Development Of Neuronal Lineages Research Articles

Spatiotemporal analysis of mRNA-protein relationships enhances transcriptome-based developmental inference

Elucidating the complex relationships between mRNA and protein expression at high spatiotemporal resolution is critical for unraveling multilevel gene regulation and enhancing mRNA-based developmental analyses. In this study, we conduct a single-cell analysis of mRNA and protein expression of transcription factors throughout C.elegans embryogenesis. Initially, cellular co-presence of mRNA and protein is low, increasing to a medium-high level (73%) upon factoring in delayed protein synthesis and long-term protein persistence. These factors substantially affect mRNA-protein concordance, leading to potential inaccuracies in mRNA-reliant gene detection and specificity characterization. Building on the learned relationship, we infer protein presence from mRNA expression and demonstrate its utility in identifying tissue-specific genes and elucidating relationships between genes and cells. This approach facilitates identifying the role of sptf-1/SP7 in neuronal lineage development. Collectively, this study provides insights into gene expression dynamics during rapid embryogenesis and approaches for improving the efficacy of transcriptome-based developmental analyses.

A patterned human neural tube model using microfluidic gradients.

The human nervous system is a highly complex but organized organ. The foundation of its complexity and organization is laid down during regional patterning of the neural tube, the embryonic precursor to the human nervous system. Historically, studies of neural tube patterning have relied on animal models to uncover underlying principles. Recently, models of neurodevelopment based on human pluripotent stem cells, including neural organoids1-5 and bioengineered neural tube development models6-10, have emerged. However, such models fail to recapitulate neural patterning along both rostral-caudal and dorsal-ventral axes in a three-dimensional tubular geometry, a hallmark of neural tube development. Here we report a human pluripotent stem cell-based, microfluidic neural tube-like structure, the development of which recapitulates several crucial aspects of neural patterning in brain and spinal cord regions and along rostral-caudal and dorsal-ventral axes. This structure was utilized for studying neuronal lineage development, which revealed pre-patterning of axial identities of neural crest progenitors and functional roles of neuromesodermal progenitors and the caudal gene CDX2 in spinal cord and trunk neural crest development. We further developed dorsal-ventral patterned microfluidic forebrain-like structures with spatially segregated dorsal and ventral regions and layered apicobasal cellular organizations that mimic development of the human forebrain pallium and subpallium, respectively. Together, these microfluidics-based neurodevelopmentmodels provide three-dimensional lumenal tissue architectures with in vivo-like spatiotemporal cell differentiation and organization, which will facilitate the study of human neurodevelopment and disease.

Identification of PAX6 and NFAT4 as the Transcriptional Regulators of the Long Noncoding RNA Mrhl in Neuronal Progenitors.

The long noncoding RNA (lncRNA) Mrhl has been shown to be involved in coordinating meiotic commitment of mouse spermatogonial progenitors and differentiation events in mouse embryonic stem cells. Here, we characterized the interplay of Mrhl with lineage-specific transcription factors during mouse neuronal lineage development. Our results demonstrate that Mrhl is expressed in the neuronal progenitor populations in mouse embryonic brains and in retinoic acid-derived radial-glia-like neuronal progenitor cells. Depletion of Mrhl leads to early differentiation of neuronal progenitors to a more committed state. A master transcription factor, PAX6, directly binds to the Mrhl promoter at a major site in the distal promoter, located at 2.9 kb upstream of the transcription start site (TSS) of Mrhl. Furthermore, NFAT4 occupies the Mrhl-proximal promoter at two sites, at 437 base pairs (bp) and 143 bp upstream of the TSS. Independent knockdown studies for PAX6 and NFAT4 confirm that they regulate Mrhl expression in neuronal progenitors. We also show that PAX6 and NFAT4 associate with each other in the same chromatin complex. NFAT4 occupies the Mrhl promoter in PAX6-bound chromatin, implying possible coregulation of Mrhl. Our studies are crucial for understanding how lncRNAs are regulated by major lineage-specific transcription factors, in order to define specific development and differentiation events.

Capicua regulates the development of adult-born neurons in the hippocampus

New neurons continuously arise from neural progenitor cells in the dentate gyrus of the adult hippocampus to support ongoing learning and memory formation. To generate functional adult-born neurons, neural progenitor cells proliferate to expand the precursor cell pool and differentiate into neurons. Newly generated cells then undergo postmitotic maturation to migrate to their final destination and develop elaborate dendritic branching, which allows them to receive input signals. Little is known about factors that regulate neuronal differentiation, migration, and dendrite maturation during adult hippocampal neurogenesis. Here, we show that the transcriptional repressor protein capicua (CIC) exhibits dynamic expression in the adult dentate gyrus. Conditional deletion of Cic from the mouse dentate gyrus compromises the adult neural progenitor cell pool without altering their proliferative potential. We further demonstrate that the loss of Cic impedes neuronal lineage development and disrupts dendritic arborization and migration of adult-born neurons. Our study uncovers a previously unrecognized role of CIC in neurogenesis of the adult dentate gyrus.

Reduced expression of Paternally Expressed Gene-3 enhances somatic cell reprogramming through mitochondrial activity perturbation

Imprinted genes control several cellular and metabolic processes in embryonic and adult tissues. In particular, paternally expressed gene-3 (Peg3) is active in the adult stem cell population and during muscle and neuronal lineage development. Here we have investigated the role of Peg3 in mouse embryonic stem cells (ESCs) and during the process of somatic cell reprogramming towards pluripotency. Our data show that Peg3 knockdown increases expression of pluripotency genes in ESCs and enhances reprogramming efficiency of both mouse embryonic fibroblasts and neural stem cells. Interestingly, we observed that altered activity of Peg3 correlates with major perturbations of mitochondrial gene expression and mitochondrial function, which drive metabolic changes during somatic cell reprogramming. Overall, our study shows that Peg3 is a regulator of pluripotent stem cells and somatic cell reprogramming.

Pooled Short Hairpin (shRNA) Library Screen Coupled with Next-Generation Sequencing Efficiently Uncover Transcriptional Network in Neural Lineage Development of Human Embryonic Stem Cells (IN8-1.009)

Objective: Our general aim is to identify genes and pathways of neural differentiation relevant for the development and function of human nervous system in health and disease. Background Large-scale, functional screening by RNAi-mediated gene-silencing is a powerful tool to unravel molecular mechanisms of normal and pathological processes. For human CNS this approach requires in vitro models. Here we describe a non-biased approach to probe molecular pathways of neuronal lineage development from human pluripotent stem cells by combining large-scale, pooled shRNA screens and massively parallel sequencing. Design/Methods: hESCs were infected with pooled lentiviral human shRNA libraries (predominantly transcription factors, targeting ∼ 1,900 genes) and differentiated neuronal progenitors (NPCs) were isolated by FACS using PSA-NCAM marker. By Illumina sequencing the abundance of the shRNA species in the different cell populations was comparatively analyzed. Results: By various ranking analyses we identified known and novel genes and pathways whose silencing either increased or decreased the relative rates of formation of NPCs. Selective depletion of shRNAs from PSA-NCAM positive cells (i.e. targeting genes required for neural differentiation) represented ∼ 5 % of all genes, > 40 % of these encoded proteins with known role in the CNS, including NSC/NPC self-renewal, fate determination and neurogenesis. Some genes/pathways involved in cortical patterning were indispensable for early commitment of pluripotent cells. Silencing of genes related to neurodevelopment disorders (i.e. MR, autism) and, unexpectedly, genes with role in late-onset neurodegenerative processes (e.g. TARDBP), also interfered with NPC differentiation. Several hits were individually reconfirmed. Conclusions: Highly quantitative, large-scale functional genetic screens by pooled shRNA libraries coupled with next-generation sequencing revealed a range of genes and networks critical in neural differentiation of hESCs in vitro. We are now utilizing this approach on patient-specific hiPSC neural differentiation models to obtain unparalleled insights into mechanisms controlling these processes in neurological disorders with monogenic or complex inheritance. Disclosure: Dr. Szekely has nothing to disclose. Dr. Zhang has nothing to disclose. Dr. Reed has nothing to disclose. Dr. Schulz has nothing to disclose. Dr. Wang has nothing to disclose. Dr. Euskirchen has nothing to disclose. Dr. Snyder has nothing to disclose. Dr. Ivanova has nothing to disclose. Dr. Weissman has nothing to disclose.

Pax6 Expression Is Sufficient to Induce a Neurogenic Fate in Glial Progenitors of the Neonatal Subventricular Zone

BackgroundThe forebrain subventricular zone (SVZ) of neonatal mammals contains a large, heterogeneous population of migratory and proliferating precursors of interneurons and glia. These cell types are produced in large numbers in the immediate postnatal period, the glioblasts populating the hemispheres with astrocytes and oligodendrocytes, the neuroblasts migrating to the olfactory bulb to become interneurons. How cell fate decisions are determined or stabilized in this mixed population is not clear, although previous studies indicate the importance of two transcription factors, Pax6 in neurons and Olig2 in glia, and suggest there may be reciprocal repression between these genes.Methodology/Principal FindingsIn examining the SVZ of neonatal mouse and rat brain, we find that the very large majority of SVZ cells express either Pax6 or Olig2, but few express both. We have used in vivo retro- and lenti-virus injections into the neonatal SVZ and in vitro gene transfer to demonstrate that pax6 over-expression is sufficient to down-regulate olig2 and to promote a neuronal lineage development and migration pattern in olig2-expressing cells. Furthermore, we provide evidence that Pax6 binds to the olig2 promoter and that an HEB sequence in the promoter is required for the Pax6 repression of olig2 transcription. Lastly, we constructed a lentivirus to target olig2-expressing cells in the SVZ to trace their fates, and found that the very large majority developed into glia.Conclusions/SignificanceWe provide evidence for a direct repression of olig2 by Pax6. Since SVZ cells can display developmental plasticity in vitro, the cross-repression promotes a stabilization of cell fates. This repression may be critical in a germinal zone in which immature cells are highly migratory and are not organized into an epithelium.

Molecular Characterization of the Mouse Superior Lateral Parabrachial Nucleus through Expression of the Transcription Factor Runx1

BackgroundThe ability to precisely identify separate neuronal populations is essential to the understanding of the development and function of different brain structures. This necessity is particularly evident in regions such as the brainstem, where the anatomy is quite complex and little is known about the identity, origin, and function of a number of distinct nuclei due to the lack of specific cellular markers. In this regard, the gene encoding the transcription factor Runx1 has emerged as a specific marker of restricted neuronal populations in the murine central and peripheral nervous systems. The aim of this study was to precisely characterize the expression of Runx1 in the developing and postnatal mouse brainstem.Methods and Principal FindingsAnatomical and immunohistochemical studies were used to characterize mouse Runx1 expression in the brainstem. It is shown here that Runx1 is expressed in a restricted population of neurons located in the dorsolateral rostral hindbrain. These neurons define a structure that is ventromedial to the dorsal nucleus of the lateral lemniscus, dorsocaudal to the medial paralemniscal nucleus and rostral to the cerebellum. Runx1 expression in these cells is first observed at approximately gestational day 12.5, persists into the adult brain, and is lost in knockout mice lacking the transcription factor Atoh1, an important regulator of the development of neuronal lineages of the rhombic lip. Runx1-expressing neurons in the rostral hindbrain produce cholecystokinin and also co-express members of the Groucho/Transducin-like Enhancer of split protein family.ConclusionBased on the anatomical and molecular characteristics of the Runx1-expressing cells in the rostral hindbrain, we propose that Runx1 expression in this region of the mouse brain defines the superior lateral parabrachial nucleus.

Temporal patterns of broad isoform expression during the development of neuronal lineages in Drosophila

BackgroundDuring the development of the central nervous system (CNS) of Drosophila, neuronal stem cells, the neuroblasts (NBs), first generate a set of highly diverse neurons, the primary neurons that mature to control larval behavior, and then more homogeneous sets of neurons that show delayed maturation and are primarily used in the adult. These latter, 'secondary' neurons show a complex pattern of expression of broad, which encodes a transcription factor usually associated with metamorphosis, where it acts as a key regulator in the transitions from larva and pupa.ResultsThe Broad-Z3 (Br-Z3) isoform appears transiently in most central neurons during embryogenesis, but persists in a subset of these cells through most of larval growth. Some of the latter are embryonic-born secondary neurons, whose development is arrested until the start of metamorphosis. However, the vast bulk of the secondary neurons are generated during larval growth and bromodeoxyuridine incorporation shows that they begin expressing Br-Z3 about 7 hours after their birth, approximately the time that they have finished outgrowth to their initial targets. By the start of metamorphosis, the oldest secondary neurons have turned off Br-Z3 expression, while the remainder, with the exception of the very youngest, maintain Br-Z3 while they are interacting with potential partners in preparation for neurite elaboration. That Br-Z3 may be involved in early sprouting is suggested by ectopically expressing this isoform in remodeling primary neurons, which do not normally express Br-Z3. These cells now sprout into ectopic locations. The expression of Br-Z3 is transient and seen in all interneurons, but two other isoforms, Br-Z4 and Br-Z1, show a more selective expression. Analysis of MARCM clones shows that the Br-Z4 isoform is expressed by neurons in virtually all lineages, but only in those cells born during a window during the transition from the second to the third larval instar. Br-Z4 expression is then maintained in this temporal cohort of cells into the adult.ConclusionThese data show the potential for diverse functions of Broad within the developing CNS. The Br-Z3 isoform appears in all interneurons, but not motoneurons, when they first begin to interact with potential targets. Its function during this early sorting phase needs to be defined. Two other Broad isoforms, by contrast, are stably expressed in cohorts of neurons in all lineages and are the first examples of persisting molecular 'time-stamps' for Drosophila postembryonic neurons.

Developmental changes in cell calcium homeostasis during neurogenesis of the embryonic rat cerebral cortex.

We quantified cytoplasmic Ca(2+) (Ca(2+)(c)) levels in cells dissociated from the embryonic (E) rat cortex during neurogenesis. Dual-recordings by flow cytometry using calcium and voltage-sensitive dyes revealed that, at the beginning of cortical development (E11-12), precursor cells exhibited either low (<100 nM), moderate (approximately 250 nM) or high (>1 microM) resting Ca(2+)(c) levels and well-polarized (-70 mV) or less-polarized (-40 mV) resting membrane potentials which reflected postmitotic or proliferative stages of the cell cycle. Ca(2+)(c) levels of all cells included a Ca(2+)(o) entry component, which was also Mn(2+)-permeant in actively proliferating precursors. Postmitotic, but not premitotic, precursors exhibited thapsigargin-sensitive intracellular Ca(2+) (Ca(2+)(i)) stores, which had similar capacities throughout neuronal lineage development. Differentiating neurons, but not precursors expressed Ca(2+)(i) stores with ryanodine and caffeine sensitivity and baseline Ca(2+)(c) levels that depended on Na(+)-Ca(2+) exchange activity. Voltage-dependent Ca(2+)(o) entry was not detected in precursors, but emerged during neuronal differentiation, with most of the neurons expressing functional L-type Ca(2+) channels. Ca(2+) imaging of individually immunoidentified cells acutely recovered in culture confirmed that precursors differentiate into neurons which stereotypically exhibit Ca(2+)(o) entry at the level of the membrane with increased Ca(2+)(i) release mechanisms on Ca(2+)(i) stores, Na(+)-Ca(2+) exchange activity and expression of voltage-dependent Ca(2+) channels.

Pan-neural Prospero terminates cell proliferation during Drosophila neurogenesis

Organogenesis requires coordination between developmental specific regulators and genes governing cell proliferation. Here we show that Drosophila prospero encodes a critical regulator of the transition from mitotically active cells to terminal differentiated neurons. Loss of pros results in aberrant expression of multiple cell-cycle regulatory genes and ectopic mitotic activity. In contrast, ectopic pros expression causes transcriptional suppression of multiple cell-cycle regulatory genes and premature termination of cell division. pros activity, hence, provides a critical regulatory link between neuronal lineage development and transcriptional regulation of cell cycle regulatory genes.

Mash1 regulates neurogenesis in the ventral telencephalon.

Previous studies have shown that mice mutant for the gene Mash1 display severe neuronal losses in the olfactory epithelium and ganglia of the autonomic nervous system, demonstrating a role for Mash1 in development of neuronal lineages in the peripheral nervous system. Here, we have begun to analyse Mash1 function in the central nervous system, focusing our studies on the ventral telencephalon where it is expressed at high levels during neurogenesis. Mash1 mutant mice present a severe loss of progenitors, particularly of neuronal precursors in the subventricular zone of the medial ganglionic eminence. Discrete neuronal populations of the basal ganglia and cerebral cortex are subsequently missing. An analysis of candidate effectors of Mash1 function revealed that the Notch ligands Dll1 and Dll3, and the target of Notch signaling Hes5, fail to be expressed in Mash1 mutant ventral telencephalon. In the lateral ganglionic eminence, loss of Notch signaling activity correlates with premature expression of a number of subventricular zone markers by ventricular zone cells. Therefore, Mash1 is an important regulator of neurogenesis in the ventral telencephalon, where it is required both to specify neuronal precursors and to control the timing of their production.

Daughterless is required for the expression of cell cycle genes in peripheral nervous system precursors of Drosophila embryos.

The function of the neuronal differentiation gene daughterless (da) is required for the proper initiation of neuronal lineage development in all PNS lineages following the selection of neuronal precursor cells. Previous studies have shown that the ubiquitously expressed da protein is required for the proper expression of neuronal precursor genes and lineage identity genes in the PNS of Drosophila melanogaster embryos. These genes are required for differentiation and cell fate determination in the developing PNS. These findings, however, did not explain the failure of the nascent PNS precursors to undergo a normal cell cycle and divide. Here we show that four genes whose products are required for various stages of the cell cycle are misexpressed in the PNS of da mutant embryos. This suggests that all aspects of PNS precursor differentiation examined so far are under the transcriptional control of da.

Regulatory interactions during early neurogenesis in Drosophila.

During neurogenesis in Drosophila, ectodermal cells are endowed with the capacity to become neuronal precursors. Following their selection, these cells initiate neuronal lineage development and differentiation. The processes of neuronal precursor specification and neuronal lineage development require the activities of several groups of genes functioning in a complex, hierarchical regulatory network. Whereas the proneural genes promote neurogenic potential, neurogenic genes restrict the acquisition of this identity to a subset of ectodermal cells. Following their selection, these cells express the pan neural neuronal precursor genes and a set of neuronal lineage identity genes. While lineage identity genes allow the various lineages to acquire specific identities, neuronal precursor genes presumably regulate functional and developmental characteristics common to all neuronal precursor cells.

Regulatory interactions during early neurogenesis in Drosophila

During neurogenesis in Drosophila, ectodermal cells are endowed with the capacity to become neuronal precursors. Following their selection, these cells initiate neuronal lineage development and differentiation. The processes of neuronal precursor specification and neuronal lineage development require the activities of several groups of genes functioning in a complex, hierarchical regulatory network. Whereas the proneural genes promote neurogenic potential, neurogenic genes restrict the acquisition of this identity to a subset of ectodermal cells. Following their selection, these cells express the pan neural neuronal precursor genes and a set of neuronal lineage identity genes. While lineage identity genes allow the various lineages to acquire specific identities, neuronal precursor genes presumably regulate functional and developmental characteristics common to all neuronal precursor cells. © 1996 Wiley-Liss, Inc.