Developmental Time Frame Research Articles

Loss of C1q alters the auditory brainstem response.

Neural circuits in the auditory brainstem compute interaural time and intensity differences used to determine the locations of sound sources. These circuits display features that are specialized for these functions. The projection from the ventral cochlear nucleus (VCN) to the medial nucleus of the trapezoid (MNTB) body travels along highly myelinated fibers and terminates in the calyx of Held. This monoinnervating synapse emerges during development as multiple inputs are eliminated. We previously demonstrated that elimination of microglia with a colony stimulating factor-1 inhibitor results in impaired synaptic pruning so that multiple calyceal terminals reside on principal cells of MNTB. This inhibitor also resulted in impaired auditory brainstem responses (ABRs), with elevated thresholds and increased peak latencies. Loss of the microglial fractalkine receptor, CX3CR1, decreased peak latencies in the ABR. The mechanisms underlying these effects are not known. One prominent microglial signaling pathway involved in synaptic pruning and plasticity during development and aging is the C1q-initiated compliment cascade. Here we investigated the classical complement pathway initiator, C1q, in auditory brainstem maturation. We found that C1q expression is detected in the MNTB by the first postnatal week. C1q levels increased with age and were detected within microglia and surrounding the soma of MNTB principal neurons. Loss of C1q did not affect microglia-dependent calyceal pruning. Excitatory and inhibitory synaptic markers in the MNTB and LSO were not altered with C1q deletion. ABRs showed that C1q KO mice had normal hearing thresholds but shortened peak latencies. Altogether this study uncovers the developmental time frame of C1q expression in the sound localization pathway and shows a subtle functional consequence of C1q knockdown.

P-208 To assess or not to assess: reconsidering day 3 embryo quality in planned freeze-all blastocyst cycles

Abstract Study question Should day 3 embryo quality assessment be consider in planned freeze-all blastocyst cycles? Summary answer Day 3 embryo assessment may be unnecessary in planned freeze-all blastocyst cycles. What is known already Day 3 embryo quality is a predictor of in vitro fertilizastion (IVF) success rates in cleavage stage embryo transfer. However, the association between day 3 embryo quality and live birth in blastocyst transfer policy is largely unknown. Study design, size, duration This retrospective study included 1074 frozen-thawed single day 5/6 blastocyst transfer between January 2019 to December 2022. Three groups were assessed depending on whether the transferred blastocyst derived from a top-quality, good-quality or poor-quality embryo at day 3. The analysis was conducted independently for each blastocyst quality group (top, good, and poor) using multivariate logistic regression. We applied factor analysis of mixed data to reduce potential collinearity between covariates used in the model. Participants/materials, setting, methods All blastocysts included in this study were obtained from first ICSI freeze-all cycles. Cleavage stage and blastocyst evaluation were performed according to the Istanbul consensus. Cleavage and blastocysts stage were assessed between 67±0.5 (day-3), 115±0.5 (day-5) and 139±0.5 (day-6) hours post-insemination (hpi), respectively. Exclusion criteria were: i) ICSI-cycles performed with surgically retrieved or frozen sperm, (ii) gamete donation cycles, (iii) cryopreserved-thawed oocyte cycles, (iv) preimplantation genetic testing. The primary outcome was live birth rate. Main results and the role of chance After adjusting for the day of transfer (day 5 or day 6) and several confounding variables, no statistical differences in live birth were observed among same quality blastocysts derived from different day 3 embryo quality groups (top=A, good=B and poor=C). The analysis was stratified for different blastocyst quality groups: 1) top quality blastocyst [day 3 quality A =reference group; day 3 quality B= OR 1.04 95% CI (0.62 – 1.74), p value=0.871; day 3 quality C= OR 0.67 95% CI (0.34 – 1.33), p value= 0.254] 2) good quality blastocyst [A =reference group; B= OR 0.80 95% CI (0.49 – 1.30), p value=0.361; C= OR 0.74 95% CI (0.44 – 1.27), p value= 0.276] 3) poor quality blastocyst [A =reference group; B= OR 0.78 95% CI (0.29 – 2.10), p value=0.622; C= OR 0.80 95% CI (0.31 – 2.07), p value= 0.650]. Limitations, reasons for caution The study is limited by its retrospective design. Embryo assessment without time-lapse technology, to assess the blastocysts at the same developmental time-frame, is another limitation; although the static observation time very close and restricted to the Istanbul consensus guidelines may mitigate such a weakness. Wider implications of the findings Our findings showed that day 3 embryo quality assessment may be unnecessary in planned freeze-all blastocyst cycles. In the context of static embryo assessment, this can reduce the stress on embryos by avoiding unnecessary retrievals from incubators. Trial registration number not applicable

Identifying vulnerable mother-infant dyads: a psychometric evaluation of two observational coding systems using varying interaction periods.

Clinical services require feasible assessments of parent-infant interaction in order to identify dyads requiring parenting intervention. We assessed the reliability and predictive validity of two observational tools and tested whether briefer forms could be identified which retain acceptable psychometric properties over short observation periods. A stratified high-risk community sample of 250 mother-infant dyads from The Wirral Child Health and Development Study completed 7-min play-based interaction at 6-8 months. Film-footage was independently coded by two trained raters using PIIOS and NICHD-SECCYD systems. Incremental predictive validity was assessed from 3, 5 and 7 min observation to attachment outcomes (Strange Situation; 14 months) and infant mental health (BITSEA; 14 and 30 months). Excellent inter-rater reliability was evident at code and subscale level for each tool and observation period. Stability of within-rater agreement was optimal after 5 min observation. ROC analysis confirmed predictive (discriminant) validity (AUCs >0.70) to top decile age 2 mental health outcomes for PIIOS total score and a brief 3-item composite from NICHD-SECCYD (sensitivity, intrusiveness, positive regard; NICHD-3), but not to attachment outcomes. Logistic regression showed dyads rated at-risk for externalizing problems using NICHD-3 were also at significantly higher risk for insecurity at 14 months (OR = 2.7, p = 0.004). PIIOS total and NICHD-3 ratings from 5 min observation are both reliable and valid tools for use in clinical practice. Findings suggest NICHD-3 may have greater utility due to its comparative brevity to train and code, with suitability for use over a broader developmental time frame (3-24 months).

Advances in 3D tissue models for neural engineering: self-assembled versus engineered tissue models.

Neural tissue engineering has emerged as a promising field that aims to create functional neural tissue for therapeutic applications, drug screening, and disease modelling. It is becoming evident in the literature that this goal requires development of three-dimensional (3D) constructs that can mimic the complex microenvironment of native neural tissue, including its biochemical, mechanical, physical, and electrical properties. These 3D models can be broadly classified as self-assembled models, which include spheroids, organoids, and assembloids, and engineered models, such as those based on decellularized or polymeric scaffolds. Self-assembled models offer advantages such as the ability to recapitulate neural development and disease processes in vitro, and the capacity to study the behaviour and interactions of different cell types in a more realistic environment. However, self-assembled constructs have limitations such as lack of standardised protocols, inability to control the cellular microenvironment, difficulty in controlling structural characteristics, reproducibility, scalability, and lengthy developmental timeframes. Integrating biomimetic materials and advanced manufacturing approaches to present cells with relevant biochemical, mechanical, physical, and electrical cues in a controlled tissue architecture requires alternate engineering approaches. Engineered scaffolds, and specifically 3D hydrogel-based constructs, have desirable properties, lower cost, higher reproducibility, long-term stability, and they can be rapidly tailored to mimic the native microenvironment and structure. This review explores 3D models in neural tissue engineering, with a particular focus on analysing the benefits and limitations of self-assembled organoids compared with hydrogel-based engineered 3D models. Moreover, this paper will focus on hydrogel based engineered models and probe their biomaterial components, tuneable properties, and fabrication techniques that allow them to mimic native neural tissue structures and environment. Finally, the current challenges and future research prospects of 3D neural models for both self-assembled and engineered models in neural tissue engineering will be discussed.

Astrocyte ensheathment of calyx-forming axons of the auditory brainstem precedes accelerated expression of myelin genes and myelination by oligodendrocytes.

Early postnatal brain development involves complex interactions among maturing neurons and glial cells that drive tissue organization. We previously analyzed gene expression in tissue from the mouse medial nucleus of the trapezoid body (MNTB) during the first postnatal week to study changes that surround rapid growth of the large calyx of Held (CH) nerve terminal. Here, we present genes that show significant changes in gene expression level during the second postnatal week, a developmental timeframe that brackets the onset of airborne sound stimulation and the early stages of myelination. Gene Ontology analysis revealed that many of these genes are related to the myelination process. Further investigation of these genes using a previously published cell type-specific bulk RNA-Seq data set in cortex and our own single-cell RNA-Seq data set in the MNTB revealed enrichment of these genes in the oligodendrocyte lineage (OL) cells. Combining the postnatal day (P)6-P14 microarray gene expression data with the previously published P0-P6 data provided fine temporal resolution to investigate the initiation and subsequent waves of gene expression related to OL cell maturation and the process of myelination. Many genes showed increasing expression levels between P2 and P6 in patterns that reflect OL cell maturation. Correspondingly, the first myelin proteins were detected by P4. Using a complementary, developmental series of electron microscopy 3D image volumes, we analyzed the temporal progression of axon wrapping and myelination in the MNTB. By employing a combination of established ultrastructural criteria to classify reconstructed early postnatal glial cells in the 3D volumes, we demonstrated for the first time that astrocytes within the mouse MNTB extensively wrap the axons of the growing CH terminal prior to OL cell wrapping and compaction of myelin. Our data revealed significant expression of several myelin genes and enrichment of multiple genes associated with lipid metabolism in astrocytes, which may subserve axon wrapping in addition to myelin formation. The transition from axon wrapping by astrocytes to OL cells occurs rapidly between P4 and P9 and identifies a potential new role of astrocytes in priming calyceal axons for subsequent myelination.

Metabolic set points of mammalian neurodevelopment

The human nervous system matures over a protracted developmental time frame relative to other species.What sets the pace of maturation has remained a mystery. In a recent publication in Science, Iwata etal. unearth critical contributions of mitochondrial metabolism in setting the pace of species-specific corticogenesis.

Genesis of a functional astrocyte syncytium in the developing mouse hippocampus.

Astrocytes are increasingly shown to operate as an isopotential syncytium in brain function. Protoplasmic astrocytes acquire this ability to functionally go beyond the single-cell level by evolving into a spongiform morphology, cytoplasmically connecting into a syncytium, and expressing a high density of K+ conductance. However, none of these cellular/functional features exist in neonatal newborn astrocytes, which imposes a basic question of when a functional syncytium evolves in the developing brain. Our results show that the spongiform morphology of individual astrocytes and their spatial organization all reach stationary levels by postnatal day (P) 15 in the hippocampal CA1 region. Functionally, astrocytes begin to uniformly express a mature level of passive K+ conductance by P11. We next used syncytial isopotentiality measurement to monitor the maturation of the astrocyte syncytium. In uncoupled P1 astrocytes, the substitution of endogenous K+ by a Na+ -electrode solution ([Na+ ]p ) resulted in the total elimination of the physiological membrane potential (VM ), and outward K+ conductance as predicted by the Goldman-Hodgkin-Katz (GHK) equation. As more astrocytes are coupled to each other through gap junctions during development, the [Na+ ]p -induced loss of physiological VM and the outward K+ conductance is progressively compensated by the neighboring astrocytes. By P15, a stably established syncytial isopotentiality (-73 mV), and a fully compensated outward K+ conductance appeared in all [Na+ ]p -recorded astrocytes. Thus, in view of the developmental timeframe wherein a singular syncytium is anatomically and functionally established for intra-syncytium K+ equilibration, an astrocyte syncytium becomes fully operational at P15 in the mouse hippocampus.

Cognitive variations following exposure to childhood adversity: evidence from a pre-registered, longitudinal study

Different methodological approaches to studying the effects and timing of childhood adversity have been proposed and tested. While childhood adversity has primarily been operationalized through specificity (effects of individual adversity types) and cumulative risk (sum of all adversities reported by an individual) models, dimensional models (probeable through latent class and other cluster analyses) have recently gained traction given that it can overcome some of the limitations of the specificity and cumulative risk approaches. On the other hand, structured lifecourse modelling is a new statistical approach that examines the effects of the timing of adversity exposure on health outcomes by comparing sensitive periods and accumulation hypotheses. In this study, we apply these sets of methodological approaches and theoretical models to better understand the complex effects of childhood adversity on cognitive outcomes. We analysed data obtained from the Avon Longitudinal Study of Parents and Children for 2965 participants (Male=1125; Female=1840). This included parental report of 11 types of childhood adversity when participants were between 8 months and 8.7 years, and performance on inhibition, working memory and emotion recognition neurocognitive tasks when participants were 24 years of age (April 1, 1992-October 31, 2017). We used latent class analysis to classify the participants into subgroups, while we used Kruskal-Wallis test to examine differences in cognitive performance among the adversity subgroups. Additionally, to test whether sensitive period or accumulation models better explain the effects of childhood adversity on cognitive functioning, we carried out separate analyses using structured lifecourse modelling approaches. Latent class analysis showed evidence of 5 classes, namely: low adversity (71.6%), dysfunctional family (9.58%); parental deprivation (9.65%); family poverty (6.07%) and global adversity (3.1%). We observed group differences in cognitive performance among the adversity classes in an inhibition control task, χ2(4)=15.624, p=0.003 and working memory task, χ2(4)=15.986, p=0.003. Pairwise comparison tests showed that participants in the family poverty class performed significantly worse than those in the low adversity class, for the inhibition control task (p=0.007) while participants in the global adversity class significantly performed worse than participants in the low adversity class (p=0.026) and dysfunctional family class (p=0.034) on the working memory task. A further analysis revealed that the associations between each individual adversity type and cognitive outcomes were mostly consistent with the observed class performance in which they co-occurred. Follow-up analyses suggested that adversity during specific sensitive periods, namely very early childhood and early childhood, explained more variability in these observed associations, compared to the accumulation of adversities. These findings suggest that dimensional approaches e.g., latent class analysis or cluster analysis could be good alternatives to studying childhood adversity. Using latent class analysis for example, can help reveal the population distribution of co-occurring adversity patterns among participants who may be at the greatest health risk and thus, enable a targeted intervention. In addition, this approach could be used to investigate specific pathways that link adversity classes to different developmental outcomes that could further complement the specificity or cumulative risk approaches to adversity. On the other hand, findings from a separate analysis using structured lifecourse modelling approaches also highlight the vital developmental timeframes in childhood during which the impact of adversity exposure on cognitive outcomes is greatest, suggesting the need to provide comprehensive academic and mental health support to individuals exposed during those specific timeframes. T.N. received funding from Cambridge Trust (University of Cambridge).

Synaptic Development in Diverse Olfactory Neuron Classes Uses Distinct Temporal and Activity-Related Programs.

Developing neurons must meet core molecular, cellular, and temporal requirements to ensure the correct formation of synapses, resulting in functional circuits. However, because of the vast diversity in neuronal class and function, it is unclear whether or not all neurons use the same organizational mechanisms to form synaptic connections and achieve functional and morphologic maturation. Moreover, it remains unknown whether neurons united in a common goal and comprising the same sensory circuit develop on similar timescales and use identical molecular approaches to ensure the formation of the correct number of synapses. To begin to answer these questions, we took advantage of the Drosophila antennal lobe (AL), a model olfactory circuit with remarkable genetic access and synapse-level resolution. Using tissue-specific genetic labeling of active zones, we performed a quantitative analysis of synapse formation in multiple classes of neurons of both sexes throughout development and adulthood. We found that olfactory receptor neurons (ORNs), projection neurons (PNs), and local interneurons (LNs) each have unique time courses of synaptic development, addition, and refinement, demonstrating that each class follows a distinct developmental program. This raised the possibility that these classes may also have distinct molecular requirements for synapse formation. We genetically altered neuronal activity in each neuronal subtype and observed differing effects on synapse number based on the neuronal class examined. Silencing neuronal activity in ORNs, PNs, and LNs impaired synaptic development but only in ORNs did enhancing neuronal activity influence synapse formation. ORNs and LNs demonstrated similar impairment of synaptic development with enhanced activity of a master kinase, GSK-3β, suggesting that neuronal activity and GSK-3β kinase activity function in a common pathway. ORNs also, however, demonstrated impaired synaptic development with GSK-3β loss-of-function, suggesting additional activity-independent roles in development. Ultimately, our results suggest that the requirements for synaptic development are not uniform across all neuronal classes with considerable diversity existing in both their developmental time frames and molecular requirements. These findings provide novel insights into the mechanisms of synaptic development and lay the foundation for future work determining their underlying etiologies.SIGNIFICANCE STATEMENT Distinct olfactory neuron classes in Drosophila develop a mature synaptic complement over unique timelines and using distinct activity-dependent and molecular programs, despite having the same generalized goal of olfactory sensation.

The paradox of family support for young mothers: An interpretive phenomenology analysis

AbstractObjectiveWe sought to illuminant the lived experiences of becoming a mother during adolescence within the context of multigenerational family caregiving.BackgroundAdolescence is a developmental time frame during which identity formation is most salient and characterized by separation from parents (Erikson, 1968). Teenage childbearing, then, presents a unique scenario during which adolescent mothers and their families may need to renegotiate autonomy and caregiving roles.MethodQualitative methods, and interpretive phenomenological analysis, were utilized to explore the lived experiences of nine women who became mothers before the age of 19 years. Mothers were recruited from rural, Central Appalachian regions in the United States.ResultsWe identified the essence of young mothers' lived experiences regarding their receipt of caregiving during the transition to parenthood as an adolescent. Mothers equated instrumental and emotional support from family with their own “lovability” and interpreted support as indicative of their evolving competence as mothers.ConclusionsYoung motherhood appeared to be a developmental paradox in that their adolescent identity conflicted with their maternal identity.ImplicationsYoung mothers would benefit from multilevel interventions. Instead, practitioners might consider supporting whole families of young mothers by providing the means to create a healthy, supportive environment for the mother and her offspring.

Developmental co-occurrence of psychopathology dimensions in childhood.

Comorbidity between psychopathologies may be attributed to genetic and environmental differences between people as well as causal processes within individuals, where one pathology increases risk for another. Disentangling between-person (co)variance from within-person processes of psychopathology dimensions across childhood may shed light on developmental causes of comorbid mental health problems. Here, we aim to determine whether and to what extent directional relationships between psychopathology dimensions within-person, and between individuals within families, play a role in comorbidity. We conducted random intercepts cross-lagged panel model (RI-CLPM) analyses to unravel the longitudinal co-occurrence of child psychopathology dimensions, jointly estimating between-person and within-person processes from childhood to early adolescence (age 7-12). We further developed an extension of the model to estimate sibling effects within-family (wf-RI-CLPM). Analyses were separately conducted in two large population-based cohorts, TEDS and NTR, including parent-rated measures of child problem behaviours based on the SDQ and CBCL scales respectively. We found evidence for strong between-person effects underlying the positive intercorrelation between problem behaviours across time. Beyond these time-varying within-person processes accounted for an increasing amount of trait variance, within- and cross-trait, overtime in both cohorts. Lastly, by accommodating family level data, we found evidence for reciprocal directional influences within sib-pairs longitudinally. Our results indicate that within-person processes partly explain the co-occurrence of psychopathology dimensions across childhood, and within sib-pairs. Analyses provided substantive results on developmental processes underlying comorbidity in behavioural problems. Future studies should consider different developmental timeframes to shed more light on the processes contributing to developmental comorbidity.

Cellular and gene expression patterns associated with root bifurcation in Selaginella.

The roots of lycophytes branch through dichotomy or bifurcation, during which the root apex splits into two daughter roots. This is morphologically distinct from lateral root (LR) branching in the extant euphyllophytes, with LRs developing along the root axis at different distances from the apex. Although the process of root bifurcation is poorly understood, such knowledge can be important, because it may represent an evolutionarily ancient strategy that roots recruited to form new stem cells or meristems. In this study, we examined root bifurcation in the lycophyte Selaginella moellendorffii. We characterized an in vitro developmental time frame based on repetitive apex bifurcations, allowing us to sample different stages of dichotomous root branching and analyze the root meristem and root branching in S. moellendorffii at the microscopic and transcriptomic level. Our results showed that, in contrast to previous assumptions, initial cells (ICs) in the root meristem are mostly not tetrahedral but rather show an irregular shape. Tracking down the early stages of root branching argues for the occurrence of a symmetric division of the single IC, resulting in two apical stem cells that initiate root meristem bifurcation. Moreover, we generated a S. moellendorffii root branching transcriptome that resulted in the delineation of a subset of core meristem genes. The occurrence of multiple putative orthologs of meristem genes in this dataset suggests the presence of conserved pathways in the control of meristem and root stem cell establishment or maintenance.

Gynoecium and fruit development in Arabidopsis.

Flowering plants produce flowers and one of the most complex floral structures is the pistil or the gynoecium. All the floral organs differentiate from the floral meristem. Various reviews exist on molecular mechanisms controlling reproductive development, but most focus on a short time window and there has been no recent review on the complete developmental time frame of gynoecium and fruit formation. Here, we highlight recent discoveries, including the players, interactions and mechanisms that govern gynoecium and fruit development in Arabidopsis. We also present the currently known gene regulatory networks from gynoecium initiation until fruit maturation.

Gut microbiomes from Gambian infants reveal the development of a non-industrialized Prevotella-based trophic network

Distinct bacterial trophic networks exist in the gut microbiota of individuals in industrialized and non-industrialized countries. In particular, non-industrialized gut microbiomes tend to be enriched with Prevotella species. To study the development of these Prevotella-rich compositions, we investigated the gut microbiota of children aged between 7 and 37 months living in rural Gambia (616 children, 1,389 stool samples, stratified by 3-month age groups). These infants, who typically eat a high-fibre, low-protein diet, were part of a double-blind, randomized iron intervention trial (NCT02941081) and here we report the secondary outcome. We found that child age was the largest discriminating factor between samples and that anthropometric indices (collection time points, season, geographic collection site, and iron supplementation) did not significantly influence the gut microbiome. Prevotella copri, Faecalibacterium prausnitzii and Prevotella stercorea were, on average, the most abundant species in these 1,389 samples (35%, 11% and 7%, respectively). Distinct bacterial trophic network clusters were identified, centred around either P.stercorea or F.prausnitzii and were found to develop steadily with age, whereas P.copri, independently of other species, rapidly became dominant after weaning. This dataset, set within a critical gut microbial developmental time frame, provides insights into the development of Prevotella-rich gut microbiomes, which are typically understudied and are underrepresented in western populations.

Atypical core-periphery brain dynamics in autism.

The intrinsic function of the human brain is dynamic, giving rise to numerous behavioral subtypes that fluctuate distinctively at multiple timescales. One of the key dynamical processes that takes place in the brain is the interaction between core-periphery brain regions, which undergoes constant fluctuations associated with developmental time frames. Core-periphery dynamical changes associated with macroscale brain network dynamics span multiple timescales and may lead to atypical behavior and clinical symptoms. For example, recent evidence suggests that brain regions with shorter intrinsic timescales are located at the periphery of brain networks (e.g., sensorimotor hand, face areas) and are implicated in perception and movement. On the contrary, brain regions with longer timescales are core hub regions. These hubs are important for regulating interactions between the brain and the body during self-related cognition and emotion. In this review, we summarize a large body of converging evidence derived from time-resolved fMRI studies in autism to characterize atypical core-periphery brain dynamics and how they relate to core and contextual sensory and cognitive profiles.

Cells to Systems Screening for Thyroid Hormone Disrupting Chemicals in the Brain

Precisely timed and regulated thyroid hormone (TH) levels are essential for vertebrate development and metabolism. To detect environmental chemicals that disrupt TH action, we developed an integrated luciferase reporter cell line in rat pituitary GH3 cells that is TH responsive through activation of endogenous thyroid hormone receptors (TRs). High throughput screening as part of the Tox21 program identified several non-TH related positive hits as RXR agonists, a supposed “silent” heterodimer partner for TRs. These data showed RXR agonists can affect TR signaling at least in pituitary cells. We extended these results to our sensitive and specific in vivo model for TH action, Xenopus laevis metamorphosis. We previously demonstrated that RXR agonists like organotins and the pharmaceutical bexarotene, strongly potentiate (and RXR antagonists inhibit) TH induced metamorphic programs, at the morphological, cellular (e.g. apoptosis or proliferation), transgenic reporter gene levels, and transcriptomic responses in tadpole tails. We have now extended this analysis to include RNA-Seq experiments over distinct time points in the tadpole brain, a common target of TH in humans and frogs, revealing specific gene sets particularly affected by TH and RXR ligands working in concert. Very few genes were affected by RXR ligands alone. The remarkable overlap between the environmental toxicant tributyltin and the synthetic and specific RXR ligand bexarotene regulated transcriptomes provides strong evidence that they have a common molecular target in multiple tissues. We have also created germline mutations in all RXR family members in Xenopus tropicalis (alpha, beta and gamma), and both copies of the duplicated TR beta gene in Xenopus laevis via genome editing approaches. This will allow us to further investigate TR-RXR heterodimer function across tissues and developmental timeframes, and in response to known and suspected TR and RXR ligands. Our studies revealed an unanticipated degree of TR and RXR ligand interactions in vitro and in vivo, highlighting a surprising role of RXRs as avenues for TH endocrine disruption, including the brain.

Trabecular bone properties in the ilium of the Middle Paleolithic/Middle Stone Age Border Cave 3 Homo sapiens infant and the onset of independent gait

The Border Cave 3 (BC3) infant skeleton has been understudied, despite its importance as an example of a well-preserved and fairly complete immature skeleton of early Homo sapiens which potentially provides a rare window into various aspects of ontogenetic development, including locomotor activity (e.g., timing of gait events). Trabecular structure in the BC3 ilium was evaluated to investigate whether it matches that of an equivalently aged infant from a postindustrialized society. Microcomputed tomography (μCT) scans were acquired from the BC3 infant and from an ontogenetic series of 25 postindustrial infants that were divided into three age classes (ACs) ranging from neonates to toddlers (<36 months). All ilia were qualitatively compared and then digitally subdivided into 10 volumes of interest (VOIs) based on anatomical reference points. The VOIs were quantified and ontogenetic differences in trabecular structure were statistically evaluated. Across the comparative ontogenetic series, trabecular architectural properties overlapped in all regions. However, trabecular thickness increased significantly after the first year of life. The BC3 infant demonstrated generally similar trabecular structure to that observed in the age-equivalent postindustrial infants (AC2), including relatively strong development of the trabecular chiasma qualitatively. However, some interesting distinctions were observed in BC3, such as low strut thickness compared with infants from the postindustrial sample, that bear further exploration in future studies. Evaluation of only one individual from the Middle Stone Age (MSA), coupled with the relatively small comparative sample, limit our ability to distinguish more meaningful biological differences in trabecular structure throughout ontogeny from idiosyncratic characteristics. Nonetheless, results of this study extend ongoing research on infant locomotor and morphological development to archeological populations in the Middle Stone Age. Further cross-cultural studies consisting of larger comparative postindustrial samples may provide additional information on trabecular structure in the infant ilium during this important developmental timeframe.

LRIG1-Mediated Inhibition of EGF Receptor Signaling Regulates Neural Precursor Cell Proliferation in the Neocortex

Here, we ask how neural stem cells (NSCs) transition in the developing neocortex from a rapidly to a slowly proliferating state, a process required to maintain lifelong stem cell pools. We identify LRIG1, known to regulate receptor tyrosine kinase signaling in other cell types, as a negative regulator of cortical NSC proliferation. LRIG1 is expressed in murine cortical NSCs as they start to proliferate more slowly during embryogenesis and then peaks postnatally when they transition to give rise to a portion of adult NSCs. Constitutive or acute loss of Lrig1 in NSCs over this developmental time frame causes stem cell expansion due to increased proliferation. LRIG1 controls NSC proliferation by associating with and negatively regulating the epidermal growth factor receptor (EGFR). These data support a model in which LRIG1 dampens the stem cell response to EGFR ligands within the cortical environment to slow their proliferation as they transition to postnatal adult NSCs.

Altered Hippocampal Epigenetic Regulation Underlying Reduced Cognitive Development in Response to Early Life Environmental Insults

The hippocampus is involved in learning and memory and undergoes significant growth and maturation during the neonatal period. Environmental insults during this developmental timeframe can have lasting effects on brain structure and function. This study assessed hippocampal DNA methylation and gene transcription from two independent studies reporting reduced cognitive development stemming from early life environmental insults (iron deficiency and porcine reproductive and respiratory syndrome virus (PRRSv) infection) using porcine biomedical models. In total, 420 differentially expressed genes (DEGs) were identified between the reduced cognition and control groups, including genes involved in neurodevelopment and function. Gene ontology (GO) terms enriched for DEGs were associated with immune responses, angiogenesis, and cellular development. In addition, 116 differentially methylated regions (DMRs) were identified, which overlapped 125 genes. While no GO terms were enriched for genes overlapping DMRs, many of these genes are known to be involved in neurodevelopment and function, angiogenesis, and immunity. The observed altered methylation and expression of genes involved in neurological function suggest reduced cognition in response to early life environmental insults is due to altered cholinergic signaling and calcium regulation. Finally, two DMRs overlapped with two DEGs, VWF and LRRC32, which are associated with blood brain barrier permeability and regulatory T-cell activation, respectively. These results support the role of altered hippocampal DNA methylation and gene expression in early life environmentally-induced reductions in cognitive development across independent studies.

Replication Stress Response and CDKN1A Engagement Constrain Fetal Hematopoietic Stem Cell Pool Size in Fanconi Anemia

Bone marrow (BM) failure is the principal source of morbidity and mortality in Fanconi Anemia (FA) patients. Recessively inherited germline mutations in one of 25 genes lead to deficits by in a pathway central to DNA crosslink repair. Functionally, FA proteins protect adult hematopoietic stem cells (HSC) from p53 mediated apoptosis elicited by alkylating agents, a range of experimental inflammatory cues or aldehyde exposure. However, these mechanisms do not seem to account for depleted hematopoietic stem and progenitor cell pools in very young FA patients, or the spontaneous, non-apoptotic and p53-independent fetal HSC deficits observed in murine models. Building on our previous observation of a quantitatively constrained fetal HSC pool in FA mice (Fancd2-/-), the current experiments reveal the specific developmental timeframe for the onset of stem cell deficits during HSC expansion in the fetal liver (FL). Cell cycle studies using an EdU/BrdU pulse chase protocol reveal delays in S-phase entry and progression at E13.5. Building on the role of FA proteins (FANCM, FANCI and FANCD2) in countering experimental replication stress (RS) in cell line models, we reasoned that rapid rates of proliferation required during expansion in the FL may similarly confer RS on the FA HSC pool. Experiments in E13.5 FL HSC confirmed the predicted increase in single stranded DNA and accumulation of nuclear replication associated protein (pRpa), along with activation of pChk1, a critical cell cycle checkpoint in cells under RS. For comparison, pChk1 in unperturbed adult cells was no different between Fancd2-/- and WT. The data are also consistent with gains in RAD51 and alkaline comet assays we previously published (Yoon et al., Stem Cell Reports 2016). The cell cycle regulator Cdkn1a (p21) is considered a canonical downstream component of the p53 response in adult FA HSC, but it also performs p53 independent functions in the RS response that coincide with its role in the nucleus. Here, we observed an increase in nuclear localization of p21 in Fancd2-/- FL HSC. TGF-β is a critical developmental morphogen that regulates p21 activity, and TGF-β inhibitors can partially reverse adult FA HSC function along with suppression of NHEJ mediated DNA repair. To test regulation of p21 in fetal HSC under RS, we first treated WT FL HSC with aphidicolin to experimentally simulate RS and found that SD208, a small molecule TGF-β-R1 inhibitor, completely rescued the p21 nuclear localization. We then went on to demonstrate that pharmacological inhibition of TGF-β signaling also reversed the nuclear p21 translocation in FA FL HSC (under physiological RS) and functionally rescued the primitive myeloid progenitor colony formation (CFU-GEMM) in vitro. Altogether, our data show that HSC deficits in FA first emerge in the fetal liver, where rapid fetal expansion causes RS that elicits pChk1 activation and nuclear p21 translocation, which restrain cell cycle progression and act as principal mechanisms limiting fetal HSC pool size in FA. Our experiments suggest a central and p53-independent role for p21 in fetal FA HSC regulation. Detailed knowledge of the physiological role of FA proteins in fetal phenotype HSC has the potential to lead to new therapies that delay or rescue hematopoietic failure in FA patients. Disclosures No relevant conflicts of interest to declare.