The diaphragm muscle is essential for breathing in mammals. Its asymmetric elevation during contraction correlates with morphological features suggestive of inherent left-right (L/R) asymmetry. Whether this asymmetry is due to L versus R differences in the muscle or in the phrenic nerve activity is unknown. Here, we have combined the analysis of genetically modified mouse models with transcriptomic analysis to show that both the diaphragm muscle and phrenic nerves have asymmetries, which can be established independently of each other during early embryogenesis in pathway instructed by Nodal, a morphogen that also conveys asymmetry in other organs. We further found that phrenic motoneurons receive an early L/R genetic imprint, with L versus R differences both in Slit/Robo signaling and MMP2 activity and in the contribution of both pathways to establish phrenic nerve asymmetry. Our study therefore demonstrates L-R imprinting of spinal motoneurons and describes how L/R modulation of axon guidance signaling helps to match neural circuit formation to organ asymmetry.
Collateral branches from axons are key components of functional neural circuits that allow neurons to connect with multiple synaptic targets. Like axon growth and guidance, formation of collateral branches depends on the regulation of microtubules, but how such regulation is coordinated to ensure proper circuit development is not known. Based on microarray analysis, we have identified a role for microtubule-associated protein 7 (MAP7) during collateral branch development of dorsal root ganglion (DRG) sensory neurons. We show that MAP7 is expressed at the onset of collateral branch formation. Perturbation of its expression by overexpression or shRNA knockdown alters axon branching in cultured DRG neurons. Localization and time-lapse imaging analysis reveals that MAP7 is enriched at branch points and colocalizes with stable microtubules, but enters the new branch with a delay, suggesting a role in branch maturation. We have also investigated a spontaneous mutant mouse that expresses a truncated MAP7 and found a gain-of-function phenotype both in vitro and in vivo Further domain analysis suggests that the amino half of MAP7 is responsible for branch formation, suggesting a mechanism that is independent of its known interaction with kinesin. Moreover, this mouse exhibits increased pain sensitivity, a phenotype that is consistent with increased collateral branch formation. Therefore, our study not only uncovers the first neuronal function of MAP7, but also demonstrates the importance of proper microtubule regulation in neural circuit development. Furthermore, our data provide new insights into microtubule regulation during axonal morphogenesis and may shed light on MAP7 function in neurological disorders.SIGNIFICANCE STATEMENT Neurons communicate with multiple targets by forming axonal branches. In search of intrinsic factors that control collateral branch development, we identified a role for microtubule-associated protein 7 (MAP7) in dorsal root ganglion sensory neurons. We show that MAP7 expression is developmentally regulated and perturbation of this expression alters branch formation. Cell biological analysis indicates that MAP7 promotes branch maturation. Analysis of a spontaneous mouse mutant suggests a molecular mechanism for branch regulation and the potential influence of collateral branches on pain sensitivity. Our studies thus establish the first neuronal function of MAP7 and demonstrate its role in branch morphogenesis and neural circuit function. These findings may help in our understanding of the contribution of MAP7 to neurological disorders and nerve regeneration.
Low-level lead (Pb) exposure is a risk factor for learning disabilities, attention deficit hyperactivity disorder (ADHD), and other neurological dysfunction. It is not known how Pb produces these behavioral deficits, but low-level exposure during development is associated with auditory temporal processing deficits in an avian model, while hearing thresholds remain normal. Similar auditory processing deficits are found in children with learning disabilities and ADHD. To identify cellular changes underlying this functional deficit, Pb-induced alterations of neurons and glia within the mammalian auditory brainstem nuclei were quantified in control and Pb-exposed mice at postnatal day 21 by using immunohistochemistry, Western blotting, and 2D gel electrophoresis. Pb-treated mice were exposed to either 0.1 mM (low) or 2 mM (high) Pb acetate throughout gestation and through 21 days postnatally. Pb exposure results in little change in glial proteins such as glial fibrillary acidic protein (GFAP), myelin basic protein (MBP), or F4/80 as determined by Western blot analysis and immunohistochemistry. In contrast, Pb exposure alters neuronal structural proteins by inducing increased phosphorylation of both the medium (NFM) and high-weight (NFH) forms of neurofilament within auditory brainstem nuclei. Axons immunolabeled for neurofilament protein show neuritic beading following Pb exposure both in vivo and in vitro, suggesting that Pb exposure also impairs axonal transport. Functional assessment shows no significant loss of peripheral function, but does reveal impairments in brainstem conduction time and temporal processing within the brainstem. These results provide evidence that Pb exposure during development alters axonal structure and function within brainstem auditory nuclei.