HCN4 ion channel function is required for early events that regulate anatomical left-right patterning in a nodal and lefty asymmetric gene expression-independent manner

Here we have identified a novel role of HCN4 channels during embryonic left-right patterning. Many previous reports indicate a critical role of ion translocators and ion fluxes in determining embryonic left-right asymmetry (Adams et al., 2006; Aw et al., 2008, 2010; Levin et al., 2002; Morokuma et al., 2008). In particular, asymmetric functions of H+/K+-ATPase and V-ATPase (Hibino et al., 2006; Kawakami et al., 2005; Levin et al., 2002; Shimeld and Levin, 2006), as well as two other potassium channels (KCNQ1 and KATP) have been implicated. For the majority of these ion translocators, their asymmetric localization and action is post 4-cell stage, but before gastrulation. Crucially, HCN4 inhibition affects asymmetry during early embryogenesis – an observation that is incompatible with potential hypotheses about roles in regulating much later events like ciliary motion at gastrulation (Basu and Brueckner, 2008) since all of those events would be targeted by inhibitor exposure starting at stage 10. The same is true of many other highly-conserved elements of the left-right symmetry breaking machinery, such as cytoskeletal proteins (Davison et al., 2016; Lobikin et al., 2012), and reinforces the importance of focusing on intracellular, biophysical events as the earliest components of left-right pattering.

We found that HCN4 channels are already present in 2-cell embryos (most likely maternally loaded) and are uniformly expressed throughout the embryo all the way through gastrulation (Fig. 3). Hence, the action of HCN4 in establishing laterality is most likely at the physiological (post-translational gating) level of its function. This is not unprecedented, as in zebrafish the H+/K+-ATPase is uniformly expressed throughout the embryo and still is involved in laterality determination by its actions at the physiological level (Kawakami et al., 2005). Moreover, HCN4 is gated by a number of ligands (e.g. cAMP) which can be differentially localized to result in differential bioelectrical activity even if HCN4 protein is ubiquitous. Future studies using fluorescent reporters of cAMP and individual ion concentrations (being developed by a number of groups but not yet available in Xenopus), as well as transgenic Xenopus in which native HCN4 is labeled with a fluorescent tag, will dissect the very early steps of HCN4 activity. New techniques for introducing material into Xenopus eggs prior to fertilization may also be useful in manipulating this process, since injections even at 1-cell stage may be attenuated in their effects by the amount of time needed to make protein from the mRNA introduced then.

Establishing invariant laterality is a fundamental aspect of most life forms across the tree of life. It is becoming clear that the mechanisms of establishing and executing laterality are redundant and highly robust to ensure correct organ situs even in presence of certain errors in the pathway. Many fascinating questions remain about the physiological processes that transmit and amplify physical chirality of intracellular cytoskeletal structures into embryo-wide programs of gene expression, and ultimately to the consistent asymmetry of organogenesis. The characterization of a novel player, the HCN4 channel, provides a new entry point into pathways which act very early during embryogenesis and then bypass the canonical asymmetric gene expression cascade of nodal-lefty-pitx2 to exert their effects much later. Moreover, the discovery of new ion channels that underlie the endogenous bioelectric signaling that is increasingly seen to be an important component of developmental (Bates, 2015; Levin, 2012; Levin and Stevenson, 2012) and regenerative (Chifflet et al., 2005; Levin, 2014b; Wang and Zhao, 2010) patterning, adds to the toolbox of available targets for understanding and control of growth and form. The investigation of the dynamic interplay between early bioelectrics, subsequent transcriptional regulation, and resultant anatomical patterning presents exciting opportunities for understanding developmental and evolutionary dynamics. It is also possible that the study of compensatory redundant pathways will reveal new approaches for harnessing the robustness of developmental mechanisms for regenerative medicine.

Xenopus laevis embryos were fertilized in vitro according to standard protocols (Sive et al., 2000) in 0.1× Marc's Modified Ringer's (MMR; 10 mM Na+, 0.2 mM K+, 10.5 mM Cl−, 0.2 mM Ca2+, pH 7.8). Xenopus embryos were housed at 14-18°C (14°C overnight after injection and subsequently at 18°C), except during drug exposure which was at 22°C, and staged according to Nieuwkoop and Faber (1967). All experiments were approved by the Tufts University Animal Research Committee (M2014-79) in accordance with the guide for care and use of laboratory animals.

Capped synthetic mRNAs generated using mMessage mMachine kit (Ambion) were dissolved in nuclease free water and injected into embryos immersed in 3% Ficoll using standard methods (Sive et al., 2000). Each injection delivered between ∼0.5-1 nl (0.5-1 ng) of mRNA (per blastomere) into the embryos, at the indicated stages into the middle of the cell in the animal pole. HCN4-DN was a mammalian (mouse) HCN4, modified as per Pitcairn et al. (2017). Briefly, a standard approach was used for generating dominant-negative channel subunit (Kuzhikandathil and Oxford, 2000; Preisig-Muller et al., 2002; Xue et al., 2002). The HCN4 channel function was abolished by altering the highly conserved cation selective sequence in the pore domain (changed from GYG349-351 to AAA349-351) to generate HCN4-(AAA)-DN mutant from HCN4-WT using primers: 5′CACATGCTGTGCATTGAGGACGAACGTCAGGCA-3′ (forward) and 5'-TGCCTGACGTTCGTCCTCAATGCACAGCATGTG-3′ (reverse). The construct was subcloned into a pCS2 vector to transcribe into mRNAs for Xenopus microinjections.

Xenopus embryos were analyzed as in Levin and Mercola (1998) for position (situs) of three organs: heart, gut and gallbladder at stage 45 (Nieuwkoop and Faber, 1967) using fiber light illumination from the ventral side. Heterotaxic embryos were defined as ones having a reversal in one or more organs. Treatments were titered to levels that gave rise to >90% embryos with normal dorso-anterior development and correctly-formed organs. All reported left-right inversions are embryos with clear (unambiguous) left-right organ situs. It is important to note that our analysis is extremely stringent – it underestimates the overall effect of any given treatment. Even if 100% of embryos are affected by some manipulation, the maximum observable effect will still be capped at 87.5% as some percentage of embryos will have all three organs randomly land in correct orientation making them indistinguishable from the wild type and hence will be scored as normal.

Xenopus embryos were incubated in pharmacological blocker of HCN4 channel ZD7288 (Tocris biosciences) (100 mM stock solution in water) dissolved in 0.1× MMR (final concentration 100 µM) during the stages indicated in respective experiments followed by several washes with 0.1× MMR. Note that these experiments were performed at 22°C since ZD7288 effect on embryonic left-right asymmetry was found stronger at 22°C and not at 14°C and 18°C (data not shown) (Yanagida et al., 2000). Untreated embryos reared at 22°C served as controls.

CC2-DMPE and DiBAC4(3) voltage reporter dyes were obtained from Invitrogen and used as per the standard protocol, including dark-field and flat-field correction (Adams and Levin, 2012). Briefly, the use of two dyes with opposite emission profiles simultaneously provides an internal control and allows ratiometric normalization. CC2-DMPE stock (5 mM) was dissolved 1:1000 in 0.1× MMR and the embryos were incubated in dark in this solution for at least 1 h followed by washes with 0.1× MMR. DiBAC4(3) stock (1.9 mM) was dissolved 1:4000 in 0.1× MMR and the CC2-DMPE-stained embryos were then incubated in dark in this solution for at least 30 min followed by visualization under the microscope. An Olympus BX-61 microscope equipped with a Hamamatsu ORCA AG CCD camera, and controlled by Metamorph software (Molecular Devices), was used to collect signal. NIH Image J software was used to quantify the fluorescence intensities of the CC2-DMPE:DiBAC signal.

Membrane potentials were measured using an oocyte clamp OC-725C amplifier (Warner Instruments, Hamden, CT, USA) with a single voltage electrode. Microelectrodes were made from thin-walled borosilicate glass pulled with a flaming/brown micropipette puller (p-97, Sutter Instruments, Novato, CA, USA) and back filled with electrode solution (2 M potassium acetate, 10 mM KCl, 5 mM HEPES pH 7.5). Tip resistances were 80-100 MΩ. Electrode penetration of ectodermal cells was by visual guidance on a fixed-stage microscope (Zeiss) using a three-axis micromanipulator.

Spatial distribution of HCN4 channel in embryos was detected by immunofluorescence for the HCN4 channel on whole embryos. Briefly, embryos were fixed overnight in MEMFA at 4°C (Sive et al., 2000). The embryos were permeabilized in PBS 0.1%Triton-X-100, blocked with 10% goat serum in PBST for 1 h at room temperature, and incubated at 4°C overnight with primary antibody (Anti-HCN4 – rabbit polyclonal; Abcam ab66501) for HCN4 at 1:500 dilution in PBST+10% goat serum (blocking buffer). Embryos were washed six times in PBST and incubated with Alexa Fluor-conjugated fluorescent secondary antibody (Invitrogen) at 1:500 dilution in PBST+10% goat serum overnight at 4°C. Embryos were washed six times in PBST and photographed using Nikon SMZ-1500 scope with Q-capture software.

Xenopus embryos were collected and fixed in MEMFA (Sive et al., 2000) and in situ hybridization was performed as previously described (Harland, 1991; Sive et al., 2000). The embryos were washed with phosphate buffered saline 0.1% Tween-20 (PBST) and transferred through series of methanol washes (25%, 50%, 75%, 100%). In situ anti-sense probes were generated in vitro from linearized templates using a DIG labeling mix (Roche). Chromogenic reaction times were optimized for signal to background ratio. Probes used were: Xnodal (Sampath et al., 1997), Xlefty (Meno et al., 1997), and Xpitx2 (Campione et al., 1999). NIH Image J software was used to quantify the in situ signal.

All statistical analysis was performed using Microsoft Excel. As appropriate for each case, data were either pooled from multiple repeat experiments, with χ2 analysis performed on them, or data from various iterations was analyzed by t-test (for 2 groups) or ANOVA (for more than two groups), as indicated with each experiment.

This work is dedicated to the memory of Amar Klar. We thank Erin Switzer, and Rakela Colon for Xenopus husbandry and general lab assistance, Dany Adams for help with microscopy, Chris Wright for Nodal and Lefty antisense probe, and H. Joseph Yost for Pitx2 antisense probes.