Developmental maturation of the hematopoietic system controlled by a Lin28b-let-7-PRC1 axis

Hematopoiesis changes over life to meet the demands of maturation and aging. Here, we find that the definitive hematopoietic stem and progenitor cell (HSPC) compartment is remodeled from gestation into adulthood, a process regulated by the heterochronic Lin28b/let-7 axis. Native fetal and neonatal HSPCs distribute with a pro-lymphoid/erythroid bias with a shift toward myeloid output in adulthood. By mining transcriptomic data comparing juvenile and adult HSPCs and reconstructing coordinately activated gene regulatory networks, we uncover the Polycomb repressor complex 1 (PRC1) component Cbx2 as an effector of Lin28b/let-7 control of hematopoietic maturation. We find that juvenile Cbx2-/- hematopoietic tissues show impairment of B-lymphopoiesis and a precocious adult-like myeloid bias and that Cbx2/PRC1 regulates developmental timing of expression of key hematopoietic transcription factors. These findings define a novel mechanism of epigenetic regulation of HSPC output as a function of age with potential impact on age-biased pediatric and adult blood disorders.

Lin28b-Let-7-PRC1 Axis Guides Developmental Maturation of the Hematopoietic System

Polycomb group (PcG) proteins are a well-studied group of chromatin modifiers belonging to one of two distinct multi-protein complexes: Polycomb repressive complex 1 (PRC1) and PRC2. With definitive hematopoiesis, PRCs contribute to many aspects of fetal and adult blood formation. However, it is largely unknown how many of the age-specific effects of PRCs in hematopoiesis are regulated. Here, we show that the definitive hematopoietic stem and progenitor cell (HSPC) compartment is remodeled from the fetus to the neonate and into young adulthood coordinated with changes in mature blood cell output. This process is in part dependent on the PRC1 component Cbx2, which is regulated by the heterochronic Lin28b/let-7 axis. First, we quantified various population of definitive hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs) using midgestation fetal liver (FL, embryonic day 14.5 (E14.5)), newborn bone marrow (BM, postnatal day 0-1), or young adult (postnatal age 6 to 8 weeks) BM. The lymphoid biased multipotent progenitor 4 (MPP4, ~0.9-fold) declined as the mice matured and aged. We also found erythroid-biased MPP2 diminished (~0.7-fold) while myeloid-biased MPP3 increased (~1.7-fold) with maturation. Using isolated long-term (LT) HSCs from these three stages, we found that E14.5 FL (~8.0-fold) and neonatal LT-HSC (~4.0-fold) showed more rapid B-cell reconstitution compared to young adult LT-HSCs upon transplantation. We found that many of these effects were regulated by Lin28b/let-7. Next, we aimed to determine the downstream mediators of Lin28/let-7's effect on HSPCs maturation. By interrogating gene regulatory subnetworks differentially active across mouse HSPC maturation and mining these subnetworks for predicted let-7 target transcripts, we found Cbx2 enriched in E14.5 FL (P=0.003) and adult HSPCs ectopically expressing LIN28B relative to wild-type adult HSPCs. In cell-based assays, we confirmed that let-7 microRNAs directly regulated CBX2 protein levels. Thus, the Lin28b/let-7 axis governs CBX2 protein levels, leading us to hypothesize that this axis exerts its wide-ranging effects on hematopoietic maturation by regulating PRC1 by controlling Cbx2 levels. As CBX2's developmental stage-specific functions have not been investigated, we generated Cbx2-/-embryos and investigated definitive FL hematopoiesis. We observed skewing of myeloerythorid progenitors to an adult-like myeloid-predominant distribution in Cbx2-/- embryos (P=0.0002), and B-cells in Cbx2-/- neonatal spleens were diminished (P=0.04). We further examined this effect using transplanted Cbx2-/- MPP4 from E14.5 FL which resulted in a decreased donor derived B-lymphoid output compared to wild-type littermates (~0.7-fold). To understand the functional role of Cbx2/PRC1 in juvenile hematopoiesis, we next investigated the role of Cbx2 in maintaining histone H2A monoubiquitinylation (H2AK119Ub) - the histone modification placed by PRC1 - in FL HSPCs. In Cbx2-/- FL HSPCs, the global distribution of H2AK119Ub localization did not change, but several specific H2AK119Ub peaks were altered. We observed differential H2AK119Ub abundance associated with a candidate enhancer within the Erg gene, suggestive of control of Erg expression by Lin28b/let-7/Cbx2. We confirmed that this enhancer activated transcription from a minimal promoter (~8-fold). Erg expression was increased in perinatal spleens of Cbx2-/- mice compared to Cbx2+/+ littermates (~4-fold). Moreover, we found that Cbx2 could repress ERG expression as well as other master HSPC transcription factors. Overall, our findings show that the Lin28b/let-7-axis controls developmental stage-specific hematopoietic output through PRC1-mediated chromatin remodeling. These findings demonstrate a key mechanism by which HSPCs alter their properties during developmental maturation with relevance to age-skewed blood disorders. Disclosures No relevant conflicts of interest to declare.

Long-Distance Descending Commissural V0v Neurons Ensure Coordinated Swimming Movements Along The Body Axis in Larval Zebrafish

Developmental maturation occurs in slow swimming behavior in larval zebrafish; older larvae acquire the ability to perform slow swimming with keeping their head stable in the yaw dimension. A class of long-distance descending commissural excitatory V0v neurons, called MCoD neurons, are known to develop at later phase of neurogenesis, and participate in slow swimming in older larvae. This led to a hypothesis that MCoD neurons play a role to coordinate the activities of trunk muscles in the diagonal dimension (e.g., the rostral left and the caudal right) to produce S-shaped swimming form, which would contribute to the stability of the head. Here, we show that MCoD neurons indeed play this role. In larvae in which MCoD neurons were laser-ablated, swimming body form often became one-sided (C-shaped) bend with reduced appearance of normal S-shaped bend. With this change of swimming form, the MCoD-ablated larvae exhibited greater degree of head yaw displacement during slow swimming. The long-distance descending commissural V0v neurons are implicated in playing roles in diagonal interlimb coordination during walking in mice. Together with this, our study suggests that the long-distance descending commissural V0v neurons form an evolutionally conserved pathway in the spinal locomotor circuits that coordinates movements of the diagonal body/limb muscles.

Adherent but Not Suspension-Cultured Embryoid Bodies Develop into Laminated Retinal Organoids

Human induced pluripotent stem cells (iPSCs) are differentiated into three-dimensional (3D) retinal organoids to study retinogenesis and diseases that would otherwise be impossible. The complexity and low yield in current protocols remain a technical challenge, particularly for inexperienced personnel. Differentiation protocols require labor-intensive and time-consuming dissection of optic vesicles (OVs). Here we compare this method with a suspension method of developing retinal organoids. iPSCs were differentiated with standard protocols but the suspension-grown method omitted the re-plating of embryoid bodies and dissection of OVs. All other media and treatments were identical between developmental methods. Developmental maturation was evaluated with RT-qPCR and immunocytochemistry. Dissection- and suspension-derived retinal organoids displayed temporal biogenesis of retinal cell types. Differences in retinal organoids generated by the two methods of differentiation included temporal developmental and the organization of neural retina layers. Retinal organoids grown in suspension showed delayed development and disorganized retinal layers compared to the dissected retinal organoids. We found that omitting the re-plating of EBs to form OVs resulted in numerous OVs that were easy to identify and matured along a retinal lineage. While more efficient, the suspension method led to retinal organoids with disorganized retinal layers compared to those obtained using conventional dissection protocols.

Age-related mushroom body expansion in male sweat bees and bumble bees

A well-documented phenomenon among social insects is that brain changes occur prior to or at the onset of certain experiences, potentially serving to prime the brain for specific tasks. This insight comes almost exclusively from studies considering developmental maturation in females. As a result, it is unclear whether age-related brain plasticity is consistent across sexes, and to what extent developmental patterns differ. Using confocal microscopy and volumetric analyses, we investigated age-related brain changes coinciding with sexual maturation in the males of the facultatively eusocial sweat bee, Megalopta genalis, and the obligately eusocial bumble bee, Bombus impatiens. We compared volumetric measurements between newly eclosed and reproductively mature males kept isolated in the lab. We found expansion of the mushroom bodies—brain regions associated with learning and memory—with maturation, which were consistent across both species. This age-related plasticity may, therefore, play a functionally-relevant role in preparing male bees for mating, and suggests that developmentally-driven neural restructuring can occur in males, even in species where it is absent in females.

Distinct functional developments of surviving and eliminated presynaptic terminals

For neuronal circuits in the brain to mature, necessary synapses must be maintained and redundant synapses eliminated through experience-dependent mechanisms. However, the functional differentiation of these synapse types during the refinement process remains elusive. Here, we addressed this issue by distinct labeling and direct recordings of presynaptic terminals fated for survival and for elimination in the somatosensory thalamus. At surviving terminals, the number of total releasable vesicles was first enlarged, and then calcium channels and fast-releasing synaptic vesicles were tightly coupled in an experience-dependent manner. By contrast, transmitter release mechanisms did not mature at terminals fated for elimination, irrespective of sensory experience. Nonetheless, terminals fated for survival and for elimination both exhibited developmental shortening of action potential waveforms that was experience independent. Thus, we dissected experience-dependent and -independent developmental maturation processes of surviving and eliminated presynaptic terminals during neuronal circuit refinement.

Dissociable mechanisms of reward learning co-mature during human adolescence as predicted by macaque lesion models

Reward-guided learning and decision-making is a fundamental adaptive ability and depends on a number of component processes. We investigate how such component processes mature during human adolescence. Our approach was guided by analyses of the effects of lateral orbitofrontal lesions in macaque monkeys, as this part of the brain shows clear developmental maturation in humans during adolescence. Using matched tasks and analyses in humans (n=388, 11-35yrs), we observe developmental changes in two key learning mechanisms as predicted from the monkey data. First, choice-reward credit assignment – the ability to link a specific outcome to a specific choice – is reduced in adolescents. Second, the effects of the global reward state – how good the environment is overall recently − exerts a distinctive pattern of influence on learning in humans compared to other primates and across adolescence this pattern becomes more pronounced. Both mechanisms were correlated across participants suggesting that associative learning of correct reward assignments and GRS based learning constitute two complementary mechanisms of reward-learning that co-mature during adolescence.

Emergence of prefrontal neuron maturation properties by training recurrent neural networks in cognitive tasks

ABSTRACTWorking memory and response inhibition are functions that mature relatively late in life, after adolescence, paralleling the maturation of the prefrontal cortex. The link between behavioral and neural maturation is not obvious, however, making it challenging to understand how neural activity underlies the maturation of cognitive function. To gain insights into the nature of observed changes in prefrontal activity between adolescence and adulthood, we investigated the progressive changes in unit activity of Recurrent Neural Networks (RNNs) as they were trained to perform working memory and response inhibition tasks. These included increased delay period activity during working memory tasks, and increased activation in antisaccade tasks. These findings reveal universal properties underlying the neuronal computations behind cognitive tasks and explicate the nature of changes that occur as the result of developmental maturation.