Table 1.
CRISPR/Cas9 molecular toolkit for individual knockout of GlyRs.
Fig 1.
Phylogenetic reconstruction of the GLR family.
Reconstruction from amino acid alignment containing 370 sites. The bottom bar represents the scale for genetic changes (number of substitutions per site). Numbers at the nodes represent bootstrap statistical values. Note that the separation between GLRA2/4 and GLRA1/3 is strongly supported. However, bootstraps are weak at the node separating GLRA2 from GLRA4 and at the node separating GLRA1 from GLRA3. Multiple factors can affect supporting values, including sequences with high similarities (which is the case here). Each GLRA gene is highlighted with a reddish sideline, while the blue sideline indicates GLRB genes.
Fig 2.
Genomic block conservation between human, spotted gar, stickleback and zebrafish.
Black lines represent a genomic region and colored arrows represent oriented genes. GLR genes are indicated by red arrows. Same genes are indicated by similar color in the different species. Spotted gar genomic regions were added for GLRA4 and GLRBB to better understand the duplication events. Hs, Homo sapiens; Lo, Lepisosteus oculatus; Dr, Danio rerio; Ga, Gasterosteus aculeatus; Chr, Chromosome.
Fig 3.
Glycine receptor subunits temporal expression.
A, Scheme of a (3α,2β) glycine receptor. Beta subunits ensure synaptic anchoring through binding to cytoplasmic gephyrin proteins. Glycine binding triggers the opening of the chloride channel. B, RT-PCR of each glycine receptor subunits from whole embryo RNA extracts at different developmental stages. Polr2d is used as a reference gene.
Fig 4.
Systemic knockout of each individual glycine receptor alpha subunit.
A, High Resolution Melting profiles of knock-out lines discriminating between wild-type (+/+), heterozygous (+/-) and homozygous (-/-) siblings. B, Genomic scheme showing the targeted exon for each knockout lines as well as the selected frameshifting deletion. C, Protein scheme showing the truncated protein caused by the mutation (in red). The glycine binding domain (in blue) and the transmembrane domains (in yellow) are shown in the wild-type protein (top).
Fig 5.
Morphology and swimming activity of GlyR alpha subunit knockouts.
A, For each knock-out, the embryos developed normally and no abnormal morphology was observed at 48 hpf. B, However, when their swimming activity was assessed at 5 dpf during 4h/4h dark/light condition, hic (glra1-/-) mutant larvae depicted a decreased activity with no startle response to light compared to their siblings. No difference was observed for the other knockout lines.
Fig 6.
Hic mutants display motor coordination dysfunction.
A, Touch-evoked escape response of hitch mutants versus their siblings at 7 dpf show a dysfunction of motor coordination of hic-/- larvae. Indeed, hic-/- larvae are not able to bend their tail in a coordinated manner in order to initiate an escape swimming response (see S1 and S2 Movies for full recordings). B, Hic-/- larvae at 7 dpf show a reduction of the trunk size as well as defects of the notochord and a change in birefringence of trunk muscles. C, Measurement of the trunk size at different age shows that the reduction in trunk length starts at 3 dpf and aggravates at 7 dpf presumably due to the motor dysfunction.