(a) Left, mean live births per litter for wild-type and Kcne2-disrupted crosses as indicated; n = 19–50 litters per group. *P<0.01. Right, genotype of surviving pups (%). n shown in parentheses.
(b) Exemplar 3-week-old pups from Kcne2^−/− × Kcne2^−/− and Kcne2^+/+ × Kcne2^+/+ crosses.
(c) Exemplar X-ray images of 5-week-old pups from Kcne2^−/− × Kcne2^−/− and Kcne2^+/+ × Kcne2^+/+ crosses.
(d) Mean body mass at 3–6 weeks of age for pups from wild-type and Kcne2-disrupted crosses as indicated; n = 15–50 pups per group. *P<0.05.
(e) Exemplar 5-week-old Kcne2^+/+ and Kcne2^−/− mice from homozygous crosses.
(f) Exemplar close-up views of skin from mice as in panel e.
(g) Exemplar micrographs of H&E-stained dermis sections from Kcne2^−/− mouse as in panel e. Scale bar = 300 µm.
(h) Exemplar 1-year-old Kcne2^+/+ and Kcne2^−/− mice from Kcne2^+/− × Kcne2^+/− crosses.
(i) Exemplar micrograph of H&E-stained dermis sections from Kcne2^−/− mouse in panel i. inset, detail from boxed region, showing border zone between normal hair and alopecia, with abrupt cessation of mature hair follicles. Scale bar = 600 µm.
(j) H&E-stained sections showing hair follicles in dermis from Kcne2^+/+ and Kcne2^−/− mice as in panel i. Scale bars = 20 µm.

(a) Immunofluorescence using antibodies raised against KCNE2, KCNQ1 and NIS in human thyrocytes in sections from individuals with Grave’s Disease. DAPI visualization of nuclei shown in blue. *Colloid. Scale bars: 4 µm.
(b) Western immunoblots (IB) using antibodies raised against KCNE2 (arrows indicate the three glycosylation states of KCNE2) and KCNQ1 (lower arrow, monomer; upper arrow, multimer) in thyroid tissue from Kcne2^+/+ and Kcne2^−/− mice (from Kcne2^+/− × Kcne2^+/− crosses).
(c) Immunofluorescence using antibodies raised against KCNE2, KCNQ1 and NIS, in thyrocytes of 3-month-old Kcne2^+/+ mice (from Kcne2^+/− × Kcne2^+/− crosses). *Colloid. Scale bars: 4 −µm.
(d) Immunofluorescence using antibodies raised against KCNE2 and KCNQ1, in thyrocytes of 3-monthold Kcne2^−/− mice (from Kcne2^+/− × Kcne2^+/− crosses). *Colloid. Scale bars: 4 µm.
(e) H & E staining of adult Kcne2^+/+ and Kcne2^−/− thyroid glands (from Kcne2^+/− × Kcne2^+/− crosses); scale bars, 20 µm.
(f) Electron micrographs of thyroid epithelium from adult Kcne2^+/+ and Kcne2^−/− mice (from Kcne2^+/− × Kcne2^+/− crosses). Scale bars, 2 µm.
(g) Western blots of membrane fractions from FRTL5 cells with and without 10 hours incubation with cAMP, using antibodies raised against KCNE2 (arrows putatively indicate the three predicted glycosylation states); and FRTL5 cells with and without 6 days TSH incubation, using antibodies raised against KCNQ1 (lower arrow, monomer; upper arrow, tetramer).
(h) Exemplar whole-cell patch-clamp recordings of XE991-sensitive currents in FRTL5 cells with or without TSH incubation. Upper left inset: voltage protocol. Zero current level indicated by dashed line.
(i) Mean whole-cell total (n = 9–16) and XE991-sensitive (n = 8–9) current relationships for FRTL5 cells with or without TSH incubation. Significant difference: *P < 0.05.

(a) Exemplar microPET images of pre-weaning Kcne2^+/+ and Kcne2^−/− pups recorded 24–96 hours after their surrogate mothers of opposite genotype received tail vein injections of ¹²⁴I. Labels: t, thyroid; s, stomach. Intensity scale shown on right. Scale bar (white) = 10 mm.
(b) Mean peak ¹²⁴I accumulation (measured as peak µCi cc⁻¹) in thyroid and stomach from imaging as in panel a, n = 7–12 pups per time-point per group, error bars indicate SEM. Measured as maximum radioactivity in each tissue minus mean background count in each mouse. *P<0.05; **P<0.01.
(c) Mean peak ¹²⁴I accumulation in thyroid relative to stomach, imaging method and pups as in panel a. Measured as ratio of maximum radioactivity in each tissue minus mean background count in each mouse. **P<0.001.
(d) Effects of pup and dam genotype on pup total thyroid and body ¹²⁴I accumulation (left), and thyroid RAIU (radioactive iodide uptake as a percentage of total body radioiodine) (right), determined using 3D regions of interest from PET analyses of pup ¹²⁴I accumulation (surrogated and non-surrogated) after tail vein injection of dams; n = 7–12. *P<0.05; **P<0.005; ***P<0.0005.
(e) Milk ejection assay. Mean mass of pups before (0 min) and after 30 min feeding period; n = 8–20; *P<0.01. ‘oxy’ indicates dams injected with oxytocin 10 minutes prior to feeding period.
(f) Milk T₄ concentration; n = 5–7; *P<0.05.
(g) Plasma I⁻ concentration in 3-week-old pups; n = 6 per genotype.

(a) Left, external view of exemplar hearts from 3-week-old Kcne2^+/+ (left) and Kcne2^−/− (right) mice from homozygous crosses. Right, mean heart mass, body mass, and heart weight/bodyweight (HW/BW; mg g⁻¹) for mice as in panel a (n = 9–14).* P < 0.000001.
(b)Exemplar transthoracic echocardiograms for a 3-week-old Kcne2^+/+ pup from a Kcne2^+/+ dam and a Kcne2^−/− pup from a Kcne2^−/− dam.
(c) Mean echocardiographic parameters from recordings as in panel b. d/s, diastolic/systolic; LVAW, left ventricular anterior wall thickness; LVID, left ventricular internal diameter; LVPW, left ventricular posterior wall thickness, n = 4 per group. * P < 0.05; ** P < 0.005; *** P < 0.0005, by one-way ANOVA.
(d) Left, mean cell capacitance for ventricular myocytes isolated from 3-week-old pups from homozygous crosses, n = 6–8; *P < 0.001. Right, exemplar whole-cell recordings from ventricular myocytes as in left. Insets: voltage protocol and scale bars.
(e) Mean peak current densities for Kcne2^+/+ (blue) and Kcne2^−/− (red) myocytes as in panel d, n = 6–8; *P < 0.05.
(f) Left, current inactivation τ values for Kcne2^+/+ (blue) versus Kcne2^−/− (red) myocytes as in panel e. Right, current amplitudes from double exponential fits for Kcne2^+/+ (solid) versus Kcne2^−/− (open) myocytes as in panel g. *P < 0.001. Current decay was best fit with two exponentials with parameters resembling I_to,f and I_K,slow, and a steady-state current, I_ss.
(g–i) Cardiac tissue from 3-week-old Kcne2^−/− mice bred from Kcne2^−/− dams.
(g) Exemplar necropsy.
(h) Left, exemplar Masson’s trichrome-stained left ventricle showing collagen (blue) indicative of fibrosis. Scale bar, 150 µm. Right, exemplar H&E-stained papillary muscle. Scale bar, 30 µm.
(i) Exemplar Masson’s trichrome stained liver showing marked perisinusoidal fibrosis (blue). Scale bar, 200 µm.
(j) Cardiac tissue from 15-month-old Kcne2^−/− mice bred from Kcne2^+/− dams; similar results were observed in 4/4 mice evaluated. Left, exemplar necropsy showing cardiomegaly; center and right, exemplar Masson’s trichrome-stained LV showing fibrosis (blue). Scale bars: center, 100 µm; right, 30 µm.

(a) Left, Serum T₄ in Kcne2^+/+ & Kcne2^−/− mice at 3 wks. Right, serum TSH in Kcne2^+/+ & Kcne2^−/− mice at 3 wks. *significantly different from Kcne2^+/+ by ANOVA, P < 0.001. Numbers in parentheses indicate n.
(b) Mean mass of thyroid glands from 12 month old Kcne2^+/+, ^+/− and ^−/− mice from Kcne2^+/− × Kcne2^+/− crosses, weighed most-mortem, numbers in parentheses indicate n. Significant differences: * P < 0.005; ** P < 1 × 10⁻⁴; ***P < 1 × 10⁻⁹.
(c) Serum T₄ in pregnant Kcne2^+/+ & Kcne2^−/− mice. *significantly different from Kcne2^+/+, P < 0.001.
(d) Exemplar 3-week-old pups from homozygous crosses surrogated (Sgt) with dams of opposite genotype
(e) Mean body mass at 3–6 weeks of age for pups from wild-type and Kcne2-disrupted crosses surrogated (Sgt), or treated (Tx) with T₃/T₄ injection (P) or by T₄ supplementation of their mothers (D); n = 9–23 pups per group. Untreated groups (/) from Figure 2 d shown for comparison.
(f) Exemplar 12-week-old Kcne2^−/− mouse before (left) and after (right) 10 days QOD T₃/T₄ administration. Results were consistent in 5/5 mice evaluated.
(g) Left, histology of mouse in panel f after T₃/T₄ treatment showing recovery of normal hair follicles. Scale bar = 200 µm. Right, percentage of mice with normal hair growth grouped according to parental genotype. Groups were untreated (/), surrogated (Sgt) with dams (genotype as indicated) or treated (Tx) directly by QOD T₃/T₄injection (P) or by T₄ supplementation of their mothers (D); n = 16–23 mice per group.
(h) Exemplar necropsies of 3-week-old Kcne2^+/+ and Kcne2^−/− mice bred from homozygous crosses, with/without surrogacy with mothers of opposite genotype.
(i) Mean heart weight/bodyweight (HW/BW; mg/g) for mice as in panel h, n = 7–9. *P<0.05 compared to values for non-surrogated pups (taken from Fig. 1c, key in Fig. 3j).
(j) Mean echocardiographic parameters for 3-week-old Kcne2^+/+ and Kcne2^−/− mice bred from homozygous crosses, with/without surrogacy with mothers of opposite genotype. d/s, diastolic/systolic; LVAW, left ventricular anterior wall thickness; LVID, left ventricular internal diameter; LVPW, left ventricular posterior wall thickness, n = 5 mice. *P<0.05 compared to values for non-surrogated pups (taken from Fig. 1c).

(a) Exemplar microPET images of lactating Kcne2^+/+ and Kcne2^−/− dams recorded during the first hour after tail vein injection of ¹²⁴I. Labels: t, thyroid; m, mammary glands. Color intensity scale on right, showing red indicates highest intensity. Scale bar (white) = 5 mm.
(b) Mean ¹²⁴I accumulation in thyroid relative to mammary gland from imaging as in panel a, n = 4 mice per group, error bars indicate SEM. Measured as ratio of maximum radioactivity in each tissue minus mean background count in each mouse. *P<1×10⁻⁸.
(c) Mean ¹²⁴I accumulation in thyroid relative to mammary gland as in panel a but from 1–72 hours after tail vein injection of ¹²⁴I, n = 4–5 mice per group, error bars indicate SEM. *P<0.05.
(d) Exemplar microPET images of pre-weaning Kcne2^+/+ and Kcne2^−/− pups recorded 24–72 hours after their mothers (same dams as in panels a–c) received tail vein injections of ¹²⁴I. Labels: t, thyroid; s, stomach. Intensity scale as in panel a. Scale bar (white) = 5 mm.
(e) Mean ¹²⁴I accumulation (in µCi) in thyroid and stomach from imaging as in panel d, n = 7–12 pups per time-point per group, error bars indicate SEM. Measured as maximum radioactivity in each tissue minus mean background count in each mouse. *P<0.05.
(f) Mean ¹²⁴I accumulation in thyroid relative to stomach, imaging method and pups as in panel e. Measured as ratio of maximum radioactivity in each tissue minus mean background count in each mouse. *P<0.0005.