Silencing the Spark: CRISPR/Cas9 Genome Editing in Weakly Electric Fish.

Electroreception and electrogenesis have changed in the evolutionary history of vertebrates. There is a striking degree of convergence in these independently derived phenotypes, which share a common genetic architecture. This is perhaps best exemplified by the numerous convergent features of gymnotiforms and mormyrids, two species-rich teleost clades that produce and detect weak electric fields and are called weakly electric fish. In the 50 years since the discovery that weakly electric fish use electricity to sense their surroundings and communicate, a growing community of scientists has gained tremendous insights into evolution of development, systems and circuits neuroscience, cellular physiology, ecology, evolutionary biology, and behavior. More recently, there has been a proliferation of genomic resources for electric fish. Use of these resources has already facilitated important insights with regards to the connection between genotype and phenotype in these species. A major obstacle to integrating genomics data with phenotypic data of weakly electric fish is a present lack of functional genomics tools. We report here a full protocol for performing CRISPR/Cas9 mutagenesis that utilizes endogenous DNA repair mechanisms in weakly electric fish. We demonstrate that this protocol is equally effective in both the mormyrid species Brienomyrus brachyistius and the gymnotiform Brachyhypopomus gauderio by using CRISPR/Cas9 to target indels and point mutations in the first exon of the sodium channel gene scn4aa. Using this protocol, embryos from both species were obtained and genotyped to confirm that the predicted mutations in the first exon of the sodium channel scn4aa were present. The knock-out success phenotype was confirmed with recordings showing reduced electric organ discharge amplitudes when compared to uninjected size-matched controls.

Post-hatching brain morphogenesis and cell proliferation in the pulse-type mormyrid Mormyrus rume proboscirostris.

The anatomical organization of African Mormyrids' brain is a clear example of departure from the average brain morphotype in teleosts, probably related to functional specialization associated to electrosensory processing and sensory-motor coordination. The brain of Mormyrids is characterized by a well-developed rhombencephalic electrosensory lobe interconnected with relatively large mesencephalic torus semicircularis and optic tectum, and a huge and complex cerebellum. This unique morphology might imply cell addition from extraventricular proliferation zones up to late developmental stages. Here we studied the ontogeny of these brain regions in Mormyrus rume proboscirostris from embryonic to adult stages by classical histological techniques and 3D reconstruction, and analyzed the spatial-temporal distribution of proliferating cells, using pulse type BrdU labeling. Brain morphogenesis and maturation progressed in rostral-caudal direction, from 4day old free embryos, through larvae, to juveniles whose brain almost attained adult morphological complexity. The change in the relative size of the telencephalon, and mesencephalic and rhombencephalic brain regions suggest a developmental shift in the relative importance of visual and electrosensory modalities. In free embryos, proliferating cells densely populated the lining of the ventricular system. During development, ventricular proliferating cells decreased in density and extension of distribution, constituting ventricular proliferation zones. The first recognizable one was found at the optic tectum of free embryos. Several extraventricular proliferation zones were found in the cerebellar divisions of larvae, persisting along life. Adult M. rume proboscirostris showed scarce ventricular but profuse cerebellar proliferation zones, particularly at the subpial layer of the valvula cerebelli, similar to lagomorphs. This might indicate that adult cerebellar proliferation is a conserved vertebrate feature.

[Post-embryonic differentiation of a cutaneous electro-receptor]. / Sur la différenciation postembryonnaire d'un électrorécepteur cutané.

In order to decrease the rate of postembryonic development of electroreceptor organs, excisions of epidermis and deafferentations were carried out in the gymnotid fish Eigenmannia virescens. Twenty-five days later, the epidermis showed electroreceptor organs without innervation. Some of these at the beginning of their development consisted of masses of identical cells, whereas others showed presumed sensory cells whose cytoplasm contained rudimentary synaptic structures. The epidermis also showed differentiated tuberous organs with a low number of sensory cells. In all these organs, radioactive thymidine was fixed in the nuclei of the platform accessory cells. Thirty-five-40 days after surgery, tuberous organs were identical to the functional organs, and thymidine was detected in the nuclei of the cavity accessory cells. These results show that the gymnotid electroreceptor organs can develop before any nervous contact occurs, and suggest that they might originate from epidermal cells.

Development of the electrosensory nervous system in Eigenmannia (Gymnotiformes): I. The peripheral nervous system.

The nerves of the anterior lateral line system in embryonic and larval stages of the weakly electric gymnotiform fish Eigenmannia were visualized by injection of the fluorescent marker DiI into the primordium of the anterior (ALLN) and posterior (PLLN) lateral line nerves. Examination of developmental series reveals that the nerve fibers that innervate the electrosensory and mechanosensory components of the anterior lateral line system are present before the first mechanoreceptors and electroreceptors have differentiated. This suggests that nerve fibers might induce the formation of lateral line receptors. Whereas the innervation of the mechanoreceptive system is already established at an early stage, the afferent innervation of electroreceptors continues to arborize in the periphery, presumably by following pioneer axon pathways. The earliest recognizable stage of the anterior lateral line nerve ganglion (ALLNG) is evident 2 days after spawning. The ganglion shows two germinal cell masses that develop into the supraorbital-infraorbital and the hyomandibular placodes. The supraorbital-infraorbital placode forms the dorsal part of the ALLNG; the hyomandibular placode forms the ventral part of the ALLNG. Counts of ALLNG cells in embryonic, larval, and adult stages of Eigenmannia show that, at each stage examined, the number of ganglion cells is always significantly larger than the number of mechanoreceptors and electroreceptor units in the periphery. During development, the distribution of ALLNG cell diameters shifts from a unimodal distribution in juveniles to a bimodal distribution in adults, peaking at 8 microns and 18 microns. These results suggest that tuberous electroreceptive organs, which are innervated by the large ALLNG cells, may not be functional prior to day 18. Our results further suggest that the number of ALLNG cells correlates with the rate of induction of lateral line receptors in the periphery.

The development of lateral-line receptors in Eigenmannia (Teleostei, Gymnotiformes). I. The mechanoreceptive lateral-line system.

The South American weakly electric fish of the genus Eigenmannia were induced to spawn by simulating the conditions of the rainy season. Whole animals were viewed using scanning electron microscopy, and skin from embryos and larvae of different ages was prepared for histological examination. Additional live fish were stained with vital dyes. Neuromasts develop within the epidermis and then rise to the surface, at which time a cupula is forming. The first neuromasts appear on the head, forming the temporal, mechanoreceptive lateral line, at 3.5 days after spawning, and 1 day later neuromasts appear on the trunk as a ventral trunk line. On day 8 all the cephalic neuromasts have appeared and a secondary, medial trunk line begins to form. A dorsal trunk line forms when the fish are juvenile. Eight neuromasts of the cephalic lines, 7 neuromasts of the medial trunk line and all neuromasts of the ventral and dorsal trunk lines remain at the surface and do not become enclosed in canals. The opercular neuromasts and 7 neuromasts of the ventral trunk line degenerate later. The formation of the head canals begins on day 17, whereas the canal of the medial trunk line starts to develop on day 25, and both head and trunk canal systems are completed by day 33. The mechanosensory system develops before the electrosensory system. Behavioral observations also indicate that the mechanoreceptive system is functional as early as day 5.

The development of lateral-line receptors in Eigenmannia (Teleostei, Gymnotiformes). II. The electroreceptive lateral-line system.

Weakly electric fish of the genus Eigenmannia were induced to spawn in conditions simulating the tropical rainy season. The skin of embryos of different ages was prepared for histological examination, and whole animals were examined by various histological methods and scanning electron microscopy. It was found that the electrosensory system develops after the first mechanoreceptive lines have formed. The tuberous and ampullary organs initially form adjacent to the lines of the lateral-line system. The tuberous organs develop at a rate 5 times higher than that of the ampullary organs. The rate of development for both classes of electroreceptors is 4 times higher on the head than on the trunk. The first tuberous organs develop on the head at day 7 and on the trunk at day 8. They increase in number and size during the growth of the fish. The ampullary organs begin to form on the head and on the most rostral part of the trunk at day 8. They are deeply sunk into the corium and have the same number of receptor cells as in adults. There are both ampullary and tuberous organs within fields of receptors that are innervated by a single nerve branch.