Pedigrees of Family A and B and Electropherograms
(A and B) Pedigree of family A (A) and B (B). Filled symbols represent affected individuals, and open symbols represent unaffected individuals.
(C–E) Partial electropherograms of heterozygous (left) and homozygous (right) patients from family A showing the c.1312 dupC, family B showing the c.707G→A (p.R236Q), and family C showing the c.971T→C (p.L324P) mutations in CNNM4, respectively.

CNNM4 Immunostaining of Mouse Lower Incisor
Immunostaining of a lower incisor of a 2-day-old mouse. CNNM4 is localized in all tissues of the mandibula, including muscle and connective tissue (B). DAPI staining of the same slide is shown in (A), and negative control without primary antibody is shown in (C). At higher magnification (400×), CNNM4 is observed in the ameloblasts (indicated by the filled triangle) and their cell body (indicated by the open triangle) as well as in the odontoblasts (indicated by the asterisk) (E). (D) shows DAPI staining of the same slide, and (F) shows negative control without secondary antibody.

Clinical Findings in Family A
(A) Fundus photograph (patient II.1) showing optic disk pallor, narrow vessels, macular atrophy with pigment mottling, and peripheral spicules.
(B) Fundus autofluorescence (patient II.1) showing markedly decreased macular autofluorescence due to atrophy as well as severe RPE changes in the inferior and nasal peripheral retina with levels of increased and decreased autofluorescence.
(C) OCT (patient II.4) showing decreased foveal and retinal thickness, loss of well-defined retinal layers suggesting extensive loss of retinal cells, and hyperreflectivity in the choroids due to the RPE and choriocapillaris atrophy.
(D) Photograph of mouth (patient II.1) showing dysplastic teeth with yellow and brown discoloration characteristic of amelogenesis imperfecta. Upper incisor has been accidentally removed.

CNNM4 Immunostaining of Mouse Cornea and Retina
CNNM4 immunostaining of cornea (A–C) and retina (D–F) of 2-month-old mouse retina. In the cornea (B), CNNM4 is mainly localized in the epithelium (epi) surrounding the nuclei, in the keratocyte present in the stroma (st), and in the endothelium (en). (A) shows DAPI staining of the same slide, and (C) shows negative control without primary antibody. In the retina (E), immunostaining is mainly localized in the ganglion cell layer (indicated by the arrow), the inner and outer plexiform layers, and the outer segments of the photoreceptors. (D) shows DAPI staining of the same slide, and (F) shows negative control of without primary antibody. Symbols are used as follows: arrow, ganglion cell layer; asterisk, inner cell layer; filled triangle, outer cell layer; triangle, retinal pigment epithelium. The rabbit polyclonal antibody raised against amino acids 21–200 of human CNNM4 was used in a 1:200 dilution on 12 μm cryosections of eyes of 2-month-old C57Bl/6 mice. Secondary antibodies conjugated to Alexa Fluor 594 (Molecular Probes, Invitrogen) were diluted at 1:1000. Sections were stained with DAPI for visualizing nuclei before mounting in Cityfluor (Cityfluor Ltd.).

Toluidine Blue-Staining Sections of a Zebrafish Eye
Toluidine blue-stained sections of 5 dpf zebrafish embryos.
(A) Wild-type embryos.
(B) Morphant embryos.
(C) Rescued embryos.
(D) Ganglion cell number was quantified in the indicated zebrafish eye and then compared to wild-type set to 1 (n = 12 eyes in each category). The following abbreviations were used: WT, wild-type; MO, morpholino-treated embryos; rescue, morpholino-treated embryos injected with CNNM4 mRNA. Morpholinos were designed for targeting the exon 2-intron 2 splice site. One hundred micromolar solutions of either cnnm4 morpholino (MO: ATTTT GCCAC TGTCC ACTCA CTGTA) or control-morpholino (ct-MO: ATaTT cCCAC TcTCC ACTgA CTcTA) were injected into the eggs at the 1–2 cell stage. In rescue experiments, the human full-length CNNM4 cDNA was subcloned in pCDNA3.1− and transcribed from the T7 promoter. Forty pg of mRNA were injected in eggs together with the MO. Embryos were evaluated at 5 dpf, and the number of ganglion cell layers (GCLs) was counted on toluidine blue-stained sections containing the optic nerve. The number of GCLs from WT embryos was set to 1, and the counts in the morphants and rescued embryos were compared. Bars represent standard errors.