Neural tube development depends on notochord-derived sonic hedgehog released into the sclerotome.

Sonic hedgehog (Shh), produced in the notochord and floor plate, is necessary for both neural and mesodermal development. To reach the myotome, Shh has to traverse the sclerotome and a reduction of sclerotomal Shh affects myotome differentiation. By investigating loss and gain of Shh function, and floor-plate deletions, we report that sclerotomal Shh is also necessary for neural tube development. Reducing the amount of Shh in the sclerotome using a membrane-tethered hedgehog-interacting protein or Patched1, but not dominant active Patched, decreased the number of Olig2+ motoneuron progenitors and Hb9+ motoneurons without a significant effect on cell survival or proliferation. These effects were a specific and direct consequence of Shh reduction in the mesoderm. In addition, grafting notochords in a basal but not apical location, vis-à-vis the tube, profoundly affected motoneuron development, suggesting that initial ligand presentation occurs at the basal side of epithelia corresponding to the sclerotome-neural tube interface. Collectively, our results reveal that the sclerotome is a potential site of a Shh gradient that coordinates the development of mesodermal and neural progenitors.

Spinal motor neurons are regenerated after mechanical lesion and genetic ablation in larval zebrafish.

In adult zebrafish, relatively quiescent progenitor cells show lesion-induced generation of motor neurons. Developmental motor neuron generation from the spinal motor neuron progenitor domain (pMN) sharply declines at 48 hours post-fertilisation (hpf). After that, mostly oligodendrocytes are generated from the same domain. We demonstrate here that within 48 h of a spinal lesion or specific genetic ablation of motor neurons at 72 hpf, the pMN domain reverts to motor neuron generation at the expense of oligodendrogenesis. By contrast, generation of dorsal Pax2-positive interneurons was not altered. Larval motor neuron regeneration can be boosted by dopaminergic drugs, similar to adult regeneration. We use larval lesions to show that pharmacological suppression of the cellular response of the innate immune system inhibits motor neuron regeneration. Hence, we have established a rapid larval regeneration paradigm. Either mechanical lesions or motor neuron ablation is sufficient to reveal a high degree of developmental flexibility of pMN progenitor cells. In addition, we show an important influence of the immune system on motor neuron regeneration from these progenitor cells.

Transcription factor expression uniquely identifies most postembryonic neuronal lineages in the Drosophila thoracic central nervous system.

Most neurons of the adult Drosophila ventral nerve cord arise from a burst of neurogenesis during the third larval instar stage. Most of this growth occurs in thoracic neuromeres, which contain 25 individually identifiable postembryonic neuronal lineages. Initially, each lineage consists of two hemilineages--'A' (Notch(On)) and 'B' (Notch(Off))--that exhibit distinct axonal trajectories or fates. No reliable method presently exists to identify these lineages or hemilineages unambiguously other than labor-intensive lineage-tracing methods. By combining mosaic analysis with a repressible cell marker (MARCM) analysis with gene expression studies, we constructed a gene expression map that enables the rapid, unambiguous identification of 23 of the 25 postembryonic lineages based on the expression of 15 transcription factors. Pilot genetic studies reveal that these transcription factors regulate the specification and differentiation of postembryonic neurons: for example, Nkx6 is necessary and sufficient to direct axonal pathway selection in lineage 3. The gene expression map thus provides a descriptive foundation for the genetic and molecular dissection of adult-specific neurogenesis and identifies many transcription factors that are likely to regulate the development and differentiation of discrete subsets of postembryonic neurons.

Genome-wide identification of Drosophila Hb9 targets reveals a pivotal role in directing the transcriptome within eight neuronal lineages, including activation of nitric oxide synthase and Fd59a/Fox-D.

Hb9 is a homeodomain-containing transcription factor that acts in combination with Nkx6, Lim3, and Tail-up (Islet) to guide the stereotyped differentiation, connectivity, and function of a subset of neurons in Drosophila. The role of Hb9 in directing neuronal differentiation is well documented, but the lineage of Hb9(+) neurons is only partly characterized, its regulation is poorly understood, and most of the downstream genes through which it acts remain at large. Here, we complete the lineage tracing of all embryonic Hb9(+) neurons (to eight neuronal lineages) and provide evidence that hb9, lim3, and tail-up are coordinately regulated by a common set of upstream factors. Through the parallel use of micro-array gene expression profiling and the Dam-ID method, we searched for Hb9-regulated genes, uncovering transcription factors as the most over-represented class of genes regulated by Hb9 (and Nkx6) in the CNS. By a nearly ten-to-one ratio, Hb9 represses rather than activates transcription factors, highlighting transcriptional repression of other transcription factors as a core mechanism by which Hb9 governs neuronal determination. From the small set of genes activated by Hb9, we characterized the expression and function of two - fd59a/foxd, which encodes a transcription factor, and Nitric oxide synthase. Under standard lab conditions, both genes are dispensable for Drosophila development, but Nos appears to inhibit hyper-active behavior and fd59a appears to act in octopaminergic neurons to control egg-laying behavior. Together our data clarify the mechanisms through which Hb9 governs neuronal specification and differentiation and provide an initial characterization of the expression and function of Nos and fd59a in the Drosophila CNS.

Modeling amyotrophic lateral sclerosis in pure human iPSc-derived motor neurons isolated by a novel FACS double selection technique.

Amyotrophic lateral sclerosis (ALS) is a severe and incurable neurodegenerative disease. Human motor neurons generated from induced pluripotent stem cells (iPSc) offer new perspectives for disease modeling and drug testing in ALS. In standard iPSc-derived cultures, however, the two major phenotypic alterations of ALS--degeneration of motor neuron cell bodies and axons--are often obscured by cell body clustering, extensive axon criss-crossing and presence of unwanted cell types. Here, we succeeded in isolating 100% pure and standardized human motor neurons by a novel FACS double selection based on a p75(NTR) surface epitope and an HB9::RFP lentivirus reporter. The p75(NTR)/HB9::RFP motor neurons survive and grow well without forming clusters or entangled axons, are electrically excitable, contain ALS-relevant motor neuron subtypes and form functional connections with co-cultured myotubes. Importantly, they undergo rapid and massive cell death and axon degeneration in response to mutant SOD1 astrocytes. These data demonstrate the potential of FACS-isolated human iPSc-derived motor neurons for improved disease modeling and drug testing in ALS and related motor neuron diseases.

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.

Pancreatic HB9 protein level is affected by early life stress in young adult rats: possible involvement of TNF-α and corticosterone.

This study examined foot shock stress effects, during weaning, on pancreatic HB9 protein expression in young adult male rats in the presence or absence of adulthood stress. The pups were divided into Control, Early life stress, Young adult stress, and Early + young adult stress groups. Plasma corticosterone, insulin, glucose, and TNF-α concentrations, and pancreatic HB9 protein expression were assessed. At 2 weeks of age, stress increased plasma corticosterone level. During young adulthood, plasma TNF-α and glucose concentrations increased, whereas plasma insulin and pancreatic HB9 protein levels decreased in Early life stress group. Whereas, Early + young adulthood stress group showed no change in the study parameters, except for plasma corticosterone and insulin concentrations. Overall, early life stress reduced pancreatic HB9 protein expression possibly by elevating plasma corticosterone and TNF-α levels in early life and adulthood, respectively. However, combined with adulthood stress, HB9 protein expression increased to the level of Control.

Standardized Reporter Systems for Purification and Imaging of Human Pluripotent Stem Cell-derived Motor Neurons and Other Cholinergic Cells.

Reliable and consistent pluripotent stem cell reporter systems for efficient purification and visualization of motor neurons are essential reagents for the study of normal motor neuron biology and for effective disease modeling. To overcome the inherent noisiness of transgene-based reporters, we developed a new series of human induced pluripotent stem cell lines by knocking in tdTomato, Cre, or CreERT2 recombinase into the HB9 (MNX1) or VACHT (SLC18A3) genomic loci. The new lines were validated by directed differentiation into spinal motor neurons and immunostaining for motor neuron markers HB9 and ISL1. To facilitate efficient purification of spinal motor neurons, we further engineered the VACHT-Cre cell line with a validated, conditional CD14-GFP construct that allows for both fluorescence-based identification of motor neurons, as well as magnetic-activated cell sorting (MACS) to isolate differentiated motor neurons at scale. The targeting strategies developed here offer a standardized platform for reproducible comparison of motor neurons across independently derived pluripotent cell lines.

Hindbrain V2a Neurons Pattern Rhythmic Activity of Motor Neurons in a Reticulospinal Coculture.

As the capacity to isolate distinct neuronal cell types has advanced over the past several decades, new two- and three-dimensional in vitro models of the interactions between different brain regions have expanded our understanding of human neurobiology and the origins of disease. These cultures develop distinctive patterns of activity, but the extent that these patterns are determined by the molecular identity of individual cell types versus the specific pattern of network connectivity is unclear. To address the question of how individual cell types interact in vitro, we developed a simplified culture using two excitatory neuronal subtypes known to participate in the in vivo reticulospinal circuit: HB9+ spinal motor neurons and Chx10+ hindbrain V2a neurons. Here, we report the emergence of cell type-specific patterns of activity in culture; on their own, Chx10+ neurons developed regular, synchronized bursts of activity that recruited neurons across the entire culture, whereas HB9+ neuron activity consisted of an irregular pattern. When these two subtypes were cocultured, HB9+ neurons developed synchronized network bursts that were precisely correlated with Chx10+ neuron activity, thereby recreating an aspect of Chx10+ neurons' role in driving motor activity. These bursts were dependent on AMPA receptors. Our results demonstrate that the molecular classification of the neurons comprising in vitro networks is a crucial determinant of their activity. It is therefore possible to improve both the reproducibility and the applicability of in vitro neurobiological and disease models by carefully controlling the constituent mixtures of neuronal subtypes.

Growth at Cold Temperature Increases the Number of Motor Neurons to Optimize Locomotor Function.

During vertebrate development, spinal neurons differentiate and connect to generate a system that performs sensorimotor functions critical for survival. Spontaneous Ca2+ activity regulates different aspects of spinal neuron differentiation. It is unclear whether environmental factors can modulate this Ca2+ activity in developing spinal neurons to alter their specialization and ultimately adjust sensorimotor behavior to fit the environment. Here, we show that growing Xenopus laevis embryos at cold temperatures results in an increase in the number of spinal motor neurons in larvae. This change in spinal cord development optimizes the escape response to gentle touch of animals raised in and tested at cold temperatures. The cold-sensitive channel TRPM8 increases Ca2+ spike frequency of developing ventral spinal neurons, which in turn regulates expression of the motor neuron master transcription factor HB9. TRPM8 is necessary for the increase in motor neuron number of animals raised in cold temperatures and for their enhanced sensorimotor behavior when tested at cold temperatures. These findings suggest the environment modulates neuronal differentiation to optimize the behavior of the developing organism.

Single-injection ex ovo transplantation method for broad spinal cord engraftment of human pluripotent stem cell-derived motor neurons.

BACKGROUND: Transplantation of human pluripotent stem cell (hPSC)-derived neurons into chick embryos is an established preliminary assay to evaluate engraftment potential. Yet, with recent advances in deriving diverse human neuronal subtypes, optimizing and standardizing such transplantation methodology for specific subtypes at their correlated anatomical sites is still required. NEW METHOD: We determined the optimal stage of hPSC-derived motor neuron (hMN) differentiation for ex ovo transplantation, and developed a single injection protocol that implants hMNs throughout the spinal cord enabling broad regional engraftment possibilities. RESULTS: A single injection into the neural tube lumen yielded a 100% chick embryo survival and successful transplantation rate with MN engraftment observed from the rostral cervical through caudal lumbar spinal cord. Transplantation of HB9+/ChAT- hMN precursors yielded the greatest amount of engraftment compared to Pax6+/Nkx6.1+/Olig2+ progenitors or mature HB9+/ChAT+ hMNs. COMPARISON WITH EXISTING METHOD(S): Our single injection hMN transplant method is the first to standardize the optimal hMN phenotype for chick embryo transplantation, provide a rubric for engraftment quantification, and enable broad engraftment throughout the spinal cord with a single surgical intervention. CONCLUSION: Transplantation of HB9+/ChAT- hMN precursors into chick embryos of Hamburger Hamilton (HH) stages 15-18 using a single luminal injection confers a high probability of embryo survival and cell engraftment in diverse regions throughout the spinal cord.

Biophysical Variation within the M1 Type of Ganglion Cell Photoreceptor.

Intrinsically photosensitive retinal ganglion cells of the M1 type encode environmental irradiance for functions that include circadian and pupillary regulation. Their distinct role, morphology, and molecular markers indicate that they are stereotyped circuit elements, but their physiological uniformity has not been investigated in a systematic fashion. We have profiled the biophysical parameters of mouse M1s and found that extreme variation is their hallmark. Most parameters span 1-3 log units, and the full range is evident in M1s that innervate brain regions serving divergent functions. Biophysical profiles differ among cells possessing similar morphology and between neighboring M1s recorded simultaneously. Variation in each parameter is largely independent of that in others, allowing for flexible individualization. Accordingly, a common stimulus drives heterogeneous spike outputs across cells. By contrast, a population of directionally selective retinal ganglion cells appeared physiologically uniform under similar conditions. Thus, M1s lack biophysical constancy and send diverse signals downstream.

Acetylcholine secretion by motor neuron-like cells from umbilical cord mesenchymal stem cells.

Umbilical cord mesenchymal stem cells were isolated by a double enzyme digestion method. The third passage of umbilical cord mesenchymal stem cells was induced with heparin and/or basic fibroblast growth factor. Results confirmed that cell morphology did not change after induction with basic fibroblast growth factor alone. However, neuronal morphology was visible, and microtubule-associated protein-2 expression and acetylcholine levels increased following induction with heparin alone or heparin combined with basic fibroblast growth factor. Hb9 and choline acetyltransferase expression was high following inductive with heparin combined with basic fibroblast growth factor. Results indicate that the inductive effect of basic fibroblast growth factor alone was not obvious. Heparin combined with basic fibroblast growth factor noticeably promoted the differentiation of umbilical cord mesenchymal stem cells into motor neuron-like cells. Simultaneously, umbilical cord mesenchymal stem cells could secrete acetylcholine.

New methods for inducing the differentiation of amniotic-derived mesenchymal stem cells into motor neuron precursor cells.

Our study investigates the differentiation of amniotic-derived mesenchymal stem cells (ADMSCs) into motor neuron (MN) precursor cells induced by a combination of extracellular matrix (ECM) and multi-cell factors. Membrane-like ECM was made by an enzymatic and chemical extraction method and exhibited good biological compatibility. Cells in the experimental group (EG) were treated with ECM and multi-cell factors in a multi-step induction process, while the control group (CG) was treated similarly, except without ECM. In the EG, after induction, the cells formed processes that connected with neighboring cells to form a net that had directionality. In these cells, neuron-specific enolase (NSE) and synaptophysin (SYN) expression levels increased and glial fibrillary acidic protein (GFAP) expression decreased. The SYN expression in the EG cells was higher compared with those in the CG. In the CG, NSE expression increased, while the expression of Nestin and SYN did not change. These were several changes in the levels of other genes: ADMSCs at passage 1 expressed Nanog, SOX2, octamer-binding transcription factor 4 (OCT4) and Nestin. In the EG, at the beginning of induction, the expression of Nanog decreased and that of SOX2 and Nestin increased. After 2 days, the cells expressed Nestin, OCT4 and SYNIII, and after 3 days, they expressed Olig2, OCT4, Nestin, SYNII and Islet1 (ISL1). Finally, at day 6, the cells expressed Nestin, SYNI, SYNIII, ISL-1, homeobox 9 (Hb9) and oligodendrocyte lineage transcription factor 2 (Olig2). In the CG, the cells never expressed SYNI, SYNII or Hb9. Our studies therefore demonstrate that the extracted ECM was capable of promoting the maturation of synapses. Human ADMSCs are composed of multiple cell subsets, including neural progenitor cells. The multi-step induction method used in this study causes human ADMSCs to differentiate into MN precursor cells.