Vertebrate Growth Research Articles

7942 Evolution of Insulin, Insulin-like Growth Factor and Their Cognate Receptors in Vertebrates, Invertebrates and Viruses

Abstract Disclosure: M. Chrudinova: None. Y.D. Dogru: None. R. Huang: None. R. Reiners: None. P. De Meyts: None. J. DaCosta: None. E. Altindis: None. The insulin/insulin-like growth factor (IGF) signaling system is an evolutionarily conserved system and controls central processes including metabolism, cellular growth, proliferation, and differentiation in vertebrates. On the other hand, invertebrate insulin-like peptides (ILPs) regulate growth, stress responses, longevity and sex-related functions. In addition, we recently showed that six viruses possess viral insulin-like peptides (VILPs) that can bind to the receptors and stimulate post-receptor signaling. The evolution of these hormones and the origin of VILPs are not completely understood. Here, we collected vertebrate and invertebrate insulin, IGF and their cognate receptor sequences to analyze their evolutionary relations. We analyzed 177 vertebrate insulins, 119 vertebrate IGF-1s, 127 vertebrate IGF-2s, 197 invertebrate ILPs and 6 viral insulin/IGF-1 like peptides. We also analyzed 214 insulin receptor (IR), 160 IGF receptor (IGF1R) and 39 invertebrate receptor sequences to identify conservation and the evolutionary trajectory to reveal conserved motifs. Through phylogenetic analyses, we observed a separate branching of invertebrate ILPs and vertebrate insulins, IGF-1s and IGF-2s with notable exceptions including VILPs. IGF-1 and IGF-2 ligands were evolutionally better conserved compared to insulin ligands. Insulin C-peptide which is processed and cleaved was not well conserved compared to the functional A- and B-chain residues. In contrast, C-peptide of IGF-1 that plays an important role in receptor binding was very well conserved. IGF-1 C-domain showed higher variability in IGF1R binding residues compared to the A and B-domains, while residues important for binding of IGF-2 to IGF1R were well conserved. Insulin residues interacting with IR binding Site 1 were more conserved than those interacting with IR binding Site 2, while residues important for insulin dimerization and hexamerization residues are well conserved across species. We also showed that VILPs contain 14 amino acids that are conserved in insulin and IGFs, indicating their functional importance. Phylogenetic relationships of the receptor sequences suggest vertebrate IRs and IGF1Rs evolved from a common ancestor during early vertebrate evolution. Interestingly, receptor subunits revealed a closer relation of amphibian IR α-subunits to Mammalia IR α-subunits than Aves and Reptilians. The conservation rate of receptor residues essential for ligand-receptor interaction revealed that these residues are best conserved in the L1 domain for IR and IGF1R, with a higher rate of variation in other domains for both receptors. Taken together, our study provides a comprehensive analysis of insulin, IGFs, VILPs and their cognate receptors’ evolution, and opens new avenues to better understand the evolution, function, and structure relations of this complex signaling system. Presentation: 6/3/2024

The metalloproteinase PAPP-A is required for IGF-dependent chondrocyte differentiation and organization

Insulin-like growth factor (IGF) signaling is required for proper growth and skeletal development in vertebrates. Consequently, its dysregulation may lead to abnormalities of growth or skeletal structures. IGF is involved in the regulation of cell proliferation and differentiation of chondrocytes. However, the availability of bioactive IGF may be controlled by antagonizing IGF binding proteins (IGFBPs) in the circulation and tissues. As the metalloproteinase PAPP-A specifically cleaves members of the IGFBP family, we hypothesized that PAPP-A activity liberates bioactive IGF in cartilage. In PAPP-A knockout mice, the femur length was reduced and the mice showed a disorganized columnar organization of growth plate chondrocytes. Similarly, zebrafish lacking pappaa showed reduced length of Meckel’s cartilage and disorganized chondrocytes, reminiscent of the mouse knockout phenotype. Expression of chondrocyte differentiation markers (sox9a, ihha, and col10a1) was markedly affected in Meckel’s cartilage of pappaa knockout zebrafish, indicating that differentiation of chondrocytes was compromised. Additionally, the zebrafish pappaa knockout phenotype was mimicked by pharmacological inhibition of IGF signaling, and it could be rescued by treatment with exogenous recombinant IGF-I. In conclusion, our data suggests that IGF activity in the growing cartilage, and hence IGF signaling in chondrocytes, requires the presence of PAPP-A. The absence of PAPP-A causes aberrant chondrocyte organization and compromised growth in both mice and zebrafish.

Sonic hedgehog (shh) gene from Pseudopleuronectes yokohamae (Teleostei: Pleuronectidae): Molecular cloning, characterization, and expression profile during early embryonic, juvenile, and adult stages.

The hedgehog signaling pathway plays an important role in early development and growth of most vertebrates. Sonic hedgehog (shh) gene is a critical regulator of embryonic development in many species, including humans. However, it is not clear what roles shh can play in the development of fish. In this paper, shh gene was cloned from Pseudopleuronectes yokohamae. The full-length complementary DNA (cDNA) of P. yokohamae sonic hedgehog gene (Pyshh) comprises 3194 bp, with a 1317-bp open reading frame (ORF) that encodes a polypeptide of 438 amino acids with a typical HH-signal domain and Hint-N domain. The conserved sequences of the protein among species were predicted by using multiple sequence comparison. The phylogenetic tree construction showed that PySHH is clustered in a branch of Pleuronectidae. To explore the expression of Pyshh gene in various tissues of P. yokohamae, we used real-time fluorescence quantitative PCR technology to detect it. The results showed that Pyshh gene is widely distributed in various tissues of P. yokohamae juveniles, different tissues of adult males and females, and is particularly expressed in immune organs. The Pyshh gene expression was higher in the muscle and brain of juvenile fish, and higher in bone, gill, and skin of male fish than that of female fish, suggesting that Pyshh might be involved in the formation of immune organs of P. yokohamae. The expression of Pyshh gene significantly upregulated from the gastrula stage to the hatching stage. Western blotting of the expression levels of PySHH during different embryonic development stages revealed that PySHH levels increased gradually during development stages from oosperm stage to hatching stage. These results indicate that Pyshh is highly conserved among species and plays a critical role in the complex process of embryonic development. Its precise regulation is essential for the proper formation of many organs and tissues in the body, and disruptions in its function may have serious consequences for the formation of immune organs in fish.

Somatostatin Receptor Gene Functions in Growth Regulation in Bivalve Scallop and Clam.

Bivalves hold an important role in marine aquaculture and the identification of growth-related genes in bivalves could contribute to a better understanding of the mechanism governing their growth, which may benefit high-yielding bivalve breeding. Somatostatin receptor (SSTR) is a conserved negative regulator of growth in vertebrates. Although SSTR genes have been identified in invertebrates, their involvement in growth regulation remains unclear. Here, we identified seven SSTRs (PySSTRs) in the Yesso scallop, Patinopecten yessoensis, which is an economically important bivalve cultured in East Asia. Among the three PySSTRs (PySSTR-1, -2, and -3) expressed in adult tissues, PySSTR-1 showed significantly lower expression in fast-growing scallops than in slow-growing scallops. Then, the function of this gene in growth regulation was evaluated in dwarf surf clams (Mulinia lateralis), a potential model bivalve cultured in the lab, via RNA interference (RNAi) through feeding the clams Escherichia coli containing plasmids expressing double-stranded RNAs (dsRNAs) targeting MlSSTR-1. Suppressing the expression of MlSSTR-1, the homolog of PySSTR-1 in M. lateralis, resulted in a significant increase in shell length, shell width, shell height, soft tissue weight, and muscle weight by 20%, 22%, 20%, 79%, and 92%, respectively. A transcriptome analysis indicated that the up-regulated genes after MlSSTR-1 expression inhibition were significantly enriched in the fat digestion and absorption pathway and the insulin pathway. In summary, we systemically identified the SSTR genes in P. yessoensis and revealed the growth-inhibitory role of SSTR-1 in bivalves. This study indicates the conserved function of somatostatin signaling in growth regulation, and ingesting dsRNA-expressing bacteria is a useful way to verify gene function in bivalves. SSTR-1 is a candidate target for gene editing in bivalves to promote growth and could be used in the breeding of fast-growing bivalves.

Loss of function in somatostatin receptor 5 has no impact on the growth of medaka fish due to compensation by the other paralogs

Somatic growth in vertebrates is regulated endocrinologically by the somatotropic axis, headed by the growth hormone (GH) and the insulin growth factor-I (IGF-I). Somatostatin (Sst), a peptide hormone synthesized in the hypothalamus, modulates GH actions through its receptors (Sstr). Four Sstr subtypes (Sstr 1–3 and 5) have been identified in teleosts. However, little is known about whether they have a specific function or tissue expression. The aim of this study was to determine the role of sstr2 and sstr5 in the growth of the medaka (Oryzias latipes). The assessed expression pattern across diverse tissues highlighted greater prevalence of sstr1 and sstr3 in brain, intestine and muscle than in pituitary or liver. The expression of sstr2 was high in all the tissues tested, while sstr5 was predominantly expressed in the pituitary gland. A CRISPR/Cas9 sstr5 mutant with loss of function (sstr5-/-) was produced. Assessment of sstr5-/- indicated no significant difference with the wild type regarding growth parameters such as standard length, body depth, or peduncle depth. Furthermore, the functional loss of sstr5 had no impact on the response to a nutritional challenge. The fact that several sstr subtypes were upregulated in different tissues in sstr5-/- medaka suggests that in the mutant fish, there may be a compensatory effect on the different tissues, predominantly by sstr1 in the liver, brain and pituitary, with sstr2 being upregulated in pituitary and liver, and sstr3 only presenting differential expression in the brain. Analysis of the sstr subtype and the sstr5-/- fish showed that sstr5 was not the only somatostatin receptor responsible for Sst-mediated Gh regulation.

New insights into the role of myostatin in fish fertility based on the findings in mstnb-deficient Nile tilapia (Oreochromis niloticus)

Myostatin (Mstn) was reported to be involved in multiple biological processes including the regulation of skeletal muscle growth, lipid metabolism and reproduction in vertebrates, therefore, it might play an essential role in the fine balance between growth and reproduction. In order to clarify the roles of Mstn in fish reproduction, we systematically investigated the gonadal development and fertility of mstnb mutants in Nile tilapia (Orochromis niloticus). In the present study, mstnb was found to be expressed in the different cell types of gonads by fluorescence in situ hybridization (FISH). Mutation of mstnb resulted in excessive proliferation of phase II follicles in the ovaries while the numbers of phase III follicles were significantly reduced at 210 days after hatching (dah). Interestingly, significant decline of cyp19a1a gene expression, the serum 17β-estradiol (E2), vitellogenin (VTG) were detected at 210 dah. At 360 dah, the ovarian development of mstnb-/- XX tilapia was partially recovered and female fish spawn regularly, however, they were subfertile and produced the poor-quality eggs with significantly lower offspring survival rate. The dramatic up-regulation of cyp19a1a gene expression and serum E2 level at 360 dah might account for the recovery of ovarian development. In mstnb-/- males, excessive proliferation of spermatogonia and retardation of testicular differentiation were found throughout the whole development stages. In mstnb-/- males, evident declines of gonadosomatic index (GSI), cyp11b2 gene expression, and 11-Ketotestosterone (11-KT) were detected at 210 dah, while the mRNA level of cyp11b2 and serum 11-KT level were significantly upregulated at 360 dah. However, mstnb-/- males were infertile since mutation of mstnb gene led to excessive increase of apoptosis of testicular cells and spermiation obstacle. Taken together, these results indicated that Mstn might be an essential regulator for the maintenance of fish fertility.

Tissue explants as tools for studying the epigenetic modulation of the GH-IGF-I axis in farmed fish.

Somatic growth in vertebrates is mainly controlled by the growth hormone (GH)/insulin-like growth factor I (IGF-I) axis. The role of epigenetic mechanisms in regulating this axis in fish is far from being understood. This work aimed to optimize and evaluate the use of short-term culture of pituitary and liver explants from a farmed fish, the gilthead seabream Sparus aurata, for studying epigenetic mechanisms involved in GH/IGF-I axis regulation. Our results on viability, structure, proliferation, and functionality of explants support their use in short-term assays. Pituitary explants showed no variation in gh expression after exposure to the DNA methylation inhibitor decitabine (5-Aza-2'-deoxycytidine; DAC), despite responding to DAC by changing dnmt3bb and tet1 expression, and TET activity, producing an increase in overall DNA hydroxymethylation. Conversely, in liver explants, DAC had no effects on dnmt s and tet s expression or activity, but modified the expression of genes from the GH-IGF-I axis. In particular, the expression of igfbp2a was increased and that of igfbp4, ghri and ghrii was decreased by DAC as well as by genistein, which is suggestive of impaired growth. While incubation of liver explants with S-adenosylmethionine (SAM) produced no clear effects, it is proposed that nutrients must ensure the methylation milieu within the liver in the fish to sustain proper growth, which need further in vivo verification. Pituitary and liver explants from S. aurata can be further used as described herein for the screening of inhibitors or activators of epigenetic regulators, as well as for assessing epigenetic mechanisms behind GH-IGF-I variation in farmed fish.

The Neuropeptide HGAP Regulates Growth, Reproduction, Metamorphosis, Tissue Damage Repair, and Response against Starvation in Pacific Abalone

Introduction: Neuropeptides regulate vital physiological processes in multicellular organisms, including growth, reproduction, metamorphosis, and feeding. Recent transcriptome analyses have revealed neuropeptide genes with potential roles in vertebrate and invertebrate growth and reproduction. Among these genes, haliotid growth-associated peptide (HGAP) was identified as a novel gene in abalone. Methods: This study focused on HGAP in Pacific abalone (Haliotis discus hannai), where the complete cDNA sequence named Hdh-HGAP was identified and characterized. Samples from different experiments, such as metamorphosis, juvenile abalone growth, gonad development stages, muscle remodeling, and starvation, were collected for mRNA expression analysis. Results: The sequence spans 552 bp, encoding 96 amino acids with a molecular weight of 10.96 kDa. Expression analysis revealed that Hdh-HGAP exhibited higher levels in muscle tissue. Notably, during metamorphosis, Hdh-HGAP exhibited greater expression in the trochophore, veliger, and juvenile stages than in the cell division stages. Regarding growth patterns, Hdh-HGAP was highly expressed during rapid growth compared to stunted, minimal, and normal growth. In gonadal development, Hdh-HGAP mRNA reached its highest expression level during the ripening stage, indicating a potential role in gonadal cell proliferation and maturation. The in vivo effects of GnRH on gonad development and the expression of the Hdh-HGAP neuropeptide indicate its involvement in regulating reproduction in Pacific abalone. While tissue remodeling is primarily governed by immune genes, Hdh-HGAP was also upregulated during muscle tissue remodeling. Conversely, Hdh-HGAP was downregulated during prolonged starvation. Conclusion: This study marks the first comprehensive exploration of the Hdh-HGAP neuropeptide gene in Pacific abalone, shedding light on its involvement in growth, reproduction, metamorphosis, tissue remodeling, and response to starvation, although regulatory mechanisms are mostly unknown.

Functional Characterization of the Almstn2 Gene and Its Association with Growth Traits in the Yellowfin Seabream Acanthopagrus latus (Hottuyn, 1782).

Myostatin (mstn), also known as GDF8, is a growth and differentiation factor of the transforming growth factor-β (TGF-β) superfamily and plays a key inhibitory effect in the regulation of skeletal muscle development and growth in vertebrates. In the present study, to comprehend the role of the mstn2 gene of the yellowfin seabream Acanthopagrus latus (Almstn2b), the genomic sequence of Almstn2b is 2359 bp, which encodes 360 amino acids and is composed of three exons and two introns, was obtained. Two typical regions, a TGF-β propeptide and TGF-β domain, constitute Almstn2b. The topology indicated that Almstn2 was grouped together with other Perciformes, such as the gilthead seabream Sparus aurata. Moreover, Almstn2b was mainly expressed in the brain, fins, and spleen. Furthermore, five SNPs, one in the exons and four in the introns, were identified in the Almstn2b gene. The allele and genotype frequencies of SNP-Almstn2b +1885 A/G were significantly related to the total weight, interorbital distance, stem length, tail length, caudal length, caudal height, body length, and total length (p < 0.05). The allele and genotype frequencies of SNP-Almstn2b +1888 A/G were significantly related to the weight, interorbital distance, long head behind the eyes, body height, tail length, caudal length, and body length. Additionally, the relationship between the SNP-Almstn2b +1915 A/G locus and weight and long head behind the eyes was significant (p < 0.05). Furthermore, the other two SNPs were not significantly associated with any traits. Thus, the SNPs identified in this study could be utilized as candidate SNPs for breeding and marker-assisted selection in A. latus.

Glycosylated queuosines in tRNAs optimize translational rate and post-embryonic growth

Transfer RNA (tRNA) modifications are critical for protein synthesis. Queuosine (Q), a 7-deaza-guanosine derivative, is present in tRNA anticodons. In vertebrate tRNAs for Tyr and Asp, Q is further glycosylated with galactose and mannose to generate galQ and manQ, respectively. However, biogenesis and physiological relevance of Q-glycosylation remain poorly understood. Here, we biochemically identified two RNA glycosylases, QTGAL and QTMAN, and successfully reconstituted Q-glycosylation of tRNAs using nucleotide diphosphate sugars. Ribosome profiling of knockout cells revealed that Q-glycosylation slowed down elongation at cognate codons, UAC and GAC (GAU), respectively. We also found that galactosylation of Q suppresses stop codon readthrough. Moreover, protein aggregates increased in cells lacking Q-glycosylation, indicating that Q-glycosylation contributes to proteostasis. Cryo-EM of human ribosome-tRNA complex revealed the molecular basis of codon recognition regulated by Q-glycosylations. Furthermore, zebrafish qtgal and qtman knockout lines displayed shortened body length, implying that Q-glycosylation is required for post-embryonic growth in vertebrates.

Novel Insights into the circRNA-Modulated Developmental Mechanism of Western Honey Bee Larval Guts.

Circular RNAs (circRNAs) are a class of novel non-coding RNAs (ncRNAs) that play essential roles in the development and growth of vertebrates through multiple manners. However, the mechanism by which circRNAs modulate the honey bee gut development is currently poorly understood. Utilizing the transcriptome data we obtained earlier, the highly expressed circRNAs in the Apis mellifera worker 4-, 5-, and 6-day-old larval guts were analyzed, which was followed by an in-depth investigation of the expression pattern of circRNAs during the process of larval guts development and the potential regulatory roles of differentially expressed circRNAs (DEcircRNAs). In total, 1728 expressed circRNAs were detected in the A. mellifera larval guts. Among the most highly expressed 10 circRNAs, seven (novel_circ_000069, novel_circ_000027, novel_circ_000438, etc.) were shared by the 4-, 5-, and 6-day-old larval guts. In addition, 21 (46) up-regulated and 22 (27) down-regulated circRNAs were, respectively, screened in the Am4 vs. Am5 (Am5 vs. Am6) comparison groups. Additionally, nine DEcircRNAs, such as novel_circ_000340, novel_circ_000758 and novel_circ_001116, were shared by these two comparison groups. These DEcircRNAs were predicted to be transcribed from 14 and 29 parental genes; these were respectively annotated to 15 and 22 GO terms such as biological regulation and catalytic activity as well as 16 and 21 KEGG pathways such as dorsoventral axis formation and apoptosis. Moreover, a complicated competing endogenous RNA (ceRNA) network was observed; novel_circ_000838 in the Am4 vs. Am5 comparison group potentially targeted ame-miR-6000a-3p, further targeting 518 mRNAs engaged in several developmental signaling pathways (e.g., TGF-beta, hedgehog, and wnt signaling pathway) and immune pathways (e.g., phagosome, lysosome, and MAPK signaling pathway). The results demonstrated that the novel_circ_000838-ame-miR-6000a-3p axis may plays a critical regulatory part in the larval gut development and immunity. Furthermore, back-splicing sites of six randomly selected DEcircRNAs were amplified and verified by PCR; an RT-qPCR assay of these six DEcircRNAs confirmed the reliability of the used high-throughput sequencing data. Our findings provide a novel insight into the honey bee gut development and pave a way for illustration of the circRNA-modulated developmental mechanisms underlying the A. mellifera worker larval guts.

Determination of thyroid hormones in lake sturgeon (Acipenser fulvescens) tissues at different developmental stages using a sensitive liquid chromatography-mass spectrometry method

Thyroid hormones (TH) are known to play an important role in the growth and development of vertebrates. In fish species, TH regulates the larval-juvenile metamorphosis, and is crucial for development during early life stages. Monitoring the variations in TH levels at different life stages can provide insights into the regulation of metamorphosis and fish development. In this study, we developed an extremely sensitive method for the quantification of thyroxine (T4), triiodothyronine (T3), and reverse-triiodothyronine (rT3), in lake sturgeon (Acipenser fulvescens) tissues from eggs, free embryos, larvae, and juveniles. The target compounds were extracted by an enzymatic digestion method, followed by protein precipitation. Further cleanup was performed by liquid–liquid extraction (LLE) and solid phase extraction (SPE) using SampliQ OPT cartridges. The liquid-chromatography tandem mass spectrometry (LC-MS/MS) method used to quantify TH compounds showed remarkably high sensitivity with the limit of detection (LOD) and the limit of quantification (LOQ) ranging from < 1 pg/mL to 10 pg/mL and linearity in the range of 10–50,000 pg/mL. This method was validated for tissue samples across several early developmental stages and was checked for intra- and inter-day accuracy (78.3−111.2 %) and precision (0.1−4.9 %), matrix effect (75.4−134.1 %), and recovery (41.2−69.0 %). The method was successfully applied for the quantification and comparison of T4, T3 and rT3 in hatchery raised lake sturgeon samples collected at unique time points (i.e., days post fertilization dpf) including fertilized eggs (11 dpf), free embryos (14 dpf), larvae (22 dpf), juveniles (40 dpf) and older juveniles (74 dpf). With modifications, this method could be applied to other species important for agriculture or conservation.

Characterization and tissue expression of twelve selenoproteins in yellow catfish Pelteobagrus fulvidraco fed diets varying in oxidized fish oil and selenium levels

BackgroundSelenium (Se) functions through selenoproteins and is essential to growth and metabolism of vertebrates. The present study was conducted to identify twelve selenoproteins genes (selenoe, selenof, selenoh, selneoi, selenom, selenok, selneon, selenoo, selenot, selenos, selenou and msrb1) from yellow catfish. Their mRNA expression patterns, as well as their response to dietary oxidized fish oils and Se addition were explored. MethodsWe use 3′and 5′ RACE PCR to clone full-length cDNA sequence of twelve selenoprotein genes from yellow catfish. Their mRNA expression patterns were assessed via quantitative real-time PCR. Yellow catfish were fed diet adequate Se+ fresh fish oil, adequate Se+ oxidized fish oil, high Se+ fresh fish oil and high Se+ oxidized fish oil, respectively, for 10 weeks. Their kidney, heart, brain and testis were used to assess the mRNA expression of twelve selenoprotein. ResultsTwelve selenoprotein genes had similar domains with mammals and the other fish. Their mRNAs were expressed widely in eleven tissues but varied with the tissues. Dietary oxidized fish oils and Se addition influenced their mRNA abundances of twelve selenoproteins in a tissue-dependent manner. ConclusionOur study demonstrated the characterization and expression of twelve selenoproteins, and elucidated their responses in yellow catfish fed diets varying in oxidized fish oils and Se addition, which increased our knowledge into the biological function and regulatory mechanism of Se and selenoproteins in fish.

Development of a time-resolved fluoroimmunoassay for salmonid insulin-like growth factor binding protein-2b

Insulin-like growth factor (IGF)-1 promotes the growth of vertebrates, and its binding proteins (IGFBPs) regulate the activity of circulating IGF-1. Three IGFBPs, IGFBP-2b, -1a, and -1b, were consistently detected in the circulatory system of salmonids. IGFBP-2b is thought to be the main carrier of IGFs and promoter of IGF-1-mediated growth in salmonids. Currently, there are no immunoassays for detecting IGFBP-2b. In this study, we developed a time-resolved fluoroimmunoassay (TR-FIA) for IGFBP-2b detection in salmonid fishes. To establish TR-FIA, we produced two recombinant trout (rt) IGFBP-2bs expressed, one with thioredoxin (Trx) and a histidine (His) tag, and the other with His-tag only. We labeled both recombinant proteins with europium (Eu). Only Eu-Trx.His.rtIGFBP-2b cross-reacted with anti-IGFBP-2b, and the addition of increasing amounts of Trx.His.rtIGFBP-2b replaced the binding, indicating its utility as a tracer and assay standard. The addition of unlabeled salmon IGF-1 did not affect the binding of the standard or sample. Serial dilution curves of sera from rainbow trout, Chinook salmon, and chum salmon were parallel to those of the standard. The assay range (ED80–ED20) of the TR-FIA was 60.4 to 251.3 ng/ml, and its minimum detection limit of this assay was 21 ng/ml. The intra- and inter-assay coefficients of variation were 5.68% and 5.65%, respectively. Circulating IGFBP-2b levels in fed rainbow trout were higher than those in fasted fish and were correlated with individual growth rates. This TR-FIA is useful for further exploring the physiological responses of circulating IGFBP-2b and evaluating the growth status of salmonids.

Seasonality and growth in tropical freshwater ectotherm vertebrates: Results from 1-year experimentation in the African gray bichir, giraffe catfish, and the West African mud turtle.

Growth in ectotherm vertebrates is strongly rhythmed by seasonal variation in environmental parameters. To track the seasonal variation in ancient times in a continental and tropical context, we aim to develop a method based on the use of the growth rate of fossil ectotherm vertebrates (actinopterygians and chelonians) influenced by seasonal environmental fluctuations they experienced in their lifetime. However, the impact of environmental parameters on growth, positive or negative, and its intensity, depends on the taxa considered, and data are scarce for tropical species. For 1 year, an experiment was conducted to better understand the effect of seasonal variation in environmental parameters (food abundance, temperature, and photoperiod) on the somatic growth rate of three species of tropical freshwater ectotherm vertebrates: the fishes Polypterus senegalus and Auchenoglanis occidentalis and the turtle Pelusios castaneus. Mimicking seasonal shifts expected to be experienced by the animals in the wild, the experiment highlighted the preponderant effect of food abundance on the growth rate of those three species. Water temperature variation had a significant effect on the growth rate of Po. senegalus and Pe. castaneus. Moreover, the photoperiod demonstrated no significant effect on the growth of the three species. The duration of application of starvation or cool water conditions, ranging from 1 to 3months, did not affect the growth rate of the animals. However, Pelusios castaneus showed a temporary sensitivity to the return of ad libitum feeding or of warm water, after a period of starvation or cool water, by a period of compensatory growth. Finally, this experiment revealed, in the three species, fluctuations in the growth rate under controlled and constant conditions. This variation, similar to the variation in precipitation and temperature observed in their native environment, could be linked to a strong effect of an internal rhythm controlling somatic growth rate.

Liver development during Xenopus tropicalis metamorphosis is controlled by T3-activation of WNT signaling

Thyroid hormone (T3) regulates vertebrate organ development, growth, and metabolism through the T3 receptor (TR). Due to maternal influence in mammals, it has been difficult to study if and how T3 regulates liver development. Liver remodeling during anuran metamorphosis resembles liver maturation in mammals and is controlled by T3. We generated Xenopus tropicalis animals with both TRα and TRβ genes knocked out and found that TR double knockout liver had developmental defects such as reduced cell proliferation and failure to undergo hepatocyte hypertrophy or activate urea cycle gene expression. RNA-seq analysis showed that T3 activated canonical Wnt pathway in the liver. Particularly, Wnt11 was activated in both fibroblasts and hepatic cells, and in turn, likely promoted the proliferation and maturation of hepatocytes. Our study offers new insights into not only how T3 regulates liver development but also on potential means to improve liver regeneration.

Wheat Germ Hemicellulose Is an Absolute Requirement for Growth and Development of the Southwestern Corn Borer1–,

The southwestern corn borer, Diatraea grandiosella Dyar, is a major pest of corn, Zea mays L., in the southern United States. To search for plant allelochemicals, chemically defined insect diets are commonly used, but southwestern corn borer larvae did not grow and develop unless a subnutritional amount of wheat germ was incorporated in the amino acid diet. Fractionation led to identification of hemicellulose as the active component which was then characterized. The hemicellulose permitted insect growth on a protein-free amino acid diet. Microfloral involvement was ruled out because larvae grew and developed free of intestinal microbes. Therefore, this insect has an absolute requirement for hemicellulose that has evidently not been demonstrated with other invertebrates or vertebrates. This finding is of general interest because hitherto, the contributions of hemicellulose to nutrition have generally been considered to be associated chiefly with microfloral conversion to assimilable carbohydrates. Investigations should be conducted to determine whether hemicelluloses are also important or essential for growth of vertebrates including mammals by mechanisms that may not have been considered.

Radial porosity profiles are a powerful tool for tracing locomotor maturation in developing limb bones.

Radial porosity profiles (RPP) are a new quantitative osteohistological parameter designed to capture the dynamic changes in the primary porosity of limb bones through ontogeny, providing insights into skeletal growth and functional development of extant and extinct vertebrates. Previous work hypothesized that RPP channelization-the intraskeletal alignment of RPPs across different bones resulting from similar cortical compaction patterns-indicates increasing locomotor performance of the developing limbs. By investigating RPPs in ontogenetic series of pheasants, pigeons and ducks representing distinct locomotor developmental strategies, we test this hypothesis here and show that RPPs are indeed powerful osteohistological correlates of locomotor ontogeny. Qualitative and quantitative analyses reveal strong association between RPP channelization and fledging, the most drastic locomotor transition in the life history of volant birds. The channelization signal is less clear in precocial leg function; however, when additional intraskeletal and intercohort RPP characteristics are considered, patterns related to leg precocity can also be identified. Thus, we demonstrate that RPPs can be used in future by palaeobiologists to generate breakthroughs in the study of the ontogeny and evolution of flight in fossil birds and pterosaurs. With further baseline data collection from modern terrestrial vertebrates, RPPs could also test hypotheses regarding ontogenetic postural shifts in dinosaurs and other terrestrial archosaurs.

L-selenomethionine affects liver development and glucolipid metabolism by inhibiting autophagy in zebrafish embryos

Selenium plays a vital role in cancer prevention, antioxidation, and the growth of humans and other vertebrates. Excessive selenium can cause liver injury and metabolic disorders, which can lead to hepatic disease, but few studies have shown the effects of excessive selenium on liver development and its mechanism in zebrafish embryos. In this study, liver development and glucolipid metabolism were investigated in selenium-stressed zebrafish embryos. Under selenium treatment, transgenic fabp10a-eGFP zebrafish embryos showed reduced liver size, and wild-type zebrafish embryos exhibited steatosis and altered lipid metabolism-related indexes and glucose metabolism-related enzyme activities. In addition, selenium-stressed embryos exhibited damaged mitochondria and inhibited autophagy in the liver. An autophagy inducer (rapamycin) alleviated selenium-induced liver injury and restored the expression of some genes related to liver development and glucolipid metabolism. In summary, our research evaluated liver developmental toxicity and metabolic disorders under selenium stress, and confirmed that autophagy and oxidative stress might involve in the selenium-induced hepatic defects.

Developmental exposure to triclosan and benzophenone-2 causes morphological alterations in zebrafish (Danio rerio) thyroid follicles and eyes

Thyroid hormones (THs) regulate a multitude of developmental and metabolic processes, which are responsible for vertebrate development, growth, and maintenance of homeostasis. THs also play a key role in neurogenesis of vertebrates and thus affect eye development, which is vital for foraging efficiency and for effective escape from predation. Currently, there are no validated test guidelines for the assessment of TH system-disrupting chemicals (THSDCs) in fish. Consequently, the present study was designed to demonstrate the suitability of novel thyroid-related endpoints in early life-stages of fish. Embryos of a transgenic zebrafish (Danio rerio) line expressing the reporter gene tg:mCherry in their thyrocytes were used to investigate the effects of the environmental THSDCs triclosan (TCS, antibacterial agent) and benzophenone-2 (BP-2, UV filter) on thyroid follicle and eye development. Both BP-2 and TCS caused thyroid follicle hyperplasia in transgenic zebrafish, thus confirming their role as THSDCs. The effect intensity on follicle size and fluorescence was comparable with a 1.7-fold increase for BP-2 and 1.6-fold for TCS. Alterations of the cellular structures of the retina indicate an impact of both substances on eye development, with a stronger impact of TCS. With respect to guideline development, results provide further evidence for the suitability of morphological changes in thyroid follicles and the eyes as novel endpoints for the sensitive assessment of THSD-related effects in fish.