Scyliorhinus torazame).

The diverse reproductive strategies of elasmobranchs (sharks, rays, and skates) have attracted research attention, but the endocrine control of reproduction is still incompletely known in elasmobranchs. By long-term monitoring of the egg-laying cycle in cloudy catsharks (Scyliorhinus torazame), we recently demonstrated a transient increase in plasma progesterone (P4) levels just prior to the appearance of the capsulated eggs in the oviducts. In the present study, we examined the in vivo effects of P4 administration in mature female cloudy catsharks. Although no capsulated eggs were observed following the implantation of P4-containing silicone tubing, we did find dark swollen oviducts in the abdominal cavity, in which clumps of long and coiled tendrils were observed. The tendril is an extension of the egg capsule, and the formation of the egg capsule begins with the tendril before main capsule formation. During the period of P4 implantation, the tendrils elongated, and their diameters were significantly increased on day 2 of treatment. Tendril formation was also confirmed on the day of endogenous P4 surge. Tendrils were not formed in catsharks implanted with estradiol-17ß or testosterone. Histological analysis of the oviducal gland revealed that P4 implantation induced the secretion of tendril materials from the secretory tubules in the baffle zone, while the tendril materials were stored in the cytoplasm of the secretory cells under low P4 condition. Morphometrically, the area of secreted luminal materials in the secretory tubules was highly correlated to the diameter of tendrils. Our results suggest that the P4 surge during the egg-laying cycle serves as a trigger for egg capsule formation in the oviducal gland of cloudy catshark, but the hormonal signals were incomplete as the main capsule was not formed. Further studies are required to identify the hormones required for ovulation and formation of the main egg capsule.

Segment-Dependent Gene Expression Profiling of the Cartilaginous Fish Nephron Using Laser Microdissection for Functional Characterization of Nephron at Segment Levels.

For adaptation to a high salinity marine environment, cartilaginous fishes have evolved a ureosmotic strategy. They have a highly elaborate "four-loop nephron" in the kidney, which is considered to be important for reabsorption of urea from the glomerular filtrate to maintain a high concentration of urea in the body. However, the function and regulation, generally, of the "four-loop nephron" are still largely unknown due to the complicated configuration of the nephron and its many subdivided segments. Laser microdissection (LMD) followed by RNA-sequencing (RNA-seq) analysis is a powerful technique to obtain segment-dependent gene expression profiles. In the present study, using the kidney of cloudy catshark, Scyliorhinus torazame, we tested several formaldehyde-free and formaldehyde-based fixatives to optimize the fixation methods. Fixation by 1% neutral buffered formalin for 15 min resulted in sufficient RNA and structural integrities, which allowed LMD clipping of specific nephron segments and subsequent RNA-seq analysis. RNA-seq from the LMD samples of the second-loop, the fourth-loop, and the five tubular segments in the bundle zone revealed a number of specific membrane transporter genes that can characterize each segment. Among them, we examined expressions of the Na + -coupled cotransporters abundantly expressed in the second loop samples. Although the proximal II segment of the second loop is known for the elimination of excess solutes, the present results imply that the PII segment is also crucial for reabsorption of valuable solutes. Looking ahead to future studies, the segment-dependent gene expression profiling will be a powerful technique for unraveling the renal mechanisms and regulation in euryhaline elasmobranchs.

Phylogenetic and functional properties of hagfish neurohypophysial hormone receptors distinct from their jawed vertebrate counterparts.

Vertebrate neurohypophysial hormones, i.e., vasopressin- and oxytocin-family peptides, exert versatile physiological actions via distinct G protein-coupled receptors. The neurohypophysial hormone receptor (NHR) family was classically categorized into four subtypes (V1aR, V1bR, V2R and OTR), while recent studies have identified seven subtypes (V1aR, V1bR, V2aR, V2bR, V2cR, V2dR and OTR; V2aR corresponds to the conventional V2R). The vertebrate NHR family were diversified via multiple gene duplication events at different scales. Despite intensive research effort in non-osteichthyes vertebrates such as cartilaginous fish and lamprey, the molecular phylogeny of the NHR family has not been fully understood. In the present study, we focused on the inshore hagfish (Eptatretus burgeri), another group of cyclostomes, and Arctic lamprey (Lethenteron camtschaticum) for comparison. Two putative NHR homologs, which were previously identified only in silico, were cloned from the hagfish and designated as ebV1R and ebV2R. In vitro, ebV1R, as well as two out of five Arctic lamprey NHRs, increased intracellular Ca2+ in response to exogenous neurohypophysial hormones. None of the examined cyclostome NHRs altered intracellular cAMP levels. Transcripts of ebV1R were detected in multiple tissues including the brain and gill, with intense hybridization signals in the hypothalamus and adenohypophysis, while ebV2R was predominantly expressed in the systemic heart. Similarly, Arctic lamprey NHRs showed distinct expression patterns, underscoring the multifunctionality of VT in the cyclostomes as in the gnathostomes. These results and exhaustive gene synteny comparisons provide new insights into the molecular and functional evolution of the neurohypophysial hormone system in vertebrates.

Molecular and morphological investigations on the renal mechanisms enabling euryhalinity of red stingray Hemitrygon akajei.

Most cartilaginous fishes live in seawater (SW), but a few exceptional elasmobranchs (sharks and rays) are euryhaline and can acclimate to freshwater (FW) environments. The plasma of elasmobranchs is high in NaCl and urea concentrations, which constrains osmotic water loss. However, these euryhaline elasmobranchs maintain high levels of plasma NaCl and urea even when acclimating to low salinity, resulting in a strong osmotic gradient from external environment to body fluid. The kidney consequently produces a large volume of dilute urine to cope with the water influx. In the present study, we investigated the molecular mechanisms of dilute urine production in the kidney of Japanese red stingray, Hemitrygon akajei, transferred from SW to low-salinity environments. We showed that red stingray maintained high plasma NaCl and urea levels by reabsorbing more osmolytes in the kidney when transferred to low salinity. RNA-seq and qPCR analyses were conducted to identify genes involved in NaCl and urea reabsorption under the low-salinity conditions, and the upregulated gene expressions of Na+-K+-Cl- cotransporter 2 (nkcc2) and Na+/K+-ATPase (nka) were found in the FW-acclimated individuals. These upregulations occurred in the early distal tubule (EDT) in the bundle zone of the kidney, which coils around the proximal and collecting tubules to form the highly convoluted structure of batoid nephron. Considering the previously proposed model for urea reabsorption, the upregulation of nkcc2 and nka not only causes the reabsorption of NaCl in the EDT, but potentially also supports enhanced urea reabsorption and eventually the production of dilute urine in FW-acclimated individuals. We propose advantageous characteristics of the batoid-type nephron that facilitate acclimation to a wide range of salinities, which might have allowed the batoids to expand their habitats.

Lotus root-like appearance in the left anterior descending artery treated with a drug-coated balloon angioplasty.

A lotus root-like appearance of the coronary artery diagnosed by optical coherence tomography (OCT) is characterized by old coronary thrombi that form small lumen channels. Herein, serial OCT images of a left anterior descending artery with a lotus root-like appearance, treated with drug-coated balloon angioplasty are described.

Long-term monitoring of egg-laying cycle using ultrasonography reveals the reproductive dynamics of circulating sex steroids in an oviparous catshark, Scyliorhinus torazame.

The many diverse reproductive strategies of elasmobranchs (sharks, skates and rays) from lecithotrophic oviparity to matrotrophic viviparity have attracted significant research attention. However, the endocrine control of elasmobranch reproduction is less well-documented largely due to their reproductive characteristics, such as a long reproductive cycle, and/or repeated internal fertilization using stored sperm in oviparous species. In the present study, for the first time, we succeeded in non-invasive monitoring of the continuing egg-laying cycle of the cloudy catshark Scyliorhinus torazame using portable ultrasound devices. Furthermore, long-term simultaneous monitoring of the egg-laying cycle and measurement of plasma sex steroids revealed cycling patterns of estradiol-17ß (E2), testosterone (T) and progesterone (P4). In particular, a decline in T followed by a reciprocal surge in plasma P4 were consistently observed prior to the appearance of the capsulated eggs, implying that P4 is likely associated with the ovulation and/or egg-case formation. While the cycling pattern of E2 was not as apparent as those of T and P4, threshold levels of E2 (>5 ng/mL) and T (>1 ng/mL) appeared to be crucial in the continuation of egg-laying cycle. The possibility to trace the dynamics of plasma sex steroids in a single individual throughout the reproductive cycles makes the catshark a useful model for regulatory and mechanistic studies of elasmobranch reproduction.

Effect of Early Initiation of Evolocumab on Lipoprotein(a) in Patients with Acute Myocardial Infarction: Sub-Analysis of a Randomized Controlled Trial.

Elevated circulating lipoprotein(a) levels are associated with an increased risk of cardiovascular events. We reported that early initiation of evolocumab, a proprotein convertase subtilisin/kexin type 9 inhibitor, in addition to a statin substantially reduced the lipoprotein(a) levels in patients with acute myocardial infarction (AMI) after primary percutaneous coronary intervention (PCI). This sub-analysis sought to investigate the effect of evolocumab on lipoprotein(a) based on baseline lipoprotein(a) levels and characteristics. This study was a prespecified analysis of a randomized controlled trial that enrolled 102 patients who underwent primary PCI for AMI. Patients received pitavastatin (2 mg/day) alone or pitavastatin and evolocumab 140 mg subcutaneously within 24 h and 2 weeks after the index PCI. The evolocumab group showed significantly suppressed lipoprotein(a) levels in patients with baseline lipoprotein(a) levels of ≤10 mg/dL, 10 < lipoprotein(a) ≤ 20 mg/dL, and >20 mg/dL compared with the control group, as well as similar reductions in lipoprotein(a) levels in all patient subgroups. Among these subgroups, evolocumab tended to show more favorable effects in patients with diabetes mellitus. In AMI patients, early initiation of evolocumab therapy within 24 h of primary PCI suppressed the increase in lipoprotein(a) levels within 4 weeks, regardless of baseline levels and characteristics.

Thyroid and endostyle development in cyclostomes provides new insights into the evolutionary history of vertebrates.

BACKGROUND: The endostyle is an epithelial exocrine gland found in non-vertebrate chordates (amphioxi and tunicates) and the larvae of modern lampreys. It is generally considered to be an evolutionary precursor of the thyroid gland of vertebrates. Transformation of the endostyle into the thyroid gland during the metamorphosis of lampreys is thus deemed to be a recapitulation of a past event in vertebrate evolution. In 1906, Stockard reported that the thyroid gland in hagfish, the sister cyclostome group of lampreys, develops through an endostyle-like primordium, strongly supporting the plesiomorphy of the lamprey endostyle. However, the findings in hagfish thyroid development were solely based on this single study, and these have not been confirmed by modern molecular, genetic, and morphological data pertaining to hagfish thyroid development over the last century. RESULTS: Here, we showed that the thyroid gland of hagfish undergoes direct development from the ventrorostral pharyngeal endoderm, where the previously described endostyle-like primordium was not found. The developmental pattern of the hagfish thyroid, including histological features and regulatory gene expression profiles, closely resembles that found in modern jawed vertebrates (gnathostomes). Meanwhile, as opposed to gnathostomes but similar to non-vertebrate chordates, lamprey and hagfish share a broad expression domain of Nkx2-1/2-4, a key regulatory gene, in the pharyngeal epithelium during early developmental stages. CONCLUSIONS: Based on the direct development of the thyroid gland both in hagfish and gnathostomes, and the shared expression profile of thyroid-related transcription factors in the cyclostomes, we challenge the plesiomorphic status of the lamprey endostyle and propose an alternative hypothesis where the lamprey endostyle could be obtained secondarily in crown lampreys.

Molecular mechanism of nutrient uptake in developing embryos of oviparous cloudy catshark (Scyliorhinus torazame).

Forms of embryonic nutrition are highly diverse in cartilaginous fishes, which contain oviparity, yolk-sac viviparity and several types of matrotrophic viviparity (histotrophy, oophagy, and placentotrophy). The molecular mechanisms of embryonic nutrition are poorly understood in these animals as few species are capable of reproducing in captivity. Oviparous cartilaginous fishes solely depend on yolk nutrients for their growth and development. In the present study, we compared the contribution to embryonic nutrition of the embryonic intestine with the yolk sac membrane (YSM). RNA-seq analysis was performed on the embryonic intestine and YSM of the oviparous cloudy catshark Scyliorhinus torazame to identify candidate genes involved in nutrient metabolism to further the understanding of nutrient utilization of developing embryos. RNA-seq discovery was subsequently confirmed by quantitative PCR analysis and we identified increases in several amino acid transporter genes (slc3a1, slc6a19, slc3a2, slc7a7) as well as genes involved in lipid absorption (apob and mtp) in the intestine after 'pre-hatching', which is a developmental event marked by an early opening of the egg case about 4 months before hatching. Although a reciprocal decrease in the nutritional role of YSM was expected after the intestine became functional, we observed similar increases in gene expression among amino acid transporters, lipid absorption molecules, and lysosomal cathepsins in the extraembryonic YSM in late developmental stages. Ultrastructure of the endodermal cells of YSM showed that yolk granules were incorporated by endocytosis, and the number of granules increased during development. Furthermore, the digestion of yolk granules in the YSM and nutrient transport through the basolateral membrane of the endodermal cells appeared to be enhanced after pre-hatching. These findings suggest that nutrient digestion and absorption is highly activated in both intestine and YSM after pre-hatching in catshark embryos, which supports the rapid growth at late developmental stages.

Regulation by Progestins, Corticosteroids, and RU486 of Transcriptional Activation of Elephant Shark and Human Progesterone Receptors: An Evolutionary Perspective.

We investigated progestin and corticosteroid activation of the progesterone receptor (PR) from elephant shark, a cartilaginous fish belonging to the oldest group of jawed vertebrates. Comparison with the human PR provides insights into the evolution of steroid activation of the human PR. At 1 nM steroid, the elephant shark PR is activated by progesterone, 17-hydroxy-progesterone, 20ß-hydroxy-progesterone, 11-deoxycorticosterone (21-hydroxyprogesterone), and 11-deoxycortisol. The human PR, in comparison, is activated at 1 nM steroid, only by progesterone and 11-deoxycorticosterone, indicating increased progestin and corticosteroid specificity during the evolution of the human PR. RU486, an important clinical antagonist of the human PR, did not inhibit progesterone activation of the elephant shark PR. Cys-528 in the elephant shark PR corresponds to Gly-722 in the human PR, which is essential for RU486 inhibition of the human PR. Confirming the importance of Cys-528 in the elephant shark PR, RU486 inhibited progesterone activation of the Cys528Gly mutant PR. To investigate the physiological relevance of Gly-722 in the human PR and Cys-528 in the elephant shark PR, we studied steroid activation of the Gly722Cys human PR and Cys528Gly elephant shark PR. Compared to the wild-type human PR, there was an increase in the activation of human Gly722Cys PR by11-deoxycortisol and a decrease in activation by corticosterone, which may have been important in selection for the mutation corresponding to the human glycine-722 PR that first evolved in the platypus PR, a basal mammal.

Fulminant myocarditis after the second dose of COVID-19 mRNA vaccination.

Myocarditis is an adverse event associated with coronavirus disease 2019 (COVID-19) mRNA vaccination. A 50-year-old man presented with dyspnea and resting chest pain after receiving the second dose of the COVID-19 mRNA vaccine and developed cardiogenic shock. Fulminant myocarditis was diagnosed by endomyocardial biopsy and treated with intravenous corticosteroids.

N-terminal domain regulates steroid activation of elephant shark glucocorticoid and mineralocorticoid receptors.

Orthologs of human glucocorticoid receptor (GR) and human mineralocorticoid receptor (MR) first appear in cartilaginous fishes. Subsequently, the MR and GR diverged to respond to different steroids: the MR to aldosterone and the GR to cortisol and corticosterone. We report that cortisol, corticosterone and aldosterone activate full-length elephant shark GR, and progesterone, which activates elephant shark MR, does not activate elephant shark GR. However, progesterone inhibits steroid binding to elephant shark GR, but not to human GR. Together, this indicates partial functional divergence of elephant shark GR from the MR. Deletion of the N-terminal domain (NTD) from elephant shark GR (truncated GR) reduced the response to corticosteroids, while truncated and full-length elephant shark MR had similar responses to corticosteroids. Swapping of NTDs of elephant shark GR and MR yielded an elephant shark MR chimera with full-length GR-like increased activation by corticosteroids and progesterone compared to full-length elephant shark MR. Elephant shark MR NTD fused to GR DBD + LBD had similar activation as full-length MR, indicating that the MR NTD lacked GR-like NTD activity. We propose that NTD activation of human GR evolved early in GR divergence from the MR.

Secular Decreasing Trend in Plasma Eicosapentaenoic and Docosahexaenoic Acids among Patients with Acute Coronary Syndrome from 2011 to 2019: A Single Center Descriptive Study.

Despite intensive lipid-lowering interventions, patients treated with statins develop atherosclerotic cardiovascular disease (ASCVD), and these patients have an increased risk of developing recurrent cardiovascular events during follow-up. Therefore, there is a need to focus on the residual risks in patients in statin therapy to further reduce ASCVD. The aim of this study was to retrospectively investigate the 10-year trend (2011-2019) regarding changes in polyunsaturated fatty acids (PUFAs) in patients with acute coronary syndrome (ACS) in a single center. We included 686 men and 203 women with ACS admitted to Kagawa Prefectural Central Hospital. Plasma PUFAs, including eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), arachidonic acid (AA), and dihomo-γ-linolenic acid (DGLA), were measured at admission for suspected ACS. A secular decreasing trend in the levels of EPA and DHA and the EPA/AA ratio, but not of AA and DGLA, was observed. The analyses based on age (>70 or <70 years) and sex showed that the decreasing trend in the levels of EPA and DHA did not depend on age and remained significant only in men. Further studies are needed to obtain robust evidence to justify that the administration of n-3 PUFA contributes to the secondary prevention of ACS.

Facilitated NaCl Uptake in the Highly Developed Bundle of the Nephron in Japanese Red Stingray Hemitrygon akajei Revealed by Comparative Anatomy and Molecular Mapping.

Batoidea (rays and skates) is a monophyletic subgroup of elasmobranchs that diverged from the common ancestor with Selachii (sharks) about 270 Mya. A larger number of batoids can adapt to low-salinity environments, in contrast to sharks, which are mostly stenohaline marine species. Among osmoregulatory organs of elasmobranchs, the kidney is known to be dedicated to urea retention in ureosmotic cartilaginous fishes. However, we know little regarding urea reabsorbing mechanisms in the kidney of batoids. Here, we performed physiological and histological investigations on the nephrons in the red stingray (Hemitrygon akajei) and two shark species. We found that the urine/plasma ratios of salt and urea concentrations in the stingray are significantly lower than those in cloudy catshark (Scyliorhinus torazame) under natural seawater, indicating that the kidney of stingray more strongly reabsorbs these osmolytes. By comparing the three-dimensional images of nephrons between stingray and banded houndshark (Triakis scyllium), we showed that the tubular bundle of stingray has a more compact configuration. In the compact tubular bundle of stingray kidney, the distal diluting tubule was highly developed and frequently coiled around the proximal and collecting tubules. Furthermore, co-expression of NKAα1 (Na+/K +-ATPase) and NKCC2 (Na+- K+-2Cl- cotransporter 2) mRNAs was prominent in the coiled diluting segment. These findings imply that NaCl reabsorption is greatly facilitated in the stingray kidney, resulting in a higher reabsorption rate of urea. Lowering the loss of osmolytes in the glomerular filtrate is likely favorable to the adaptability of batoids to a wide range of environmental salinity.

Morphological and functional development of the spiral intestine in cloudy catshark (Scyliorhinus torazame).

Cartilaginous fish have a comparatively short intestine known as the spiral intestine that consists of a helical spiral of intestinal mucosa. However, morphological and functional development of the spiral intestine has not been fully described. Unlike teleosts, cartilaginous fish are characterized by an extremely long developmental period in ovo or in utero; for example, in the oviparous cloudy catshark (Scyliorhinus torazame), the developing fish remains inside the egg capsule for up to 6 months, suggesting that the embryonic intestine may become functional prior to hatching. In the present study, we describe the morphological and functional development of the spiral intestine in the developing catshark embryo. Spiral formation of embryonic intestine was completed at the middle of stage 31, prior to 'pre-hatching', which is a developmental event characterized by the opening of the egg case at the end of the first third of development. Within 48 h of the pre-hatching event, egg yolk began to flow from the external yolk sac into the embryonic intestine via the yolk stalk. At the same time, there was a rapid increase in mRNA expression of the peptide transporter pept1 and neutral amino acid transporter slc6a19 Secondary folds in the intestinal mucosa and microvilli on the apical membrane appeared after pre-hatching, further supporting the onset of nutrient absorption in the developing intestine at this time. We demonstrate the acquisition of intestinal nutrient absorption at the pre-hatching stage of an oviparous elasmobranch.

Transcriptional activation of elephant shark mineralocorticoid receptor by corticosteroids, progesterone, and spironolactone.

The mineralocorticoid receptor (MR) is a nuclear receptor and part of a large and diverse family of transcription factors that also includes receptors for glucocorticoids, progesterone, androgens, and estrogens. The corticosteroid aldosterone is the physiological activator of the MR in humans and other terrestrial vertebrates; however, its activator is not known in cartilaginous fish, the oldest group of extant jawed vertebrates. Here, we analyzed the ability of corticosteroids and progesterone to activate the full-length MR from the elephant shark (Callorhinchus milii). On the basis of their measured activities, aldosterone, cortisol, 11-deoxycorticosterone, corticosterone, 11-deoxcortisol, progesterone, and 19-norprogesterone are potential physiological mineralocorticoids. However, aldosterone, the physiological mineralocorticoid in humans and other terrestrial vertebrates, is not found in cartilaginous or ray-finned fish. Although progesterone activates MRs in ray-finned fish, progesterone does not activate MRs in humans, amphibians, or alligator, suggesting that during the transition to terrestrial vertebrates, progesterone lost the ability to activate the MR. Both elephant shark MR and human MR are expressed in the brain, heart, ovary, testis, and other nonepithelial tissues, suggesting that MR expression in diverse tissues evolved in the common ancestor of jawed vertebrates. Our data suggest that 19-norprogesterone- and progesterone-activated MR may have unappreciated functions in reproductive physiology.

Comprehensive analysis of genes contributing to euryhalinity in the bull shark, Carcharhinus leucas; Na+-Cl- co-transporter is one of the key renal factors upregulated in acclimation to low-salinity environment.

Most cartilaginous fishes live principally in seawater (SW) environments, but a limited number of species including the bull shark, Carcharhinus leucas, inhabit both SW and freshwater (FW) environments during their life cycle. Euryhaline elasmobranchs maintain high internal urea and ion levels even in FW environments, but little is known about the osmoregulatory mechanisms that enable them to maintain internal homeostasis in hypoosmotic environments. In the present study, we focused on the kidney because this is the only organ that can excrete excess water from the body in a hypoosmotic environment. We conducted a transfer experiment of bull sharks from SW to FW and performed differential gene expression analysis between the two conditions using RNA-sequencing. A search for genes upregulated in the FW-acclimated bull shark kidney indicated that the expression of the Na+-Cl- cotransporter (NCC; Slc12a3) was 10 times higher in the FW-acclimated sharks compared with that in SW sharks. In the kidney, apically located NCC was observed in the late distal tubule and in the anterior half of the collecting tubule, where basolateral Na+/K+-ATPase was also expressed, implying that these segments contribute to NaCl reabsorption from the filtrate for diluting the urine. This expression pattern was not observed in the houndshark, Triakis scyllium, which had been transferred to 30% SW; this species cannot survive in FW environments. The salinity transfer experiment combined with a comprehensive gene screening approach demonstrates that NCC is a key renal protein that contributes to the remarkable euryhaline ability of the bull shark.

Inner ear development in cyclostomes and evolution of the vertebrate semicircular canals.

Jawed vertebrates have inner ears with three semicircular canals, the presence of which has been used as a key to understanding evolutionary relationships. Ostracoderms, the jawless stem gnathostomes, had only two canals and lacked the lateral canal1-3. Lampreys, which are modern cyclostomes, are generally thought to possess two semicircular canals whereas the hagfishes-which are also cyclostomes-have only a single canal, which used to be regarded as a more primitive trait1,4. However, recent molecular and developmental analyses have strongly supported the monophyly of cyclostomes5-7, which has left the evolutionary trajectory of the vertebrate inner ear unclear8. Here we show the differentiation of the otic vesicle of the lamprey Lethenteron camtschaticum and inshore hagfish Eptatretus burgeri. This is the first time, to our knowledge, that the development of the hagfish inner ear is reported. We found that canal development in the lamprey starts with two depressions-which is reminiscent of the early developmental pattern of the inner ear in modern gnathostomes. These cyclostome otic vesicles show a pattern of expression of regulatory genes, including OTX genes, that is comparable to that of gnathosomes. Although two depressions appear in the lamprey vesicle, they subsequently fuse to form a single canal that is similar to that of hagfishes. Complete separation of the depressions results in anterior and posterior canals in gnathostomes. The single depression of the vesicle in hagfishes thus appears to be a secondarily derived trait. Furthermore, the lateral canal in crown gnathostomes was acquired secondarily-not by de novo acquisition of an OTX expression domain, but by the evolution of a developmental program downstream of the OTX genes.