Duplicated 4.1B genes are differentially expressed in the zebrafish trunk.

(A) Analysis of 4.1B expression levels during various stages of development. 4.1Ba and 4.1Bb are expressed throughout development. From left to right, cDNA samples were derived from wild-type (AB/Tübingen) zebrafish at the 16-cell (1.5 hpf), 90% epiboly (9 hpf), 3-somite (11 hpf), 16-, 24-, 48-, 72 hpf, and 7 days post fertilization (dpf) stages. (B) in situ hybridization showing 4.1Ba and 4.1Bb expression in the brain and trunk of the developing zebrafish embryo at 26 hpf. Both 4.1Ba and 4.1Bb are expressed in the telencephalon (Te), the mesencephalon (Me), and retina (Rt). Only 4.1Bb was seen in muscle (Mu). (C) Cross section of the trunk at 26 hpf further demonstrates only 4.1Ba is expressed in the spinal cord (dotted ellipse), whereas 4.1Bb is expressed in muscle. (D—G) Fluorescent in situ hybridization of the trunk confirming expression of 4.1Ba, but not 4.1Bb in CaPs (E) and CoPA cells (F), and 4.1Bb, but not 4.1Ba, in muscle (G) at 26 hpf. White boxes in D indicate cells shown in E. Scale Bar in B: 500 μm; C: 100 μm; D: 20 μm; E, F: 5 μm; G: 50 μm; Abbreviations: bg = background from chicken anti-GFP antibody.

Knockdown of 4.1Ba causes a reduction in the number of synapses at CaPs.

(A) Labeling of 4.1Ba-GFP puncta colocalized with the presynaptic marker Synapsin 1/2 (Syn) and the postsynaptic marker panMAGUK (PM) at CaPs. Arrows indicate sites of colocalization. (B) Representative images of Syn and PM in Ctl MO, 4.1Ba MO, and 4.1Ba MO + 4.1Ba RNA (4.1Ba Res) injected embryos. (C) Knockdown of 4.1Ba reduced the number of synapses at CaPs. The phenotype was rescued with 4.1Ba and 4.1Bb mRNA (Rescue). Misexpression of 4.1Ba did not affect synapse number. (D—E) Quantification of SV2 puncta (D) and Synaptotagmin 2b puncta (E) at CaPs. Both presynaptic proteins are reduced in the absence of 4.1Ba. (F) Cell specific rescue of 4.1Ba in CaPs. Embryos were collected from a tg(mnx1:Gal4:UAS:GFP) and tg(mnx1:Gal4) cross and injected with the Ctl MO, 4.1Ba MO, or the 4.1Ba MO + UAS:4.1Ba-GFP plasmid. Embryos were analyzed based on GFP expression. Expression of 4.1Ba-GFP restored the number of synapses specifically in CaPs. N = 10 for all conditions and experiments. Significance was evaluated with a One-way ANOVA followed by Tukey’s post-hoc analysis. All experiments were repeated in triplicate. Error bars represent s.e.m. * p < 0.05; ** p< 0.01. Scale bars in A—B, D—F: 5 μm. Abbreviations: Ctl MO = control morpholino; 4.1Ba MO = 4.1Ba morpholino; 4.1Ba Rescue = 4.1Ba MO + 4.1Ba mRNA; 4.1Bb Rescue = 4.1Ba MO + 4.1Bb mRNA; 4.1Ba GFP Rescue = 4.1Ba MO + UAS:4.1Ba GFP DNA.

4.1Ba knockdown causes kinetic differences during touch-evoked coiling.

(A) Example of touch-evoked coiling in Ctl MO, 4.1Ba MO, and 4.1Ba Rescue embryos at 26 hpf. The time series depicts the full response used in our analysis to quantify the latency, L, and velocity, V, of C-bend responses. 4.1Ba knockdown increased the latency (B) and decreased the velocity (C) of C-bend responses which was rescued with 4.1Ba and 4.1Bb RNA. (D) Correlation of touch-evoked response duration, defined as the latency plus time in motion, with synapse number for Ctl MO, 4.1Ba MO, 4.1Ba mRNA, and 4.1Ba and 4.1Bb rescues. (E) Latency and (F) velocity for touch-evoked coiling after inhibition of NMDA receptors with MK-801. No additive effect was seen when 4.1Ba was knocked down and NMDA receptors were inhibited. N = 30 per condition for each experiment. Data represents three separate experiments normalized to 1 and evaluated with a one-way ANOVA followed by Tukey’s post-hoc analysis. Error bars represent s.e.m. * p < 0.05. Abbreviations: Ctl MO = control morpholino; 4.1Ba MO = 4.1Ba morpholino; 4.1Ba Rescue = 4.1Ba MO + 4.1Ba mRNA; 4.1Bb Rescue = 4.1Ba MO + 4.1Bb mRNA.

4.1Ba MO specifically affects glutamatergic synapses at CaPs.

(A) Schematic of 4.1Ba knockdown. The 4.1Ba MO (blue box) is designed between exon 1 (E1) and intron 1 to incorporate intron 1 in the mRNA. Intron 1 contains an early stop codon (purple line), leading to a truncated, nonfunctional protein. Primers designed in E1 (green arrow) and E2 (red arrow) were used for RT-PCR and sequencing products. (B) Confirmation of 4.1Ba knockdown by RT-PCR. RT-PCR products were larger by the expected size of intron 1 in MO injected animals. The 4.1Ba MO caused an 86% reduction in properly spliced transcripts that were confirmed by sequencing. (C) Acridine Orange staining demonstrates no difference in cell death between Ctl MO and 41Ba MO. Red box represents the spinal cord region examined. Dotted lines represent the edges of the spinal cord (SC). (D) Representation and (E) quantification of Syn and PM labeling at CoPA cells. No changes in the number of pre- or postsynaptic markers were found. (F) Representation and (G) quantification of Syn and PM labeling after knockdown of both 4.1B proteins, 4.1Ba and 4.1Bb alone, and misexpression of 4.1Bb. 4.1Bb knockdown did not affect synapse number, nor did it enhance the phenotype associated with 4.1Ba knockdown. No effect was seen with 4.1Bb misexpression. (H) Representation and (I) quantification of Gephyrin labeling at CaPs. Loss of 4.1Ba did not affect the number of inhibitory synapses at CaPs. N = 10 for all conditions and experiments. Significance was evaluated with a One-way ANOVA followed by Tukey’s post-hoc analysis. All experiments were repeated in triplicate. Error bars represent s.e.m. * p < 0.05; ** p< 0.01. Scale bar in B, D: 5 μm. Abbreviations: Ctl MO = control morpholino; 4.1Ba MO = 4.1Ba morpholino; 4.1Ba Rescue = 4.1Ba MO + 4.1Ba mRNA; 4.1Bb MO = 4.1Bb morpholino.

4.1Ba MO does not alter structures involved in the sensorimotor circuit.

(A) Lateral view of CaPs, the myotome, and the NMJ in the trunk of 26 hpf Ctl MO and 4.1Ba MO injected embryos. (B—D) No differences were found in the number of branch points and terminal points (B), number of processes per cell (C), or the length of the processes (D). (E) Quantification of acetylcholine puncta at the neuromuscular junction. There were no changes in the number of acetylcholine puncta at the NMJ. (F) Failure rate of touch-evoked responses represented as a percentage of stimuli. No effect was seen in the ability of the embryos to respond to a tactile stimulus. (G) Frequency of spontaneous coils represented as the number of spontaneous coils per minute at 19 hpf. (H) Kinematic analysis of individual C-tail bends at 19 hpf. No differences were seen in the frequency or velocity of spontaneous coils. Error bars: s.e.m. Scale bar in A: 10 μm—CaPs; 50 μm—muscle and NMJ.