In Vivo Modeling of the Morbid Human Genome using Danio rerio

- [Narrator] The overall goal of the following experiment is to assay the pathogenicity of clinically significant non-synonymous changes using in vivo complementation in zebrafish. This is achieved by first generating mutant human mRNA to recapitulate human genetic lesions. Next, human wild-type and mutant mRNA and morpholinos are injected into zebrafish embryos and translated into functional protein or used to block translation respectively. Then, the embryos are phenotyped in order to evaluate the effects of the mutant protein on development. Results are obtained that show the deleterious effects of mutations on human protein sequences that can be confirmed based on the rescue capability of mutant human mRNA.

- The main advantage of this technique over existing methods like the mouse model, is that potentially damaging alleles can be more rapidly assayed at a medium to high throughput level, and also, allelic series within a single gene can be evaluated to better understand directional effect at a cellular level, such as loss of function or gain of function.

- [Narrator] To begin, determine if the human gene of interest has a zebrafish ortholog, and if so, how many copies. We recommend reciprocal BLAST of the human protein against the zebrafish genome, and subsequent BLAST of the zebrafish hit against the human genome. True orthologs will be the best hit in each instance. Obtain or generate a construct using the human Open Reading Frame for the desired gene less than six kilobases in length as well as a 5' SP6 transcription site and a 3' polyA signal. Next, design a morpholino to block splicing or translation of the targeted zebrafish gene. Using zfin.org determine if the zebrafish ortholog is expressed in a spatio-temporal context relevant to the phenotypic readout. Alternatively, conduct RT-PCR using cDNA from whole zebrafish embryos or in situ hybridization. To carry out site directed mutagenesis, begin by designing mutagenesis primers 25 to 45 bases in length with the desired mutation in the center that will anneal to opposite strands of the plasmid. The primer melting temperature should be greater than or equal to 78 degrees Celsius. Assemble the mutagenesis reaction with a high-fidelity polymerase, and cycle using the program shown here. When the PCR reaction is complete, add one microliter of DpnI restriction endonuclease per reaction to remove the dam methylated template. Incubate at 37 degrees Celsius for two hours. Transform two microliters of the mutagenesis reaction into 20 microliters of competent cells according to standard protocols. Once the mutation of interest, and the sequence of the entire ORF are confirmed from overnight cultures, linearize the pCS2+ template and generate capped mRNA. After determining the mRNA concentration, and verifying its integrity by gel electrophoresis, store the samples in three or more aliquots at negative 80 degrees Celsius until ready to use. For variant loss of function experiments, obtain embryos from natural zebrafish matings, and maintain them at 28 degrees Celsius in embryo water in six or ten centimeter dishes. Conduct a morpholino Dose Response Curve by injecting at least three different concentrations between one to ten nanograms into 50 to 100 zebrafish embryos per dose. Efficient morpholinos should give rise to dose dependent increases in the proportion of affected embryos in a batch. When performing quantitative or qualitative analysis, if scoring embryos at 24h post fertilization or later, treat the embryos with phenylthiourea at 24h post fertilization to reduce melanocyte formation. For splice blocking morpholinos, test morpholino deficiency by extracting total RNA from whole embryo lysates at the time point of phenotypic scoring. Generate cDNA and conduct RT-PCR of the target gene using primers flanking the morpholino target site. To verify the suppression efficiency of a translation blocking morpholino, or TBMO, if an antibody that cross-reacts with the zebrafish protein is available, harvest whole embryo protein lysates, and conduct immunoblotting to compare levels of the targeted protein versus control. If an antibody is not available, co-inject the wild-type mRNA with the TBMO to show that it rescues the phenotype, or demonstrate a dosage dependency as described earlier. If a qualitative phenotype was observed, select a morpholino dose in which 50% to 75% of embryos are affected. For quantitative phenotypes, choose a dose in which the phenotypic measure is significantly different from the wild-type. Next, inject new batches of embryos with a cocktail containing the assay dose of morpholino, and the dosage curve of human wild-type mRNA. Then, conduct masked scoring of injection batches to determine the wild-type mRNA dose with the most significant rescue in comparison to MO alone. This will be the assay dose of mRNA. Inject new batches of embryos with the assay doses of morpholino and mutant human mRNA. Phenotype the embryos at the appropriate stage, and using a chi-squared or t-test, compare the results to a rescue with wild-type human mRNA. If no loss of function phenotype is observed from morpholino injection, or if mutant mRNA gives rise to phenotypes not significantly different from MO alone, inject a dosage curve of wild-type human mRNA into batches of embryos. Conduct phenotypic scoring and determine the highest dose at which there is not a statistically significant number of dead and or affected embryos in comparison to uninjected controls. This is the assay dose. Inject the assay dose of mutant human mRNA, phenotype the embryos and compare the results to the scoring from the injection of the assay dose of wild-type human mRNA or the assay morpholino concentration. If the results are indistinguishable from morpholino, titrate mutant mRNA with wild-type mRNA and compare to human wild-type and mutant mRNA injections. Improvement of phenotypes with mutant plus wild-type mRNA injected batches indicates a dominant negative. No improvement indicates a gain of function. Finally, to integrate zebrafish in vivo pathogenicity data with other lines of evidence, compare it to genetic data within the pedigree, control population frequency data and in vitro cell-based protein studies. Shown here is a quantitative and qualitative evaluation of human MKS1 mutations modeled in zebrafish embryos. Based on severity, phenotypes are classified into 3 groups: Morpholino injected embryos with Class I phenotypes had grossly normal morphology, but were shorter with excessive embryonic tissue on the yolk compared to control injected embryos. Class II morphants were thinned, short, and had poorly developed head and tail structures, and additionally, lacked somitic definition and symmetry. Class III embryos were severely delayed with poorly developed and misshapen somites, undulated notochords and typically did not survive past the 10=somite stage. Co-injection of human and KS1 mRNA rescued each of these defects, demonstrating specificity of the phenotypes to MKS1 suppression. Phenotypes were quantified by measuring the distance from the first to the last appreciable somite in embryos stained with krox20, pax2, and myoD riboprobes at the 11-somite stage. Seen here is a zebrafish model of human craniofacial dismorphology. Control and mutant mRNA injected embryos were stained with Alcian blue at five days post-fertilization. The mutant embryos displayed notably smaller and misshapen heads, and a general disorganization of the cartilaginous craniofacial skeleton, including splayed branchial arches and missing or malformed structures. At five days post-fertilization, a macrocephaly morphant injected embryo displays widening of the head as seen by the space between the eyes. In this example of reduced vascular integrity at two days post-fertilization, morphants display impaired sprouting of intersegmental vessels and other vascular structures. In situ hybridization of uninjected wild-type embryos shows spaw expression in the left lateral plate mesoderm, a control for correct heart looping. Ccdc39 morphant embryos showed bilateral or, in most cases, undetectable spaw expression. Shown here, is an example of an Ift80 morphant that develops large kidney cysts, pericardial edema, and a curled tail. To visualize reduced glomerular filtration, rhodamine dextran was injected into the heart and the fluorescence was visualized 24 hours later. Fluorescence is absent from the control embryo, where it disperses throughout the vascular system and is almost completely evacuated by the kidney. The Ift80 morphant embryo displays a persistent fluorescent dextran signal suggesting reduced glomerular filtration. In this model of muscular dystrophy, wild-type injected embryos show normal slow myofibers spanning the somites between adjacent myosepta, as determined by immunostaining using an anti-slow myosin antibody. Mutant injected embryos showed partial to complete detachment of myofibers from myosepta in one or multiple somites. Shown here are live lateral views of an uninjected control and a Kif7 morphant at 30 hours post-fertilization. As seen by the comparison of somite angle, morphants display abnormally shaped somites, attributable to ectopic Hedgehog signaling in the zebrafish myotome.

- After watching this video, you should have a good idea of how to develop physiologically relevant, sensitive and specific in vivo assays for interpreting human variation in pathology using the zebrafish as a model of organism.