Natterin-like depletion by CRISPR/Cas9 impairs zebrafish (Danio rerio) embryonic development

Abstract

Background The Natterin protein family was first discovered in the venom of the medically significant fish Thalassophryne nattereri, and over the last decade natterin-like genes have been identified in various organisms, notably performing immune-related functions. Previous findings support natterin-like genes as effector defense molecules able to activate multiprotein complexes driving the host innate immune response, notably due to the pore-forming function of the aerolysin superfamily members. Herein, employing a combination of the CRISPR/Cas9 depletion system, phenotype-based screening, and morphometric methods, we evaluated the role of one family member, LOC795232, in the embryonic development of zebrafish since it might be implicated in multiple roles and characterization of the null mutant is central for analysis of gene activity. Results Multiple sequence alignment revealed that the candidate natterin-like has the highest similarity to zebrafish aep1, a putative and better characterized fish-specific defense molecule from the same family. Compared to other species, zebrafish have many natterin-like copies. Whole-mount in situ hybridization confirmed the knockout and mutant embryos exhibited epiboly delay, growth retardation, yolk sac and heart edema, absent or diminished swim bladder, spinal defects, small eyes and head, heart dysfunction, and behavioral impairment. As previously demonstrated, ribonucleoproteins composed of Cas9 and duplex guide RNAs are effective at inducing mutations in the F0 zebrafish. Conclusions The considerably high natterin-like copies in zebrafish compared to other species might be due to the teleost-specific whole genome duplication and followed by subfunctionalization or neofunctionalization. In the present work, we described some of the natterin-like features in the zebrafish development and infer that natterin-like proteins potentially contribute to the embryonary development and immune response.

Prednisolone Accelerates Embryonic Development of Zebrafish via Glucocorticoid Receptor Signaling at Low Concentrations

“…To determine whether PREL significantly accelerates zebrafish embryonic development, alterations in further parameters, including somite numbers, head-trunk angle, and the area of the yolk sac, were additionally measured. These parameters are widely accepted as markers for the estimation of the growth and developmental process of fish embryos. , As shown in Figure A, PREL significantly and dose-dependently increased the number of somites by 1.6, 2.1, 2.2, and 1.7 at concentrations of 0.010, 0.096, 0.95, and 9.26 μg/L, respectively. This result was quite consistent with increases in the frequency of yolk extension formation (Figure B).…”

“…These parameters are widely accepted as markers for the estimation of the growth and developmental process of fish embryos. 35,40 As shown in Figure 2A, PREL significantly and dosedependently increased the number of somites by 1.6, 2.1, 2.2, and 1.7 at concentrations of 0.010, 0.096, 0.95, and 9.26 μg/L, respectively. This result was quite consistent with increases in the frequency of yolk extension formation (Figure 1B).…”

Tissue-specific proteome profile analysis reveals regulatory and stress responsive networks in passion fruit during storage

“…These were first discovered in the venom of the medically significant fish Thalassophyrne nattereri and have since then been found in various organisms performing immune-related functions. They have been found to activate multiprotein complexes driving the host’s innate immune responses and have been found to be involved in embryogenesis where a CRISPR knockout caused mutations in growth retardation, spinal defects, heart dysfunction, and more 55 . Three major pru ar 1-like proteins were identified in passion fruit, which may be the major cause of allergies upon the fruit consumption.…”

In Search of a Target Gene for a Desirable Phenotype in Aquaculture: Genome Editing of Cyprinidae and Salmonidae Species

“…The data presented here and below on gene functions were processed using genome editing methods. High embryo lethality may also be linked to impaired organogenesis and immunity, as evidenced by mutants in knockout genes such as LOC795232 [ 225 ], sphk1 [ 226 ], asap1a , asap1b [ 227 ], and zrsr2 [ 228 ]. The regulation of cartilage and bone formation, bone mineralization, and osteoblast differentiation involves sox9 [ 229 ], vwa1 [ 230 ], tmem38b [ 231 ], ppp2r3b [ 68 ], col11a2 [ 69 ], and wnt16 [ 232 ] genes.…”