An Fgf-Shh positive feedback loop drives growth in developing unpaired fins.

The origin and diversification of appendage types is a central question in vertebrate evolution. Understanding the genetic mechanisms that underlie fin and limb development can reveal relationships between different appendages. Here we demonstrate, using chemical genetics, a mutually agonistic interaction between Fgf and Shh genes in the developing dorsal fin of the channel catfish, Ictalurus punctatus. We also find that Fgf8 and Shh orthologs are expressed in the apical ectodermal ridge and zone of polarizing activity, respectively, in the median fins of representatives from other major vertebrate lineages. These findings demonstrate the importance of this feedback loop in median fins and offer developmental evidence for a median fin-first scenario for vertebrate paired appendage origins.

Deep Transcriptomic Analysis Reveals the Dynamic Developmental Progression during Early Development of Channel Catfish (Ictalurus punctatus).

The transition from fertilized egg to larva in fish is accompanied with various biological processes. We selected seven early developmental stages in channel catfish, Ictalurus punctatus, for transcriptome analysis, and covered 22,635 genes with 590 million high-quality RNA-sequencing (seq) reads. Differential expression analysis between neighboring developmental timepoints revealed significantly enriched biological categories associated with growth, development and morphogenesis, which was most evident at 2 vs. 5 days post fertilization (dpf) and 5 vs. 6 dpf. A gene co-expression network was constructed using the Weighted Gene Co-expression Network Analysis (WGCNA) approach and four critical modules were identified. Among candidate hub genes, GDF10, FOXA2, HCEA and SYCE3 were involved in head formation, egg development and the transverse central element of synaptonemal complexes. CK1, OAZ2, DARS1 and UBE2V2 were mainly associated with regulation of cell cycle, growth, brain development, differentiation and proliferation of enterocytes. IFI44L and ZIP10 were critical for the regulation of immune activity and ion transport. Additionally, TCK1 and TGFB1 were related to phosphate transport and regulating cell proliferation. All these genes play vital roles in embryogenesis and regulation of early development. These results serve as a rich dataset for functional genomic studies. Our work reveals new insights of the underlying mechanisms in channel catfish early development.

Gene Editing in Channel Catfish via Double Electroporation of Zinc-Finger Nucleases.

The traditional approach for gene editing with zinc-finger nucleases (ZFNs) in fish has been microinjection of mRNA. Here, we develop and describe an alternative protocol in which ZFN plasmids are electroporated to channel catfish, Ictalurus punctatus, sperm, and embryos. Briefly, plasmids were propagated to supply a sufficient quantity for electroporation. Sperm cells were prepared in saline solution, electroporated with plasmids, and then used for fertilization. Embryos were incubated with the plasmids before performing electroporation just prior to first cell division. Plasmids were then transcribed and translated by embryonic cells to produce ZFNs for gene editing, resulting in mutated fry.

Molecular characterization of the insulin-like growth factor-I (IGF-I) gene in channel catfish (Ictalurus punctatus).

The insulin-like growth factor I (IGF-I) gene was characterized in channel catfish. Partial cDNA sequence, missing exon 1 and part of exon 2, was obtained in 5'- and 3'-RACE experiments. Direct sequencing of two bacterial artificial chromosome clones revealed gene structure and provided sequence from 640 bp upstream of the initiator methionine to 136 bp beyond the polyadenylation site. Genomic sequence contained a putative TATA box 506 bp upstream of the initiator methionine. The 477-bp reading frame within five exons encoded a 159-amino acid (aa) pre-propeptide highly similar to IGF-I in higher vertebrates. The sequence encoding the signal peptide was unique in catfish and contained 70% G+C content with the potential for a stable stem-loop structure. Full-length cDNA was only maintained in recombination-deficient (DH10B) strain E. coli. Levels of IGF-I mRNA were highest in liver, followed by brain and muscle, then heart and kidney (P<0.05). A CT/GA dinucleotide microsatellite in intron 1 was highly polymorphic in commercial channel catfish, and permitted placement of the IGF-I gene on the catfish genetic map. However, specific IGF-I alleles were not correlated with differences in growth rate from 100 to 130 days post-hatch in USDA103 line catfish.

Fluctuating asymmetry and condition in golden shiner (Notemigonus crysoleucas) and channel catfish (Ictalurus punctatus) reared in sublethal concentrations of isopropyl methylphosphonic acid.

Fluctuating asymmetry (FA; small, random differences between the left and right sides of a bilateral character) has been related to embryological stress during development. Some populations exhibit positive relations between FA and exposure to toxicants during development. The United States has agreed to demilitarize chemical warfare agents in accordance with the Chemical Weapons Convention Treaty. Isopropyl methylphosphonic acid (IMPA), the main hydrolysis product of sarin, could be introduced accidentally into the environment during demilitarization. The present study examined the use of FA as a biomarker of developmental stress caused by sublethal exposures to IMPA. A suite of morphometric and meristic traits were measured in two fish species exposed to IMPA. Significant differences were found in composite FA among groups of channel catfish (Ictalurus punctatus) exposed to sublethal concentrations of IMPA during development. No differences were found in composite FA among groups of golden shiner (Notemigonus crysoleucas) exposed to IMPA. No relation was found between individual relative condition and FA in channel catfish or golden shiner.

Taste bud development in the channel catfish.

Taste bud formation in channel catfish is first seen to occur in stage 39 embryos, when taste bud primordia (stage 1), consisting of three to five cells, including a single calretinin-positive cell, can be recognized within the oropharyngeal cavity and maxillary barbels. Within a short time (stage 40), stage 2 taste bud primordia are apparent and include two or three calretinin-positive cells. The number of calretinin-positive cells continues to increase (stage 3), and the primordia begin to erupt as mature taste buds (stage 4) by embryonic stage 48. This same pattern of taste bud development characterizes other regions of the head, with calretinin-positive cells first detected around the mouth and on the other barbels by stage 41 and on the rest of the head by stage 48. The development of trunk taste buds lags far behind that of the head, with the first calretinin-positive cells occurring on the lobes of the caudal fin by stage 48 and on the remaining fins by stage 50. Taste bud primordia on the trunk proper do not begin to appear until stage 53, when the larvae begin to feed, and these receptors begin to erupt only in 1-week-old larvae. Fibers of the facial nerve, which innervate all external taste buds, ramify within the ectoderm prior to the first appearance of taste bud primordia or their precursors.

Differential gene expression of IGF-I, IGF-II, and toll-like receptors 3 and 5 during embryogenesis in hybrid (channel x blue) and channel catfish.

Insulin-like growth factors-I and-II (IGF-I and IGF-II) play important roles in growth and development of mammals. Toll-like receptors (TLRs) are pattern recognition molecules that orchestrate the induction of early innate immune response by recognition of specific sequences. Evidence is growing that suggests a relationship between growth and immune function. The objective of the study was to examine changes in gene expression of IGF-I, IGF-II, TLR3, and TLR5 during embryogenesis and early larval development in hybrid (channel catfishxblue catfish) and channel catfish. Egg samples were taken pre- and post-fertilization; embryos were collected at two stages of embryogenesis, at hatch, and at swim-up. All genes were detected in unfertilized catfish eggs. Expression levels of TLR5 and IGF-I mRNA in channel catfish and expression levels of TLR3, IGF-I, and IGF-II mRNA in hybrids increased over time (P<0.01). Effect of time was not significant for expression of IGF-II or TLR3 mRNA in channel catfish and for TLR5 mRNA in hybrid catfish. Results of this study suggest growth (IGF-I and IGF-II) and immune (TLR3 and TLR5) associated genes could be functional and play important roles during embryogenesis and early development of hybrid and channel catfish.

Influence of dihydrotestosterone on sex determination in channel catfish and blue catfish: period of developmental sensitivity.

Treatment of channel catfish with 0.2, 20, or 200 mg/liter of dihydrotestosterone (DHT) in the water during the egg stage or during egg and sac-fry stages did not alter the expected 1:1 sex ratio of the progeny. Feeding DHT at 200 mg/kg of feed for the first 21 days after yolk sac absorption resulted in 80% females; this proportion was increased by combining feeding with treatment of 200 mg DHT/liter in the sac-fry stage (90%) or in the egg and sac-fry stage (97%). In contrast, treatment of blue catfish sac-fry with 200 mg DHT/liter, with or without the combination of feeding DHT at 200 mg/kg food, resulted in 100% female populations. Neither clomiphene citrate, an estrogen-receptor blocking agent, nor clofibrate, an inhibitor of hepatic synthesis of cholesterol, affected the sex ratio of channel catfish, and neither of these compounds altered the feminizing effect of 200 mg DHT/kg when fed in combination with DHT. The nonaromatizable androgen DHT is not as effective as many other androgens in producing paradoxical female populations of channel catfish. However, feminization of blue catfish by treatment of sac-fry indicates that this species is more susceptible to hormonal manipulation and that the period of sex determination may occur earlier in development than in channel catfish.

Development of catfish lateral line organs: electroreceptors require innervation, although mechanoreceptors do not.

In amphibians the lateral line sense organs, i.e., mechanoreceptors (neuromasts) and electroreceptors, develop autonomously from placodal tissue, with no need for innervating nerve fibers. The present study deals with the question whether or not the mechano- and the (ampullary) electroreceptors develop in the same manner in teleosts. On the tail of larval catfish, Silurus, the first mechano- and electroreceptors appear several days after hatching in two longitudinal rows, one along the outgrowing main branch of the lateral line nerve and one along its ventral branch. Prevention of outgrowth of both nerve branches by repeated nerve sectioning, before the receptors form, shows (1) that the mechanoreceptors can develop without innervation as in amphibians, and (2) that the electroreceptors do not develop without innervation, unlike amphibian electroreceptors. These results are discussed with regard to the placodal origin and the phylogeny of the two sense organs.

Hormonal sex manipulation and evidence for female homogamety in channel catfish.

The mechanism of sex determination in channel catfish Ictalurus punctatus was evaluated by hormonal and genetic methods. Aromatizable and nonaromatizable androgens, as well as an estrogen, caused feminization in fish fed steroids for 21 days after yolk-sac absorption. The effectiveness of 60 micrograms of ethynyltestosterone/g food decreased markedly when the experimental feeding period was shortened and was ineffective when the treatment lasted less than 12 days. Females from all-female populations produced by treatment with sex hormones were mated with normal males resulting in nine spawns with a sex ratio different from 1:1. The sex ratios were statistically similar to 3 male: 1 female in five spawns, both 2:1 and 3:1 in two spawns, and 2:1 in two spawns. These data are consistent with a model for female homogametic sex determination in channel catfish and suggest that the YY equivalent genotype is viable.