Multiple sequence alignment of the zebrafish natterin-like proteins evidencing the identity among residues within the aerolysin domain. The zebrafish natterin-like protein sequences were obtained from National Center for Biotechnology Information (NCBI) and aligned through Clustal Omega (European Molecular Biology Laboratory - EMBL/European Bioinformatics Institute - EBI). The alignment is displayed in MView evidencing the coverage (cov), percentage of identity (pid), and consensus among proteins. The highlight in yellow→indicates the reference natterin protein studied herein coded by LOC795232 and the Natterin family conserved residues (AGIP) are highlighted in the red square

Qualitative evaluation of natterin-like gene expression by whole-mount in situ Hybridization (WISH). Larvae after 24 h post-fertilization (hpf) were fixed, dehydrated, permeabilized and hybridized with the DIG-labeled probe, complementing the natterin-like RNA sequence (Qiagen, #339500 LCD0168623-BKG). Probe hybridization was determined by binding the AP-labeled anti-DIG antibody (#11093274910, Roche Diagnostics) and revealed through chromogenic reaction by NBT/BCIP solution. The images were obtained in the stereomicroscope STEREO LUMAR-V-12 Carl Zeiss. The picture snippets indicate the area of the larva’s body where the blue-purple precipitate (indicating gene product) can be observed in the non-depleted wild-type (WT) larvae (left), indicative of the presence of the natterin-like transcripts; and in the depleted (KO) larvae by CRISPR/Cas9 system (crRNA natterin-like WD07479944, Sigma) (right). The embryos body changed the shape due to long-lasting high temperature incubation in conical-bottom microtubes, not representing developmental malformations

Zebrafish epiboly progression. The differences in epiboly progression are evidenced in wild-type (WT) (left) and CRISPR/Cas9 natterin-like KO (right) embryos at 11 and 19 h post-fertilization (hpf). The analysis was performed using a stereomicroscope Leica M205C (LAS V4.11 software) in a 27x magnification. The asterisks (*) represent the progress of blastopore until its closure at the end of gastrulation period. Representative figures from a sample of 20 embryos

Survival rate, sublethal and teratogenic abnormalities of zebrafish after depletion of the natterin-like gene by the CRISPR/Cas9 system. The depletion of the natterin-like gene LOC795232 in 0 h post-fertilization (hpf) zebrafish embryos was done by the synthetic duplex guide RNA (tracrRNA + natterin-like crRNA, 50 μg.nL^− 1) with Cas9 (250 μg.nL^− 1). The survival of the depleted larvae (KO) and wild-type (WT) group (A), sublethality (B), and teratogenicity (C) endpoints were analyzed at 24, 48, 72, 96, 120, and 144 hpf (20 embryos per group). The dashed line indicates the percentage of mortality acceptable by the OECD of 30%. The lines indicate the average of abnormalities in the KO group at each time measured. The asterisks (*) represent a significant difference with the WT (p < 0.05), which is represented in the complementary graph in the bottom of B and C. See additional file 2 for raw data of 20 embryos per group behind the figures

Analysis of aberrant phenotypes in natterin-like depleted (KO) zebrafish compared to wild-type (WT). The larvae where the natterin-like gene LOC795232 was depleted (KO) by CRISPR/Cas9 system or WT were photographed in stereomicroscope Leica M205C (LAS V4.11 software, 27x magnification) at 24, 48, 72, 96, 120, and 144 h post-fertilization (hpf) (top to bottom) and analyzed for malformations: SD = spinal defect, YSE = yolk sac edema, PE = pericardial edema, ASB = absence of swimming bladder, DD = development delay, PL = pigmentation loss, CNSN = central nervous system necrosis, TN = tail necrosis, SE = small eyes, SH = small head. Representative figures from a sample of 20 embryos, see additional file for more examples

Depletion of natterin-like protein (LOC795232) led to kinetic alterations in zebrafish development. The natterin-like gene depleted (KO) or the non-depleted larvae (WT) were analyzed at 24, 48, 72, 96, 120, and 144 h post-fertilization (hpf). The larvae were aligned and photographed (20 per group) by Leica M205C stereomicroscope and the measurements of body size (μm, from the top of the head to the end of the tail) (A), eye size (μm) (B), yolk sac area (μm²) (C), swimming bladder area (μm²) (D), head angle (in degrees, given by the measure of the opening of the head in relation to the yolk sac) (E), and head size (μm, the antero-caudal measurement of the forebrain to the end of the hindbrain) (F) were evaluated in the ImageJ software. The lines represent the average of abnormalities at each designated time. The asterisks (*) represent a significant difference with the WT (p < 0.05)

Cardiac alterations of natterin-like depleted (KO) zebrafish larvae. The KO or the wild-type (WT) larvae had the heart area and heartbeat rate measured at 24, 48, 72, 96, 120, and 144 h post-fertilization (hpf; 20 larvae per time). Pericardial measurement was evaluated using the ImageJ software from images obtained in a Leica M205C stereomicroscope. The data represent the average of each measurement at the designated time (A; 20 larvae per group). The beat rate was counted in 15 s videos acquired on the Leica M205C stereomicroscope at 50x magnification (LAS V4.11 software). Dot plot data show heart rate individually and the bars represent the mean of each group plus the standard deviation (B). The asterisks (*) represent a significant difference with the WT control (p < 0.05)

Locomotor analysis of natterin-like depleted (KO) zebrafish larvae. The 144 h post-fertilization (hpf) KO or wild-type (WT) larvae were distributed in a 96-well plate (1 larva/well; 20 larvae per group) in 100 μL of medium and the locomotor activity represented by the distance moved was analyzed through the Zebrabox system (ViewPoint). Larvae were exposed to an acclimatization period of 20 min in the dark followed by 5 min of alternated 25 s light cycles (15% light stimulus) interspersed with 5 s dark cycles (0% light stimulus) to induce visual and neurological stimulation