Molecular components underlying nongenomic thyroid hormone signaling in embryonic zebrafish neurons.
BACKGROUND
Neurodevelopment requires thyroid hormone, yet the mechanisms and targets of thyroid hormone action during embryonic stages remain ill-defined. We previously showed that the thyroid hormone thyroxine (T4) rapidly increases voltage-gated sodium current in zebrafish Rohon-Beard cells (RBs), a primary sensory neuron subtype present during embryonic development. Here, we determined essential components of the rapid T4 signaling pathway by identifying the involved intracellular messengers, the targeted sodium channel isotype, and the spatial and temporal expression pattern of the nongenomic alphaVbeta3 integrin T4 receptor.


RESULTS
We first tested which signaling pathways mediate T4's rapid modulation of sodium current (I(Na)) by perturbing specific pathways associated with nongenomic thyroid hormone signaling. We found that pharmacological blockade of protein phosphatase 1 and the mitogen-activated protein kinase p38 isoform decreased and increased tonic sodium current amplitudes, respectively, and blockade of either occluded rapid responses to acute T4 application. We next tested for the ion channel target of rapid T4 signaling via morpholino knock-down of specific sodium channel isotypes. We found that selective knock-down of the sodium channel alpha-subunit Na(v)1.6a, but not Na(v)1.1la, occluded T4's acute effects. We also determined the spatial and temporal distribution of a nongenomic T4 receptor, integrin alphaVbeta3. At 24 hours post fertilization (hpf), immunofluorescent assays showed no specific integrin alphaVbeta3 immunoreactivity in wild-type zebrafish embryos. However, by 48 hpf, embryos expressed integrin alphaVbeta3 in RBs and primary motoneurons. Consistent with this temporal expression, T4 modulated RB I(Na) at 48 but not 24 hpf. We next tested whether T4 rapidly modulated I(Na) of caudal primary motoneurons, which express the receptor (alphaVbeta3) and target (Na(v)1.6a) of rapid T4 signaling. In response to T4, caudal primary motoneurons rapidly increased sodium current peak amplitude 1.3-fold.


CONCLUSIONS
T4's nongenomic regulation of sodium current occurs in different neuronal subtypes, requires the activity of specific phosphorylation pathways, and requires both integrin alphaVbeta3 and Na(v)1.6a. Our in vivo analyses identify molecules required for T4's rapid regulation of voltage-gated sodium current.
Yonkers
Marc A
MA
Department of Physiology and Biophysics, University of Colorado Denver at Anschutz Medical Center, Aurora, Colorado 80045, USA. marc.yonkers@ucdenver.edu
Ribera
Angeles B
AB
D000704:Analysis of Variance; D000818:Animals; D030801:Animals, Genetically Modified; D004625:Embryo, Nonmammalian; D004791:Enzyme Inhibitors / Q000494:pharmacology; D049452:Green Fluorescent Proteins / Q000235:genetics; D006727:Hormone Antagonists / Q000494:pharmacology; D039302:Integrin alphaVbeta3 / Q000378:metabolism; D008564:Membrane Potentials / Q000187:drug effects; D062557:NAV1.6 Voltage-Gated Sodium Channel; D009474:Neurons / Q000187:drug effects* / Q000502:physiology; D020319:Oligodeoxyribonucleotides, Antisense / Q000494:pharmacology; D018408:Patch-Clamp Techniques / Q000379:methods; D011988:Receptors, Thyroid Hormone / Q000502:physiology*; D015398:Signal Transduction / Q000187:drug effects* / Q000502:physiology*; D015222:Sodium Channels / Q000235:genetics; D012965:Sodium Chloride / Q000494:pharmacology; D013116:Spinal Cord / Q000166:cytology; D013974:Thyroxine / Q000494:pharmacology*; D015027:Zebrafish / Q000033:anatomy & histology / Q000196:embryology; D029961:Zebrafish Proteins / Q000037:antagonists & inhibitors / Q000235:genetics
D004791:Enzyme Inhibitors; D006727:Hormone Antagonists; D039302:Integrin alphaVbeta3; D062557:NAV1.6 Voltage-Gated Sodium Channel; D020319:Oligodeoxyribonucleotides, Antisense; D011988:Receptors, Thyroid Hormone; D015222:Sodium Channels; D029961:Zebrafish Proteins; C515010:scn8aa protein, zebrafish; D049452:Green Fluorescent Proteins; D012965:Sodium Chloride; D013974:Thyroxine