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Sox9 is a multifunctional gene and plays crucial roles in vertebrate development including chondrogensis. In teleost, due to the genome duplication event, there are two co-orthologs sox9a and sox9b. In this study, CRISPR/Cas9 technology was performed to disrupt the function of either sox9a or sox9b. All sox9a mutants (sox9aΔ10 and sox9aΔ67) and sox9b mutants (sox9bΔ11 and sox9bΔ20) lost HMG domain and Q/S domain, however, only sox9a mutant larvae had mis-shaped pectoral fins and lacked the scapulocoracoid cartilage. sox9b mutant larvae showed normal cartilages similar to wild type larvae. The results suggested that sox9a, not sox9b was required for cartilage development in zebrafish, which was different from the sox9b-mutant phenotype induced by N-ethyl-N-nitrosourea (ENU) treatment, gamma radiation treatment or morpholino injection. This study confirmed that ancestral sox9 gene functions partitioned between the two paralogs in zebrafish.

The two major hallmarks of Alzheimer's disease (AD) are beta-amyloid plaques and neurofibrillary tangles. Amyloid peptide aggregations in the brain cause loss of synaptic connections and subsequent neurotoxicity leading to neurodegeneration and memory deficits. However, the physiological effects of beta-amyloid on early embryonic development still remain unclear. Administration of human beta-amyloid peptide (1–42) through cerebrospinal ventricular injection was carried out at 24 hpf (hours post fertilization) and it was uptaken into the cellular layers of the early ventricular development without any plaque aggregation. Whole-mount Immunostaining of zebrafish embryos injected with the beta-amyloid at 60 hpf revealed the delay in Sox9a expression. Decreased level of cartilage to bone transformation rate in 15 dpf (days post fertilization) zebrafish was observed by differential staining. These results suggest the possible existence of a genetic relationship between extrinsic amyloid peptide and Sox9a expression. Thus, our results demonstrated that the human beta-amyloid influences bone development through Sox9a expression during osteogenesis in zebrafish. [ABSTRACT FROM AUTHOR]

Our aim was to study the relationship between the sex-determining genes, sox9a and cyp19a, and water temperature in Gadus chalcogrammus. We assessed the sex ratio based on the expression levels of sox9a and cyp19a at different water temperatures (5, 8, 11, and 14 °C) and at different stages of walleye pollock development (embryos, larvae, and juveniles). Next, we used immature walleye pollock to assess sox9a expression in males and cyp19a and vitellogenin (VTG) expression in females at different water temperatures. Males expressed sox9a in the gonadal tissues, while females expressed cyp19a in the gonadal tissues and VTG in the blood plasma. In the first experiment, cyp19a expression was higher at 5 °C and 8 °C, and sox9a expression was higher at 11 and 14 °C. In the second experiment, sox9a expression remained relatively stable, but cyp19a expression decreased with increasing temperature, decreasing significantly after 14 °C. Similar patterns were also observed for VTG expression. These results indicate that lower water temperatures increase cyp19a expression, which increases the female ratio. Higher water temperatures increase sox9a expression, which increases the male ratio. Therefore, this study highlights the potential of the sex-determining genes and the influence of water temperature.

Steroidogenesis is an important biological process for gonadal differentiation and development. In mammals, 3β-hydroxysteroid dehydrogenase 7 (HSD3B7) could convert 3β-hydroxy of 7α-hydroxycholesterol into a ketone and form 7α-hydroxy-4-cholesten-3-one, which may affect steroidogenesis. However, in fish, the study of Hsd3b7 is still lacking. In this study, Hsd3b7 was identified in the olive flounder Paralichthys olivaceus , an important mariculture fish. According to bioinformatics analysis, Hsd3b7 belongs to a Rossmann-fold NAD(P)(+)-binding protein and can interact in a predictable manner with Hsd17b2, −3, and − 4, which play a role in steroidogenesis. In the adult flounder, Hsd3b7 was expressed in various tissues, at particularly high level in male muscle. The expression levels of Hsd3b7 at gonadal development stages I-V initially increased and then decreased, with an inflection point in the ovary at stage III and in the testis at stage IV. At stage III, the expression level of Hsd3b7 was significantly higher in the ovary than in the testis (P < 0.01). The results of in situ hybridization (ISH) revealed that it was mainly expressed in oocytes of phases I-IV or around oocytes of phases IV-V in the ovaries and around spermatid lobules at stages IV-V in the testes. Three regulatory sites of SRY-box transcription factor 9 (Sox9), a transcription factor involved in steroidogenesis and gonadal differentiation, were predicted in the promoter of Hsd3b7. After intraperitoneal injection with the recombination flounder Sox9a, the expression of Hsd3b7 was significantly up-regulated (P < 0.01). During the flounder gonadal differentiation, 17β-estradiol (E2, 5 μg/g feed) and 17α-methyltestosterone (T, 5 μg/g feed) were used to obtain the phenotypic female or male flounder, and the results showed that in the E2 group, Hsd3b7 expression was highest at 2 cm TL, the primordial gonad stage, which was significantly higher than that at 12 cm TL (P < 0.05). In the T group, Hsd3b7 expression level was also highest at 2 cm TL and significantly higher than at 10 and 12 cm TL (P < 0.05). Moreover, Hsd3b7 was detected to be localized mainly around oogonia and spermatogonia during the differentiated gonads with ISH. These findings first introduce the expression characteristics of Hsd3b7 and the effect of Sox9a on its expression, which contribute to our understanding of the function of Hsd3b7 in fish gonads. [Display omitted] • The sequence of flounder Hsd3b7 contains the domain of the NADB_Rossmann super family. • Hsd3b7 was widely distributed in the tissues of both male and female flounder. • Hsd3b7 expressed in or around oocytes in the ovary, and around spermatid lobules in the testis. • Injection of the transcription factor Sox9a strongly stimulated the expression of Hsd3b7 in the ovary. • The expression of Hsd3b7 was the highest in the primordial gonads. [ABSTRACT FROM AUTHOR]

Abstract Anti-Müllerian hormone (amh) is one of the earliest functional genes expressed during testicular differentiation. It has been suggested that androgen signaling regulates critical genes for the differentiation and development of the testis. To elucidate the exact regulatory mechanisms involved in amh transcription mediated by androgen signaling, androgen signaling was manipulated in zebrafish by cytochrome P450 17a1 (cyp17a1) knockout and Flutamide treatment. In cyp17a1 -deficient and Flutamide-treated testes, up-regulated sry- box9 a (sox9a) and down-regulated amh were observed. Moreover, a physical association of the zebrafish androgen receptor (AR) and SOX9A was found. The interaction between AR and SOX9A was mediated via the DNA binding domain (DBD) of AR and the transactivation domain (TA) of SOX9A, and was further enhanced by 5-alpha dihydrotestosterone (DHT), one of the most potent androgens. Intriguingly, together with SOX9A, androgen signaling synergistically promoted amh transcription, mainly through the proximal 1 kb of the amh promoter region. Taken together, our data demonstrate a critical mechanism underlying the direct synergy of androgen signaling and SOX9A in the regulation of amh transcription. [ABSTRACT FROM AUTHOR]

Gynogenesis is a form of asexual reproduction that is used to obtain all-female fish stocks. In this study, we were interested in studying gender-specific differences in gene expression profiles in gynogenetic teleosts, using a carp species. The four-month old gynogenetic Pengze crucian carp FI (Carassius auratus var. pengzensis, Pcc) showed a high ratio of males under laboratory culture condition. The present study aimed to investigate the differences between males and females. The gonadosomatic index of the females was significantly higher than that of the males. Moreover, the hepatosomatic index of the females was significantly lower than that of the males. Vitellogenin B mRNA was abnormally highly expressed in male hepatopancreas and testes compared to females. Similarly, zona pellucida 2 expressed at a significantly high level in the testes. For the sex related genes, dosage-sensitive sex reversal, adrenal hypoplasia congenital critical region on the X-chromosome gene 1, doublesex and mab-3 related transcription factor la, nuclear receptor subfamily 5, group A, member lb and SRYbox containing gene 9a had significantly higher expression levels in the males than in the females, whereas there was no difference in expression of anti-Mullerian hormone, cytochrome P450 family 19 subfamily A member 1A and forkhead box L2 transcripts between the two genders. The females showed higher levels of estrogen but no significant difference in testosterone compared to the males. The data suggest remarkable differences between the two genders of the Pengze crucian carp. [ABSTRACT FROM AUTHOR]

Highlights: [•] Zebrafish synuclein-γ2 (sncgb) expressed specifically in the notochord. [•] ZNF219L associated with Sox9a through its sixth or ninth zinc finger domain. [•] Knockdown of znf219L and sox9a caused a synergistic decrease of sncgb expression. [•] ZNF219L and Sox9a are involved in regulation of sncgb gene expression. [ABSTRACT FROM AUTHOR]

Dithiocarbamates (DTCs) have a wide variety of applications in diverse fields ranging from agriculture to medicine. DTCs are teratogenic to vertebrates but the mechanisms by which they exert these effects are poorly understood. Here, we show that low nanomolar exposure to three DTCs, tetraethylthiuram (thiram), tetramethylthiuram (disulfiram), and sodium metam (metam), leads to craniofacial abnormalities in developing zebrafish embryos that are reminiscent of DTC-induced abnormalities found in higher vertebrates. In order to better understand the molecular events underlying DTC teratogenesis, we exposed embryonic zebrafish (PAC2) cells to thiram and disulfiram and measured changes in gene expression with microarrays. We found differential expression of 166 genes that were specific for exposure to DTCs and identified a network of genes related to connective tissue development and function. Additionally, we found eight downregulated genes related to transforming growth factor β-1 (TGF-β1) signaling, including an essential transcription factor for zebrafish craniofacial development, SRY-box–containing gene 9a (sox9a). Finally, we show that sox9a expression is perturbed in the ceratobranchial arches of DTC-exposed zebrafish, suggesting that this is an important event in the development of DTC-induced craniofacial abnormalities. Together, we provide evidence for a novel teratogenic endpoint and a molecular basis for a better understanding of DTC-induced teratogenesis in vertebrates. [ABSTRACT FROM PUBLISHER]

To investigate the evolutional significance of Sox9 in fish, we isolated and characterized Sox9a cDNA and genomic clones in triploid crucian carp. The cDNA encoded a protein of 457 amino acids with an HMG box and showed more than 60% amino acid sequence identity with known vertebrate Sox9 proteins. Triploid crucian carp and vertebrate Sox9s showed similar gene structure, and two introns in the coding region were located at conserved positions. On the basis of the amino acid sequences, Sox9a can be categorized into the same subgroup of Sox-E proteins as Sox8, 9, and 10. Interestingly, the expression of triploid crucian carp Sox9a was predominantly observed not in the ovary but in the testis by Northern blot and RT-PCR analysis. The expression analysis of Sox9a suggested that it may seldom contribute to the formation of normal functions of spermatozoa, but it may play an important role in the development of testicular tubules. Besides the testicular expression, Sox9a was also shown to be expressed in many other tissues including the brain, kidney, and heart of triploid crucian carp, indicating that Sox9 may have unique functions in some specific tissues during development. [ABSTRACT FROM AUTHOR]

Abstract: Endothelin1 (Edn1) signaling promotes ventral character to the facial skeleton. In zebrafish edn1 mutants, the ventral jaw structures are severely reduced and fused to their dorsal counterparts, with a loss of joints that normally form at an intermediate dorsal–ventral position. Loss of function at another locus, sturgeon, also yields joint losses, but only mild reductions in the ventral jaw structures. We show that sturgeon encodes one of two orthologs of Furin present in zebrafish, and that both furin genes may function partially redundantly to activate Edn1 signaling. Supporting this hypothesis, early expression of edn1-dependent genes is downregulated in sturgeon (furinA) mutants. Later in development, expression of most of these genes recovers to near wild-type levels in furinA mutants but not in edn1 mutants. The recovery explains the less severe furinA mutant skeletal phenotype and suggests that late gene expression is dependent on a critical level of Edn1 signaling not present in the more severe edn1 mutants. However, expression defects in the intermediate joint-forming domains in both mutants persist, explaining the joint losses observed later in both mutants. We further show that in both mutants the arches fail to correctly undergo ventral elongation before skeletogenesis begins and propose a model in which this failure is largely responsible for the loss of an Edn1-dependent compartmentation of the arch into the intermediate and ventral domains. [Copyright &y& Elsevier]

Abstract: The role of Anti-Müllerian hormone (Amh) during gonad development has been studied extensively in mammals, but is less well understood in other vertebrates. In male mammalian embryos, Sox9 activates expression of Amh, which initiates the regression of the Müllerian ducts and inhibits the expression of aromatase (Cyp19a1), the enzyme that converts androgens to estrogens. To better understand shared features of vertebrate gonadogenesis, we cloned amh cDNA from zebrafish, characterized its genomic structure, mapped it, analyzed conserved syntenies, studied its expression pattern in embryos, larvae, juveniles, and adults, and compared it to the expression patterns of sox9a, sox9b and cyp19a1a. We found that the onset of amh expression occurred while gonads were still undifferentiated and sox9a and cyp19a1a were already expressed. In differentiated gonads of juveniles, amh showed a sexually dimorphic expression pattern. In 31 days post-fertilization juveniles, testes expressed amh and sox9a, but not cyp19a1a, while ovaries expressed cyp19a1a and sox9b, but not amh. In adult testes, amh and sox9a were expressed in presumptive Sertoli cells. In adult ovaries, amh and cyp19a1a were expressed in granulosa cells surrounding the oocytes, and sox9b was expressed in a complementary fashion in the ooplasm of oocytes. The observed expression patterns of amh, sox9a, sox9b, and cyp19a1a in zebrafish correspond to the patterns expected if their regulatory interactions have been conserved with mammals. The finding that zebrafish sox9b and sox8 were not co-expressed with amh in oocytes excludes the possibility that amh expression in zebrafish granulosa cells is directly regulated by either of these two genes. [Copyright &y& Elsevier]

The vertebrate inner ear arises from an ectodermal thickening, the otic placode, that forms adjacent to the presumptive hindbrain. Previous studies have suggested that competent ectodermal cells respond to Fgf signals from adjacent tissues and express two highly related paired box transcription factors Pax2a and Pax8 in the developing placode. We show that compromising the functions of both Pax2a and Pax8 together blocks zebrafish ear development, leaving only a few residual otic cells. This suggests that Pax2a and Pax8 are the main effectors downstream of Fgf signals. Our results further provide evidence that pax8 expression and pax2a expression are regulated by two independent factors, Foxil and DIx3b, respectively. Combined loss of both factors eliminates all indications of otic specification. We suggest that the Foxil-Pax8 pathway provides an early 'jumpstart' of otic specification that is maintained by the Dlx3b-Pax2a pathway. [ABSTRACT FROM AUTHOR]

Our aim was to study the relationship between the sex-determining genes, sox9a and cyp19a, and water temperature in Gadus chalcogrammus. We assessed the sex ratio based on the expression levels of sox9a and cyp19a at different water temperatures (5, 8, 11, and 14 °C) and at different stages of walleye pollock development (embryos, larvae, and juveniles). Next, we used immature walleye pollock to assess sox9a expression in males and cyp19a and vitellogenin (VTG) expression in females at different water temperatures. Males expressed sox9a in the gonadal tissues, while females expressed cyp19a in the gonadal tissues and VTG in the blood plasma. In the first experiment, cyp19a expression was higher at 5 °C and 8 °C, and sox9a expression was higher at 11 and 14 °C. In the second experiment, sox9a expression remained relatively stable, but cyp19a expression decreased with increasing temperature, decreasing significantly after 14 °C. Similar patterns were also observed for VTG expression. These results indicate that lower water temperatures increase cyp19a expression, which increases the female ratio. Higher water temperatures increase sox9a expression, which increases the male ratio. Therefore, this study highlights the potential of the sex-determining genes and the influence of water temperature. [ABSTRACT FROM AUTHOR]

The molecular mechanisms governing sex determination and differentiation in the zebrafish (Danio rerio) are not fully understood. To gain more insights into the function of specific genes in these complex processes, the expression of multiple candidates needs to be assessed, preferably on the protein level. Here, we developed a targeted proteomics method based on selected reaction monitoring (SRM) to study the candidate sex-related proteins in zebrafish which were selected based on a global proteomics analysis of adult gonads and representational difference analysis of male and female DNA, as well as on published information on zebrafish and other vertebrates. We employed the developed SRM protocols to acquire time-resolved protein expression profiles during the gonad differentiation period in vas::EGFP transgenic zebrafish. Evidence on protein expression was obtained for the first time for several candidate genes previously studied only on the mRNA level or suggested by bioinformatic predictions. Tuba1b (tubulin alpha 1b), initially included in the study as one of the potential housekeeping proteins, was found to be preferentially expressed in the adult testis with nearly absent expression in the ovary. The revealed changes in protein expression patterns associated with gonad differentiation suggest that several of the examined proteins, especially Ilf2 and Ilf3 (interleukin enhancer-binding factors 2 and 3), Raldh3 (retinaldehyde dehydrogenase type 3), Zgc:195027 (low density lipoprotein-related receptor protein 3) and Sept5a (septin 5a), may play a specific role in the sexual differentiation in zebrafish., (Copyright © 2013 Elsevier Inc. All rights reserved.)