Genome-based discovery of pachysiphine synthases in Tabernaemontana elegans.

Plant-specialized metabolism represents an inexhaustible source of active molecules, some of which have been used in human health for decades. Among these, monoterpene indole alkaloids (MIAs) include a wide range of valuable compounds with anticancer, antihypertensive, or neuroactive properties. This is particularly the case for the pachysiphine derivatives which show interesting antitumor and anti-Alzheimer activities but accumulate at very low levels in several Tabernaemontana species. Unfortunately, genome data in Tabernaemontanaceae are lacking and knowledge on the biogenesis of pachysiphine-related MIAs in planta remains scarce, limiting the prospects for the biotechnological supply of many pachysiphine-derived biopharmaceuticals. Here, we report a raw version of the toad tree (Tabernaemontana elegans) genome sequence. These new genomic resources led to the identification and characterization of a couple of genes encoding cytochrome P450 with pachysiphine synthase activity. Our phylogenomic and docking analyses highlight the different evolutionary processes that have been recruited to epoxidize the pachysiphine precursor tabersonine at a specific position and in a dedicated orientation, thus enriching our understanding of the diversification and speciation of the MIA metabolism in plants. These gene discoveries also allowed us to engineer the synthesis of MIAs in yeast through the combinatorial association of metabolic enzymes resulting in the tailor-made synthesis of non-natural MIAs. Overall, this work represents a step forward for the future supply of pachysiphine-derived drugs by microbial cell factories.

The Madagascar palm genome provides new insights on the evolution of Apocynaceae specialized metabolism.

Specialized metabolites possess diverse interesting biological activities and some cardenolides- and monoterpene indole alkaloids- (MIAs) derived pharmaceuticals are currently used to treat human diseases such as cancers or hypertension. While these two families of biocompounds are produced by specific subfamilies of Apocynaceae, one member of this medicinal plant family, the succulent tree Pachypodium lamerei Drake (also known as Madagascar palm), does not produce such specialized metabolites. To explore the evolutionary paths that have led to the emergence and loss of cardenolide and MIA biosynthesis in Apocynaceae, we sequenced and assembled the P. lamerei genome by combining Oxford Nanopore Technologies long-reads and Illumina short-reads. Phylogenomics revealed that, among the Apocynaceae whose genomes have been sequenced, the Madagascar palm is so far the species closest to the common ancestor between MIA producers/non-MIA producers. Transposable elements, constituting 72.48% of the genome, emerge as potential key players in shaping genomic architecture and influencing specialized metabolic pathways. The absence of crucial MIA biosynthetic genes such as strictosidine synthase in P. lamerei and non-Rauvolfioideae species hints at a transposon-mediated mechanism behind gene loss. Phylogenetic analysis not only showcases the evolutionary divergence of specialized metabolite biosynthesis within Apocynaceae but also underscores the role of transposable elements in this intricate process. Moreover, we shed light on the low conservation of enzymes involved in the final stages of MIA biosynthesis in the distinct MIA-producing plant families, inferring independent gains of these specialized enzymes along the evolution of these medicinal plant clades. Overall, this study marks a leap forward in understanding the genomic dynamics underpinning the evolution of specialized metabolites biosynthesis in the Apocynaceae family, with transposons emerging as potential architects of genomics restructuring and gene loss.

Development of Potent Mcl-1 Inhibitors: Structural Investigations on Macrocycles Originating from a DNA-Encoded Chemical Library Screen.

Evasion of apoptosis is critical for the development and growth of tumors. The pro-survival protein myeloid cell leukemia 1 (Mcl-1) is an antiapoptotic member of the Bcl-2 family, associated with tumor aggressiveness, poor survival, and drug resistance. Development of Mcl-1 inhibitors implies blocking of protein-protein interactions, generally requiring a lengthy optimization process of large, complex molecules. Herein, we describe the use of DNA-encoded chemical library synthesis and screening to directly generate complex, yet conformationally privileged macrocyclic hits that serve as Mcl-1 inhibitors. By applying a conceptual combination of conformational analysis and structure-based design in combination with a robust synthetic platform allowing rapid analoging, we optimized in vitro potency of a lead series into the low nanomolar regime. Additionally, we demonstrate fine-tuning of the physicochemical properties of the macrocyclic compounds, resulting in the identification of lead candidates 57/59 with a balanced profile, which are suitable for future development toward therapeutic use.

An updated version of the Madagascar periwinkle genome.

The Madagascar periwinkle, Catharanthus roseus, belongs to the Apocynaceae family. This medicinal plant, endemic to Madagascar, produces many important drugs including the monoterpene indole alkaloids (MIA) vincristine and vinblastine used to treat cancer worldwide. Here, we provide a new version of the C. roseus genome sequence obtained through the combination of Oxford Nanopore Technologies long-reads and Illumina short-reads. This more contiguous assembly consists of 173 scaffolds with a total length of 581.128 Mb and an N50 of 12.241 Mb. Using publicly available RNAseq data, 21,061 protein coding genes were predicted and functionally annotated. A total of 42.87% of the genome was annotated as transposable elements, most of them being long-terminal repeats. Together with the increasing access to MIA-producing plant genomes, this updated version should ease evolutionary studies leading to a better understanding of MIA biosynthetic pathway evolution.

Genome and transcriptome analysis of the beet armyworm Spodoptera exigua reveals targets for pest control.

The genus Spodoptera (Lepidoptera: Noctuidae) includes some of the most infamous insect pests of cultivated plants including Spodoptera frugiperda, Spodoptera litura, and Spodoptera exigua. To effectively develop targeted pest control strategies for diverse Spodoptera species, genomic resources are highly desired. To this aim, we provide the genome assembly and developmental transcriptome comprising all major life stages of S. exigua, the beet armyworm. Spodoptera exigua is a polyphagous herbivore that can feed on > 130 host plants, including several economically important crops. The 419 Mb beet armyworm genome was sequenced from a female S. exigua pupa. Using a hybrid genome sequencing approach (Nanopore long-read data and Illumina short read), a high-quality genome assembly was achieved (N50 = 1.1 Mb). An official gene set (18,477 transcripts) was generated by automatic annotation and by using transcriptomic RNA-seq datasets of 18 S. exigua samples as supporting evidence. In-depth analyses of developmental stage-specific expression combined with gene tree analyses of identified homologous genes across Lepidoptera genomes revealed four potential genes of interest (three of them Spodoptera-specific) upregulated during first- and third-instar larval stages for targeted pest-outbreak management. The beet armyworm genome sequence and developmental transcriptome covering all major developmental stages provide critical insights into the biology of this devastating polyphagous insect pest species worldwide. In addition, comparative genomic analyses across Lepidoptera significantly advance our knowledge to further control other invasive Spodoptera species and reveals potential lineage-specific target genes for pest control strategies.

A 180 Myr-old female-specific genome region in sturgeon reveals the oldest known vertebrate sex determining system with undifferentiated sex chromosomes.

Several hypotheses explain the prevalence of undifferentiated sex chromosomes in poikilothermic vertebrates. Turnovers change the master sex determination gene, the sex chromosome or the sex determination system (e.g. XY to WZ). Jumping master genes stay main triggers but translocate to other chromosomes. Occasional recombination (e.g. in sex-reversed females) prevents sex chromosome degeneration. Recent research has uncovered conserved heteromorphic or even homomorphic sex chromosomes in several clades of non-avian and non-mammalian vertebrates. Sex determination in sturgeons (Acipenseridae) has been a long-standing basic biological question, linked to economical demands by the caviar-producing aquaculture. Here, we report the discovery of a sex-specific sequence from sterlet (Acipenser ruthenus). Using chromosome-scale assemblies and pool-sequencing, we first identified an approximately 16 kb female-specific region. We developed a PCR-genotyping test, yielding female-specific products in six species, spanning the entire phylogeny with the most divergent extant lineages (A. sturio, A. oxyrinchus versus A. ruthenus, Huso huso), stemming from an ancient tetraploidization. Similar results were obtained in two octoploid species (A. gueldenstaedtii, A. baerii). Conservation of a female-specific sequence for a long period, representing 180 Myr of sturgeon evolution, and across at least one polyploidization event, raises many interesting biological questions. We discuss a conserved undifferentiated sex chromosome system with a ZZ/ZW-mode of sex determination and potential alternatives. This article is part of the theme issue 'Challenging the paradigm in sex chromosome evolution: empirical and theoretical insights with a focus on vertebrates (Part I)'.

Swimming under elevated hydrostatic pressure increases glycolytic activity in gas gland cells of the European eel.

In spite of many decades of research, the spawning migration of the European eel Anguilla anguilla from the European coast to the Sargasso Sea remains a mystery. In particular, the role of the swimbladder as a buoyancy regulating structure is not yet understood. In this study, we exercised silver eels in a swim tunnel under elevated hydrostatic pressure. The transcriptome of gas gland tissue of these exercised eels was then compared to the known transcriptome of not exercised (control) silver eel gas gland cells. Due to the high infection rate of the eel population with the swimbladder parasite Anguillicola crassus, the comparison also included an exercised group of silver eels with a heavily damaged swimbladder, and we compared the previously published transcriptome of not exercised silver eels with a highly damaged swimbladder with the exercised group of silver eels with a heavily damaged swimbladder. The comparisons of unexercised (control) silver eels with exercised silver eels with functional swimbladder (EF), as well as with exercised silver eels with damaged swimbladder (ED), both showed a significant elevation in transcripts related to glycolytic enzymes. This could also be observed within the comparison of unexercised silver eels with a highly infected swimbladder with exercised eels with a damaged swimbladder (DED). In contrast to EF, in ED a significant elevation in transcript numbers of mitochondrial NADH dehydrogenase was observed. While in EF the transcriptional changes suggested that acid production and secretion was enhanced, in ED these changes appeared to be related to thickened tissue and thus elevated diffusion distances. The remarkable number of differentially expressed transcripts coding for proteins connected to cAMP-dependent signaling pathways indicated that metabolic control in gas gland cells includes cAMP-dependent pathways. In contrast to ED, in EF significant transcriptional changes could be related to the reconstruction of the extracellular matrix, while in ED tissue repair and inflammation was more pronounced. Surprisingly, in exercised eels hypoxia inducible transcription factor expression was elevated. In EF, a large number of genes related to the circadian clock were transcriptionally modified, which may be connected to the circadian vertical migrations observed during the spawning migration.

Functional divergence of thyrotropin beta-subunit paralogs gives new insights into salmon smoltification metamorphosis.

Smoltification is a metamorphic event in salmon life history, which initiates downstream migration and pre-adapts juvenile salmon for seawater entry. While a number of reports concern thyroid hormones and smoltification, few and inconclusive studies have addressed the potential role of thyrotropin (TSH). TSH is composed of a α-subunit common to gonadotropins, and a ß-subunit conferring hormone specificity. We report the presence and functional divergence of duplicated TSH ß-subunit paralogs (tshßa and tshßb) in Atlantic salmon. Phylogeny and synteny analyses allowed us to infer that they originated from teleost-specific whole genome duplication. Expression profiles of both paralogs in the pituitary were measured by qPCR throughout smoltification in Atlantic salmon from the endangered Loire-Allier population raised in a conservation hatchery. This revealed a striking peak of tshßb expression in April, concomitant with downstream migration initiation, while tshßa expression remained relatively constant. In situ hybridization showed two distinct pituitary cell populations, tshßa cells in the anterior adenohypophysis, and tshßb cells near to the pituitary stalk, a location comparable to the pars tuberalis TSH cells involved in seasonal physiology and behaviour in birds and mammals. Functional divergence of tshß paralogs in Atlantic salmon supports a specific role of tshßb in smoltification.

Anguillicola crassus infection affects mRNA expression levels in gas gland tissue of European yellow and silver eel.

Using Illumina sequencing, we investigated transcriptional changes caused by the nematode Anguillicola crassus within yellow and silver eels by comparing swimbladder samples of uninfected yellow with infected yellow eels, and uninfected silver with infected silver eels, respectively. In yellow eel gas gland, the infection caused a modification of steady state mRNA levels of 1675 genes, most of them being upregulated. Functional annotation analysis based on GO terms was used to categorize identified genes with regard to swimbladder metabolism or response to the infection. In yellow eels, the most prominent category was 'immune response', including various inflammatory components, complement proteins, and immunoglobulins. The elevated expression of several glucose and monocarboxylate transporters indicated an attempt to maintain the level of glucose metabolism, even in due to the infection thickened swimbladder tissue. In silver eel swimbladder tissue, on the contrary, the mRNA levels of only 291 genes were affected. Genes in the categories 'glucose metabolism' and 'ROS metabolism' barely responded to the infection and even the reaction of the immune system was much less pronounced compared to infected yellow eels. However, in the category 'extracellular matrix', the mRNA levels of several mucin genes were strongly elevated, suggesting increased mucus production as a defense reaction against the parasite. The present study revealed a strong reaction to an Anguillicola crassus infection on mRNA expression levels in swimbladder tissue of yellow eels, whereas in silver eels the changes ware almost negligible. A possible explanation for this difference is that the silvering process requires so much energy that there is not much scope to cope with the additional challenge of a nematode infection. Another possible explanation could be that gas-secreting activity of the silver eel swimbladder was largely reduced, which could coincide with a reduced responsiveness to other challenges, like a nematode infection.

Genomic and transcriptomic approaches to study immunology in cyprinids: What is next?

Accelerated by the introduction of Next-Generation Sequencing (NGS), a number of genomes of cyprinid fish species have been drafted, leading to a highly valuable collective resource of comparative genome information on cyprinids (Cyprinidae). In addition, NGS-based transcriptome analyses of different developmental stages, organs, or cell types, increasingly contribute to the understanding of complex physiological processes, including immune responses. Cyprinids are a highly interesting family because they comprise one of the most-diversified families of teleosts and because of their variation in ploidy level, with diploid, triploid, tetraploid, hexaploid and sometimes even octoploid species. The wealth of data obtained from NGS technologies provides both challenges and opportunities for immunological research, which will be discussed here. Correct interpretation of ploidy effects on immune responses requires knowledge of the degree of functional divergence between duplicated genes, which can differ even between closely-related cyprinid fish species. We summarize NGS-based progress in analysing immune responses and discuss the importance of respecting the presence of (multiple) duplicated gene sequences when performing transcriptome analyses for detailed understanding of complex physiological processes. Progressively, advances in NGS technology are providing workable methods to further elucidate the implications of gene duplication events and functional divergence of duplicates genes and proteins involved in immune responses in cyprinids. We conclude with discussing how future applications of NGS technologies and analysis methods could enhance immunological research and understanding.

Anguillicola crassus Infection Significantly Affects the Silvering Related Modifications in Steady State mRNA Levels in Gas Gland Tissue of the European Eel.

Using Illumina sequencing, transcriptional changes occurring during silvering in swimbladder tissue of the European eel have been analyzed by comparison of yellow and silver eel tissue samples. Functional annotation analysis based on GO terms revealed significant expression changes in a number of genes related to the extracellular matrix, important for the control of gas permeability of the swimbladder, and to reactive oxygen species (ROS) defense, important to cope with ROS generated under hyperbaric oxygen partial pressures. Focusing on swimbladder tissue metabolism, levels of several mRNA species encoding glucose transport proteins were several-fold higher in silver eels, while enzymes of the glycolytic pathway were not affected. The significantly higher steady state level of a transcript encoding for membrane bound carbonic anhydrase, however, suggested that CO2 production in the pentose phosphate shunt and diffusion of CO2 was of particular importance in silver eel swimbladder. In addition, the mRNA level of a large number of genes related to immune response and to sexual maturation was significantly modified in the silver eel swimbladder. The modification of several processes related to protein metabolism and transport, cell cycle, and apoptosis suggested that these changes in swimbladder metabolism and permeability were achieved by increasing cell turn-over. The impact of an infection of the swimbladder with the nematode Anguillicola crassus has been assessed by comparing these expression changes with expression changes observed between uninfected yellow eel swimbladder tissue and infected silver eel swimbladder tissue. In contrast to uninfected silver eel swimbladder tissue, in infected tissue the mRNA level of several glycolytic enzymes was significantly elevated, and with respect to extracellular matrix, several mucin genes were many-fold higher in their mRNA level. Modification of many immune related genes and of the functional categories "response to DNA damage stimulus" and "cellular response to stress" illustrated the damaging effect of the nematode infection. This study has identified a range of cellular processes in the swimbladder of silver eels that appear to be altered by nematode infection. These altered cellular processes could contribute to detrimental changes in swimbladder function that, in turn, may lead to impairment of spawning migration.

Temperature modulates testis steroidogenesis in European eel.

This study evaluates the effects of temperature on hCG-induced spermatogenesis in European eel (Anguilla anguilla), subjected to three thermal regimes: T10: 10°C (first 4weeks), 15°C (next 3weeks) and 20°C (last 6weeks); T15: 15°C (first 4weeks) and 20°C (last 9weeks); and T20: constant 20°C for the duration of the experiment. At 10°C, maturation stopped in the A spermatogonial stage (SPG1), and no further maturation was observed until the temperature was ≥15°C. With the aim of explaining these results, the influence of temperature on steroidogenic enzyme gene expression and steroid synthesis was tested. The initial synthesis of androgens (T and 11-KT) increased at SPG1, and was not influenced by temperature. Likewise, the gene expression of the steroidogenic enzymes linked to androgen synthesis (aacyp11a1, aacyp17-I and aa11ßHSD) also increased at SPG1. In contrast, no correlation was seen between the increase in E2 and the aacyp19a1 gene expression peak in the testes, with E2 increasing as a consequence of the seawater acclimation carried out before hormonal treatment, and peaking the aacyp19a1 gene expression at B spermatogonial stage (SPG2). Aacyp21 gene expression was also higher at SPG2, and this stage was only reached when the rearing temperature was ≥15°C. In conclusion, androgen synthesis is not dependent on temperature, but further maturation requires higher temperatures in order to induce a change in the steroidogenic pathway towards estrogen and progestin synthesis. This study demonstrates that temperature plays a crucial role in European eel maturation, even perhaps controlling gonad development during the reproductive migration.

Changes in ovarian gene expression profiles and plasma hormone levels in maturing European eel (Anguilla anguilla); Biomarkers for broodstock selection.

Complete sexual maturation of European eels (Anguilla anguilla) in captivity can only be achieved via injections with gonadotropins. For female eels this procedure takes 4-6months and the response ranges from "unresponsive" to final maturation and ovulation. Reproductive success could be significantly increased via early selection of responders based on predictive markers and minimally invasive sampling methods. To get a better understanding of the genetic background of ovarian maturation of the European eel we performed a pilot deep-sequencing transcriptome analysis of ovarian tissue derived from a yellow eel, a prepubertal silver eel and a post-spawning matured eel. Two key players in steroidogenesis were strongly correlated with advanced sexual maturation, namely P450c17 and liver receptor homolog-1, suggesting that blood plasma steroids might qualify as minimally invasive markers for early detection of responders. Since the predictive value of plasma sex steroid levels for final maturation of the European eel had not yet been carefully examined, we performed an extensive artificial maturation trial. Farmed silver eels were treated with pituitary extracts and sampled at multiple time intervals. Expression of steroidogenesis-related genes in ovarian tissue of responding and non-responding eels after four weekly injections with pituitary extract was compared using a custom-built microarray and RNAseq. Increased expression of 17ß-hsd1 was strongly linked to sexual maturation. Blood plasma levels of sex steroids were measured using ELISAs. We show that a 2.5-fold increase in blood-plasma estradiol level after 4 weekly pituitary extract injections is a strong predictor of final sexual maturation of female European eel.

The Olfactory Transcriptome and Progression of Sexual Maturation in Homing Chum Salmon Oncorhynchus keta.

Reproductive homing migration of salmonids requires accurate interaction between the reception of external olfactory cues for navigation to the spawning grounds and the regulation of sexual maturation processes. This study aimed at providing insights into the hypothesized functional link between olfactory sensing of the spawning ground and final sexual maturation. We have therefore assessed the presence and expression levels of olfactory genes by RNA sequencing (RNAseq) of the olfactory rosettes in homing chum salmon Oncorhynchus keta Walbaum from the coastal sea to 75 km upstream the rivers at the pre-spawning ground. The progression of sexual maturation along the brain-pituitary-gonadal axis was assessed through determination of plasma steroid levels by time-resolved fluoroimmunoassays (TR-FIA), pituitary gonadotropin subunit expression and salmon gonadotropin-releasing hormone (sgnrh) expression in the brain by quantitative real-time PCR. RNAseq revealed the expression of 75 known and 27 unknown salmonid olfactory genes of which 13 genes were differentially expressed between fish from the pre-spawning area and from the coastal area, suggesting an important role of these genes in homing. A clear progression towards final maturation was characterised by higher plasma 17α,20ß-dihydroxy-4-pregnen-3-one (DHP) levels, increased pituitary luteinizing hormone ß subunit (lhß) expression and sgnrh expression in the post brain, and lower plasma testosterone (T) and 17ß-estradiol (E2) levels. Olfactomedins and ependymin are candidates among the differentially expressed genes that may connect olfactory reception to the expression of sgnrh to regulate final maturation.

Robotic injection of zebrafish embryos for high-throughput screening in disease models.

The increasing use of zebrafish larvae for biomedical research applications is resulting in versatile models for a variety of human diseases. These models exploit the optical transparency of zebrafish larvae and the availability of a large genetic tool box. Here we present detailed protocols for the robotic injection of zebrafish embryos at very high accuracy with a speed of up to 2000 embryos per hour. These protocols are benchmarked for several applications: (1) the injection of DNA for obtaining transgenic animals, (2) the injection of antisense morpholinos that can be used for gene knock-down, (3) the injection of microbes for studying infectious disease, and (4) the injection of human cancer cells as a model for tumor progression. We show examples of how the injected embryos can be screened at high-throughput level using fluorescence analysis. Our methods open up new avenues for the use of zebrafish larvae for large compound screens in the search for new medicines.

Multiple kisspeptin receptors in early osteichthyans provide new insights into the evolution of this receptor family.

Deorphanization of GPR54 receptor a decade ago led to the characterization of the kisspeptin receptor (Kissr) in mammals and the discovery of its major role in the brain control of reproduction. While a single gene encodes for Kissr in eutherian mammals including human, other vertebrates present a variable number of Kissr genes, from none in birds, one or two in teleosts, to three in an amphibian, xenopus. In order to get more insight into the evolution of Kissr gene family, we investigated the presence of Kissr in osteichthyans of key-phylogenetical positions: the coelacanth, a representative of early sarcopterygians, the spotted gar, a non-teleost actinopterygian, and the European eel, a member of an early group of teleosts (elopomorphs). We report the occurrence of three Kissr for the first time in a teleost, the eel. As measured by quantitative RT-PCR, the three eel Kissr were differentially expressed in the brain-pituitary-gonadal axis, and differentially regulated in experimentally matured eels, as compared to prepubertal controls. Subfunctionalisation, as shown by these differences in tissue distribution and regulation, may have represented significant evolutionary constraints for the conservation of multiple Kissr paralogs in this species. Furthermore, we identified four Kissr in both coelacanth and spotted gar genomes, providing the first evidence for the presence of four Kissr in vertebrates. Phylogenetic and syntenic analyses supported the existence of four Kissr paralogs in osteichthyans and allowed to propose a clarified nomenclature of Kissr (Kissr-1 to -4) based on these paralogs. Syntenic analysis suggested that the four Kissr paralogs arose through the two rounds of whole genome duplication (1R and 2R) in early vertebrates, followed by multiple gene loss events in the actinopterygian and sarcopterygian lineages. Due to gene loss there was no impact of the teleost-specific whole genome duplication (3R) on the number of Kissr paralogs in current teleosts.

A DNAJB chaperone subfamily with HDAC-dependent activities suppresses toxic protein aggregation.

Misfolding and aggregation are associated with cytotoxicity in several protein folding diseases. A large network of molecular chaperones ensures protein quality control. Here, we show that within the Hsp70, Hsp110, and Hsp40 (DNAJ) chaperone families, members of a subclass of the DNAJB family (particularly DNAJB6b and DNAJB8) are superior suppressors of aggregation and toxicity of disease-associated polyglutamine proteins. The antiaggregation activity is largely independent of the N-terminal Hsp70-interacting J-domain. Rather, a C-terminal serine-rich (SSF-SST) region and the C-terminal tail are essential. The SSF-SST region is involved in substrate binding, formation of polydisperse oligomeric complexes, and interaction with histone deacetylases (HDAC4, HDAC6, SIRT2). Inhibiting HDAC4 reduced DNAJB8 function. DNAJB8 is (de)acetylated at two conserved C-terminal lysines that are not involved in substrate binding, but do play a role in suppressing protein aggregation. Combined, our data provide a functional link between HDACs and DNAJs in suppressing cytotoxic protein aggregation.

Differential neuroendocrine expression of multiple brain-derived neurotrophic factor transcripts.

Brain-derived neurotrophic factor (BDNF) is a neurotrophin with important growth-promoting properties. We report here the first characterization of a BDNF gene in an amphibian, Xenopus laevis, and demonstrate that environmental factors can activate this gene in a promoter-specific fashion. The Xenopus BDNF gene contains six promoter-specific 5'-exons and one 3'-protein-encoding exon. We examined the expression of promoter-specific transcripts in Xenopus neuroendocrine melanotrope cells. These cells make a good model to study how environmental factors control gene expression. In animals placed on a black background melanotrope cells more actively produce and release alphaMSH than in animals on a white background. BDNF is cosequestered and coreleased with alphaMSH and stimulates biosynthesis of proopiomelanocortin (POMC), the precursor protein for alphaMSH. Our analysis of the expression of the BDNF transcripts revealed that there is differential use of some BDNF promoters in melanotrope cells, depending on the adaptation state of the frog. During black-background adaptation, stimulation of expression of BDNF transcript IV preceded that of the POMC transcript, suggesting the BDNF gene is an effector gene for POMC expression. The possible mechanisms regulating expression of the various transcripts are discussed on the basis of the potential calcium- and cAMP-responsive elements in the promoter region of exon IV. Finally, we show that the upstream open reading frames of BDNF transcripts I and IV markedly decrease BDNF translation efficiency, giving the first indication for a functional role of untranslated BDNF exons.