Multiple parallel cell lineages in the developing mammalian cerebral cortex.

Cortical neurogenesis follows a simple lineage: apical radial glia cells (RGCs) generate basal progenitors, and these produce neurons. How this occurs in species with expanded germinal zones and a folded cortex, such as human, remains unclear. We used single-cell RNA sequencing from individual cortical germinal zones in ferret and barcoded lineage tracking to determine the molecular diversity of progenitor cells and their lineages. We identified multiple RGC classes that initiate parallel lineages, converging onto a common class of newborn neuron. Parallel RGC classes and transcriptomic trajectories were repeated across germinal zones and conserved in ferret and human, but not in mouse. Neurons followed parallel differentiation trajectories in the gyrus and sulcus, with different expressions of human cortical malformation genes. Progenitor cell lineage multiplicity is conserved in the folded mammalian cerebral cortex.

Vitamin A resolves lineage plasticity to orchestrate stem cell lineage choices.

Lineage plasticity-a state of dual fate expression-is required to release stem cells from their niche constraints and redirect them to tissue compartments where they are most needed. In this work, we found that without resolving lineage plasticity, skin stem cells cannot effectively generate each lineage in vitro nor regrow hair and repair wounded epidermis in vivo. A small-molecule screen unearthed retinoic acid as a critical regulator. Combining high-throughput approaches, cell culture, and in vivo mouse genetics, we dissected its roles in tissue regeneration. We found that retinoic acid is made locally in hair follicle stem cell niches, where its levels determine identity and usage. Our findings have therapeutic implications for hair growth as well as chronic wounds and cancers, where lineage plasticity is unresolved.

Seven-up acts in neuroblasts to specify adult central complex neuron identity and initiate neuroblast decommissioning.

An unanswered question in neurobiology is how are diverse neuron cell types generated from a small number of neural stem cells? In the Drosophila larval central brain, there are eight bilateral Type 2 neuroblast (T2NB) lineages that express a suite of early temporal factors followed by a different set of late temporal factors and generate the majority of the central complex (CX) neurons. The early-to-late switch is triggered by the orphan nuclear hormone receptor Seven-up (Svp), yet little is known about how this Svp-dependent switch is involved in specifying CX neuron identities. Here, we: (1) birth date the CX neurons P-EN and P-FN (early and late, respectively); (2) show that Svp is transiently expressed in all early T2NBs; and (3) show that loss of Svp expands the population of early born P-EN neurons at the expense of late born P-FN neurons. Furthermore, in the absence of Svp, T2NBs fail decommissioning and abnormally extend their lineage into week-old adults. We conclude that Svp is required to specify CX neuron identity, as well as to initiate T2NB decommissioning.

Out of Line or Altered States? Neural Progenitors as a Target in a Polygenic Neurodevelopmental Disorder.

The genesis of a mature complement of neurons is thought to require, at least in part, precursor cell lineages in which neural progenitors have distinct identities recognized by exclusive expression of one or a few molecular markers. Nevertheless, limited progenitor types distinguished by specific markers and lineal progression through such subclasses cannot easily yield the magnitude of neuronal diversity in most regions of the nervous system. The late Verne Caviness, to whom this edition of Developmental Neuroscience is dedicated, recognized this mismatch. In his pioneering work on the histogenesis of the cerebral cortex, he acknowledged the additional flexibility required to generate multiple classes of cortical projection and interneurons. This flexibility may be accomplished by establishing cell states in which levels rather than binary expression or repression of individual genes vary across each progenitor's shared transcriptome. Such states may reflect local, stochastic signaling via soluble factors or coincidence of cell surface ligand/receptor pairs in subsets of neighboring progenitors. This probabilistic, rather than determined, signaling could modify transcription levels via multiple pathways within an apparently uniform population of progenitors. Progenitor states, therefore, rather than lineal relationships between types may underlie the generation of neuronal diversity in most regions of the nervous system. Moreover, mechanisms that influence variation required for flexible progenitor states may be targets for pathological changes in a broad range of neurodevelopmental disorders, especially those with polygenic origins.

Multipotent progenitors instruct ontogeny of the superior colliculus.

The superior colliculus (SC) in the mammalian midbrain is essential for multisensory integration and is composed of a rich diversity of excitatory and inhibitory neurons and glia. However, the developmental principles directing the generation of SC cell-type diversity are not understood. Here, we pursued systematic cell lineage tracing in silico and in vivo, preserving full spatial information, using genetic mosaic analysis with double markers (MADM)-based clonal analysis with single-cell sequencing (MADM-CloneSeq). The analysis of clonally related cell lineages revealed that radial glial progenitors (RGPs) in SC are exceptionally multipotent. Individual resident RGPs have the capacity to produce all excitatory and inhibitory SC neuron types, even at the stage of terminal division. While individual clonal units show no pre-defined cellular composition, the establishment of appropriate relative proportions of distinct neuronal types occurs in a PTEN-dependent manner. Collectively, our findings provide an inaugural framework at the single-RGP/-cell level of the mammalian SC ontogeny.

Imp is expressed in INPs and newborn neurons where it regulates neuropil targeting in the central complex.

The generation of neuronal diversity remains incompletely understood. In Drosophila, the central brain is populated by neural stem cells derived from progenitors called neuroblasts (NBs). There are two types of NBs, type 1 and 2. T1NBs have a relatively simple lineage, whereas T2NBs expand and diversify the neural population with the generation of intermediate neural progenitors (INPs), contributing many neurons to the adult central complex, a brain region essential for navigation. However, it is not fully understood how neural diversity is created in T2NB and INP lineages. Imp, an RNA-binding protein, is expressed in T2NBs in a high-to-low temporal gradient, while the RNA-binding protein Syncrip forms an opposing gradient. It remains unknown if Imp expression is carried into INPs; whether it forms a gradient similar to NBs; and whether INP expression of Imp is required for generating neuronal identity or morphology. Here, we show that Imp/Syp are both present in INPs, but not always in opposing gradients. We find that newborn INPs adopt their Imp/Syp levels from their parental T2NBs; that Imp and Syp are expressed in stage-specific high-to-low gradients in INPs. In addition, there is a late INP pulse of Imp. We find that neurons born from old INPs (E-PG and PF-R neurons) have altered morphology following both Imp knock-down and Imp overexpression. We conclude that Imp functions in INPs and newborn neurons to determine proper neuronal morphology and central complex neuropil organization.

Mechanisms of lineage specification in Caenorhabditis elegans.

The studies of cell fate and lineage specification are fundamental to our understanding of the development of multicellular organisms. Caenorhabditis elegans has been one of the premiere systems for studying cell fate specification mechanisms at single cell resolution, due to its transparent nature, the invariant cell lineage, and fixed number of somatic cells. We discuss the general themes and regulatory mechanisms that have emerged from these studies, with a focus on somatic lineages and cell fates. We next review the key factors and pathways that regulate the specification of discrete cells and lineages during embryogenesis and postembryonic development; we focus on transcription factors and include numerous lineage diagrams that depict the expression of key factors that specify embryonic founder cells and postembryonic blast cells, and the diverse somatic cell fates they generate. We end by discussing some future perspectives in cell and lineage specification.

Disruption of lineage integrity as a precursor to breast tumor initiation.

Increase in lineage infidelity and/or imbalance is frequently observed around the earliest stage of breast tumor initiation. In response to disruption of homeostasis, differentiated cells can partially lose their identity and gain cellular plasticity, a process involving epigenome landscape remodeling. This increase of cellular plasticity may promote the malignant transformation of breast tumors and fuel their heterogeneity. Here, we review recent studies that have yield insights into important regulators of lineage integrity and mechanisms that trigger mammary epithelial lineage derail, and evaluate their impacts on breast tumor development.

Principles of neural stem cell lineage progression: Insights from developing cerebral cortex.

How to generate a brain of correct size and with appropriate cell-type diversity during development is a major question in Neuroscience. In the developing neocortex, radial glial progenitor (RGP) cells are the main neural stem cells that produce cortical excitatory projection neurons, glial cells, and establish the prospective postnatal stem cell niche in the lateral ventricles. RGPs follow a tightly orchestrated developmental program that when disrupted can result in severe cortical malformations such as microcephaly and megalencephaly. The precise cellular and molecular mechanisms instructing faithful RGP lineage progression are however not well understood. This review will summarize recent conceptual advances that contribute to our understanding of the general principles of RGP lineage progression.

TEMPO enables sequential genetic labeling and manipulation of vertebrate cell lineages.

During development, regulatory factors appear in a precise order to determine cell fates over time. Consequently, to investigate complex tissue development, it is necessary to visualize and manipulate cell lineages with temporal control. Current strategies for tracing vertebrate cell lineages lack genetic access to sequentially produced cells. Here, we present TEMPO (Temporal Encoding and Manipulation in a Predefined Order), an imaging-readable genetic tool allowing differential labeling and manipulation of consecutive cell generations in vertebrates. TEMPO is based on CRISPR and powered by a cascade of gRNAs that drive orderly activation and inactivation of reporters and/or effectors. Using TEMPO to visualize zebrafish and mouse neurogenesis, we recapitulated birth-order-dependent neuronal fates. Temporally manipulating cell-cycle regulators in mouse cortex progenitors altered the proportion and distribution of neurons and glia, revealing the effects of temporal gene perturbation on serial cell fates. Thus, TEMPO enables sequential manipulation of molecular factors, crucial to study cell-type specification.

Paracrine interactions through FGFR1 and FGFR2 receptors regulate the development of preimplantation mouse chimaeric embryo.

The preimplantation mammalian embryo has the potential to self-organize, allowing the formation of a correctly patterned embryo despite experimental perturbation. To better understand the mechanisms controlling the developmental plasticity of the early mouse embryo, we used chimaeras composed of an embryonic day (E)3.5 or E4.5 inner cell mass (ICM) and cleaving 8-cell embryo. We revealed that the restricted potential of the ICM can be compensated for by uncommitted 8-cell embryo-derived blastomeres, thus leading to the formation of a normal chimaeric blastocyst that can undergo full development. However, whether such chimaeras maintain developmental competence depends on the presence or specific orientation of the polarized primitive endoderm layer in the ICM component. We also demonstrated that downregulated FGFR1 and FGFR2 expression in 8-cell embryos disturbs intercellular interactions between both components and results in an inverse proportion of primitive endoderm and epiblast within the resulting ICM and abnormal embryo development. This finding suggests that FGF signalling is a key part of the regulatory mechanism that assigns cells to a given lineage and ensures the proper composition of the blastocyst, which is a prerequisite for its successful implantation in the uterus and for further development.

Transcription Factor Hb9 Is Expressed in Glial Cell Lineages in the Developing Mouse Spinal Cord.

Hb9 (Mnx1) is a transcription factor described as a spinal cord motor neuron (MN)-specific marker and critical factor for the postmitotic specification of these cells. To date, expression of Hb9 in other cell types has not been reported. We performed a fate-mapping approach to examine distributions of Hb9-expressing cells and their progeny ("Hb9-lineage cells") within the embryonic and adult spinal cord of Hb9cre;Ai14 mice. We found that Hb9-lineage cells are distributed in a gradient of increasing abundance throughout the rostrocaudal spinal cord axis during embryonic and postnatal stages. Furthermore, although the majority of Hb9-lineage cells at cervical spinal cord levels are MNs, at more caudal levels, Hb9-lineage cells include small-diameter dorsal horn neurons, astrocytes, and oligodendrocytes. In the peripheral nervous system, we observed a similar phenomenon with more abundant Hb9-lineage Schwann cells in muscles of the lower body versus upper body muscles. We cultured spinal cord progenitors in vitro and found that gliogenesis was increased by treatment with the caudalizing factor FGF-8B, while glial tdTomato expression was increased by treatment with both FGF-8B and GDF-11. Together, these observations suggest that early and transient expression of Hb9 in spinal cord neural progenitors may be induced by caudalizing factors such as FGF and GDF signaling. Furthermore, our work raises the possibility that early Hb9 expression may influence the development of spinal cord macroglia and Schwann cells, especially at caudal regions. Together, these findings highlight the importance of using caution when designing experiments using Hb9cre mice to perform spinal cord MN-specific manipulations.

A pendulum of induction between the epiblast and extra-embryonic endoderm supports post-implantation progression.

Embryogenesis is supported by dynamic loops of cellular interactions. Here, we create a partial mouse embryo model to elucidate the principles of epiblast (Epi) and extra-embryonic endoderm co-development (XEn). We trigger naive mouse embryonic stem cells to form a blastocyst-stage niche of Epi-like cells and XEn-like cells (3D, hydrogel free and serum free). Once established, these two lineages autonomously progress in minimal medium to form an inner pro-amniotic-like cavity surrounded by polarized Epi-like cells covered with visceral endoderm (VE)-like cells. The progression occurs through reciprocal inductions by which the Epi supports the primitive endoderm (PrE) to produce a basal lamina that subsequently regulates Epi polarization and/or cavitation, which, in return, channels the transcriptomic progression to VE. This VE then contributes to Epi bifurcation into anterior- and posterior-like states. Similarly, boosting the formation of PrE-like cells within blastoids supports developmental progression. We argue that self-organization can arise from lineage bifurcation followed by a pendulum of induction that propagates over time.

Single cell RNA-seq analysis reveals temporally-regulated and quiescence-regulated gene expression in Drosophila larval neuroblasts.

The mechanisms that generate neural diversity during development remains largely unknown. Here, we use scRNA-seq methodology to discover new features of the Drosophila larval CNS across several key developmental timepoints. We identify multiple progenitor subtypes - both stem cell-like neuroblasts and intermediate progenitors - that change gene expression across larval development, and report on new candidate markers for each class of progenitors. We identify a pool of quiescent neuroblasts in newly hatched larvae and show that they are transcriptionally primed to respond to the insulin signaling pathway to exit from quiescence, including relevant pathway components in the adjacent glial signaling cell type. We identify candidate "temporal transcription factors" (TTFs) that are expressed at different times in progenitor lineages. Our work identifies many cell type specific genes that are candidates for functional roles, and generates new insight into the differentiation trajectory of larval neurons.

Hunchback prevents notch-induced apoptosis in the serotonergic lineage of Drosophila Melanogaster.

The serotonergic lineage (NB7-3) in the Drosophila ventral nerve cord produces six cells during neurogenesis. Four of the cells differentiate into neurons: EW1, EW2, EW3 and GW. The other two cells undergo apoptosis. This simple lineage provides an opportunity to examine genes that are required to induce or repress apoptosis during cell specification. Previous studies have shown that Notch signaling induces apoptosis within the NB7-3 lineage. The three EW neurons are protected from Notch-induced apoptosis by asymmetric distribution of Numb protein, an inhibitor of Notch signaling. In a numb1 mutant EW2 and EW3 undergo apoptosis. The EW1 and GW neurons survive even in a numb1 mutant background suggesting that these cells are protected from Notch-induced apoptosis by some factor other than Numb. The EW1 and GW neurons are mitotic sister cells, and uniquely express the transcription factor Hunchback. We present evidence that Hunchback prevents apoptosis in the NB7-3 lineage during normal CNS development and can rescue the two apoptotic cells in the lineage when it is ectopically expressed. We show that hunchback overexpression produces ectopic cells that express markers similar to the EW2 neuron and changes the expression pattern of the EW3 neuron to a EW2 neuron. In addition we show that hunchback overexpression can override apoptosis that is genetically induced by the pro-apoptotic genes grim and hid.

Molecular divergence of mammalian astrocyte progenitor cells at early gliogenesis.

During mammalian brain development, how different astrocytes are specified from progenitor cells is not well understood. In particular, whether astrocyte progenitor cells (APCs) start as a relatively homogenous population or whether there is early heterogeneity remains unclear. Here, we have dissected subpopulations of embryonic mouse forebrain progenitors using single-cell transcriptome analyses. Our sequencing data revealed two molecularly distinct APC subgroups at the start of gliogenesis from both dorsal and ventral forebrains. The two APC subgroups were marked, respectively, by specific expression of Sparc and Sparcl1, which are known to function in mature astrocytes with opposing activities for regulating synapse formation. Expression analyses showed that SPARC and SPARCL1 mark APC subgroups that display distinct temporal and spatial patterns, correlating with major waves of astrogliogenesis during development. Our results uncover an early molecular divergence of APCs in the mammalian brain and provide a useful transcriptome resource for the study of glial cell specification.

Cell surface fluctuations regulate early embryonic lineage sorting.

In development, lineage segregation is coordinated in time and space. An important example is the mammalian inner cell mass, in which the primitive endoderm (PrE, founder of the yolk sac) physically segregates from the epiblast (EPI, founder of the fetus). While the molecular requirements have been well studied, the physical mechanisms determining spatial segregation between EPI and PrE remain elusive. Here, we investigate the mechanical basis of EPI and PrE sorting. We find that rather than the differences in static cell surface mechanical parameters as in classical sorting models, it is the differences in surface fluctuations that robustly ensure physical lineage sorting. These differential surface fluctuations systematically correlate with differential cellular fluidity, which we propose together constitute a non-equilibrium sorting mechanism for EPI and PrE lineages. By combining experiments and modeling, we identify cell surface dynamics as a key factor orchestrating the correct spatial segregation of the founder embryonic lineages.

Warm and cold temperatures have distinct germline stem cell lineage effects during Drosophila oogenesis.

Despite their medical and economic relevance, it remains largely unknown how suboptimal temperatures affect adult insect reproduction. Here, we report an in-depth analysis of how chronic adult exposure to suboptimal temperatures affects oogenesis using the model insect Drosophila melanogaster. In adult females maintained at 18°C (cold) or 29°C (warm), relative to females at the 25°C control temperature, egg production was reduced through distinct cellular mechanisms. Chronic 18°C exposure improved germline stem cell maintenance, survival of early germline cysts and oocyte quality, but reduced follicle growth with no obvious effect on vitellogenesis. By contrast, in females at 29°C, germline stem cell numbers and follicle growth were similar to those at 25°C, while early germline cyst death and degeneration of vitellogenic follicles were markedly increased and oocyte quality plummeted over time. Finally, we also show that these effects are largely independent of diet, male factors or canonical temperature sensors. These findings are relevant not only to cold-blooded organisms, which have limited thermoregulation, but also potentially to warm-blooded organisms, which are susceptible to hypothermia, heatstroke and fever.

Matrix remodeling controls a nuclear lamin A/C-emerin network that directs Wnt-regulated stem cell fate.

Skeletal stem cells (SSCs) reside within a three-dimensional extracellular matrix (ECM) compartment and differentiate into multiple cell lineages, thereby controlling tissue maintenance and regeneration. Within this environment, SSCs can proteolytically remodel the surrounding ECM in response to growth factors that direct lineage commitment via undefined mechanisms. Here, we report that Mmp14-dependent ECM remodeling coordinates canonical Wnt signaling and guides stem cell fate by triggering an integrin-activated reorganization of the SCC cytoskeleton that controls nuclear lamin A/C levels via the linker of nucleoskeleton and cytoskeleton (LINC) complexes. In turn, SSC lamin A/C levels dictate the localization of emerin, an inner nuclear membrane protein whose ability to regulate ß-catenin activity modulates Wnt signaling while directing lineage commitment in vitro and in vivo. These findings define a previously undescribed axis wherein SSCs use Mmp14-dependent ECM remodeling to control cytoskeletal and nucleoskeletal organization, thereby governing Wnt-dependent stem cell fate decisions.

Spatiotemporal patterns of neuronal subtype genesis suggest hierarchical development of retinal diversity.

How do neuronal subtypes emerge during development? Recent molecular studies have profiled existing neuronal diversity, but neuronal subtype genesis remains elusive. The 15 types of mouse retinal bipolar interneurons are characterized by distinct functions, morphologies, and transcriptional profiles. Here, we develop a comprehensive spatiotemporal map of bipolar subtype genesis in the murine retina. Combining multiplexed detection of 16 RNA markers with timed delivery of 5-ethynyl uridine (EdU) and bromodeoxyuridine (BrdU), we analyze more than 30,000 single cells in full retinal sections to classify all bipolar subtypes and their birthdates. We find that bipolar subtype birthdates are ordered and follow a centrifugal developmental axis. Spatial analysis reveals a striking wave pattern of bipolar subtype birthdates, and lineage analyses suggest clonal restriction on homotypic subtype production. These results inspire a hierarchical developmental model, with ordered subtype genesis within lineages. Our results provide insight into neuronal subtype development and establish a framework for studying subtype diversification.