Key Developmental Stages Research Articles

The human blood fluke, Schistosoma mansoni, harbours bacteria throughout the parasite's life cycle.

Symbiotic relationships between invertebrates and bacteria have been extensively described; nevertheless, studies of microbial communities inhabiting parasitic worms remain scarce. Exploring the microbiota associated with helminths responsible for major infectious diseases will inform on parasite biology, host-pathogen interactions, and disease pathophysiology. We investigated the presence of microorganisms inhabiting tissues of the human parasite Schistosoma mansoni. In situ hybridisation using a pan-bacterial 16S rRNA gene probe revealed bacteria colonising key developmental stages that were successfully removed after antibiotic treatment of live parasites. Understanding the composition and function of S. mansoni-associated microbiota may lead to the development of novel microbiome-targeted control strategies.

Weighted Gene Co-Expression Network Analysis of red body color formation of crimson snapper, Lutjanus erythropterus

Crimson snapper (Lutjanus erythropterus) is a highly valued economic fish species, with its red body color being a crucial economic characteristic. A systematic study of skin pigmentation could offer insights into the mechanism of red coloration. In this study, we conducted the first network analysis of snapper transcriptomes during key developmental stages of pigmentation, identifying a cascade of cellular development and responsive gene modules and key genes, including eight developmental-specific modules. Among the four candidate hub genes (TNNT2, ROBO2, APBA2, and APOD), APOD was involved in pigmentation development. The network further revealed some key genes, such as TYRP1a, TYRP1b, PMEL, BCO1, BCO2, CYP2J2, CYP2J6, CYP3A4, PNPLA2, FABP6, ENPP7, and FFAR3, associated with melanin synthesis, carotenoids, and lipid metabolism. Our findings showed that pteridine and carotenoids were involved in yellow coloration after initial feeding, while yellow pigmentation-related genes (e.g., GCH1, PTS, SPR, CBR1, SCARB1, CD36, ABCA1, NpC1L1, etc.) had the same expression pattern at three days post-hatching (3 dph). At the red coloration emergence stage (45 dph), we observed an RNA level signal indicating the conversion of coloring substances from pteridine to carotenoids. These results substantially enhance understanding of the molecular mechanisms underlying pigmentation and skin color variation in L. erythropterus, providing a theoretical basis for improving of body coloration in economically cultured fish and genetic breeding of ornamental fish.

How do you build a nectar spur? A transcriptomic comparison of nectar spur development in Linaria vulgaris and gibba development in Antirrhinum majus.

Nectar spurs (tubular outgrowths of floral organs) have long fascinated biologists. However, given that no model species possess nectar spurs, there is still much to learn about their development. In this study we combined morphological analysis with comparative transcriptomics to gain a global insight into the morphological and molecular basis of spur outgrowth in Linaria. Whole transcriptome sequencing was performed on two related species at three key developmental stages (identified by our morphological analysis), one with a spur (Linaria vulgaris), and one without a spur (Antirrhinum majus). A list of spur-specific genes was selected, on which we performed a gene enrichment analysis. Results from our RNA-seq analysis agreed with our morphological observations. We describe gene activity during spur development and provide a catalogue of spur-specific genes. Our list of spur-specific genes was enriched for genes connected to the plant hormones cytokinin, auxin and gibberellin. We present a global view of the genes involved in spur development in L. vulgaris, and define a suite of genes which are specific to spur development. This work provides candidate genes for spur outgrowth and development in L. vulgaris which can be investigated in future studies.

Generation of a novel Stra8-driven Cre recombinase strain for use in pre-meiotic germ cells in mice†.

The development of oocytes occurs over a broad time frame, starting at the earliest stages of embryogenesis and continuing into adulthood. Conditional knockout technologies such as the Cre/loxP recombination system are useful for analyzing oocyte development at specific stages, but not every time frame has appropriate Cre drivers, for instance, during oocyte meiotic initiation through early prophase I in the embryo. Here, we generated a novel knockin mouse line that produces a bicistronic transcript from the endogenous Stra8 locus that includes a "self-cleaving" 2A peptide upstream of cre. This allows for high efficiency cleavage and production of both proteins individually and results in expression of cre in both male and female gonads at the biologically relevant stage. Fluorescent reporter analysis confirms that this line recapitulates endogenous Stra8 expression in both sexes and does not affect fertility of heterozygous nor homozygous mice. This line, named Stra8P2Acre, adds to the repertoire of germ-cell specific cre driver lines and, importantly, allows for deletion of target genes during key embryonic oocyte developmental stages, including early events in meiosis. Summary Sentence Generation of a novel cre recombinase knockin to the Stra8 locus allows production of Stra8 and cre without affecting fertility.

Genetic models for lineage tracing in musculoskeletal development, injury, and healing

Musculoskeletal development and later post-natal homeostasis are highly dynamic processes, marked by rapid structural and functional changes across very short periods of time. Adult anatomy and physiology are derived from pre-existing cellular and biochemical states. Consequently, these early developmental states guide and predict the future of the system as a whole. Tools have been developed to mark, trace, and follow specific cells and their progeny either from one developmental state to the next or between circumstances of health and disease. There are now many such technologies alongside a library of molecular markers which may be utilized in conjunction to allow for precise development of unique cell ‘lineages’. In this review, we first describe the development of the musculoskeletal system beginning as an embryonic germ layer and at each of the key developmental stages that follow. We then discuss these structures in the context of adult tissues during homeostasis, injury, and repair. Special focus is given in each of these sections to the key genes involved which may serve as markers of lineage or later in post-natal tissues. We then finish with a technical assessment of lineage tracing and the techniques and technologies currently used to mark cells, tissues, and structures within the musculoskeletal system.

Modeling human skeletal development using human pluripotent stem cells

Chondrocytes and osteoblasts differentiated from induced pluripotent stem cells (iPSCs) will provide insights into skeletal development and genetic skeletal disorders and will generate cells for regenerative medicine applications. Here, we describe a method that directs iPSC-derived sclerotome to chondroprogenitors in 3D pellet culture then to articular chondrocytes or, alternatively, along the growth plate cartilage pathway to become hypertrophic chondrocytes that can transition to osteoblasts. Osteogenic organoids deposit and mineralize a collagen I extracellular matrix (ECM), mirroring invivo endochondral bone formation. We have identified gene expression signatures at key developmental stages including chondrocyte maturation, hypertrophy, and transition to osteoblasts and show that this system can be used to model genetic cartilage and bone disorders.

Programming of metabolism by adipokines during development.

The intrauterine and early postnatal periods represent key developmental stages in which an organism is highly susceptible to being permanently influenced by maternal factors and nutritional status. Strong evidence indicates that either undernutrition or overnutrition during development can predispose individuals to disease later in life, especially type 2 diabetes mellitus and obesity, aconcept known as metabolic programming. Adipose tissue produces important signalling molecules that control energy and glucose homeostasis, including leptin and adiponectin. In addition to their well-characterized metabolic effects in adults, adipokines have been associated with metabolic programming by affecting different aspects of development. Therefore, alterations in the secretion or signalling of adipokines, caused by nutritional insults in early life, might lead to metabolic diseases in adulthood. This Review summarizes and discusses the potential role of several adipokines in inducing metabolic programming through their effects during development. The identification of the endocrine factors that act in early life to permanently influence metabolism represents a key step in understanding the mechanisms behind metabolic programming. Thus, future strategies aiming to prevent and treat these metabolic diseases can be designed, taking into consideration the relationship between adipokines and the developmental origins of health and disease.

Clinical Relevance of Interferon Regulatory Family-4 (IRF4) Expression in Newly Diagnosed Patients with Multiple Myeloma

Multiple myeloma (MM) is a malignant plasma cell neoplasm with complex biology and heterogenous course. Interferon regulatory factor 4 (IRF4) transcription factor, important key developmental stages of hematopoiesis, represents an excellent potential therapeutic target. The present work aimed to investigate the expression status of IRF4 in the diagnostic bone marrow biopsy (BMB) cores of MM patients. This prospective study included 62 newly diagnosed MM patients. The expression of IRF4 was assessed in the BMB by immunohistochemistry (IHC). The data were correlated to the patients’ clinico-pathological features, response to treatment and survival rates. IRF4 expression was observed in 50% of MM patients (31/62). IRF-4 positive patients were more frequently male patients (P = 0.018), have immunoglobulin heavy chain (IgH) translocations (P = 0.05) and tended to present with a higher platelets count (P = 0.07). Multiple myeloma patients presenting with urine M-protein had worse overall survival (OS) than negative cases (P = 0.012). Normocellular BM aspirate (BMA) was associated with better OS than hypercellular and hypocellular BMA (P = 0.006). Patchy distribution of plasma cells in BMB was associated with better disease-free survival (DFS) while diffuse infiltration had the worst (P = 0.019). Of note, after treatment, MM patients had significantly lower percentage of BMA plasma cells, platelet count, β2 microglobulin and creatinine levels (P = 0.037, < 0.001, 0.022 and 0.026, respectively). Had higher albumin level (P = 0.007), compared to initial investigations. No significant association was found between IRF4 expression and the patients’clinical outcomes. Patterns of plasma cells distribution in BMB, BMA cellularity and urine M-protein are prognostically relevant in MM.

A Robust Pipeline for the Multi-Stage Accelerated Differentiation of Functional 3D Cortical Organoids from Human Pluripotent Stem Cells.

Disordered cellular development, abnormal neuroanatomical formations, and dysfunction of neuronal circuitry are among the pathological manifestations of cortical regions in the brain that are often implicated in complex neurodevelopmental disorders. With the advancement of stem cell methodologies such as cerebral organoid generation, it is possible to study these processes in vitro using 3D cellular platforms that mirror key developmental stages occurring throughout embryonic neurogenesis. Patterning-based stem cell models of directed neuronal development offer one approach to accomplish this, but these protocols often require protracted periods of cell culture to generate diverse cell types and current methods are plagued by a lack of specificity, reproducibility, and temporal control over cell derivation. Although ectopic expression of transcription factors offers another avenue to rapidly generate neurons, this process of direct lineage conversion bypasses critical junctures of neurodevelopment during which disease-relevant manifestations may occur. Here, we present a directed differentiation approach for generating human pluripotent stem cell (hPSC)-derived cortical organoids with accelerated lineage specification to generate functionally mature cortical neurons in a shorter timeline than previously established protocols. This novel protocol provides precise guidance for the specification of neuronal cell type identity as well as temporal control over the pace at which cortical lineage trajectories are established. Furthermore, we present assays that can be used as tools to interrogate stage-specific developmental signaling mechanisms. By recapitulating major components of embryonic neurogenesis, this protocol allows for improved in vitro modeling of cortical development while providing a platform that can be utilized to uncover disease-specific mechanisms of disordered development at various stages across the differentiation timeline. © 2023 Wiley Periodicals LLC. Basic Protocol 1: 3D hPSC neural induction Support Protocol 1: Neural rosette formation assay Support Protocol 2: Neurosphere generation Support Protocol 3: Enzymatic dissociation, NSC expansion, and cryopreservation Basic Protocol 2: 3D neural progenitor expansion Basic Protocol 3: 3D accelerated cortical lineage patterning and terminal differentiation.

Relation between Nasal Septum Deviation and Facial Asymmetry: An Ontogenetic Analysis from Infants to Children Using Geometric Morphometrics

The nasal septum has been postulated to have an intrinsic growth power and act as a pacemaker for facial development, its interactions with local craniofacial structures likely to influence facial anatomy and morphology. Recent studies have begun to investigate the link between nasal septum deviation and facial asymmetry; however, the magnitude and mechanisms of this relation are still unclear. This study aimed to analyse the degree of nasal septum deviation in a sample of infants and children (males and females from 0 to 8 years old) and its correlation with the three-dimensional structure of the facial skeleton. The scope was to test whether septal deviation is linked, and might cause, the development of a more asymmetric face. For this aim, 41 3D landmarks (homologous points) were collected on the nasal septum and cranial surface of 46 specimens extracted from medical CT-scans and were analysed using Geometric Morphometrics, Multiple Linear regressions, Multivariate ANOVAs, and Principal Component Analysis (PCA). Results showed no significant correlation between magnitude of septal deviation and the ontogeny (changes in age) or sex of the sample, but a significant association was found between side of deviation and septal deviation magnitude and frequency. The asymmetric PCA reveals that most of the asymmetry identified is fluctuating, and that changes in the asymmetric morphology of the face are not associated to a specific side of septal deviation. In addition, a series of Multivariate ANOVAs showed that age, sex, and septal deviation have no impact on facial asymmetry, with only age impacting the symmetric development of the facial morphology. When looking at factors impacting the general morphology of the face, age is again the only major driving component, with fluctuating asymmetry and sex only approaching significance. These results could imply a certain degree of dissociation between the mechanisms of facial and septal growth and development; however, an investigation of other key developmental stages in facial morphology is needed to further understand the relation between septal deviation and facial growth.

The first week following insemination is the period of major pregnancy failure in pasture-grazed dairy cows.

A 60% pregnancy success for inseminations is targeted to optimize production efficiency for dairy cows within a seasonal, pasture-grazed system. Routine measures of pregnancy success are widely available but are limited, in practice, to a gestation stage beyond the first 28 d. Although some historical data exist on embryonic mortality before this stage, productivity of dairy systems and genetics of the cows have advanced significantly in recent decades. Accordingly, the aim was to construct an updated estimate of pregnancy success at key developmental stages during the first 70 d after insemination. Blood samples were collected for progesterone concentrations on d 0 and 7. A temporal series of 4 groups spanning fertilization through d 70 were conducted on 4 seasonal, pasture-grazed dairy farms (n = 1,467 cows) during the first 21 d of the seasonal breeding period. Morphological examination was undertaken on embryos collected on d 7 (group E7) and 15 (group E15), and pregnancy was diagnosed via ultrasonography on approximately d 28 and 35 (group E35) as well as d 70 (group E70). Fertilization, embryo, and fetal evaluation for viability established a pregnancy success pattern. Additionally, cow and on-farm risk factor variables associated with pregnancy success were evaluated. We estimated pregnancy success rates of 70.9%, 59.1%, 63.8%, 62.3%, and 56.7% at d 7, 15, 28, 35, and 70, respectively. Fertilization failure (15.8%) and embryonic arrest before the morula stage (10.3%) were the major developmental events contributing to first-week pregnancy failures. Embryo elongation failure of 7% contributed to pregnancy failure during the second week. The risk factors for pregnancy success that were related to the cows included interval between calving and insemination, and d-7 plasma progesterone concentrations, whereas insemination sire was associated with pregnancy outcome. Most pregnancy failure occurs during the first week among seasonal-calving pasture-grazed dairy cows.

Low cell number ChIP-seq reveals chromatin state-based regulation of gene transcription in the rice male meiocytes.

Low cell number ChIP-seq reveals chromatin state-based regulation of gene transcription in the rice male meiocytes.

Transcriptomic Analyses of Grapevine Leafroll-Associated Virus 3 Infection in Leaves and Berries of 'Cabernet Franc'.

Grapevine leafroll-associated virus 3 (GLRaV-3) is one of the most important viruses affecting global grape and wine production. GLRaV-3 is the chief agent associated with grapevine leafroll disease (GLRD), the most prevalent and economically destructive grapevine viral disease complex. Response of grapevine to GLRaV-3 infection at the gene expression level is poorly characterized, limiting the understanding of GLRaV-3 pathogenesis and viral-associated symptom development. In this research, we used RNA-Seq to profile the changes in global gene expression of Cabernet franc, a premium red wine grape, analyzing leaf and berry tissues at three key different developmental stages. We have identified 1457 differentially expressed genes (DEGs) in leaves and 1181 DEGs in berries. The expression profiles of a subset of DEGs were validated through RT-qPCR, including those involved in photosynthesis (VvPSBP1), carbohydrate partitioning (VvSUT2, VvHT5, VvGBSS1, and VvSUS), flavonoid biosynthesis (VvUFGT, VvLAR1, and VvFLS), defense response (VvPR-10.3, and VvPR-10.7), and mitochondrial activities (ETFB, TIM13, and NDUFA1). GLRaV-3 infection altered source–sink relationship between leaves and berries. Photosynthesis and photosynthate assimilation were inhibited in mature leaves while increased in young berries. The expression of genes involved in anthocyanin biosynthesis increased in GLRaV-3-infected leaves, correlating with interveinal tissue reddening, a hallmark of GLRD symptoms. Notably, we identified changes in gene expression that suggest a compromised sugar export and increased sugar retrieval in GLRaV-3-infected leaves. Genes associated with mitochondria were down-regulated in both leaves and berries of Cabernet franc infected with GLRaV-3. Results of the present study suggest that GLRaV-3 infection may disrupt mitochondrial function in grapevine leaves, leading to repressed sugar export and accumulation of sugar in mature leaf tissues. The excessive sugar accumulation in GLRaV-3-infected leaves may trigger downstream GLRD symptom development and negatively impact berry quality. We propose a working model to account for the molecular events underlying the pathogenesis of GLRaV-3 and symptom development.

Examining the New Zealand school food environment: what needs to change?

Habitual dietary intakes and nutrition behaviours developed during childhood and adolescence pave the way for similar behaviours to manifest in adulthood. Childhood obesity rates have now reached a point where one in six children globally are classified as overweight or obese. Schools have the unique ability to reach almost all children during key developmental stages, making them an ideal setting for influencing children's nutrition behaviours. Evidence suggests the school food environment is not always conducive to healthy food choices and may be obesogenic. The aim of this narrative review is to explore factors that influence the healthy food and drink environment in and around schools in New Zealand. The review focused on evidence from New Zealand and Australia given the close resemblance in education systems and school food guidance. Using the Analysis Grid for Environments Linked to Obesity (ANGELO) framework, the school food environment was categorised into the following domains: economic, political, physical and socio-cultural factors. Findings suggest that food policies are not utilised within schools, and guidelines to improve the school food environment are not well implemented. Canteen profit models, lack of staff support and resources, and higher availability of low-cost unhealthy foods are among barriers that hinder implementation. This review highlights recommendations from existing evidence, including canteen pricing strategies, restriction of unhealthy foods and using peer modelling in a time-scarce curriculum to improve the school food environment. Key areas for improvement, opportunities to enhance policy implementation and untapped avenues to improve the food and nutrition behaviours of children are highlighted.

High-throughput transcriptome sequencing reveals the key stages of cardiovascular development in zebrafish embryos

BackgroundThe cardiovascular developmental process is a tightly regulated network involving multiple genes. The current understanding of the molecular mechanism behind cardiovascular development is insufficient and requires further research.ResultsTranscriptome sequencing of three developmental stages in zebrafish embryos was performed and revealed three key cardiovascular developmental stages. Then, the differentially expressed genes (DEGs) involved in cardiovascular development were screened out. The three developmental stages were 18 (T1), 24 (T2), and 42 h post fertilization (hpf) (T3), and the three stages were confirmed by detecting differences in expression between cardiomyocyte and endothelial marker genes (cmlc2, fli1) using in situ hybridization, which represents the characteristics of cardiovascular development. Thousands of DEGs were identified using transcriptome analysis. Of them, 2605 DEGs were in T1-vs-T2, including 2003 up-regulated and 602 down-regulated genes, 6446 DEGs were in T1-vs-T3, consisting of 4608 up-regulated and 1838 down-regulated genes, and 3275 DEGs were in T2-vs-T3, including 2420 up-regulated and 855 down-regulated genes. There were 644 common DEGs and 167 common five-fold higher differentially expressed genes (HDEGs) identified, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID). Significant differences was observed in the levels of gene expression among different developmental stages in multiple GO terms and KEGG pathways, such as cell migration to the midline involved in heart development, cardiovascular system development, circulatory system process for biological processes of GO terms; and cardiac muscle contraction, adrenergic signaling in cardiomyocytes for KEGG pathways. These results demonstrated that these three stages were important period for the development of the cardiovascular system. Lastly, we used quantitative real-time PCR (qPCR) to validate the reliability of RNA-sequencing by selecting 21 DEGs.ConclusionsThese results demonstrated that these three stages represented the important periods for cardiovascular system development of zebrafish and some candidate genes was obtained and provided a solid foundation for additional functional studies of the DEGs.

Identification of AP2/ERF transcription factors in Tetrastigma hemsleyanum revealed the specific roles of ERF46 under cold stress.

Tetrastigma hemsleyanum (T. hemsleyanum) is a traditional medicinal plant that is widely used in China. Cultivated T. hemsleyanum usually encounters cold stress, limiting its growth and quality at key developmental stages. APETALA2 (AP2)/ethylene-responsive factor (ERF) transcription factors (TFs) comprise one of the largest gene superfamilies in plants and are widely involved in biotic and abiotic stresses. To reveal the roles of AP2/ERF TFs during T. hemsleyanum development, 70 AP2/ERF TFs were identified in T. hemsleyanum. Among them, 18 and 2 TFs were classified into the AP2 and RAV families, respectively. The other 50 TFs belonged to the ERF family and were further divided into the ERF and (dehydration reaction element binding factor) DREB subfamilies. The ERF subfamily contained 46 TFs, while the DREB subfamily contained 4 TFs. Phylogenetic analysis indicated that AP2/ERF TFs could be classified into five groups, in which 10 conserved motifs were confirmed. Several motifs were group- or subgroup-specific, implying that they were significant for the functions of the AP2/ERF TFs of these clades. In addition, 70 AP2/ERF TFs from the five groups were used for an expression pattern analysis under three low-temperature levels, namely, –4, 0, and 4°C. The majority of these AP2/ERF TFs exhibited a positive response to cold stress conditions. Specifically, ThERF5, ThERF31, ThERF46, and ThERF55 demonstrated a more sensitive response to cold stress. Moreover, AP2/ERF TFs exhibited specific expression patterns under cold stress. Transient overexpression and RNA interference indicated that ThERF46 has a specific tolerance to cold stress. These new insights provide the basis for further studies on the roles of AP2/ERF TFs in cold stress tolerance in T. hemsleyanum.

Did decades of glyphosate use have selected for resistant amphibians in agricultural habitats?

Glyphosate-based herbicides are used worldwide, and glyphosate's primary metabolite (aminomethylphosphonic acid: AMPA), is globally retrieved in surface waters. AMPA induces various adverse effects on aquatic wildlife, including selective mortality, which suggests that glyphosate exposure may have selected for AMPA-resistant individuals. We tested this hypothesis using spined toads (Bufo spinosus), an amphibian found in a variety of habitats, from AMPA-exposed agricultural lands to AMPA-free forested areas. We predicted that the offspring of individuals originating from agricultural habitats would develop AMPA-resistance - and be less prone to develop adverse effects from- AMPA exposure. To investigate this question, we performed a common garden brood-rearing experiment. The embryos and larvae of 40 spined toad pairs captured in agricultural and forest ponds were exposed either to an environmental relevant concentration of AMPA (0.4 μg L−1) or to control conditions (n = 8160 embryos, n = 240 tadpoles). We monitored development durations, developmental abnormalities and morphology, measured across key developmental stages. Although we observed significant effects of AMPA on fitness parameters in each group, these effects were not exacerbated in individuals from AMPA-free habitats. We suggest that temporal and/or spatial dynamics of contamination, as well as gene flow between exposed and preserved populations, may hinder adaptive divergence between populations. Yet, we show strong adverse effects of AMPA exposure at early developmental stages. AMPA could therefore be one of the numerous causes of declining wild amphibian populations.

Retinoschisin Deficiency Induces Persistent Aberrant Waves of Activity Affecting Neuroglial Signaling in the Retina.

Genetic disorders that present during development make treatment strategies particularly challenging because there is a need to disentangle primary pathophysiology from downstream dysfunction caused at key developmental stages. To provide a deeper insight into this question, we studied a mouse model of X-linked juvenile retinoschisis, an early-onset inherited condition caused by mutations in the Rs1 gene encoding retinoschisin (RS1) and characterized by cystic retinal lesions and early visual deficits. Using an unbiased approach in expressing the fast intracellular calcium indicator GCaMP6f in neuronal, glial, and vascular cells of the retina of RS1-deficient male mice, we found that initial cyst formation is paralleled by the appearance of aberrant spontaneous neuroglial signals as early as postnatal day 15, when eyes normally open. These presented as glutamate-driven wavelets of neuronal activity and sporadic radial bursts of activity by Müller glia, spanning all retinal layers and disrupting light-induced signaling. This study confers a role to RS1 beyond its function as an adhesion molecule, identifies an early onset for dysfunction in the course of disease, establishing a potential window for disease diagnosis and therapeutic intervention.SIGNIFICANCE STATEMENT Developmental disorders make it difficult to distinguish pathophysiology due to ongoing disease from pathophysiology due to disrupted development. Here, we investigated a mouse model for X-linked retinoschisis, a well defined monogenic degenerative disease caused by mutations in the Rs1 gene, which codes for the protein retinoschisin. We evaluated the spontaneous activity of explanted retinas lacking retinoschisin at key stages of development using the unbiased approach of ubiquitously expressing GCaMP6f in all retinal neurons, vasculature, and glia. In mice lacking RS1, we found that an array of novel phenotypes, which present around eye opening, are linked to glutamatergic neurotransmission and affect visual processing. These data identify a novel pathophysiology linked to RS1, and define a window where treatments might be best targeted.

Transcriptome Analysis and HPLC Profiling of Flavonoid Biosynthesis in Citrus aurantium L. during Its Key Developmental Stages

Simple SummaryFlavonoid is an important secondary metabolite with rich biological activity and pharmacological activity, and Citrus aurantium L. is of value in this regard. Citrus aurantium L. fruits are rich in flavonoid, but research information on flavonoid biosynthesis of Citrus aurantium L. is rare. Therefore, analysis of the key developmental stage and genes for flavonoid biosynthesis is essential for breeding Citrus aurantium L. varieties with high flavonoid content. Here, we report the profile of flavonoid and key flavonoid biosynthesis genes in the growth period of Citrus aurantium L. based on transcriptome analysis. We found that total flavonoid content decreased gradually during the fruit development stage, and that neohesperidin was the main flavonoid in the early development stage but with the progression of the development stage, naringin content increased rapidly and became the main flavonoid component. In addition, the key genes related to flavonoid biosynthesis in Citrus aurantium L. were identified. These results will lay the foundation for the mechanism underlying flavonoid biosynthesis in Citrus aurantium L. fruits.Citrus aurantium L. (sour orange) is a significant Chinese medicinal and fruit crop rich in flavonoids. However, the pathways and genes involved in flavonoid biosynthesis at the key developmental stages of Citrus aurantium L. are not fully understood. This study found that the total flavonoid concentration gradually decreased as the fruit developed. Additionally, it showed that neohesperidin was the main flavonoid in the early stages of sour orange fruit development. However, as the development stage progressed, naringin content increased rapidly and emerged as the main flavonoid component. From 27 cDNA libraries, RNA sequencing yielded 16.64 billion clean bases, including 8989 differentially expressed genes. We identified 74 flavonoid related unigenes mapped to the phenylalanine, tyrosine, and phenylpropanoid biosynthesis pathways. A total of 152 UDP-glucuronosyltransferase genes (UGTs) were identified from C. aurantium L. transcriptome database, in which 22 key flavonoid-correlated UGTs were divided into five main AtGT groups: E, G, I, L, M. We observed that the ethylene responsive factors (ERF) and myeloblastosis (MYB) family mainly regulated the key genes involved in flavonoid biosynthesis. Overall, our study generated extensive and detailed transcriptome data on the development of C. aurantium L. and characterized the flavonoid biosynthesis pattern during its fruit developmental stages. These results will benefit genetic modification or selection to increase the flavonoid content in sour oranges.

Comparative Transcriptome Analysis Identified Key Pathways and Genes Regulating Differentiated Stigma Color in Melon (Cucumis melo L.)

Stigma color is an important morphological trait in many flowering plants. Visual observations in different field experiments have shown that a green stigma in melons is more attractive to natural pollinators than a yellow one. In the current study, we evaluated the characterization of two contrasted melon lines (MR-1 with a green stigma and M4-7 with a yellow stigma). Endogenous quantification showed that the chlorophyll and carotenoid content in the MR-1 stigmas was higher compared to the M4-7 stigmas. The primary differences in the chloroplast ultrastructure at different developmental stages depicted that the stigmas of both melon lines were mainly enriched with granum, plastoglobulus, and starch grains. Further, comparative transcriptomic analysis was performed to identify the candidate pathways and genes regulating melon stigma color during key developmental stages (S1–S3). The obtained results indicated similar biological processes involved in the three stages, but major differences were observed in light reactions and chloroplast pathways. The weighted gene co-expression network analysis (WGCNA) of differentially expressed genes (DEGs) uncovered a “black” network module (655 out of 5302 genes), mainly corresponding to light reactions, light harvesting, the chlorophyll metabolic process, and the chlorophyll biosynthetic process, and exhibited a significant contribution to stigma color. Overall, the expression of five key genes of the chlorophyll synthesis pathway—CAO (MELO03C010624), CHLH (MELO03C007233), CRD (MELO03C026802), HEMA (MELO03C011113), POR (MELO03C016714)—were checked at different stages of stigma development in both melon lines using quantitative real time polymerase chain reaction (qRT-PCR). The results exhibited that the expression of these genes gradually increased during the stigma development of the MR-1 line but decreased in the M4-7 line at S2. In addition, the expression trends in different stages were the same as RNA-seq, indicating data accuracy. To sum up, our research reveals an in-depth molecular mechanism of stigma coloration and suggests that chlorophyll and related biological activity play an important role in differentiating melon stigma color.