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

Role of muscle spindle feedback in regulating muscle activity strength during walking at different speed in mice

Terrestrial animals increase their walking speed by increasing the activity of the extensor muscles. However, the mechanism underlying this speed dependent amplitude modulation is achieved remain obscure. Previous studies have shown that group Ib afferent feedback from Golgi tendon organs that signal force is one of the major regulators of the strength of muscle activity during walking in cats and humans. In contrast, the contribution of group Ia/II afferent feedback from muscle spindle stretch receptors which signal angular displacement of leg joints is unclear. Some studies indicate that group II afferent feedback may be important for amplitude regulation in humans, but the role of muscle spindle feedback in regulation of muscle activity strength in quadrupedal animals is very poorly understood. To examine the role of feedback from muscle spindles, we combined in vivo electrophysiology and motion analysis with mouse genetics and gene delivery with adeno associated virus. We provide evidence that proprioceptive sensory feedback from muscle spindles is important for the regulation of the muscle activity strength and speed dependent amplitude modulation. Furthermore, our data suggest that feedback from the muscle spindles of the ankle extensor muscles, the triceps surae, are the main source for this mechanism. In contrast, muscle spindle feedback from the knee extensor muscles, the quadriceps femoris, has no influence on speed dependent amplitude modulation. We provide evidence that proprioceptive feedback from ankle extensor muscles is critical for regulating muscle activity strength as gait speed increases

Floor plate and motor neuron induction by different concentrations of the amino-terminal cleavage product of sonic hedgehog autoproteolysis

AbstractThe differentiation of floor plate cells and motor neurons can be induced by Sonic hedgehog (SHH), a secreted signaling protein that undergoes autoproteolytic cleavage to generate amino- and carboxy-terminal products. We have found that both floor plate cells and motor neurons are induced by the aminoterminal cleavage product of SHH (SHH-N). The threshold concentration of SHH-N required for motor neuron induction is about 5-fold lower than that required for floor plate induction. Higher concentrations of SHH-N can induce floor plate cells at the expense of motor neuron differentiation. Our results suggest that the induction of floor plate cells and motor neurons by the notochord in vivo is mediated by exposure of neural plate cells to different concentrations of the amino-terminal product of SHH autoproteolytic cleavage

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

Neural development: Patterning cascades in the neural tube

The vertebrate central nervous system comprises an intricate array of neurons generated in a highly organized way. Examination of the genes expressed and required at early stages of neural differentiation reveals that a coordinated signalling cascade transforms progenitor cells into discrete neuronal subsets

Adaptive hybrid optimization strategy for calibration and parameter estimation of physical models

A new adaptive hybrid optimization strategy, entitled squads, is proposed for complex inverse analysis of computationally intensive physical models. The new strategy is designed to be computationally efficient and robust in identification of the global optimum (e.g. maximum or minimum value of an objective function). It integrates a global Adaptive Particle Swarm Optimization (APSO) strategy with a local Levenberg-Marquardt (LM) optimization strategy using adaptive rules based on runtime performance. The global strategy optimizes the location of a set of solutions (particles) in the parameter space. The LM strategy is applied only to a subset of the particles at different stages of the optimization based on the adaptive rules. After the LM adjustment of the subset of particle positions, the updated particles are returned to the APSO strategy. The advantages of coupling APSO and LM in the manner implemented in squads is demonstrated by comparisons of squads performance against Levenberg-Marquardt (LM), Particle Swarm Optimization (PSO), Adaptive Particle Swarm Optimization (APSO; the TRIBES strategy), and an existing hybrid optimization strategy (hPSO). All the strategies are tested on 2D, 5D and 10D Rosenbrock and Griewank polynomial test functions and a synthetic hydrogeologic application to identify the source of a contaminant plume in an aquifer. Tests are performed using a series of runs with random initial guesses for the estimated (function/model) parameters. Squads is observed to have the best performance when both robustness and efficiency are taken into consideration than the other strategies for all test functions and the hydrogeologic application