FIGURE SUMMARY
Title

CNNM2 Mutations Cause Impaired Brain Development and Seizures in Patients with Hypomagnesemia

Authors
Arjona, F.J., de Baaij, J.H., Schlingmann, K.P., Lameris, A.L., van Wijk, E., Flik, G., Regele, S., Korenke, G.C., Neophytou, B., Rust, S., Reintjes, N., Konrad, M., Bindels, R.J., Hoenderop, J.G.
Source
Full text @ PLoS Genet.

Knockdown of cnnm2a results in Mg wasting in zebrafish larvae (5 dpf).

(A) mRNA expression of cnnm2a in developing zebrafish. Expression patterns were analysed by RT-qPCR (n = 6 per time point). (B) Survival curve at 5 dpf (n = 3 per experimental condition). The dose of zero represents injection with control-MO. (C) Morphological phenotypes in zebrafish larvae (5 dpf) in cnnm2a knockdown experiments. (D) Distribution of morphological phenotypes in zebrafish larvae (5 dpf) untreated (wild-type) or injected with different doses of cnnm2a-MO or control-MO. Numbers on top of the bars indicate the number of animals in each experimental condition. (E) Distribution of morphological phenotypes in zebrafish larvae at 5 dpf in rescue experiments. The wild-type phenotype (class I) was restored in morphants by co-injection of cnnm2a-MO (2 ng MO/embryo) with wild-type (WT) CNNM2 cRNA (50 pg cRNA/embryo), but not with mutant (MT, p.Glu357Lys) CNNM2 cRNA (50 pg cRNA/embryo). (F) Magnesium content in zebrafish injected with different doses of cnnm2a-MO, the dose of zero represents injection with control-MO (n = 10 per experimental condition except in 8 ng MO-injected zebrafish where n = 5). (G) Rescue of Mg wasting in morphant zebrafish by co-injection of cnnm2a-MO (2 ng MO/embryo) with cRNA encoding for wild-type (WT) CNNM2 (50 pg cRNA/embryo). Co-injection with cRNA encoding for mutant (MT, p.Glu357Lys) CNNM2 (50 pg cRNA/embryo) did not restore Mg levels (n = 10 per experimental condition). Data are presented as mean ± SEM. Different letters indicate significant differences between mean values in experimental groups (P<0.05).

EXPRESSION / LABELING:
Gene:
Fish:
Anatomical Term:
Stage Range: Shield to Day 5
PHENOTYPE:
Fish:
Knockdown Reagent:
Observed In:
Stage: Day 5

Knockdown of cnnm2b results in Mg wasting and brain malformations in zebrafish larvae (5 dpf).

(A) mRNA expression of cnnm2b in developing zebrafish. Expression patterns were analysed by RT-qPCR (n = 6 per time point). (B) Survival curve at 5 dpf (n = 3 per experimental condition). The dose of zero represents injection with control-MO. (C) Morphological phenotypes in zebrafish larvae (5 dpf) in cnnm2b knockdown experiments. (D) Distribution of morphological phenotypes in zebrafish larvae (5 dpf) untreated (wild-type) or injected with different doses of cnnm2b-MO or control-MO. Brain malformations (widened cerebrospinal fluid spaces, class IV phenotype) are prominent in morphants injected with 4–8 ng MO/embryo. Numbers on top of the bars indicate the number of animals in each experimental condition. (E) Distribution of morphological phenotypes in zebrafish larvae at 5 dpf in rescue experiments. The wild-type phenotype (class I) was restored in morphants by co-injection of cnnm2b-MO (8 ng MO/embryo) with wild-type (WT) CNNM2 cRNA (50 pg cRNA/embryo), but not with mutant (MT, p.Glu357Lys) CNNM2 cRNA (50 pg cRNA/embryo). (F) Magnesium content in zebrafish injected with different doses of cnnm2b-MO. The dose of zero represents injection with control-MO (n = 10 per experimental condition). (G) Rescue of Mg wasting in morphant zebrafish by co-injection of cnnm2b-MO (8 ng MO/embryo) with cRNA encoding for wild-type (WT) CNNM2 (50 pg cRNA/embryo). Co-injection with cRNA encoding for mutant (MT, p.Glu357Lys) CNNM2 (50 pg cRNA/embryo) did not restore Mg levels (n = 10 per experimental condition). Data are presented as mean ± SEM. Different letters indicate significant differences between mean values in experimental groups (P<0.05).

EXPRESSION / LABELING:
Gene:
Fish:
Anatomical Term:
Stage Range: Shield to Day 5
PHENOTYPE:
Fish:
Knockdown Reagent:
Observed In:
Stage: Day 5

Dysfunctional cnnm2a causes brain abnormalities and increased spontaneous contractions in zebrafish embryos (25 hpf).

(A) Phenotypes in zebrafish embryos untreated (wild-type) or following treatment with cnnm2a-MO (2 ng MO/embryo) or control-MO. Abbreviations indicate the following parts in the zebrafish embryonic brain: M, midbrain; T, tectum; MHB, midbrain-hindbrain boundary; FV, fourth ventricle; and H, hindbrain. (B) Distribution of phenotypes and (C) Mg content (n = 10 per experimental condition) in zebrafish embryos untreated (wild-type) or injected with 2 ng of cnnm2a-MO or control-MO and exposed to a medium with a concentration of Mg2+ of 0.33 or 25 mM. Numbers on top of the bars indicate the number of animals in each experimental condition. (D) Restoration of normal brain development by co-injection of cnnm2a-MO (2 ng MO/embryo) with cRNA encoding for wild-type (WT) CNNM2 (50 pg cRNA/embryo), and not by co-injection with cRNA encoding for mutant (MT, p.Glu357Lys) CNNM2 (50 pg cRNA/embryo). (E) Spontaneous contractions in zebrafish embryos untreated (wild-type) or injected with 2 ng of cnnm2a-MO or control-MO and exposed to a medium with a concentration of Mg2+ of 0.33 or 25 mM (n = 30 per experimental condition). (F) Restoration of normal spontaneous contraction activity (n = 30 per experimental condition) by co-injection of cnnm2a-MO (2 ng MO/embryo) with cRNA encoding for wild-type (WT) CNNM2 (50 pg cRNA/embryo), and not by co-injection with cRNA encoding for mutant (MT, p.Glu357Lys) CNNM2 (50 pg cRNA/embryo). Data are presented as mean ± SEM. *P<0.05 versus wild-type and control. #P<0.05 versus Mg2+-normal (0.33 mM Mg2+) medium. Data are presented as mean ± SEM. Different letters indicate significant differences between mean values in experimental groups (P<0.05).

Dysfunctional cnnm2b causes brain abnormalities and increased spontaneous contractions in zebrafish embryos (25 hpf).

(A) Phenotypes in zebrafish embryos untreated (wild-type) or following treatment with cnnm2b-MO (8 ng MO/embryo) or control-MO. See Figure 6 for an explanation of the abbreviations shown. (B) Distribution of phenotypes and (C) Mg content (n = 10 per experimental condition) in zebrafish embryos untreated (wild-type) or injected with 8 ng of cnnm2b-MO or control-MO and exposed to a medium with a concentration of Mg2+ of 0.33 or 25 mM. Numbers on top of the bars indicate the number of animals in each experimental condition. (D) Restoration of normal brain development by co-injection of cnnm2b-MO (8 ng MO/embryo) with cRNA encoding for wild-type (WT) CNNM2 (50 pg cRNA/embryo), and not by co-injection with cRNA encoding for mutant (MT, p.Glu357Lys) CNNM2 (50 pg cRNA/embryo). (E) Spontaneous contractions in zebrafish embryos untreated (wild-type) or injected with 8 ng of cnnm2b-MO or control-MO and exposed to a medium with a concentration of Mg2+ of 0.33 or 25 mM (n = 30 per experimental condition). (F) Restoration of normal spontaneous contraction activity (n = 30 per experimental condition) by co-injection of cnnm2b-MO (8 ng MO/embryo) with cRNA encoding for wild-type (WT) CNNM2 (50 pg cRNA/embryo), and not by co-injection with cRNA encoding for mutant (MT, p.Glu357Lys) CNNM2 (50 pg cRNA/embryo). Data are presented as mean ± SEM. *P<0.05 versus wild-type and control. #P<0.05 versus Mg2+-normal (0.33 Mm Mg2+) medium. Data are presented as mean ± SEM. Different letters indicate significant differences between mean values in experimental groups (P<0.05).

Impairment of touch-evoked escape behaviour in cnnm2a morphant zebrafish. Touch-evoked escape behaviour score in zebrafish cnnm2a morphants at 5 dpf after injection of 2 ng control-MO/embryo, 2 ng cnnm2a-MO/embryo, 2 ng cnnm2a-MO/embryo+50 pg wild-type (WT) CNNM2 cRNA/embryo, or 2 ng cnnm2a-MO/embryo+50 pg mutant (MT, p.Glu357Lys) CNNM2 cRNA/embryo. Three categories were distinguished, responders, late responders and non-responders, to which the following scores were given: 3 points for responders: fish quickly react (swimming or flicking the tail) to the stimuli after 1 or 2 twitches; 2 points for late responders: fish react (swimming or flicking the tail) to the stimuli after 3, 4 or 5 twitches; and 1 point for non-responders: fish do not react to the stimuli after more than 5 twitches. The upper part of the figure shows frames of videos showing touch-evoked escape contractions at 5 dpf of control and morphant zebrafish larvae. Time of each video frame is indicated in centisenconds (cs). Data are shown as mean ± SEM (n = 30). Different letters indicate significant differences between mean values in experimental groups (P<0.05).

Impairment of touch-evoked escape behaviour in cnnm2b morphant zebrafish. Touch-evoked escape behaviour score in zebrafish cnnm2b morphants at 5 dpf after injection of 8 ng control-MO/embryo, 8 ng cnnm2b-MO/embryo, 8 ng cnnm2b-MO/embryo+50 pg wild-type (WT) CNNM2 cRNA/embryo, or 2 ng cnnm2b-MO/embryo+50 pg mutant (MT, p.Glu357Lys) CNNM2 cRNA/embryo. Three categories were distinguished, responders, late responders and non-responders, to which the following scores were given: 3 points for responders: fish quickly react (swimming or flicking the tail) to the stimuli after 1 or 2 twitches; 2 points for late responders: fish react (swimming or flicking the tail) to the stimuli after 3, 4 or 5 twitches; and 1 point for non-responders: fish do not react to the stimuli after more than 5 twitches. The upper part of the figure shows frames of videos showing touch-evoked escape contractions at 5 dpf of control and morphant zebrafish larvae. Time of each video frame is indicated in centisenconds (cs). Data are shown as mean ± SEM (n = 30). Different letters indicate significant differences between mean values in experimental groups (P<0.05).

Acknowledgments
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