Early Developmental Expression Research Articles

Adaptive tail-length evolution in deer mice is associated with differential Hoxd13 expression in early development

Variation in the size and number of axial segments underlies much of the diversity in animal body plans. Here we investigate the evolutionary, genetic and developmental mechanisms driving tail-length differences between forest and prairie ecotypes of deer mice (Peromyscus maniculatus). We first show that long-tailed forest mice perform better in an arboreal locomotion assay, consistent with tails being important for balance during climbing. We then identify six genomic regions that contribute to differences in tail length, three of which associate with caudal vertebra length and the other three with vertebra number. For all six loci, the forest allele increases tail length, indicative of the cumulative effect of natural selection. Two of the genomic regions associated with variation in vertebra number contain Hox gene clusters. Of those, we find an allele-specific decrease in Hoxd13 expression in the embryonic tail bud of long-tailed forest mice, consistent with its role in axial elongation. Additionally, we find that forest embryos have more presomitic mesoderm than prairie embryos and that this correlates with an increase in the number of neuromesodermal progenitors, which are modulated by Hox13 paralogues. Together, these results suggest a role for Hoxd13 in the development of natural variation in adaptive morphology on a microevolutionary timescale.

Identification of a Chondrocyte-Specific Enhancer in the Hoxc8 Gene.

Hox genes encode transcription factors whose roles in patterning animal body plans during embryonic development are well-documented. Multiple studies demonstrate that Hox genes continue to act in adult cells, in normal differentiation, in regenerative processes, and, with abnormal expression, in diverse types of cancers. However, surprisingly little is known about the regulatory mechanisms that govern Hox gene expression in specific cell types, as they differentiate during late embryonic development, and in the adult organism. The murine Hoxc8 gene determines the identity of multiple skeletal elements in the lower thoracic and lumbar region and continues to play a role in the proliferation and differentiation of cells in cartilage as the skeleton matures. This study was undertaken to identify regulatory elements in the Hoxc8 gene that control transcriptional activity, specifically in cartilage-producing chondrocytes. We report that an enhancer comprising two 416 and 224 bps long interacting DNA elements produces reporter gene activity when assayed on a heterologous transcriptional promoter in transgenic mice. This enhancer is distinct in spatial, temporal, and molecular regulation from previously identified regulatory sequences in the Hoxc8 gene that control its expression in early development. The identification of a tissue-specific Hox gene regulatory element now allows mechanistic investigations into Hox transcription factor expression and function in differentiating cell types and adult tissues and to specifically target these cells during repair processes and regeneration.

Multiomics of Bohring-Opitz syndrome truncating ASXL1 mutations identify canonical and noncanonical Wnt signaling dysregulation.

ASXL1 (additional sex combs-like 1) plays key roles in epigenetic regulation of early developmental gene expression. De novo protein-truncating mutations in ASXL1 cause Bohring-Opitz syndrome (BOS; OMIM #605039), a rare neurodevelopmental condition characterized by severe intellectual disabilities, distinctive facial features, hypertrichosis, increased risk of Wilms tumor, and variable congenital anomalies, including heart defects and severe skeletal defects giving rise to a typical BOS posture. These BOS-causing ASXL1 variants are also high-prevalence somatic driver mutations in acute myeloid leukemia. We used primary cells from individuals with BOS (n = 18) and controls (n = 49) to dissect gene regulatory changes caused by ASXL1 mutations using comprehensive multiomics assays for chromatin accessibility (ATAC-seq), DNA methylation, histone methylation binding, and transcriptome in peripheral blood and skin fibroblasts. Our data show that regardless of cell type, ASXL1 mutations drive strong cross-tissue effects that disrupt multiple layers of the epigenome. The data showed a broad activation of canonical Wnt signaling at the transcriptional and protein levels and upregulation of VANGL2, which encodes a planar cell polarity pathway protein that acts through noncanonical Wnt signaling to direct tissue patterning and cell migration. This multiomics approach identifies the core impact of ASXL1 mutations and therapeutic targets for BOS and myeloid leukemias.

CRISPR/Cas9-Mediated Knockout of tnfaip1 in Zebrafish Plays a Role in Early Development.

TNF α-induced protein 1 (TNFAIP1) was first identified in human umbilical vein endothelial cells and can be induced by tumor necrosis factor α (TNFα). Early studies have found that TNFAIP1 is involved in the development of many tumors and is closely associated with the neurological disorder Alzheimer's disease. However, little is known about the expression pattern of TNFAIP1 under physiological conditions and its function during embryonic development. In this study, we used zebrafish as a model to illustrate the early developmental expression pattern of tnfaip1 and its role in early development. First, we examined the expression pattern of tnfaip1 during early zebrafish development using quantitative real-time PCR and whole mount in situ hybridization and found that tnfaip1 was highly expressed in early embryonic development and, subsequently, expression became localized to anterior embryonic structures. To investigate the function of tnfaip1 during early development, we constructed a model of a stably inherited tnfaip1 mutant using the CRISPR/Cas9 system. Tnfaip1 mutant embryos showed significant developmental delays as well as microcephaly and microphthalmia. At the same time, we found decreased expression of the neuronal marker genes tuba1b, neurod1, and ccnd1 in tnfaip1 mutants. Analysis of transcriptome sequencing data revealed altered expression of the embryonic development related genes dhx40, hspa13, tnfrsf19, nppa, lrp2b, hspb9, clul1, zbtb47a, cryba1a, and adgrg4a in the tnfaip1 mutants. These findings suggest an important role for tnfaip1 in the early development of zebrafish.

TCF7L2 acts as a molecular switch in midbrain to control mammal vocalization through its DNA binding domain but not transcription activation domain

Vocalization is an essential medium for social signaling in birds and mammals. Periaqueductal gray (PAG) a conserved midbrain structure is believed to be responsible for innate vocalizations, but its molecular regulation remains largely unknown. Here, through a mouse forward genetic screening we identified one of the key Wnt/β-catenin effectors TCF7L2/TCF4 controls ultrasonic vocalization (USV) production and syllable complexity during maternal deprivation and sexual encounter. Early developmental expression of TCF7L2 in PAG excitatory neurons is necessary for the complex trait, while TCF7L2 loss reduces neuronal gene expressions and synaptic transmission in PAG. TCF7L2-mediated vocal control is independent of its β-catenin-binding domain but dependent of its DNA binding ability. Patient mutations associated with developmental disorders, including autism spectrum disorders, disrupt the transcriptional repression effect of TCF7L2, while mice carrying those mutations display severe USV impairments. Therefore, we conclude that TCF7L2 orchestrates gene expression in midbrain to control vocal production through its DNA binding but not transcription activation domain.

Transcriptional Ontogeny Platform of Sex Determination/Sex Differentiation Genes in Almaco Jack Larvae

AbstractThe fish sex determination/sex differentiation (SD) system is still intriguing in many species, such as Seriola fish. Marine amberjacks have a conserved ZW/ZZ SD system. We aimed to identify how early SD genes were expressed in larval Almaco Jack Seriola rivoliana by RNA sequencing and to determine whether there was an early developmental differential expression up to the onset of exogenous feeding. Transcriptome analysis identified sox5 and sox7 (female) and sox3 and sox8 (male) genes; all of the known female SD genes, including hsd17b1 (master sex gene in Seriola), foxl2, and cyp19a, plus genes involved in female sexual development and maintenance, estrogen signaling, and osteoclast proliferation; and all of the known male SD genes, including dmrt1, nr5a1, gsdf, amhy, and amhr2. The expression of most SD genes occurred on each day during larval development. However, most of them showed low abundance levels and null differential expression except for the downregulation of the sox and foxl2 genes, which might have served an unknown function or were maternally derived rather than autonomously downregulated. We suggest that the concomitant expression of female and male SD genes in early larval stages is a biological transcriptional ontogeny sex platform that ensures posterior sex‐specific expression and sex differentiation. A single missense single‐nucleotide polymorphism (SNP) near the catalytic center of hsd17b1 has been suggested to differentiate phenotypic sex in Seriola by an endocrine regulation on estradiol synthesis. In that case, the ontogeny sex platform would be directed by the SNP but still critical to differentiate gonads.

Antisense oligonucleotide therapy rescues disturbed brain rhythms and sleep in juvenile and adult mouse models of Angelman syndrome.

UBE3A encodes ubiquitin protein ligase E3A, and in neurons its expression from the paternal allele is repressed by the UBE3A antisense transcript (UBE3A-ATS). This leaves neurons susceptible to loss-of-function of maternal UBE3A. Indeed, Angelman syndrome, a severe neurodevelopmental disorder, is caused by maternal UBE3A deficiency. A promising therapeutic approach to treating Angelman syndrome is to reactivate the intact paternal UBE3A by suppressing UBE3A-ATS. Prior studies show that many neurological phenotypes of maternal Ube3a knockout mice can only be rescued by reinstating Ube3a expression in early development, indicating a restricted therapeutic window for Angelman syndrome. Here, we report that reducing Ube3a-ATS by antisense oligonucleotides in juvenile or adult maternal Ube3a knockout mice rescues the abnormal electroencephalogram (EEG) rhythms and sleep disturbance, two prominent clinical features of Angelman syndrome. Importantly, the degree of phenotypic improvement correlates with the increase of Ube3a protein levels. These results indicate that the therapeutic window of genetic therapies for Angelman syndrome is broader than previously thought, and EEG power spectrum and sleep architecture should be used to evaluate the clinical efficacy of therapies.

Differential nuclear import sets the timing of protein access to the embryonic genome

The development of a fertilized egg to an embryo requires the proper temporal control of gene expression. During cell differentiation, timing is often controlled via cascades of transcription factors (TFs). However, in early development, transcription is often inactive, and many TF levels stay constant, suggesting that alternative mechanisms govern the observed rapid and ordered onset of gene expression. Here, we find that in early embryonic development access of maternally deposited nuclear proteins to the genome is temporally ordered via importin affinities, thereby timing the expression of downstream targets. We quantify changes in the nuclear proteome during early development and find that nuclear proteins, such as TFs and RNA polymerases, enter the nucleus sequentially. Moreover, we find that the timing of nuclear proteins’ access to the genome corresponds to the timing of downstream gene activation. We show that the affinity of proteins to importin is a major determinant in the timing of protein entry into embryonic nuclei. Thus, we propose a mechanism by which embryos encode the timing of gene expression in early development via biochemical affinities. This process could be critical for embryos to organize themselves before deploying the regulatory cascades that control cell identities.

Genetic variation as a long-distance modulator of RAD21 expression in humans

Somatic mutations and changes in expression of RAD21 are common in many types of cancer. Moreover, sub-optimal levels of RAD21 expression in early development can result in cohesinopathies. Altered RAD21 levels can result directly from mutations in the RAD21 gene. However, whether DNA variants outside of the RAD21 gene could control its expression and thereby contribute to cancer and developmental disease is unknown. In this study, we searched for genomic variants that modify RAD21expression to determine their potential to contribute to development or cancer by RAD21 dysregulation. We searched 42,953,834 genomic variants for a spatial-eQTL association with the transcription of RAD21. We identified 123 significant associations (FDR < 0.05), which are local (cis) or long-distance (trans) regulators of RAD21 expression. The 123 variants co-regulate a further seven genes (AARD, AKAP11, GRID1, KCNIP4, RCN1, TRIOBP, and USP32), enriched for having Sp2 transcription factor binding sites in their promoter regions. The Sp2 transcription factor and six of the seven genes had previously been associated with cancer onset, progression, and metastasis. Our results suggest that genome-wide variation in non-coding regions impacts on RAD21 transcript levels in addition to other genes, which then could impact on oncogenesis and the process of ubiquitination. This identification of distant co-regulation of oncogenes represents a strategy for discovery of novel genetic regions influencing cancer onset and a potential for diagnostics.

Abstract 1531: CD146 couples and sequesters regulatory kinases to control breast cancer cell state plasticity

Abstract CD146 (Mcam; Muc18; s-endo; and gicerin) is an integral membrane glycoprotein originally identified in a human malignant melanoma, but mis-expressed in a wider variety of malignancies including breast cancer, where it has been implicated in controlling tumor growth and dissemination. The mechanisms by which CD146 conveys extracellular cues into malignant cellular outputs is not well understood, but its high-level expression in early development and normal mouse mammary stem cells, as well as expression in highly plastic adult pericytes (formerly known as mesenchymal stem cells) suggested to us that it may control cell state plasticity. We tested the role of CD146 in a plastic, stem cell-like mouse mammary tumor cell line derived from the MMTV-PyMT mouse (PY230). We identified domains in the CD146 cytoplasmic tail essential for controlling substrate utilization by upstream kinases in Akt, Mapk, Stat3, and integrin-related signaling pathways that alter cell state plasticity and tumorigenicity. This work suggests that MCAM plays a key role in controlling lineage plasticity in normal and neoplastic mammary cells with relevance for understanding tumor cell plasticity, progression and evasion of targeted cancer therapies. Citation Format: Ozlen Balcioglu, Brooke L. Gates, David W. Freeman, Berhane M. Hagos, Benjamin T. Spike. CD146 couples and sequesters regulatory kinases to control breast cancer cell state plasticity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1531.

Early Developmental PMCA2b Expression Protects From Ketamine-Induced Apoptosis and GABA Impairments in Differentiating Hippocampal Progenitor Cells.

PMCA2 is not expressed until the late embryonic state when the control of subtle Ca2+ fluxes becomes important for neuronal specialization. During this period, immature neurons are especially vulnerable to degenerative insults induced by the N-methyl-D-aspartate (NMDA) receptor blocker, ketamine. As H19-7 hippocampal progenitor cells isolated from E17 do not express the PMCA2 isoform, they constitute a valuable model for studying its role in neuronal development. In this study, we demonstrated that heterologous expression of PMCA2b enhanced the differentiation of H19-7 cells and protected from ketamine-induced death. PMCA2b did not affect resting [Ca2+]c in the presence or absence of ketamine and had no effect on the rate of Ca2+ clearance following membrane depolarization in the presence of the drug. The upregulation of endogenous PMCA1 demonstrated in response to PMCA2b expression as well as ketamine-induced PMCA4 depletion were indifferent to the rate of Ca2+ clearance in the presence of ketamine. Yet, co-expression of PMCA4b and PMCA2b was able to partially restore Ca2+ extrusion diminished by ketamine. The profiling of NMDA receptor expression showed upregulation of the NMDAR1 subunit in PMCA2b-expressing cells and increased co-immunoprecipitation of both proteins following ketamine treatment. Further microarray screening demonstrated a significant influence of PMCA2b on GABA signaling in differentiating progenitor cells, manifested by the unique regulation of several genes key to the GABAergic transmission. The overall activity of glutamate decarboxylase remained unchanged, but Ca2+-induced GABA release was inhibited in the presence of ketamine. Interestingly, PMCA2b expression was able to reverse this effect. The mechanism of GABA secretion normalization in the presence of ketamine may involve PMCA2b-mediated inhibition of GABA transaminase, thus shifting GABA utilization from energetic purposes to neurosecretion. In this study, we show for the first time that developmentally controlled PMCA expression may dictate the pattern of differentiation of hippocampal progenitor cells. Moreover, the appearance of PMCA2 early in development has long-standing consequences for GABA metabolism with yet an unpredictable influence on GABAergic neurotransmission during later stages of brain maturation. In contrast, the presence of PMCA2b seems to be protective for differentiating progenitor cells from ketamine-induced apoptotic death.

Effects of the Selective Serotonin Reuptake Inhibitor Fluoxetine on Developing Neural Circuits in a Model of the Human Fetal Cortex.

The developing prenatal brain is particularly susceptible to environmental disturbances. During prenatal brain development, synapses form between neurons, resulting in neural circuits that support complex cognitive functions. In utero exposure to environmental factors such as pharmaceuticals that alter the process of synapse formation increases the risk of neurodevelopmental abnormalities. However, there is a lack of research into how specific environmental factors directly impact the developing neural circuitry of the human brain. For example, selective serotonin reuptake inhibitors are commonly used throughout pregnancy to treat depression, yet their impact on the developing fetal brain remains unclear. Recently, human brain models have provided unprecedented access to the critical window of prenatal brain development. In the present study, we used human neurons and cortical spheroids to determine whether the selective serotonin reuptake inhibitor fluoxetine alters neurite and synapse formation and the development of spontaneous activity within neural circuits. We demonstrate that cortical spheroids express serotonin transporter, thus recapitulating the early developmental expression of serotonin transporter associated with cortical pyramidal neurons. Cortical spheroids also appropriately express serotonin receptors, such as synaptic 5-HT2A and glial 5-HT5A. To determine whether fluoxetine can affect developing neural circuits independent of serotonergic innervation from the dorsal and medial raphe nuclei, we treated cortical neurons and spheroids with fluoxetine. Fluoxetine alters neurite formation in a dose-dependent fashion. Intriguingly, in cortical spheroids, neither acute nor chronic fluoxetine significantly altered excitatory synapse formation. However, only acute, but not chronic fluoxetine exposure altered inhibitory synaptogenesis. Finally, fluoxetine reversibly suppresses neuronal activity in a dose-dependent manner. These results demonstrate that fluoxetine can acutely alter synaptic function in developing neural circuits, but the effects were not long-lasting. This work provides a foundation for future studies to combine serotonergic innervation with cortical spheroids and assess the contributions of fluoxetine-induced alterations in serotonin levels to brain development.

Copper promoting oyster larval growth and settlement: Molecular insights from RNA-seq

As a cofactor of key enzymes, Cu is required in living organisms, although Cu levels in the natural environment are typically low. In this study, the promotion of growth and settlement on the larvae of oyster Crassostrea angulata was observed at an environmentally relevant concentration (10 μg/L Cu). Interestingly, Cu accumulation in the soft tissue of oyster larvae increased during the larval development and exhibited a sharp increase at the late pelagic stage. With the help of RNA-seq, we constructed a high-quality transcriptional database of the oyster C. angulata larvae (24,257 genes with an average length of 1594 bp) via de novo assembly, which provided the basic molecular changes during the larval development. Network analysis of five early developmental stages and differential expression under Cu exposure were integrated to examine the roles of Cu in oyster larvae. Our molecular analysis demonstrated that both ion channels and organic transporters contributed to Cu internalization from the external environment, including zinc transporters and amino acid transporters. The followed distribution of Cu across cells was achieved by ATP7A, the circulatory system, and the Cu transporters (CTRs). Cu exposure enhanced the ribosome and the calcium binding proteins with a higher rate of translation and shell formation, giving rise to faster growth of oyster larvae. Furthermore, Cu facilitated the settling process by upregulating the chitin binding genes and then promoting the formation of the proteinaceous matrix between larvae and substrate. Our study presents the molecular basis for Cu promotion (i.e., hormesis) effects on oyster larval growth and settlement.

Integrative analysis of genome-wide association studies identifies novel loci associated with neuropsychiatric disorders

Neuropsychiatric disorders, such as autism spectrum disorder (ASD), attention deficit hyperactivity disorder (ADHD), schizophrenia (SCZ), bipolar disorder (BIP), and major depressive disorder (MDD) share common clinical presentations, suggesting etiologic overlap. A substantial proportion of SNP-based heritability for neuropsychiatric disorders is attributable to genetic components, and genome-wide association studies (GWASs) focusing on individual diseases have identified multiple genetic loci shared between these diseases. Here, we aimed at identifying novel genetic loci associated with individual neuropsychiatric diseases and genetic loci shared by neuropsychiatric diseases. We performed multi-trait joint analyses and meta-analysis across five neuropsychiatric disorders based on their summary statistics from the Psychiatric Genomics Consortium (PGC), and further carried out a replication study of ADHD among 2726 cases and 16299 controls in an independent pediatric cohort. In the multi-trait joint analyses, we found five novel genome-wide significant loci for ADHD, one novel locus for BIP, and ten novel loci for MDD. We further achieved modest replication in our independent pediatric dataset. We conducted fine-mapping and functional annotation through an integrative multi-omics approach and identified causal variants and potential target genes at each novel locus. Gene expression profile and gene-set enrichment analysis further suggested early developmental stage expression pattern and postsynaptic membrane compartment enrichment of candidate genes at the genome-wide significant loci of these neuropsychiatric disorders. Therefore, through a multi-omics approach, we identified novel genetic loci associated with the five neuropsychiatric disorders which may help to better understand the underlying molecular mechanism of neuropsychiatric diseases.

Advances in early identification of children and adolescents at risk for psychiatric illness.

Childhood and adolescence are a crucial time window for the early identification of perspectival risk for adult psychopathology. This article reviews current advances in the field. Converging developmentally focused and predictive approaches are rapidly expanding in the field of early identification of children and adolescents at risk for psychiatric illness. Although the former emphasizes early phenotypic trajectories emerging in childhood and adolescence, the latter operate in terms of clinical risk staging. Decisive advances in the field include the metaanalytical evidence of the predictive value of clinical high-risk criteria in childhood-adolescence; the systematization of premorbid manifestations of psychotic vulnerability and bipolar prodromes; the corroboration of certain sets of anomalous subjective experiences as transdiagnostic index of familial genetic risk (i.e., basic symptoms) and early developmental expression of schizophrenia spectrum vulnerability (i.e., self-disorders). Childhood phenotypic manifestations of liability to mental disorders are often cooccurrent and undergo a plastic, heterotypic modification along the transition to adolescence. In general, they represent epiphenomenic flags of a broad vulnerability for the subsequent structuration of mental illness. This perspective coheres with the p-factor model of psychopathology and complements the clinical staging model which informs the early detection paradigm of severe mental disorders.

Spatiotemporal Patterning of ABC Multidrug‐Resistance Transporters During Germ Layer Specification

Xenobiotic transporters are important for the protection of differentiated somatic cells, yet less is known about the role of these proteins in rapidly changing embryonic cells. A surprisingly large number of ABC transporters (80%) are expressed from fertilized egg to gastrulation, but their spatial and temporal patterns of expression during early embryogenesis remain incompletely understood. Here I report a pattern of ABC transporter expression that emerges during the early differentiation of mesoderm, endoderm, and ectoderm of the sea urchin. These included orthologs of six major xenobiotic transporters, including ABCB1, ABCB4, ABCC1, ABCC4, ABCC5, and ABCG2. In‐situ hybridization (ISH) revealed that transporters followed one of three patterns of early developmental expression: The first, seen with protective and homeostatic genes such as ABCB1 and ABCC1, was the early and ubiquitous expression across all three germ layers, and later enrichment in ectoderm‐derived sensory and feeding structures of the larva. The second, seen with the transporters ABCC4 and ‐C5 that are known to transport signal molecules, is a spatially restricted expression domain within a ring of mesodermal cells of the early blastula; ABCC4 then becomes restricted to primordial germ cells (PGCs) and their embryonic niche during gastrulation. Third, ABCB4 and ABCG2 show expression only in endoderm fated cells, particularly in the hindgut (presumptive intestines) at the onset of gut development; both genes eventually expand to the midgut region (presumptive stomach). To further refine the ISH map, I used fluorescent substrates to assay the functional outputs of transporter mRNA localization. ABCC1 and ‐C4 specific substrates are strongly effluxed out of the ring of mesodermal cells. This overlaps with robust ABCC5 transport activity in blastulae, suggesting a potential ABCC‐mediated signaling center in early mesoderm, where the PGCs are born. In the endoderm, ABCB4 and ABCG2‐specific efflux activity begins several days before the larva feeds. Collectively, this work is the first systems‐level description of the emergence of xenobiotic transporter expression patterns across the germ layers during embryogenesis. The resulting map of transport reveals unanticipated developmental functions for these genes and provides a framework for understanding the genetic regulatory controls of transporter expression. This will have additional impact on predicting and mitigating the effects of xenobiotic exposures during vulnerable windows of early development.Support or Funding InformationSupported by NIH R01ES027921 and R01ES030318 to AH, and NIH F32ES029843 to CSS.

Shaken, rattled and rolled: The effects of hatchery-rearing techniques on endogenous cortisol production, stress-related gene expression, growth and survival in larval Lake Sturgeon, Acipenser fulvescens

Sturgeon, one of the most endangered families of vertebrates, are of high conservation concern with population restoration efforts worldwide. Sturgeon species are often raised in conservation hatcheries to supplement wild populations with little understanding of effects of hatchery-related stressors on early development. Lake Sturgeon hatcheries deviate from natural conditions by coating adhesive embryos in clay to prevent clumping, embryos are then added to tumbling jars, colloquially referred to as McDonald Jars; a cone or cylinder-shaped container with constant flow, causing the embryos to roll and move throughout the water column. In the wild, Lake Sturgeon embryos adhere to substrate in fast-flowing rivers, where orientation is maintained throughout embryogenesis. In this study, we examined the effects of hatchery early-rearing environment, i.e. the use of tumbling jars, on gene expression, endogenous cortisol production, growth, and survival in larval Lake Sturgeon. We compared pre- and post-hatch tumbled larvae to non-tumbled larvae that were allowed to adhere to substrate throughout embryogenesis. We hypothesized that deviating from natural rearing conditions, with the use of tumbling jars, would result in changes to cortisol production, growth and mortality in post-hatch, tumbled larvae. Endogenous cortisol production was delayed one week for fish in the tumbled treatment (two-factor ANOVA; P < .05) when compared to non-tumbled fish. Tumbled fish were also smaller throughout development, with lasting effects on growth 62 days after hatching (P < .05). The tumbled fish had 23% mortality post-hatch compared to 6% in the non-tumbled group (Cox Proportional-Hazards Model; P < .0001). There were differences in gene expression in early development at specific time points, between the two treatment groups, in StAR and hsp70 (P < .05). The observed developmental effects on endogenous cortisol production and growth in the tumbled embryos could be counterproductive to the goal of producing a hatchery-reared fish that can survive in the wild. As conservation hatcheries aim to release individuals that will supplement and reproduce with wild populations, changes to developmental timing, growth and survival should be avoided. The findings of this study suggest that tumbling jars may not be appropriate in conservation hatcheries for Lake Sturgeon as they may have long-term negative consequences on individual fitness.

The peptide transporter 1a of the zebrafish Danio rerio, an emerging model in nutrigenomics and nutrition research: molecular characterization, functional properties, and expression analysis

BackgroundPeptide transporter 1 (PepT1, alias Slc15a1) mediates the uptake of dietary di/tripeptides in all vertebrates. However, in teleost fish, more than one PepT1-type transporter might function, due to specific whole genome duplication event(s) that occurred during their evolution leading to a more complex paralogue gene repertoire than in higher vertebrates (tetrapods).ResultsHere, we describe a novel di/tripeptide transporter in the zebrafish (Danio rerio), i.e., the zebrafish peptide transporter 1a (PepT1a; also known as Solute carrier family 15 member a1, Slc15a1a), which is a paralogue (78% similarity, 62% identity at the amino acid level) of the previously described zebrafish peptide transporter 1b (PepT1b, alias PepT1; also known as Solute carrier family 15 member 1b, Slc15a1b). Also, we report a basic analysis of the pept1a (slc15a1a) mRNA expression levels in zebrafish adult tissues/organs and embryonic/early larval developmental stages. As assessed by expression in Xenopus laevis oocytes and two-electrode voltage clamp measurements, zebrafish PepT1a, as PepT1b, is electrogenic, Na+-independent, and pH-dependent and functions as a low-affinity system, with K0.5 values for Gly-Gln at − 60 mV of 6.92 mmol/L at pH 7.6 and 0.24 mmol/L at pH 6.5 and at − 120 mV of 3.61 mmol/L at pH 7.6 and 0.45 mmol/L at pH 6.5. Zebrafish pept1a mRNA is highly expressed in the intestine and ovary of the adult fish, while its expression in early development undergoes a complex trend over time, with pept1a mRNA being detected 1 and 2 days post-fertilization (dpf), possibly due to its occurrence in the RNA maternal pool, decreasing at 3 dpf (~ 0.5-fold) and increasing above the 1–2 dpf levels at 4 to 7 dpf, with a peak (~ 7-fold) at 6 dpf.ConclusionsWe show that the zebrafish PepT1a-type transporter is functional and co-expressed with pept1b (slc15a1b) in the adult fish intestine. Its expression is also confirmed during the early phases of development when the yolk syncytial layer is present and yolk protein resorption processes are active. While completing the missing information on PepT1-type transporters function in the zebrafish, these results open to future investigations on the similar/differential role(s) of PepT1a/PepT1b in zebrafish and teleost fish physiology.

Actin genes and their expression in pacific white shrimp, Litopenaeus vannamei.

Actin is a multi-functional gene family that can be divided into muscle-type actins and non-muscle-type actins. In this study, 37 unigenes encoding actins were identified from RNA-Seq data of Pacific white shrimp, Litopenaeus vannamei. According to phylogenetic analysis, four and three cDNAs belong to cytoplasmic- and heart-type actins and were named LvActinCT and LvActinHT, respectively. 10 cDNAs belong to the slow-type skeletal muscle actins, and 18 belong to the fast-type skeletal muscle actins; they were designated LvActinSSK and LvActinFSK, respectively. Some muscle actin genes formed gene clusters in the genome. Multiple alternative transcription starts sites (ATSSs) were found for LvActinCT1. Based on the early developmental expression profile, almost all LvActins were highly expressed between the early limb bud and post-larval stages. Using LvActinSSK5 as probes, slow-type muscle was localized in pleopod muscle and superficial ventral muscle. We also found three actin genes that were down-regulated in the hemocytes of white spot syndrome virus (WSSV)- and Vibrio parahaemolyticus-infected L. vannamei. This study provides valuable information on the actin gene structure of shrimp, furthers our understanding of the shrimp muscle system and helps us develop strategies for disease control and sustainable shrimp farming.

Tissue distribution and early developmental expression patterns of aldolase A, B, and C in grass carp Ctenopharyngodon idellus.

Aldolase is a key enzyme involved in glycolysis, gluconeogenesis, and the pentose phosphate pathway. To establish the expression patterns of all three aldolase isozyme genes in different tissues and during early embryogenesis in lower vertebrates, as well as to explore the functional differences between these three isozymes, the grass carp was selected as a model owing to its relatively high glucose-metabolizing capability. Based on the cDNA sequences of the aldolase A, B, and C genes, the expression patterns of these three isozymes were analyzed in different tissues and during early embryogenesis using quantitative real-time polymerase chain reaction (qRT-PCR). Sequence analysis of cDNAs indicated that aldolase A, B, and C (GenBank accession numbers: KM192250, KM192251, and KM192252) consist of 364, 364, and 363 amino acids, respectively. The qRT-PCR results showed that the expression levels of aldolase A, B, and C were highest in the muscle, liver, and brain, respectively. Aldolase A and C exhibited similar expression patterns during embryogenesis, with high levels observed in unfertilized and fertilized eggs and at the blastocyst stage, followed by a decline and then increase after organogenesis. In contrast, aldolase B transcript was not detected during the unfertilized egg stage, and appeared only from gastrulation; the expression increased markedly during the feeding period (72 h after hatching), at which point the level was higher than those of aldolase A and C. These data suggest that the glucose content of grass carp starter feed should be adjusted according to the metabolic activity of aldolase B.