Induction of motor neurons by Sonic hedgehog is independent of floor plate differentiation

AbstractBackground: The differentiation of floor plate cells and motor neurons in the vertebrate neural tube appears to be induced by signals from the notochord. The secreted protein encoded by the Sonic hedgehog (Shh) gene is expressed by axial midline cells and can induce floor plate cells in vivo and in vitro. Motor neurons can also be induced in vitro by cells that synthesize Sonic hedgehog protein (Shh). It remains unclear, however, if the motor-neuron-inducing activity of Shh depends on the synthesis of a distinct signaling molecule by floor plate cells. To resolve this issue, we have developed an in vitro assay which uncouples the notochord-mediated induction of motor neurons from floor plate differentiation, and have used this assay to examine whether Shh induces motor neurons in the absence of floor plate differentiation.Results Floor plate cells and motor neurons were induced in neural plate explants grown in contact with the notochord, but only motor neurons were induced when explants were separated from the notochord. COS cells transfected with Shh induced both floor plate cells and motor neurons when grown in contact with neural plate explants, whereas only motor neurons were induced when the explants were grown at a distance from Shh-transfected COS cells. Direct transfection of neural plate cells with an Shh-expression construct induced both floor plate cells and motor neurons, with motor neuron differentiation occurring prior to, or coincidentally with, floor plate differentiation. The induction of motor neurons appears, therefore, not to depend on floor plate differentiation.Conclusion The induction of motor neurons by Shh does not depend on distinct floor-plate-derived signaling molecules. Shh can, therefore, initiate the differentiation of two cell types that are generated in the ventral region of the neural tube. These results show that the early development of motor neurons involves the inductive action of Shh, whereas the survival of motor neurons at later stages of embryonic development requires neurotrophic factors

Mapping Sensory Circuits by Anterograde Transsynaptic Transfer of Recombinant Rabies Virus

SummaryPrimary sensory neurons convey information from the external world to relay circuits within the CNS, but the identity and organization of the neurons that process incoming sensory information remains sketchy. Within the CNS, viral tracing techniques that rely on retrograde transsynaptic transfer provide a powerful tool for delineating circuit organization. Viral tracing of the circuits engaged by primary sensory neurons has, however, been hampered by the absence of a genetically tractable anterograde transfer system. In this study, we demonstrate that rabies virus can infect sensory neurons in the somatosensory system, is subject to anterograde transsynaptic transfer from primary sensory to spinal target neurons, and can delineate output connectivity with third-order neurons. Anterograde transsynaptic transfer is a feature shared by other classes of primary sensory neurons, permitting the identification and potentially the manipulation of neural circuits processing sensory feedback within the mammalian CNS

Complementary Domains of Retinoic Acid Production and Degradation in the Early Chick Embryo

AbstractExcess retinoids as well as retinoid deprivation cause abnormal development, suggesting that retinoid homeostasis is critical for proper morphogenesis. RALDH-2 and CYP26, two key enzymes that carry out retinoic acid (RA) synthesis and degradation, respectively, were cloned from the chick and show significant homology with their orthologs in other vertebrates. Expression patterns of RALDH-2 and CYP26 genes were determined in the early chick embryo by in situ hybridization. During gastrulation and neurulation RALDH-2 and CYP26 were expressed in nonoverlapping regions, with RALDH-2 transcripts localized to the presumptive presomitic and lateral plate mesoderm and CYP26 mRNA to the presumptive mid- and forebrain. The two domains of expression were separated by an approximately 300-μm-wide gap, encompassing the presumptive hindbrain. In the limb region, a similar spatial segregation of RALDH-2 and CYP26 expression was found at stages 14 and 15. Limb region mesoderm expressed RALDH-2, whereas the overlying limb ectoderm expressed CYP26. RA-synthesizing and -degrading enzymatic activities were measured biochemically in regions expressing RALDH-2 or CYP26. Regions expressing RALDH-2 generated RA efficiently from precursor retinal but degraded RA only inefficiently. Conversely, tissue expressing CYP26 efficiently degraded but did not synthesize RA. Localized regions of RA synthesis and degradation mediated by these two enzymes may therefore provide a mechanism to regulate RA homeostasis spatially in vertebrate embryos

Genetic Targeting of Adult Renshaw Cells Using a Calbindin 1 Destabilized Cre Allele for Intersection With Parvalbumin or Engrailed1

Renshaw cells (RCs) are one of the most studied spinal interneurons; however, their roles in motor control remain enigmatic in part due to the lack of experimental models to interfere with RC function, specifically in adults. To overcome this limitation, we leveraged the distinct temporal regulation of Calbindin (Calb1) expression in RCs to create genetic models for timed RC manipulation. We used a Calb1 allele expressing a destabilized Cre (dgCre) theoretically active only upon trimethoprim (TMP) administration. TMP timing and dose influenced RC targeting efficiency, which was highest within the first three postnatal weeks, but specificity was low with many other spinal neurons also targeted. In addition, dgCre showed TMP-independent activity resulting in spontaneous recombination events that accumulated with age. Combining Calb1-dgCre with Parvalbumin (Pvalb) or Engrailed1 (En1) Flpo alleles in dual conditional systems increased cellular and timing specificity. Under optimal conditions, Calb1-dgCre/Pvalb-Flpo mice targeted 90% of RCs and few dorsal horn neurons; Calb1-dgCre/En1-Flpo mice showed higher specificity, but only a maximum of 70% of RCs targeted. Both models targeted neurons throughout the brain. Restricted spinal expression was obtained by injecting intraspinally AAVs carrying dual conditional genes. These results describe the first models to genetically target RCs bypassing development

Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus

BACKGROUND: Several Cre reporter strains of mice have been described, in which a lacZ gene is turned on in cells expressing Cre recombinase, as well as their daughter cells, following Cre-mediated excision of a loxP-flanked transcriptional "stop" sequence. These mice are useful for cell lineage tracing experiments as well as for monitoring the expression of Cre transgenes. The green fluorescent protein (GFP) and variants such as EYFP and ECFP offer an advantage over lacZ as a reporter, in that they can be easily visualized without recourse to the vital substrates required to visualize β-gal in living tissue. RESULTS: In view of the general utility of targeting the ubiquitously expressed ROSA26 locus, we constructed a generic ROSA26 targeting vector. We then generated two reporter lines of mice by inserting EYFP or ECFP cDNAs into the ROSA26 locus, preceded by a loxP-flanked stop sequence. These strains were tested by crossing them with transgenic strains expressing Cre in a ubiquitous (β-actin-Cre) or a cell-specific (Isl1-Cre and En1-Cre) pattern. The resulting EYFP or ECFP expression patterns indicated that the reporter strains function as faithful monitors of Cre activity. CONCLUSIONS: In contrast to existing lacZ reporter lines, where lacZ expression cannot easily be detected in living tissue, the EYFP and ECFP reporter strains are useful for monitoring the expression of Cre and tracing the lineage of these cells and their descendants in cultured embryos or organs. The non-overlapping emission spectra of EYFP and ECFP make them ideal for double labeling studies in living tissues

Stringent Specificity in the Construction of a GABAergic Presynaptic Inhibitory Circuit

SummaryGABAergic interneurons are key elements in neural coding, but the mechanisms that assemble inhibitory circuits remain unclear. In the spinal cord, the transfer of sensory signals to motor neurons is filtered by GABAergic interneurons that act presynaptically to inhibit sensory transmitter release and postsynaptically to inhibit motor neuron excitability. We show here that the connectivity and synaptic differentiation of GABAergic interneurons that mediate presynaptic inhibition is directed by their sensory targets. In the absence of sensory terminals these GABAergic neurons shun other available targets, fail to undergo presynaptic differentiation, and withdraw axons from the ventral spinal cord. A sensory-specific source of brain derived neurotrophic factor induces synaptic expression of the GABA synthetic enzyme GAD65 – a defining biochemical feature of this set of interneurons. The organization of a GABAergic circuit that mediates presynaptic inhibition in the mammalian CNS is therefore controlled by a stringent program of sensory recognition and signaling

Integration of geoscientific uncertainty into geophysical inversion by means of local gradient regularization

We introduce a workflow integrating geological modelling uncertainty information to constrain gravity inversions. We test and apply this approach to the Yerrida Basin (Western Australia), where we focus on prospective greenstone belts beneath sedimentary cover. Geological uncertainty information is extracted from the results of a probabilistic geological modelling process using geological field data and their inferred accuracy as inputs. The uncertainty information is utilized to locally adjust the weights of a minimum-structure gradient-based regularization function constraining geophysical inversion. Our results demonstrate that this technique allows geophysical inversion to update the model preferentially in geologically less certain areas. It also indicates that inverted models are consistent with both the probabilistic geological model and geophysical data of the area, reducing interpretation uncertainty. The interpretation of inverted models reveals that the recovered greenstone belts may be shallower and thinner than previously thought.</p

Differential loss of spinal interneurons in a mouse model of ALS

Amyotrophic lateral sclerosis (ALS) leads to a loss of specific motor neuron populations in the spinal cord and cortex. Emerging evidence suggests that interneurons may also be affected, but a detailed characterization of interneuron loss and its potential impacts on motor neuron loss and disease progression is lacking. To examine this issue, the fate of V1 inhibitory neurons during ALS was assessed in the ventral spinal cord using the SODG93A mouse model. The V1 population makes up ∼30% of all ventral inhibitory neurons, ∼50% of direct inhibitory synaptic contacts onto motor neuron cell bodies, and is thought to play a key role in modulating motor output, in part through recurrent and reciprocal inhibitory circuits. We find that approximately half of V1 inhibitory neurons are lost in SODG93A mice at late disease stages, but that this loss is delayed relative to the loss of motor neurons and V2a excitatory neurons. We further identify V1 subpopulations based on transcription factor expression that are differentially susceptible to degeneration in SODG93A mice. At an early disease stage, we show that V1 synaptic contacts with motor neuron cell bodies increase, suggesting an upregulation of inhibition before V1 neurons are lost in substantial numbers. These data support a model in which progressive changes in V1 synaptic contacts early in disease, and in select V1 subpopulations at later stages, represent a compensatory upregulation and then deleterious breakdown of specific interneuron circuits within the spinal cord

Utilisation of probabilistic magnetotelluric modelling to constrain magnetic data inversion: proof-of-concept and field application

We propose, test and apply a methodology integrating 1D magnetotelluric (MT) and magnetic data inversion, with a focus on the characterisation of the cover–basement interface. It consists of a cooperative inversion workflow relying on standalone inversion codes. Probabilistic information about the presence of rock units is derived from MT and passed on to magnetic inversion through constraints combining structural constraints with petrophysical prior information. First, we perform the 1D probabilistic inversion of MT data for all sites and recover the respective probabilities of observing the cover–basement interface, which we interpolate to the rest of the study area. We then calculate the probabilities of observing the different rock units and partition the model into domains defined by combinations of rock units with non-zero probabilities. Third, we combine these domains with petrophysical information to apply spatially varying, disjoint interval bound constraints (DIBC) to least-squares magnetic data inversion using the alternating direction method of multipliers (or ADMM). We demonstrate the proof-of-concept using a realistic synthetic model reproducing features from the Mansfield area (Victoria, Australia) using a series of uncertainty indicators. We then apply the workflow to field data from the prospective mining region of Cloncurry (Queensland, Australia). Results indicate that our integration methodology efficiently leverages the complementarity between separate MT and magnetic data modelling approaches and can improve our capability to image the cover–basement interface. In the field application case, our findings also suggest that the proposed workflow may be useful to refine existing geological interpretations and to infer lateral variations within the basement.</p

Onecut-dependent Nkx6.2 transcription factor expression is required for proper formation and activity of spinal locomotor circuits.

In the developing spinal cord, Onecut transcription factors control the diversification of motor neurons into distinct neuronal subsets by ensuring the maintenance of Isl1 expression during differentiation. However, other genes downstream of the Onecut proteins and involved in motor neuron diversification have remained unidentified. In the present study, we generated conditional mutant embryos carrying specific inactivation of Onecut genes in the developing motor neurons, performed RNA-sequencing to identify factors downstream of Onecut proteins in this neuron population, and employed additional transgenic mouse models to assess the role of one specific Onecut-downstream target, the transcription factor Nkx6.2. Nkx6.2 expression was up-regulated in Onecut-deficient motor neurons, but strongly downregulated in Onecut-deficient V2a interneurons, indicating an opposite regulation of Nkx6.2 by Onecut factors in distinct spinal neuron populations. Nkx6.2-null embryos, neonates and adult mice exhibited alterations of locomotor pattern and spinal locomotor network activity, likely resulting from defective survival of a subset of limb-innervating motor neurons and abnormal migration of V2a interneurons. Taken together, our results indicate that Nkx6.2 regulates the development of spinal neuronal populations and the formation of the spinal locomotor circuits downstream of the Onecut transcription factors