Investigating the basis of lineage decisions and developmental trajectories in the dorsal spinal cord through pseudotime analyses.

Dorsal interneurons (dIs) in the spinal cord encode the perception of touch, pain, heat, itchiness and proprioception. Previous studies using genetic strategies in animal models have revealed important insights into dI development, but the molecular details of how dIs arise as distinct populations of neurons remain incomplete. We have developed a resource to investigate dI fate specification by combining a single-cell RNA-Seq atlas of mouse embryonic stem cell-derived dIs with pseudotime analyses. To validate this in silico resource as a useful tool, we used it to first identify genes that are candidates for directing the transition states that lead to distinct dI lineage trajectories, and then validated them using in situ hybridization analyses in the developing mouse spinal cord in vivo. We have also identified an endpoint of the dI5 lineage trajectory and found that dIs become more transcriptionally homogeneous during terminal differentiation. This study introduces a valuable tool for further discovery about the timing of gene expression during dI differentiation and demonstrates its utility in clarifying dI lineage relationships.

Investigating the basis of lineage decisions and developmental trajectories in the dorsal spinal cord through pseudotime analyses.

Dorsal interneurons (dIs) in the spinal cord encode the perception of touch, pain, heat, itch, and proprioception. While previous studies using genetic strategies in animal models have revealed important insights into dI development, the molecular details by which dIs arise as distinct populations of neurons remain incomplete. We have developed a resource to investigate dI fate specification by combining a single-cell RNA-Seq atlas of mouse ESC-derived dIs with pseudotime analyses. To validate this in silico resource as a useful tool, we used it to first identify novel genes that are candidates for directing the transition states that lead to distinct dI lineage trajectories, and then validated them using in situ hybridization analyses in the developing mouse spinal cord in vivo . We have also identified a novel endpoint of the dI5 lineage trajectory and found that dIs become more transcriptionally homogenous during terminal differentiation. Together, this study introduces a valuable tool for further discovery about the timing of gene expression during dI differentiation and demonstrates its utility clarifying dI lineage relationships. Summary statement: Pseudotime analyses of embryonic stem cell-derived dorsal spinal interneurons reveals both novel regulators and lineage relationships between different interneuron populations.

Netrin1 patterns the dorsal spinal cord through modulation of Bmp signaling.

We have identified an unexpected role for netrin1 as a suppressor of bone morphogenetic protein (Bmp) signaling in the developing dorsal spinal cord. Using a combination of gain- and loss-of-function approaches in chicken, embryonic stem cell (ESC), and mouse models, we have observed that manipulating the level of netrin1 specifically alters the patterning of the Bmp-dependent dorsal interneurons (dIs), dI1-dI3. Altered netrin1 levels also change Bmp signaling activity, as measured by bioinformatics, and monitoring phosophoSmad1/5/8 activation, the canonical intermediate of Bmp signaling, and Id levels, a known Bmp target. Together, these studies support the hypothesis that netrin1 acts from the intermediate spinal cord to regionally confine Bmp signaling to the dorsal spinal cord. Thus, netrin1 has reiterative activities shaping dorsal spinal circuits, first by regulating cell fate decisions and then acting as a guidance cue to direct axon extension.

In vitro atlas of dorsal spinal interneurons reveals Wnt signaling as a critical regulator of progenitor expansion.

Restoring sensation after injury or disease requires a reproducible method for generating large quantities of bona fide somatosensory interneurons. Toward this goal, we assess the mechanisms by which dorsal spinal interneurons (dIs; dI1-dI6) can be derived from mouse embryonic stem cells (mESCs). Using two developmentally relevant growth factors, retinoic acid (RA) and bone morphogenetic protein (BMP) 4, we recapitulate the complete in vivo program of dI differentiation through a neuromesodermal intermediate. Transcriptional profiling reveals that mESC-derived dIs strikingly resemble endogenous dIs, with the correct molecular and functional signatures. We further demonstrate that RA specifies dI4-dI6 fates through a default multipotential state, while the addition of BMP4 induces dI1-dI3 fates and activates Wnt signaling to enhance progenitor proliferation. Constitutively activating Wnt signaling permits the dramatic expansion of neural progenitor cultures. These cultures retain the capacity to differentiate into diverse populations of dIs, thereby providing a method of increasing neuronal yield.

Getting in touch with your senses: Mechanisms specifying sensory interneurons in the dorsal spinal cord.

The spinal cord is functionally and anatomically divided into ventrally derived motor circuits and dorsally derived somatosensory circuits. Sensory stimuli originating either at the periphery of the body, or internally, are relayed to the dorsal spinal cord where they are processed by distinct classes of sensory dorsal interneurons (dIs). dIs convey sensory information, such as pain, heat or itch, either to the brain, and/or to the motor circuits to initiate the appropriate response. They also regulate the intensity of sensory information and are the major target for the opioid analgesics. While the developmental mechanisms directing ventral and dorsal cell fates have been hypothesized to be similar, more recent research has suggested that dI fates are specified by novel mechanisms. In this review, we will discuss the molecular events that specify dorsal neuronal patterning in the spinal cord, thereby generating diverse dI identities. We will then discuss how this molecular understanding has led to the development of robust stem cell methods to derive multiple spinal cell types, including the dIs, and the implication of these studies for treating spinal cord injuries and neurodegenerative diseases. This article is categorized under: Neurological Diseases > Stem Cells and Development.

Dorsal commissural axon guidance in the developing spinal cord.

Commissural axons have been a key model system for identifying axon guidance signals in vertebrates. This review summarizes the current thinking about the molecular and cellular mechanisms that establish a specific commissural neural circuit: the dI1 neurons in the developing spinal cord. We assess the contribution of long- and short-range signaling while sequentially following the developmental timeline from the birth of dI1 neurons, to the extension of commissural axons first circumferentially and then contralaterally into the ventral funiculus.

Derivation of dorsal spinal sensory interneurons from human pluripotent stem cells.

We describe two differentiation protocols to derive sensory spinal interneurons (INs) from human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). In protocol 1, we use retinoic acid (RA) to induce pain, itch, and heat mediating dI4/dI6 interneurons, and in protocol 2, RA with bone morphogenetic protein 4 (RA+BMP4) is used to induce proprioceptive dI1s and mechanosensory dI3s in hPSC cultures. These protocols provide an important step toward developing therapies for regaining sensation in spinal cord injury patients. For complete details on the use and execution of this protocol, please refer to Gupta et al. (2018).

Apcdd1 is a dual BMP/Wnt inhibitor in the developing nervous system and skin.

Animal development and homeostasis depend on precise temporal and spatial intercellular signaling. Components shared between signaling pathways, generally thought to decrease specificity, paradoxically can also provide a solution to pathway coordination. Here we show that the Bone Morphogenetic Protein (BMP) and Wnt signaling pathways share Apcdd1 as a common inhibitor and that Apcdd1 is a taxon-restricted gene with novel domains and signaling functions. Previously, we showed that Apcdd1 inhibits Wnt signaling (Shimomura et al., 2010), here we find that Apcdd1 potently inhibits BMP signaling in body axis formation and neural differentiation in chicken, frog, zebrafish. Furthermore, we find that Apcdd1 has an evolutionarily novel protein domain. Our results from experiments and modeling suggest that Apcdd1 may coordinate the outputs of two signaling pathways that are central to animal development and human disease.

The Cofilin/Limk1 Pathway Controls the Growth Rate of Both Developing and Regenerating Motor Axons.

Regenerating axons often have to grow considerable distances to reestablish circuits, making functional recovery a lengthy process. One solution to this problem would be to co-opt the "temporal" guidance mechanisms that control the rate of axon growth during development to accelerate the rate at which nerves regenerate in adults. We have previously found that the loss of Limk1, a negative regulator of cofilin, accelerates the rate of spinal commissural axon growth. Here, we use mouse models to show that spinal motor axon outgrowth is similarly promoted by the loss of Limk1, suggesting that temporal guidance mechanisms are widely used during development. Furthermore, we find that the regulation of cofilin activity is an acute response to nerve injury in the peripheral nervous system. Within hours of a sciatic nerve injury, the level of phosphorylated cofilin dramatically increases at the lesion site, in a Limk1-dependent manner. This response may be a major constraint on the rate of peripheral nerve regeneration. Proof-of-principle experiments show that elevating cofilin activity, through the loss of Limk1, results in faster sciatic nerve growth, and improved recovery of some sensory and motor function.SIGNIFICANCE STATEMENT The studies shed light on an endogenous, shared mechanism that controls the rate at which developing and regenerating axons grow. An understanding of these mechanisms is key for developing therapies to reduce painful recovery times for nerve-injury patients, by accelerating the rate at which damaged nerves reconnect with their synaptic targets.

New perspectives on the mechanisms establishing the dorsal-ventral axis of the spinal cord.

Distinct classes of neurons arise at different positions along the dorsal-ventral axis of the spinal cord leading to spinal neurons being segregated along this axis according to their physiological properties and functions. Thus, the neurons associated with motor control are generally located in, or adjacent to, the ventral horn whereas the interneurons (INs) that mediate sensory activities are present within the dorsal horn. Here, we review classic and recent studies examining the developmental mechanisms that establish the dorsal-ventral axis in the embryonic spinal cord. Intriguingly, while the cellular organization of the dorsal and ventral halves of the spinal cord looks superficially similar during early development, the underlying molecular mechanisms that establish dorsal vs ventral patterning are markedly distinct. For example, the ventral spinal cord is patterned by the actions of a single growth factor, sonic hedgehog (Shh) acting as a morphogen, i.e., concentration-dependent signal. Recent studies have shed light on the mechanisms by which the spatial and temporal gradient of Shh is transduced by cells to elicit the generation of different classes of ventral INs, and motor neurons (MNs). In contrast, the dorsal spinal cord is patterned by the action of multiple factors, most notably by members of the bone morphogenetic protein (BMP) and Wnt families. While less is known about dorsal patterning, recent studies have suggested that the BMPs do not act as morphogens to specify dorsal IN identities as previously proposed, rather each BMP has signal-specific activities. Finally, we consider the promise that elucidation of these mechanisms holds for neural repair.

Deriving Dorsal Spinal Sensory Interneurons from Human Pluripotent Stem Cells.

Cellular replacement therapies for neurological conditions use human embryonic stem cell (hESC)- or induced pluripotent stem cell (hiPSC)-derived neurons to replace damaged or diseased populations of neurons. For the spinal cord, significant progress has been made generating the in-vitro-derived motor neurons required to restore coordinated movement. However, there is as yet no protocol to generate in-vitro-derived sensory interneurons (INs), which permit perception of the environment. Here, we report on the development of a directed differentiation protocol to derive sensory INs for both hESCs and hiPSCs. Two developmentally relevant factors, retinoic acid in combination with bone morphogenetic protein 4, can be used to generate three classes of sensory INs: the proprioceptive dI1s, the dI2s, and mechanosensory dI3s. Critical to this protocol is the competence state of the neural progenitors, which changes over time. This protocol will facilitate developing cellular replacement therapies to reestablish sensory connections in injured patients.

BMPs direct sensory interneuron identity in the developing spinal cord using signal-specific not morphogenic activities.

The Bone Morphogenetic Protein (BMP) family reiteratively signals to direct disparate cellular fates throughout embryogenesis. In the developing dorsal spinal cord, multiple BMPs are required to specify sensory interneurons (INs). Previous studies suggested that the BMPs act as concentration-dependent morphogens to direct IN identity, analogous to the manner in which sonic hedgehog patterns the ventral spinal cord. However, it remains unresolved how multiple BMPs would cooperate to establish a unified morphogen gradient. Our studies support an alternative model: BMPs have signal-specific activities directing particular IN fates. Using chicken and mouse models, we show that the identity, not concentration, of the BMP ligand directs distinct dorsal identities. Individual BMPs promote progenitor patterning or neuronal differentiation by their activation of different type I BMP receptors and distinct modulations of the cell cycle. Together, this study shows that a 'mix and match' code of BMP signaling results in distinct classes of sensory INs.

Netrin1 establishes multiple boundaries for axon growth in the developing spinal cord.

The canonical model for netrin1 function proposed that it acted as a long-range chemotropic axon guidance cue. In the developing spinal cord, floor-plate (FP)-derived netrin1 was thought to act as a diffusible attractant to draw commissural axons to the ventral midline. However, our recent studies have shown that netrin1 is dispensable in the FP for axon guidance. We have rather found that netrin1 acts locally: netrin1 is produced by neural progenitor cells (NPCs) in the ventricular zone (VZ), and deposited on the pial surface as a haptotactic adhesive substrate that guides Dcc+ axon growth. Here, we further demonstrate that this netrin1 pial-substrate has an early role orienting pioneering spinal axons, directing them to extend ventrally. However, as development proceeds, commissural axons choose to grow around a boundary of netrin1 expressing cells in VZ, instead of continuing to extend alongside the netrin1 pial-substrate in the ventral spinal cord. This observation suggests netrin1 may supply a more complex activity than pure adhesion, with netrin1-expressing cells also supplying a growth boundary for axons. Supporting this possibility, we have observed that additional domains of netrin1 expression arise adjacent to the dorsal root entry zone (DREZ) in E12.5 mice that are also required to sculpt axonal growth. Together, our studies suggest that netrin1 provides "hederal" boundaries: a local growth substrate that promotes axon extension, while also preventing local innervation of netrin1-expressing domains.

Netrin1 Produced by Neural Progenitors, Not Floor Plate Cells, Is Required for Axon Guidance in the Spinal Cord.

Netrin1 has been proposed to act from the floor plate (FP) as a long-range diffusible chemoattractant for commissural axons in the embryonic spinal cord. However, netrin1 mRNA and protein are also present in neural progenitors within the ventricular zone (VZ), raising the question of which source of netrin1 promotes ventrally directed axon growth. Here, we use genetic approaches in mice to selectively remove netrin from different regions of the spinal cord. Our analyses show that the FP is not the source of netrin1 directing axons to the ventral midline, while local VZ-supplied netrin1 is required for this step. Furthermore, rather than being present in a gradient, netrin1 protein accumulates on the pial surface adjacent to the path of commissural axon extension. Thus, netrin1 does not act as a long-range secreted chemoattractant for commissural spinal axons but instead promotes ventrally directed axon outgrowth by haptotaxis, i.e., directed growth along an adhesive surface.

From classical to current: analyzing peripheral nervous system and spinal cord lineage and fate.

During vertebrate development, the central (CNS) and peripheral nervous systems (PNS) arise from the neural plate. Cells at the margin of the neural plate give rise to neural crest cells, which migrate extensively throughout the embryo, contributing to the majority of neurons and all of the glia of the PNS. The rest of the neural plate invaginates to form the neural tube, which expands to form the brain and spinal cord. The emergence of molecular cloning techniques and identification of fluorophores like Green Fluorescent Protein (GFP), together with transgenic and electroporation technologies, have made it possible to easily visualize the cellular and molecular events in play during nervous system formation. These lineage-tracing techniques have precisely demonstrated the migratory pathways followed by neural crest cells and increased knowledge about their differentiation into PNS derivatives. Similarly, in the spinal cord, lineage-tracing techniques have led to a greater understanding of the regional organization of multiple classes of neural progenitor and post-mitotic neurons along the different axes of the spinal cord and how these distinct classes of neurons assemble into the specific neural circuits required to realize their various functions. Here, we review how both classical and modern lineage and marker analyses have expanded our knowledge of early peripheral nervous system and spinal cord development.

Neuronal organization: unsticking the cadherin code.

Hindbrain cranial motor neurons are organized into discrete functional clusters. A new study demonstrates that coalescence of these nuclei is driven by the expression of distinct combinations of cadherin adhesion molecules by each motor neuron group.

Smad1 and 5 but not Smad8 establish stem cell quiescence which is critical to transform the premature hair follicle during morphogenesis toward the postnatal state.

Hair follicles (HFs) are regenerative miniorgans that offer a highly informative model system to study the regulatory mechanisms of hair follicle stem cells (hfSCs) homeostasis and differentiation. Bone morphogenetic protein (BMP) signaling is key in both of these processes, governing hfSCs quiescence in the bulge and differentiation of matrix progenitors. However, whether canonical or noncanonical pathways of BMP signaling are responsible for these processes remains unresolved. Here, we conditionally ablated two canonical effectors of BMP signaling, Smad1 and Smad5 during hair morphogenesis and postnatal cycling in mouse skin. Deletion of Smad1 and Smad5 (dKO) in the epidermis during morphogenesis resulted in neonatal lethality with lack of visible whiskers. Interestingly, distinct patterns of phospho-Smads (pSmads) activation were detected with pSmad8 restricted to epidermis and pSmad1 and pSmad5 exclusively activated in HFs. Engraftment of dKO skin revealed retarded hair morphogenesis and failure to differentiate into visible hair. The formation of the prebulge and bulge reservoir for quiescent hfSCs was precluded in dKO HFs which remained in prolonged anagen. Surprisingly, in postnatal telogen HFs, pSmad8 expression was no longer limited to epidermis and was also present in dKO bulge hfSCs and matrix progenitors. Although pSmad8 activity alone could not prevent dKO hfSCs precocious anagen activation, it sustained efficient postnatal differentiation and regeneration of visible hairs. Together, our data suggest a pivotal role for canonical BMP signaling demonstrating distinguished nonoverlapping function of pSmad8 with pSmad1 and pSmad5 in hfSCs regulation and hair morphogenesis but a redundant role in adult hair progenitors differentiation.

PLZF regulates fibroblast growth factor responsiveness and maintenance of neural progenitors.

Distinct classes of neurons and glial cells in the developing spinal cord arise at specific times and in specific quantities from spatially discrete neural progenitor domains. Thus, adjacent domains can exhibit marked differences in their proliferative potential and timing of differentiation. However, remarkably little is known about the mechanisms that account for this regional control. Here, we show that the transcription factor Promyelocytic Leukemia Zinc Finger (PLZF) plays a critical role shaping patterns of neuronal differentiation by gating the expression of Fibroblast Growth Factor (FGF) Receptor 3 and responsiveness of progenitors to FGFs. PLZF elevation increases FGFR3 expression and STAT3 pathway activity, suppresses neurogenesis, and biases progenitors towards glial cell production. In contrast, PLZF loss reduces FGFR3 levels, leading to premature neuronal differentiation. Together, these findings reveal a novel transcriptional strategy for spatially tuning the responsiveness of distinct neural progenitor groups to broadly distributed mitogenic signals in the embryonic environment.

My brain told me to do it.

Voluntary motor control requires circuits in the brain to develop synchronously with spinal motor circuitry. In this issue of Developmental Cell,Reimer et al. (2013) demonstrate that this process is coordinated in zebrafish: dopamine released from descending projections modulates formation of motor neurons by attenuating the response of progenitors to Shh signaling.