Biology subjects

Burgess, S. M.

Publications and source records attributed to Burgess, S. M..

27 records · Page 2Linked to original sources

bioRxiv2019

Sex chromosome and sex locus characterization in the goldfish, Carassius auratus

BackgroundGoldfish is an important model for various areas of research, including neural development and behavior and a species of significant importance in aquaculture, especially as an ornamental species. It has a male heterogametic (XX/XY) sex determination system that relies on both genetic and environmental factors, with high temperatures being able to produce female-to-male sex reversal. Little, however, is currently known on the molecular basis of genetic sex determination in this important cyprinid model. We used sequencing approaches to better characterize sex determination and sex-chromosomes in goldfish. ResultsOur results confirmed that sex determination in goldfish is a mix of environmental and genetic factors and that its sex determination system is male heterogametic (XX/XY). Using reduced representation (RAD-seq) and whole genome (pool-seq) approaches, we characterized sex-linked polymorphisms and developed male specific genetic markers. These male specific markers were used to distinguish sex-reversed XX neomales from XY males and to demonstrate that XX female-to-male sex reversal could even occur at a relatively low rearing temperature (18{degrees}C), for which sex reversal has been previously shown to be close to zero. We also characterized a relatively large non-recombining region ([~]11.7 Mb) on goldfish linkage group 22 (LG22) that contained a high-density of male-biased genetic polymorphisms. This large LG22 region harbors 373 genes, including a single candidate as a potential master sex gene, i.e., the anti-Mullerian hormone gene (amh). However, no sex-linked polymorphisms were detected in the goldfish amh gene or its 5 kb proximal promoter sequence. ConclusionsThese results show that goldfish have a relatively large sex locus on LG22, which is likely the goldfish Y chromosome. The presence of a few XX males even at low temperature also suggests that other environmental factors in addition to temperature could trigger female-to-male sex reversal. Finally, we also developed sex-linked genetic markers in goldfish, which will be important for future research on sex determination and aquaculture applications in this species.

genomics↗

bioRxiv2019

Amyloid precursor protein-b facilitates cell adhesion during early development in zebrafish

Understanding the biological function of amyloid beta (A{beta}) precursor protein (APP) beyond its role in Alzheimers disease is emerging. Yet, its function during embryonic development is poorly understood. The zebrafish APP homologue, Appb, is strongly expressed during early development but thus far has only been studied via morpholino-mediated knockdown. Zebrafish enables analysis of cellular processes in an ontogenic context, which is limited in many other vertebrates. We characterized zebrafish carrying a homozygous mutation that introduces a premature stop in exon 2 of the appb gene. We report that appb mutants are significantly smaller until 2dpf and display perturbed enveloping layer (EVL) integrity and cell protrusions at the blastula stage.\n\nMoreover, appb mutants surviving beyond 48 hpf exhibited no behavioral defects at 6 dpf and developed into healthy and fertile adults. The expression of the app-family members, appa and aplp2, was found to be altered in appb mutants. Taken together, we show that appb orchestrates the initial development by supporting the integrity of the EVL, likely by mediating cell adhesion properties. The loss of Appb might be compensated for by other app family members to be able to implement continued normal development.

developmental biology↗

bioRxiv2019

A subset of SMN complex members have a specific role in tissue regeneration via ERBB pathway-mediated proliferation

Spinal Muscular Atrophy (SMA) is the most common genetic disease in childhood. SMA is generally caused by mutations in SMN1. The Survival of Motor Neurons (SMN) complex consists of SMN1, Gemins (2-8) and Strap/Unrip. We previously demonstrated smn1 and gemin5 inhibited tissue regeneration in zebrafish. Here we investigated each individual SMN complex member and identified gemin3 as another regeneration-essential gene. These three genes are likely pan-regenerative since they affect the regeneration of hair cells, liver and caudal fin. RNA-Seq and miRNA-Seq analyses reveal that smn1, gemin3, and gemin5 are linked to a common set of genetic pathways, including the tp53 and ErbB pathways. Additional studies indicated all three genes facilitate regeneration by inhibiting the ErbB pathway, thereby allowing cell proliferation in the injured neuromasts. This study provides a new understanding of the SMN complex and a potential etiology for SMA and potentially other rare unidentified genetic diseases with similar symptoms.

genetics↗

bioRxiv2019

A New Role for Telomerase in Promoting Meiotic Homolog Pairing Fidelity

We report a role for telomerase, beyond its known function of telomeric DNA end extension, in maintaining normal chromosome dynamics during meiosis in Saccharomyces cerevisiae. When telomerase at telomeres was reduced by various genetic means, increased frequencies of crossover and noncrossover recombination events occurred. To investigate the mechanism of this increased meiotic recombination, we examined the kinetics of meiosis events, and tracked the movement of chromosomes in live cells during meiotic prophase. Cytoskeletal forces acting on telomeres during meiosis have been shown to promote active chromosome motion needed to pair homologous chromosomes. Here we show that changes in telomerase interaction with telomeres using a tlc1-11 mutant result in altered meiotic motion. Specifically, reduction in telomerase at telomeres leads to a decreased frequency of high velocity chromosome pulls. In the tlc1-11 mutant, we see earlier synapsis and increased genome-wide recombination for the majority of the cells and lower gamete viability. Notably, homologous pairing is not delayed unlike other telomere binding mutants. Although synapsis initiates earlier, the overall timing of synapsis remains the same, except for a subset of cells that do not exit meiosis I. Together, these results suggest that the strong pulling component of the active chromosome motion promotes homolog pairing fidelity, likely by pulling apart improperly associated regions. Our combined observations are consistent with a model in which telomerase-mediated telomeric anchoring to the nuclear envelope helps engage and properly transmit cytoskeletal forces to chromosomes. Thus, telomerase contributes to efficient chromosome movements leading to normal gamete viability.

cell biology↗

bioRxiv2019

The Vertebrate Codex Gene Breaking Protein Trap Library For Genomic Discovery and Disease Modeling Applications

The zebrafish is a powerful model to explore the molecular genetics and expression of the vertebrate genome. The gene break transposon (GBT) is a unique insertional mutagen that reports the expression of the tagged member of the proteome while generating Cre-revertible genetic alleles. This 1000+ locus collection represents novel codex expression data from the illuminated mRFP protein trap, with 36% and 87% of the cloned lines showcasing to our knowledge the first described expression of these genes at day 2 and day 4 of development, respectively. Analyses of 183 molecularly characterized loci indicate a rich mix of genes involved in diverse cellular processes from cell signaling to DNA repair. The mutagenicity of the GBT cassette is very high as assessed using both forward and reverse genetic approaches. Sampling over 150 lines for visible phenotypes after 5dpf shows a similar rate of discovery of embryonic phenotypes as ENU and retroviral mutagenesis. Furthermore, five cloned insertions were in loci with previously described phenotypes; embryos homozygous for each of the corresponding GBT alleles displayed strong loss of function phenotypes comparable to published mutants using other mutagenesis strategies (ryr1b, fras1, tnnt2a, edar and hmcn1). Using molecular assessment after positional cloning, to date nearly all alleles cause at least a 99+% knockdown of the tagged gene. Interestingly, over 35% of the cloned loci represent 68 mutants in zebrafish orthologs of human disease loci, including nervous, cardiovascular, endocrine, digestive, musculoskeletal, immune and integument systems. The GBT protein trapping system enabled the construction of a comprehensive protein codex including novel expression annotation, identifying new functional roles of the vertebrate genome and generating a diverse collection of potential models of human disease.

genomics↗

bioRxiv2018

tbx5 and tbx15 mediate zebrafish posterior lateral line migration and development via a negative feedback loop

The zebrafish posterior lateral line (pLL) is a mechanosensory organ enabling detection of movement in the aqueous environment. Evidence suggests that two T-box transcription factors, tbx5 and tbx15, participate in pLL migration and development. Lateral line migration and deposition defects are observed following tbx5 or tbx15 morpholino knockdown. Additional studies demonstrate that the tbx5 phenotype is partially rescued when tbx15 is also knocked down. pLL defects similar to those of tbx5 are noted following knockdown of camKII-{beta}2, a known downstream target of Tbx5, suggesting a potential mechanistic pathway. Ectopic expression of human CamKII-{partial}C partially rescues the tbx5 phenotype but exacerbates the tbx15 phenotype. These results, combined with in situ hybridization and qRT-PCR profiling, indicate a negative feedback loop controlling multiple primordium patterning markers, including cxcr4b, cxcr7b, fgf10a and notch3, which ultimately affects pLL neuromast migration and deposition.

developmental biology↗

bioRxiv2018

The telomere bouquet is a hub where meiotic double-strand breaks, synapsis, and stable homolog juxtaposition are coordinated in the zebrafish, Danio rerio

Meiosis is a cellular program that generates haploid gametes for sexual reproduction. While chromosome events that contribute to reducing ploidy (homologous chromosome pairing, synapsis, and recombination) are well conserved, their execution varies across species and even between sexes of the same species. The telomere bouquet is a conserved feature of meiosis that was first described nearly a century ago, yet its role is still debated. Here we took advantage of the prominent telomere bouquet in zebrafish, Danio rerio, and super-resolution microscopy to show that axis morphogenesis, synapsis, and the formation of double-strand breaks (DSBs) all take place within the immediate vicinity of telomeres. We established a coherent timeline of events and tested the dependence of each event on the formation of Spo11-induced DSBs. First, we found that the axis protein Sycp3 loads adjacent to telomeres and extends inward, suggesting a specific feature common to all telomeres seeds the development of the axis. Second, we found that newly formed axes near telomeres engage in presynaptic co-alignment by a mechanism that depends on DSBs, even when stable juxtaposition of homologous chromosomes at interstitial regions is not yet evident. Third, we were surprised to discover that ~30% of telomeres in early prophase I engage in associations between two or more chromosome ends and these interactions decrease in later stages. Finally, while pairing and synapsis were disrupted in both spo11 males and females, their reproductive phenotypes were starkly different; spo11 mutant males failed to produce sperm while females produced offspring with severe developmental defects. Our results support zebrafish as an important vertebrate model for meiosis with implications for differences in fertility and genetically derived birth defects in males and females.\n\nAuthor SummaryInherent to reproduction is the transmission of genetic information from one generation to the next. In sexually reproducing organisms, each parent contributes an equal amount of genetic information, packaged in chromosomes, to the offspring. Diploid organisms, like humans, have two copies of every chromosome, while their haploid gametes (e.g. eggs and sperm) have only one. This reduction in ploidy depends on the segregation of chromosomes during meiosis, resulting in gametes with one copy of each chromosome. Missegregation of the chromosomes in the parents leads to abnormal chromosome numbers in the offspring, which is usually lethal or has detrimental developmental effects. While it has been known for over a century that homologous chromosomes pair and recombine to facilitate proper segregation, how homologs find their partners has remained elusive. A structure that has been central to the discussion of homolog pairing is the bouquet, or the dynamic clustering of telomeres during early stages of meiosis. Here we use zebrafish to show that the telomere bouquet is the site where key events leading to homologous chromosome pairing are coordinated. Furthermore, we show that deletion of spo11, a gene required for proper recombination in most studied organisms, resulted in very different effects in males and females where males were sterile while females produced deformed progeny.

genetics↗

bioRxiv2018

De Novo assembly of the goldfish (Carassius auratus) genome and the evolution of genes after whole genome duplication

For over a thousand years throughout Asia, the common goldfish (Carassius auratus) was raised for both food and as an ornamental pet. Selective breeding over more than 500 years has created a wide array of body and pigmentation variation particularly valued by ornamental fish enthusiasts. As a very close relative of the common carp (Cyprinus carpio), goldfish shares the recent genome duplication that occurred approximately 14-16 million years ago (mya) in their common ancestor. The combination of centuries of breeding and a wide array of interesting body morphologies is an exciting opportunity to link genotype to phenotype as well as understanding the dynamics of genome evolution and speciation. Here we generated a high-quality draft sequence of a \"Wakin\" goldfish using 71X PacBio long-reads. We identified 70,324 coding genes and more than 11,000 non-coding transcripts. We found that the two sub-genomes in goldfish retained extensive synteny and collinearity between goldfish and zebrafish. However, \"ohnologous\" genes were lost quickly after the carp whole-genome duplication, and the expression of 30% of the retained duplicated gene diverged significantly across seven tissues sampled. Loss of sequence identity and/or exons determined the divergence of the expression across all tissues, while loss of conserved, non-coding elements determined expression variance between different tissues. This draft assembly also provides an important resource for comparative genomics with the very commonly used zebrafish model (Danio rerio), and for understanding the underlying genetic causes of goldfish variants.

genomics↗

bioRxiv2016

Highly-efficient Cpf1-mediated gene targeting in mice following high concentration pronuclear injection

Cpf1 has emerged as an alternative to the Cas9 RNA-guided nuclease. Here we show that gene targeting rates in mice using Cpf1 can meet or even surpass Cas9 targeting rates (approaching 100% targeting) but require higher concentrations of mRNA and guide. We also demonstrate that co-injecting two guides with close targeting sites can result in synergistic genomic cutting, even if one of the guides has minimal cutting activity.

genetics↗