FIGURE

Fig. 7

ID

ZDB-FIG-220802-61

Publication

Chen et al., 2022 - Autism-Risk Gene necab2 Regulates Psychomotor and Social Behavior as a Neuronal Modulator of mGluR1 Signaling

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Fig. 7

Necab2 co-localizes with mGluR1 and promotes PIP2-PLC-PKC signaling. (A–A″,B–B″) NECAB2 and mGluR1 colocalize at the cell membrane of HeLa cells (arrowheads). The HeLa cells were transiently transfected with EGFP-NECAB2 and mGluR1-mCherry and live imaging was conducted with the confocal microscope. Scale bar = 10 μm. (C–C″′) Co-immunofluorescent staining of anti-Necab2 with anti-Pvalb7 in 7 dpf larvae showed that Necab2 was expressed in the Purkinje cells in the cerebellum (asterisks). Scale bar = 20 μm (C). The region in the dashed white box (C) is shown at higher magnification on the right (C′–C″′). Scale bar = 5 μm. (D–D″′) NECAB2 and mGluR1 co-localized in the cerebellum of the zebrafish larvae (arrowheads). The necab2^+/+ larvae at 7 dpf were processed for anti-Necab2 and anti-mGluR1 immunofluorescence staining following fixation. Scale bar = 20 μm (D). The region in the dashed white box (D) was shown at higher magnification on the right (D′–D″′). Scale bar = 5 μm. (E–E″′) Live imaging of necab2^+/+ larvae at 7 dpf in Tg(hsp70:plc-ph-mCherry) and Tg(Pnecab2:EGFP) background by confocal microscope. Z-stack, Maximum intensity projection. Scale bar = 100 μm (E′). The cerebral region (in the dashed white box) was shown at higher magnification on the right (E′–E″″). Scale bar = 20 μm. (F–F″,G–G″) The necab2^–/^– larvae accumulated more PLC-PH-mCherry at the cell membrane of the necab2-expressing cells (asterisks) than that of the necab2^+/+ larvae. Genotyping of the 7 dpf larvae derived from heterozygous necab2^+/– in the Tg (Pnecab2:EGFP) and Tg(hsp70:plc-ph-mCherry) background mating was done after live imaging with the confocal microscope. Scale bar = 10 μm. (H) The quantification of the PLC-PH-mCherry intensity in the necab2^–/^– larvae (n = 6) and necab2^+/+ (n = 4) larvae by calculating the fluorescent intensity of plasma/cytosol. Two different sections per larva and three independent necab2-expressing cells per section were analyzed. (***p < 0.001, Mann–Whitney U test). (I) The summary of Necab2 interacting with mGluR1. Necab2 bound mGluR1 through the NHR domain. Necab2 was likely to facilitate the mGluR1 mediated PLC-PIP2-PKC signaling. dpf, day post-fertilization; Tel, telencephalon; Ha, habenula; OT, optic tectum; Ce, cerebellum; HB, hindbrain.

Expression Data

Genes:	EGFP mCherry
Antibodies:	Ab1-Necab2 Ab2-pvalb7
Fish:	AB s4304Tg/s4304Tg; s4305Tg/s4305Tg necab2^s4301/s4301; s4304Tg/s4304Tg; s4305Tg/s4305Tg
Anatomical Terms:	cerebellum cytoplasm cerebellum plasma membrane cerebellum Purkinje cell
Stage:	Days 7-13

Expression Detail

Antibody Labeling

Phenotype Data

Fish:	s4305Tg/s4305Tg necab2^s4301/s4301; s4304Tg/s4304Tg; s4305Tg/s4305Tg
Observed In:	cerebellum cytoplasm cerebellum glutamate receptor binding cerebellum plasma membrane
Stage:	Days 7-13

Phenotype Detail

Acknowledgments

This image is the copyrighted work of the attributed author or publisher, and ZFIN has permission only to display this image to its users. Additional permissions should be obtained from the applicable author or publisher of the image. Full text @ Front. Mol. Neurosci.