Bioelectric signalling via potassium channels: a mechanism for craniofacial dysmorphogenesis in KCNJ2‐associated Andersen–Tawil Syndrome

Abstract

Key pointsr Xenopus laevis craniofacial development is a good system for the study of Andersen-Tawil Syndrome (ATS)-associated craniofacial anomalies (CFAs) because (1) Kcnj2 is expressed in the nascent face; (2) molecular-genetic and biophysical techniques are available for the study of ion-dependent signalling during craniofacial morphogenesis; (3) as in humans, expression of variant Kcnj2 forms in embryos causes a muscle phenotype; and (4) variant forms of Kcnj2 found in human patients, when injected into frog embryos, cause CFAs in the same cell lineages. r Expression of other potassium channels and two different light-activated channels, all of which have an effect on V mem , causes CFAs like those induced by injection of Kcnj2 variants. In contrast, expression of Slc9A (NHE3), an electroneutral ion channel, and of GlyR, an inactive Cl − channel, do not cause CFAs, demonstrating that correct craniofacial development depends on a pattern of bioelectric states, not on ion-or channel-specific signalling.r Using optogenetics to control both the location and the timing of ion flux in developing embryos, we show that affecting V mem of the ectoderm and no other cell layers is sufficient to cause CFAs, but only during early neurula stages. Changes in V mem induced late in neurulation do not affect craniofacial development.r We interpret these data as strong evidence, consistent with our hypothesis, that ATS-associated CFAs are caused by the effect of variant Kcnj2 on the V mem of ectodermal cells of the developing face. We predict that the critical time is early during neurulation, and the critical cells are the ectodermal cranial neural crest and placode lineages. This points to the potential utility of extant, ion flux-modifying drugs as treatments to prevent CFAs associated with channelopathies such as ATS.Abstract Variants in potassium channel KCNJ2 cause Andersen-Tawil Syndrome (ATS); the induced craniofacial anomalies (CFAs) are entirely unexplained. We show that KCNJ2 is expressed in Xenopus and mouse during the earliest stages of craniofacial development. resting potentials using other ion translocators, we show that a change in ectodermal voltage, not tied to a specific protein or ion, is sufficient to cause CFAs. By adapting optogenetics for use in non-neural cells in embryos, we show that developmentally patterned K + flux is required for correct regionalization of the resting potentials and for establishment of endogenous early gene expression domains in the anterior ectoderm, and that variants in KCNJ2 disrupt this regionalization, leading to the CFAs seen in ATS patients.

Systems biology of facial development: contributions of ectoderm and mesenchyme

“…First, we detected expression of the voltage-gated potassium channel Kcnh7 and the A-type current regulator Kcnip1 in this late ectoderm program. This finding is consistent with a role for potassium currents in craniofacial patterning (Adams et al, 2016; Dahal et al, 2012), and provides a specific temporal focus for electrical excitability in patterning the ectoderm. Second, the Gaba receptor A, Pi subunit (Gabrp) was highly expressed in ectoderm from E11.5 and E12.5 (e.g.…”

Modeling Human Craniofacial Disorders in Xenopus

“…The specific role of Kir 2.1 during craniofacial development was addressed more recently. In Xenopus , kcnj2 is initially expressed at early neurula stage (NF stage 14) in a broad dorso-anterior domain, which later becomes regionalized anteriorly, consistent with a potential role in craniofacial morphogenesis [92]. Previous studies from this group showed that there is a unique pattern of membrane resting potential (V mem ) in the ectoderm of the embryo that is critical to maintain gene expression domains as development progresses [93].…”

“…Injection of either wild-type (WT) Kcnj2 or a variant carrying one of the known ATS mutations led to changes in the normal pattern of V mem at the neurula stage, and resulted in craniofacial anomalies at the tadpole stage. However, Kcnj2 mutant proteins caused defects at a higher frequency than the WT [92]. The craniofacial anomalies observed in these tadpoles included misshapen eyes, small or bent Meckel’s cartilage, undersized branchial arches and pigmented optic nerve.…”

“…Interestingly, misexpression of other channels that also alter the V mem caused similar craniofacial anomalies. This effect was not seen upon overexpression of an electroneutral ion channel, indicating that rather than the specific identity of the channel, it is the maintenance of the bioelectric state of the embryo that is critical for craniofacial morphogenesis [92]. …”

“…Finally, using optogenetics to spatially and temporally alter the V mem of the embryo, the work showed that maintaining a normal pattern of V mem in the ectoderm during early neurula stages is especially critical for normal craniofacial development - altering the V mem during late neurulation did not impact craniofacial development [92]. …”