Function Of Developmental Stage Research Articles

Peer cybervictimization and cyberaggression as a function of developmental stage during adolescence: A preliminary study

Peer cybervictimization and cyberaggression are educational and social concerns which have been extensively studied during adolescence but there is less research conducted specifically across all stages of adolescence (early, middle, late, and emerging adulthood). The objective was to analyse the prevalence of cybervictimization and cyberaggression, the roles, and the associated behaviors, depending on the stages of adolescence. The sample was composed of 7295 non-university Spanish adolescents, between 11 and 22 years old from 47 schools. The frequency of cybervictimization and cyberaggression was 22.5 % and 15 %, respectively. The highest frequencies are found in late adolescence and the lowest in emerging adulthood. Mainly, involvement increases from early to late adolescence and decreases in emerging adulthood. The magnitude of cybervictimization and cyberaggression behaviors for the roles of pure cybervictim and pure cyberaggressor is similar through all stages.

Microtransplantation of Postmortem Native Synaptic mGluRs Receptors into Xenopus Oocytes for Their Functional Analysis.

Metabotropic glutamate receptors (mGluRs) are membrane receptors that play a central role in the modulation of synaptic transmission and neuronal excitability and whose dysregulation is implicated in diverse neurological disorders. Most current understanding about the electrophysiological properties of such receptors has been determined using recombinant proteins. However, recombinant receptors do not necessarily recapitulate the properties of native receptors due to the lack of obligated accessory proteins, some of which are differentially expressed as function of developmental stage and brain region. To overcome this limitation, we sought to microtransplant entire synaptosome membranes from frozen rat cortex into Xenopus oocytes, and directly analyze the responses elicited by native mGluRs. We recorded ion currents elicited by 1 mM glutamate using two electrodes voltage clamp. Glutamate produced a fast ionotropic response (6 ± 0.3 nA) in all microtransplanted oocytes (n = 218 oocytes) and a delayed oscillatory response (52 ± 7 nA) in 73% of them. The participation of Group 1 mGluRs was confirmed by the presence of metabotropic oscillations during the administration of (±)-1-Aminocyclopentane-trans-1,3-dicarboxylic acid (ACPD; Group 1 mGluR agonist), and the absence of oscillations during co-administration of N-(1-adamantyl)quinoxaline-2-carboxamide (NPS 2390; Group 1 mGluR antagonist). Since both mGluR1 and mGluR5 belong to Group 1 mGluRs, further investigation revealed that mGluR1 antagonism with LY 456236 has little effect on metabotropic oscillations, while mGluR5 antagonism with 100 µM AZD 9272 has significant reduction of metabotropic currents elicited by ACPD and glutamate. We confirmed the expression of mGluR1 and mGluR5 in native synaptosomes by immunoblots, both of which are enhanced when compared to their counterpart proteins in rat cortex tissue lysates. Finally, these results demonstrate the merit of using microtransplantation of native synaptosomes for the study of mGluRs and the contribution of mGluR5 to the metabotropic glutamate signaling, providing a better tool for the understanding of the role of these receptors in neurological disorders.

Design and validation of a modular micro-robotic system for the mechanical characterization of soft tissues

The mechanical properties of tissues are critical design parameters for biomaterials and regenerative therapies seeking to restore functionality after disease or injury. Characterizing the mechanical properties of native tissues and extracellular matrix throughout embryonic development helps us understand the microenvironments that promote growth and remodeling, activities critical for biomaterials to support. The mechanical characterization of small, soft materials like the embryonic tissues of the mouse, an established mammalian model for development, is challenging due to difficulties in handling minute geometries and resolving forces of low magnitude. While uniaxial tensile testing is the physiologically relevant modality to characterize tissues that are loaded in tension in vivo, there are no commercially available instruments that can simultaneously measure sufficiently low tensile force magnitudes, directly measure sample deformation, keep samples hydrated throughout testing, and effectively grip minute geometries to test small tissues. To address this gap, we developed a micromanipulator and spring system that can mechanically characterize small, soft materials under tension. We demonstrate the capability of this system to measure the force contribution of soft materials, silicone, fibronectin sheets, and fibrin gels with a 5 nN – 50 µN force resolution and perform a variety of mechanical tests. Additionally, we investigated murine embryonic tendon mechanics, demonstrating the instrument can measure differences in mechanics of small, soft tissues as a function of developmental stage. This system can be further utilized to mechanically characterize soft biomaterials and small tissues and provide physiologically relevant parameters for designing scaffolds that seek to emulate native tissue mechanics. Statement of significanceThe mechanical properties of cellular microenvironments are critical parameters that contribute to the modulation of tissue growth and remodeling. The field of tissue engineering endeavors to recapitulate these microenvironments in order to construct tissues de novo. Therefore, it is crucial to uncover the mechanical properties of the cellular microenvironment during tissue formation. Here, we present a system capable of acquiring microscale forces and optically measuring sample deformation to calculate the stress-strain response of soft, embryonic tissues under tension, and easily adaptable to accommodate biomaterials of various sizes and stiffnesses. Altogether, this modular system enables researchers to probe the unknown mechanical properties of soft tissues throughout development to inform the engineering of physiologically relevant microenvironments.

Temperature affects transition timing and phenotype between key developmental stages in white sturgeon Acipenser transmontanus yolk-sac larvae

Temperature differentially influences developmental trajectories of traits during early life stages that can affect survival and recruitment. Experiments were conducted to quantify temperature-induced developmental responses of White Sturgeon (Acipenser transmontanus) yolk-sac larvae (YSL) reared at temperatures encountered across the species’ range (12.5, 14.0, 15.5, and 17.0 °C). We quantified effects of temperature on timing of transitions between sequential developmental stages from hatch to initiation of exogenous feeding. Rate of development significantly increased at warmer compared to cooler temperatures; no significant difference was observed between 15.5 and 17.0 °C or 12.5 and 14.0 °C. When standardized by relative timing of development (RTi), developmental rate was not significantly different among treatments. Morphological traits (total length; body area; yolk-sac area; head area; gill filament area; mouth area; pectoral fin area) were measured daily, though only data for YSL reared at 12.5 and 17.0 °C was used to quantify phenotypic variation. Morphological traits (excluding yolk-sac area) were generally larger 48+ hours post hatch for YSL reared at 17. 0 °C compared to 12.5 °C. In contrast, these same traits, with the exception of gill filament area, were larger in 12.5 °C reared YSL when considered as a function of developmental stage. These opposing results suggest trade-offs associated with allocating resources to a particular trait depended on rearing temperature. Our results provide the ability to estimate timing of critical early life stages (i.e., hatch, emergence) as a function of temperature which is an important management tool to understand how early life development contributes to recruitment processes and adaptability in thermally altered systems.

Emerging roles for carbonic anhydrase in mesophyll conductance and photosynthesis.

Carbonic anhydrase (CA) is an abundant protein in most photosynthesizing organisms and higher plants. This review paper considers the physiological importance of the more abundant CA isoforms in photosynthesis, through their effects on CO2 diffusion and other processes in photosynthetic organisms. In plants, CA has multiple isoforms in three different families (α, β and γ) and is mainly known to catalyze the CO2 equilibrium. This reversible conversion has a clear role in photosynthesis, primarily through sustaining the CO2 concentration at the site of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). Despite showing the same major reaction mechanism, the three main CA families are evolutionarily distinct. For different CA isoforms, cellular localization and total gene expression as a function of developmental stage are predicted to determine the role of each family in relation to the net assimilation rate. Reaction-diffusion modeling and observational evidence support a role for CA activity in reducing resistance to CO2 diffusion inside mesophyll cells by facilitating CO2 transfer in both gas and liquid phases. In addition, physical and/or biochemical interactions between CAs and other membrane-bound compartments, for example aquaporins, are suggested to trigger a CO2 -sensing response by stomatal movement. In response to environmental stresses, changes in the expression level of CAs and/or stimulated deactivation of CAs may correspond with lower photosynthetic capacity. We suggest that further studies should focus on the dynamics of the relationship between the activity of CAs (with different subcellular localization, abundance and gene expression) and limitations due to CO2 diffusivity through the mesophyll and supply of CO2 to photosynthetic reactions.

Asymmetric developmental change regarding the effect of reward and punishment on response inhibition

Reward and punishment influence inhibitory performance, but developmental changes in these effects are not well understood. Our aim was to understand the effects of potential reward gains and losses (as indices of reward and punishment) on response inhibition among children and adolescents. We conducted financial and non-financial go/no-go tasks with 40 boys (8- to 15-year-olds). Participants gained or lost money depending on their performance on the financial task, and score rankings were compared to participants on the non-financial task. We found that adolescents’ inhibitory control, as reflected in their reaction times when they made inhibitory errors, was lower in the reward-present condition than in the reward-absent condition, although accuracy was higher when the reward was available for all participants. Additionally, inhibitory control, specifically among adolescents, was higher for financial feedback than for non-financial feedback. These results suggest that the effects of reward and feedback type on motor impulsivity differ as a function of developmental stage. We discuss the theoretical implications of the present findings in terms of the interaction between emotional feedback and response inhibition among children and adolescents.

Novel insights from actigraphy: Anxiety is associated with sleep quantity but not quality during childhood.

Anxiety and sleep function change dynamically across development, and sleep dysfunction has emerged as a correlate and predictor of anxiety in pediatric clinical samples. Despite this, previous research has not investigated how associations between qualitative and quantitative measures of sleep function change with anxiety across development, specifically from childhood to adolescence. The present study used actigraphy collection to examine whether associations between quantitative and qualitative sleep function and anxiety differed as a function of developmental stage in a community pediatric sample (8-17 years old; N = 92). Age moderated the association between anxiety and sleep quantity, but not sleep quality. Contrary to hypotheses, higher anxiety was related to increased sleep for children, but not adolescents. Results suggest age-related changes in the association between sleep function and anxiety across development, with implications for targeting sleep-related interventions for youth with anxiety.

Reference gene identification and validation for quantitative real-time PCR studies in developing Xenopus laevis

Reference genes are essential for gene expression analysis when using real-time quantitative PCR (RT-qPCR). Xenopus laevis is a popular amphibian model for studying vertebrate embryogenesis and development. Further, X. laevis is ideal for studying thyroid signaling due to its thyroid dependent metamorphosis, a stage comparable to birth in humans. When using PCR based studies, a primary concern is the choice of reference genes. Commonly used references are eef1a1, odc1, rpl8, and actnB, although there is a lack of ad hoc reference genes for X. laevis. Here, we used previously published RNA-seq data on different X. laevis stages and identified the top 14 candidate genes with respect to their expression levels as a function of developmental stage and degree of variation. We further evaluated the stability of these and other candidate genes using RT-qPCR on various stages including the unfertilised eggs, whole embryos during early development and brains during late development. We used four different statistical software packages: deltaCT, geNorm, NormFinder and BestKeeper. We report optimized reference gene pair combinations for studying development (early whole embryos), brains at later stages (metamorphosis and adult), and thyroid signalling. These reference gene pairs are suitable for studying different aspects of X. laevis development and organogenesis.

Phosphorylation Dynamics Dominate the Regulated Proteome during Early Xenopus Development

The earliest stages of animal development are largely controlled by changes in protein phosphorylation mediated by signaling pathways and cyclin-dependent kinases. In order to decipher these complex networks and to discover new aspects of regulation by this post-translational modification, we undertook an analysis of the X. laevis phosphoproteome at seven developmental stages beginning with stage VI oocytes and ending with two-cell embryos. Concurrent measurement of the proteome and phosphoproteome enabled measurement of phosphosite occupancy as a function of developmental stage. We observed little change in protein expression levels during this period. We detected the expected phosphorylation of MAP kinases, translational regulatory proteins, and subunits of APC/C that validate the accuracy of our measurements. We find that more than half the identified proteins possess multiple sites of phosphorylation that are often clustered, where kinases work together in a hierarchical manner to create stretches of phosphorylated residues, which may be a means to amplify signals or stabilize a particular protein conformation. Conversely, other proteins have opposing sites of phosphorylation that seemingly reflect distinct changes in activity during this developmental timeline.

Screening and identification of major phytochemical compounds in seeds, sprouts and leaves of Tuscan black kale Brassica oleracea (L.) ssp acephala (DC) var. sabellica L.

We report the spectrophotometric determination of total polyphenols, flavonoids, glucosinolates and antioxidant activity in seeds, seedlings and leaves of Tuscan black kale. The highest content of phytochemicals was observed in 10 days sprouts and antioxidant activity was maximum in 2, 4 days seedlings. Identification and characterisation of phytochemicals were performed by mass spectrometry (MS), high resolution and tandem MS with electrospray ionisation mode. Low-molecular-weight metabolites were evidenced in seeds while metabolites at high m/z range were detected in cotyledons and leaves. MS spectra evidenced different phenolic compounds (flavonoid caffeoyl glucose, hydroxycinnamic acid sinapine) and glucosinolates (glucoerucin, glucobrassicin and glucoraphanin) in function of developmental stage; galactolipids ω3 and ω6 were observed in leaves. Identification of stages with the highest phytochemicals content encourages the consumption of black kale sprouts and young leaves. Our research can support food and pharmaceutical industries for production of health promoting products from black kale.

Doublesex alters aggressiveness as a function of social context and sex in the polyphenic beetle Onthophagus taurus

Despite sharing nearly the same genome, individuals within the same species can vary drastically in both morphology and behaviour as a function of developmental stage, sex or developmental plasticity. Thus, regulatory processes must exist that enable the stage-, sex- or environment-specific expression of traits and their integration during ontogeny, yet exactly how trait complexes are co-regulated and integrated is poorly understood. In this study, we explore the developmental genetic basis of the regulation and integration of environment-dependent sexual dimorphism in behaviour and morphology in the horn-polyphenic dung beetle Onthophagus taurus through the experimental manipulation of the transcription factor doublesex (dsx). The gene dsx plays a profound role in the developmental regulation of morphological differences between sexes as well as alternative male morphs by inhibiting horn formation in females but enabling nutrition-responsive horn growth in males. Specifically, we investigated whether experimental downregulation of dsx expression affects male and female aggressive and courtship behaviours in two social contexts: interactions between individuals of the same sex and interactions between males and females. We find that dsx downregulation significantly alters aggressiveness in both males and females, yet does so differently for both sexes as a function of social context: dsxRNAi males exhibited elevated aggression towards males but showed reduced aggression towards females, whereas dsxRNAi females became more aggressive towards males, while their aggressiveness towards other females was unaffected. Moreover, we document unexpectedly high levels of female aggression independent of dsx treatment in both wild-type and control-injected individuals. Lastly, we found no effects of dsxRNAi on courtship and mating behaviours. We discuss the role of dsx in the regulation of sex-specific and plastic behaviours, the unexpectedly high levels of aggression of hornless dsxRNAi males in relation to the well-established description of the hornless sneaker phenotype and the potential ecological function of high female aggression.

Whole Transcriptome Analysis of Notochord-Derived Cells during Embryonic Formation of the Nucleus Pulposus

Recapitulation of developmental signals represents a promising strategy for treating intervertebral disc degeneration. During development, embryonic notochord-derived cells (NDCs) are the direct progenitors of cells that populate the adult nucleus pulposus (NP) and are an important source of secreted signaling molecules. The objective of this study was to define global gene expression profiles of NDCs at key stages of embryonic disc formation. NDCs were isolated from Shh-cre;ROSA:YFP mice at embryonic day 12.5 and postnatal day 0, representing opposite ends of the notochord to NP transformation. Differences in global mRNA abundance across this developmental window were established using RNA-Seq. Protein expression of selected molecules was confirmed using immunohistochemistry. Principal component analysis revealed clustering of gene expression at each developmental stage with more than 5000 genes significantly differentially expressed between E12.5 and P0. There was significantly lower mRNA abundance of sonic hedgehog pathway elements at P0 vs E12.5, while abundance of elements of the transforming growth factor-beta and insulin-like growth factors pathways, and extracellular matrix components including collagen 6 and aggrecan, were significantly higher at P0. This study represents the first transcriptome-wide analysis of embryonic NDCs. Results suggest signaling and biosynthesis of NDCs change dramatically as a function of developmental stage.

Gene Regulation in Developing Chloroplasts Disentangled.

Gene Regulation in Developing Chloroplasts Disentangled.

Differential thyroid hormone sensitivity of fast cycling progenitors in the neurogenic niches of tadpoles and juvenile frogs

Adult neurogenesis occurs in neural stem cell (NSC) niches where slow cycling stem cells give rise to faster cycling progenitors. In the adult mouse NSC niche thyroid hormone, T3, and its receptor TRα act as a neurogenic switch promoting progenitor cell cycle completion and neuronal differentiation. Little is known about whether and how T3 controls proliferation of differentially cycling cells during xenopus neurogenesis. To address this question, we first used Sox3 as a marker of stem cell and progenitor populations and then applied pulse-chase EdU/IdU incorporation experiments to identify Sox3-expressing slow cycling (NSC) and fast cycling progenitor cells. We focused on the lateral ventricle of Xenopus laevis and two distinct stages of development: late embryonic development (pre-metamorphic) and juvenile frogs (post-metamorphic). These stages were selected for their relatively stable thyroid hormone availability, either side of the major dynamic phase represented by metamorphosis. TRα expression was found in both pre and post-metamorphic neurogenic regions. However, exogenous T3 treatment only increased proliferation of the fast cycling Sox3+ cell population in post-metamorphic juveniles, having no detectable effect on proliferation in pre-metamorphic tadpoles. We hypothesised that the resistance of proliferative cells to exogenous T3 in pre-metamorphic tadpoles could be related to T3 inactivation by the inactivating Deiodinase 3 enzyme. Expression of dio3 was widespread in the tadpole neurogenic niche, but not in the juvenile neurogenic niche. Use of a T3-reporter transgenic line showed that in juveniles, T3 had a direct transcriptional effect on rapid cycling progenitors. Thus, the fast cycling progenitor cells in the neurogenic niche of tadpoles and juvenile frogs respond differentially to T3 as a function of developmental stage.

Actin cytoskeleton contributes to the elastic modulus of embryonic tendon during early development.

Tendon injuries are common and heal poorly. Strategies to regenerate or replace injured tendons are challenged by an incomplete understanding of normal tendon development. Our previous study showed that embryonic tendon elastic modulus increases as a function of developmental stage. Inhibition of enzymatic collagen crosslink formation abrogated increases in tendon elastic modulus at late developmental stages, but did not affect increases in elastic modulus of early stage embryonic tendons. Here, we aimed to identify potential contributors to the mechanical properties of these early stage embryonic tendons. We characterized tendon progenitor cells in early stage embryonic tendons, and the influence of actin cytoskeleton disruption on tissue elastic modulus. Cells were closely packed in embryonic tendons, and did not change in density during early development. We observed an organized network of actin filaments that seemed contiguous between adjacent cells. The actin filaments exhibited a crimp pattern with a period and amplitude that matched the crimp of collagen fibers at each developmental stage. Chemical disruption of the actin cytoskeleton decreased tendon tissue elastic modulus, measured by atomic force microscopy. Our results demonstrate that early developmental stage embryonic tendons possess a well organized actin cytoskeleton network that contributes significantly to tendon tissue mechanical properties.

Lysyl oxidase-mediated collagen crosslinks may be assessed as markers of functional properties of tendon tissue formation

Mechanical property elaboration of engineered tissues is often assumed on the basis of gene and protein characterizations, rather than mechanical testing. However, we recently demonstrated that mechanical properties are not consistently correlated with matrix content and organization during embryonic tissue development. Based on this, mechanical properties should be assessed independently during natural or engineered tissue formation. Unfortunately, mechanical testing is destructive, and thus alternative means of assessing these properties are desirable. In this study, we examined lysyl oxidase (LOX)-mediated crosslinks as markers for mechanical properties during embryonic tendon formation and the potential to detect them non-destructively. We used tandem mass spectrometry (LC-MS/MS) to quantify changes in hydroxylysyl pyridinoline (HP) and lysyl pyridinoline (LP) crosslink density in embryonic chick tendon as a function of developmental stage. In addition, we assessed a multiphoton imaging approach that exploits the natural fluorescence of HP and LP. With both techniques, we quantified crosslink density in normal and LOX-inhibited tendons, and correlated measurements with mechanical properties. HP and LP crosslink density varied as a function of developmental stage, with HP-to-dry mass ratio correlating highly to elastic modulus, even when enzymatic crosslink formation was inhibited. Multiphoton optical imaging corroborated LC-MS/MS data, identifying significant reductions in crosslink density from LOX inhibition. Taken together, crosslink density may be useful as a marker of tissue mechanical properties that could be assessed with imaging non-destructively and perhaps non-invasively. These outcomes could have significant scientific and clinical implications, enabling continuous and long-term monitoring of mechanical properties of collagen-crosslinked tissues or engineered constructs.

Embryonic mechanical and soluble cues regulate tendon progenitor cell gene expression as a function of developmental stage and anatomical origin

Stem cell-based engineering strategies for tendons have yet to yield a normal functional tissue, due in part to a need for tenogenic factors. Additionally, the ability to evaluate differentiation has been challenged by a lack of markers for differentiation. We propose to inform tendon regeneration with developmental cues involved in normal tissue formation and with phenotypic markers that are characteristic of differentiating tendon progenitor cells (TPCs). Mechanical forces, fibroblast growth factor (FGF)-4 and transforming growth factor (TGF)-β2 are implicated in embryonic tendon development, yet the isolated effects of these factors on differentiating TPCs are unknown. Additionally, developmental mechanisms vary between limb and axial tendons, suggesting the respective cell types are programmed to respond uniquely to exogenous factors. To characterize developmental cues and benchmarks for differentiation toward limb vs. axial phenotypes, we dynamically loaded and treated TPCs with growth factors and assessed gene expression profiles as a function of developmental stage and anatomical origin. Based on scleraxis expression, TGFβ2 was tenogenic for TPCs at all stages, while loading was for late-stage cells only, and FGF4 had no effect despite regulation of other genes. When factors were combined, TGFβ2 continued to be tenogenic, while FGF4 appeared anti-tenogenic. Various treatments elicited distinct responses by axial vs. limb TPCs of specific stages. These results identified tenogenic factors, suggest tendon engineering strategies should be customized for tissues by anatomical origin, and provide stage-specific gene expression profiles of limb and axial TPCs as benchmarks with which to monitor tenogenic differentiation of stem cells.

A Lifespan Developmental-Stage Approach to Tobacco and Other Drug Abuse Prevention.

At least by informal design, tobacco and other drug abuse prevention programs are tailored to human developmental stage. However, few papers have been written to examine how programming has been formulated as a function of developmental stage throughout the lifespan. In this paper, I briefly define lifespan development, how it pertains to etiology of tobacco and other drug use, and how prevention programming might be constructed by five developmental stages: (a) young child, (b) older child, (c) young teen, (d) older teen, and (e) adult (emerging, young-to-middle and older adult substages). A search of the literature on tobacco and other drug abuse prevention by developmental stage was conducted, and multiple examples of programs are provided for each stage. A total of 34 programs are described as examples of each stage (five-young children, 12-older children, eight-young teens, four-older teens, and five-adults). Implications for future program development research are stated. In particular, I suggest that programming continue to be developed for all stages in the lifespan, as opposed to focusing on a single stage and that developmentally appropriate features continues to be pursued to maximize program impact.

Urinary Erythropoietin Concentrations after Early Short-Term Infusion of High-Dose Recombinant Epo for Neuroprotection in Preterm Neonates

Background: High-dose recombinant human erythropoietin (rEpo) has first been administered in clinical trials for neuroprotection in very preterm neonates at high risk of brain injury and in (near-) term neonates with hypoxic-ischemic encephalopathy. However, recent trials in adults raised concerns about the safety of high-dose rEpo for neuro- and cardioprotection. Objectives: To evaluate the putative accumulation or renal leakage of Epo as a function of developmental stage after repetitive early short-term infusion of high-dose rEpo (3 × 3,000 U/kg within 42 h after birth; NCT00413946) for neuroprotection in very preterm infants. Methods: Epo concentrations were measured using the ELISA technique in the first two consecutive urine specimens after each rEpo infusion. Results: Renal Epo excretion was significantly higher in preterm infants with gestational ages <29 weeks than in more mature infants and reached up to 23% of the administered rEpo within 8 h after each infusion. The urinary Epo concentration did not increase after three repetitive infusions of high-dose rEpo. The ratio of urinary Epo to total protein concentrations was the same in infants with gestational ages <29 weeks and in those with gestational ages ≥29 weeks. Conclusions: Our data suggest that the higher renal Epo excretion in more immature infants may be attributed to a higher glomerular filtration leakage due to the lower maturation of the kidneys and argue against saturation kinetics after multiple doses of 3,000 U/kg rEpo. This information should be considered in future trials on the use of rEpo for neuroprotection in neonates.

Single-cell analysis of murine long-term hematopoietic stem cells reveals distinct patterns of gene expression during fetal migration.

BackgroundLong-term hematopoietic stem cells (LT-HSCs) migrate from the fetal liver (FL) to the fetal bone marrow (FBM) during development. Various adhesion and chemotactic receptor genes have been implicated in the migration of adult LT-HSCs. However, their role in the migration of fetal LT-HSCs is not clearly understood due, in part, to the rare number of these cells in fetal tissues, which preclude classical gene expression analysis. The aim of this study is to characterize the expression of migration related genes in fetal LT-HSC across different anatomical locations during development.Methodology/Principal FindingsWe isolated fetal LT-HSC from different developmental stages, as well as different anatomical locations, and performed single-cell multiplex RT-qPCR and flow cytometry analysis of eight molecules involved in adult LT-HSC migration. Our results show that the gene expression of the chemokine receptor Cxcr4 in LT-HSC varies across developmental microenvironments and times, while the cadherin Cdh2 (Ncad) and the calcium receptor Casr show higher gene expression and variability only in FBM at 17.5 days post coitum (dpc). The cadherin Cdh5 (Vecad) maintains high expression variability only during fetal development, while the integrin subunit Itga5 (α5) increases its variability after 14.5 dpc. The integrin subunits Itga4 (α4) and Itgal (Lfa1), as well as the selectin ligand Selplg (Psgl1), did not show differences in their expression in single LT-HSCs irrespective of the developmental times or anatomical microenvironments studied.Conclusions/SignificanceOur data demonstrate that the expression pattern of phenotypically identical, single LT-HSCs fluctuates as a function of developmental stage and anatomical microenvironment. This is the first exhaustive gene expression comparison of migration-related molecules in fetal tissues across developmental times, enhancing the understanding of LT-HSC migration fate decisions during development.