Two vascular networks nourish the embryonic eye as it develops - the hyaloid vasculature, located at the anterior of the eye between the retina and lens, and the choroidal vasculature, located at the posterior of the eye, surrounding the optic cup. Little is known about hyaloid development and morphogenesis, however. To begin to identify the morphogenetic underpinnings of hyaloid formation, we utilized in vivo time-lapse confocal imaging to characterize morphogenesis of the zebrafish hyaloid through 5 days post fertilization (dpf). Our data segregate hyaloid formation into three distinct morphogenetic stages: Stage I: Arrival of hyaloid cells at the lens and formation of the hyaloid loop; Stage II: Formation of a branched hyaloid network; Stage III: Refinement of the hyaloid network. Utilizing fixed and dissected tissues, distinct Stage II and Stage III aspects of hyaloid formation were quantified over time. Combining in vivo imaging with microangiography, we demonstrate that the hyaloid system becomes fully enclosed by 5dpf. To begin to identify the molecular and cellular mechanisms underlying hyaloid morphogenesis, we identified a recessive mutation in the mab21l2 gene, and in a subset of mab21l2 mutants the lens does not form. Utilizing these “lens-less” mutants, we determined whether the lens was required for hyaloid morphogenesis. Our data demonstrate that the lens is not required for Stage I of hyaloid formation; however, Stages II and III of hyaloid formation are disrupted in the absence of a lens, supporting a role for the lens in hyaloid maturation and maintenance. Taken together, this study provides a foundation on which the cellular, molecular and embryologic mechanisms underlying hyaloid morphogenesis can be elucidated.
The application of new technologies for gene editing in horses may allow the generation of improved sportive individuals. Here, we aimed to knock out the myostatin gene (MSTN), a negative regulator of muscle mass development, using CRISPR/Cas9 and to generate edited embryos for the first time in horses. We nucleofected horse fetal fibroblasts with 1, 2 or 5 µg of 2 different gRNA/Cas9 plasmids targeting the first exon of MSTN. We observed that increasing plasmid concentrations improved mutation efficiency. The average efficiency was 63.6% for gRNA1 (14/22 edited clonal cell lines) and 96.2% for gRNA2 (25/26 edited clonal cell lines). Three clonal cell lines were chosen for embryo generation by somatic cell nuclear transfer: one with a monoallelic edition, one with biallelic heterozygous editions and one with a biallelic homozygous edition, which rendered edited blastocysts in each case. Both MSTN editions and off-targets were analyzed in the embryos. In conclusion, CRISPR/Cas9 proved an efficient method to edit the horse genome in a dose dependent manner with high specificity. Adapting this technology sport advantageous alleles could be generated, and a precision breeding program could be developed.
Somatic cell nuclear transfer (SCNT) is an asexual reproductive technique where cloned offspring contain the same genetic material as the original donor. Although this technique preserves the sex of the original animal, the birth of sex-reversed offspring has been reported in some species. Here, we report for the first time the birth of a female foal generated by SCNT of a male nuclear donor. After a single SCNT procedure, 16 blastocysts were obtained and transferred to eight recipient mares, resulting in the birth of two clones: one male and one female. Both animals had identical genetic profiles, as observed in the analysis of 15-horse microsatellite marker panel, which confirmed they are indeed clones of the same animal. Cytogenetic analysis and fluorescent in situ hybridization using X and Y specific probes revealed a 63,X chromosome set in the female offspring, suggesting a spontaneous Y chromosome loss. The identity of the lost chromosome in the female was further confirmed through PCR by observing the presence of X-linked markers and absence of Y-linked markers. Moreover, cytogenetic and molecular profiles were analyzed in blood and skin samples to detect a possible mosaicism in the female, but results showed identical chromosomal constitutions. Although the cause of the spontaneous chromosome loss remains unknown, the possibility of equine sex reversal by SCNT holds great potential for the preservation of endangered species, development of novel breeding techniques, and sportive purposes.
Historically, livestock improvement by selective breeding was the principal selection force in animal production and welfare, but the desired phenotype may involve more than 1 generation. Nowadays, new technologies such as CRISPR/Cas9 could overpass these limits and improve animal quality by insertion or modification of the desired genotype. In this work, we aim to knock out the myostatin (MSTN) gene, a negative regulator of muscle mass development, in equine cells and to generate equine cloned embryos with this modified genotype. To achieve this, 1×105 equine fibroblasts were nucleofected with 5, 2, or 1μg of the plasmid hspCas9-2A-Puro V2.0 that codified for the Cas 9 nuclease and a single guide RNA (gRNA). Two different gRNA (gRNA 1 and gRNA 2) complementary to the first exon of the MSTN gene were designed and evaluated. Cells were also nucleofected with the enhanced green fluorescent protein-N1 plasmid in order to determine the transfection efficiency, obtaining more than 90% of enhanced green fluorescent protein+ cells in the 3 conditions. Forty-eight hours after nucleofection, cells were treated with 2.5μg mL−1 of puromycin for 48h to isolate cells that incorporated the plasmid. After that, clonal culture was achieved by plating individual cells in 96-well plates. The clones were then expanded individually and genomic DNA was isolated from each one, genotyped for the MSTN exon 1 locus by PCR amplification, and Sanger sequenced. Both gRNA had mutational activity, with 96% efficiency (24/25 clones) for gRNA 1 and 55.5% mutation activity (10/18 clones) for gRNA2. We obtained different genotypes depending on the gRNA and the dose that was used-gRNA 1: 1μg=57% wt/wt, 14% wt/mutX, and 29% mutX/mutX; 2μg=33.3% wt/wt, 33.3% mutX/mutY, and 33.3% mutX/mutX; 5μg=40% wt/wt and 60% mutX/mutY; gRNA 2: 1μg=17% wt/wt, 17% wt/mutX, 50% mutX/mutY, and 17% mutX/mutX; 2μg=67% mutX/mutY and 34% mutX/mutX; 5μg=54% mutX/mutY and 46% mutX/mutX. Two of the gRNA2 mutated cell lines were used for embryo generation by NT, 1 wt/mutX line (gRNA2-1ug-C2, heterozygote clone) and 1 mutX/mutX line (gRNA2-5ug-C13, homozygote clone). Before that, to assess specificity, the first 2 highly ranked off-target sites of gRNA2 were checked by Sanger sequencing in the selected clones, not observing modifications in their sequences. In both cases, we could generate edited MSTN equine cloned blastocysts: 3/153 (2%), 3/155 (2%), 8/140 (6%), and 9/73 (12%) for gRNA2-1μg-C2, gRNA2-5μg-C13, control fibroblasts, and control mesenchymal cells, respectively. In conclusion, genome edition by CRISPR/Cas9 is an efficient method to edit the genome of horse fibroblasts in a dose-dependent manner with apparent high specificity. Moreover, equine embryos can be generated with these cells with lower blastocyst rates than control fibroblasts of the same cell line or mesenchymal cells, probably due to higher cell passages needed for cell clone isolation and expansion. To the best of our knowledge, this is the first report of genome edited horse embryos.
Sir Francis Burdett was one of the most important reformers of his period. A controversial figure, particularly in his early career, Burdett played a significant and leading role in several of the most popular agitations of the early nineteenth century.
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