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IntroductionIn the developing brain, neurons extend an axonal process through a complex and changing environment to form synaptic connections with the correct targets in response to extracellular cues. Microtubule and actin filaments provide mechanical support and drive axon growth in the correct direction. The axonal cytoskeleton responds to extracellular guidance cues. Netrin-1 is a multifunctional guidance cue that can induce alternate responses based on the bound receptor. The mechanism by which actin responds to Netrin-1 is well described. However, how Netrin-1 influences the microtubule cytoskeleton is less understood. Appropriate microtubule function is required for axon pathfinding, as mutations in tubulin phenocopy axon crossing defects of Netrin-1 and DCC mutants. Microtubule stabilization is required for attractive guidance cue response. The C-terminal tails of microtubules can be post-translationally modified. Post-translational modifications (PTMs) help control the microtubule cytoskeleton. MethodsWe measured polyglutamylation in cultured primary mouse cortical neurons before and after Netrin-1 stimulation. We used immunohistochemistry to measure how Netrin-1 stimulation alters microtubule-associated protein localization. Next, we manipulated TTLL1 to determine if Netrin-1-induced axon growth and MAP localization depend on polyglutamylation levels. ResultsIn this study, we investigated if Netrin-1 signaling alters microtubule PTMs in the axon. We found that microtubule polyglutamylation increases after Netrin-1 stimulation. This change in polyglutamylation is necessary for Netrin-1-induced axonal growth rate increases. We next determined that MAP1B and DCX localization changes in response to Netrin-1. These proteins can both stabilize the microtubule cytoskeleton and may be responsible for Netrin-1-induced growth response in neurons. The changes in DCX and MAP1B depend on TTLL1, a protein responsible for microtubule polyglutamylation. 

Smoking cigarettes during pregnancy is associated with adverse effects on infants including low birth weight, defective lung development, and skeletal abnormalities. Pregnant women are increasingly turning to vaping [use of electronic (e)-cigarettes] as a perceived safer alternative to cigarettes. However, nicotine disrupts fetal development, suggesting that like cigarette smoking, nicotine vaping may be detrimental to the fetus. To test the impact of maternal vaping on fetal lung and skeletal development in mice, pregnant dams were exposed to e-cigarette vapor throughout gestation. At embryonic day (E)18.5, vape exposed litter sizes were reduced and some embryos exhibited growth restriction compared to air exposed controls. Fetal lungs were collected for histology and whole transcriptome sequencing. Maternally nicotine vaped embryos exhibited histological and transcriptional changes consistent with impaired distal lung development. Embryonic lung gene expression changes mimicked transcriptional changes observed in adult mouse lungs exposed to cigarette smoke, suggesting that the developmental defects may be due to direct nicotine exposure. Fetal skeletons were analyzed for craniofacial and long bone lengths. Nicotine directly binds and inhibits the Kcnj2 potassium channel which is important for bone development. The length of the maxilla, palatal shelves, humerus, and femur were reduced in vaped embryos, which was further exacerbated by loss of one copy of the Kcnj2 gene. Nicotine vapor exposed Kcnj2KO/+ embryos also had significantly lower birth weights than unexposed animals of either genotype. Kcnj2 mutants had severely defective lungs with and without vape exposure, suggesting that potassium channels may be broadly involved in mediating the detrimental developmental effects of nicotine vaping. These data indicate that intrauterine nicotine exposure disrupts fetal lung and skeletal development likely through inhibition of Kcnj2. 

The role of ion channels in neurons and muscles has been well characterized. However, recent work has demonstrated both the presence and necessity of ion channels in diverse cell types for morphological development. For example, mutations that disrupt ion channels give rise to abnormal structural development in species of flies, frogs, fish, mice, and humans. Furthermore, medications and recreational drugs that target ion channels are associated with higher incidence of birth defects in humans. In this review we establish the effects of several teratogens on development including epilepsy treatment drugs (topiramate, valproate, ethosuximide, phenobarbital, phenytoin, and carbamazepine), nicotine, heat, and cannabinoids. We then propose potential links between these teratogenic agents and ion channels with mechanistic insights from model organisms. Finally, we talk about the role of a particular ion channel, Kir2.1, in the formation and development of bone as an example of how ion channels can be used to uncover important processes in morphogenesis. Because ion channels are common targets of many currently used medications, understanding how ion channels impact morphological development will be important for prevention of birth defects. It is becoming increasingly clear that ion channels have functional roles outside of tissues that have been classically considered excitable.