Achieving single-cell-resolution lineage tracing in zebrafish by continuous barcoding mutations during embryogenesis

Unraveling the lineage relationships of all descendants from a zygote is fundamental to advancing our understanding of developmental and stem cell biology.However,existing cell barcoding technologies in zebrafish lack the resolution to capture the majority of cell divisions during embryogenesis.A recently developed method,a substitution mutation-aided lineage-tracing system(SMALT),successfully reconstructed high-resolution cell phylogenetic trees for Drosophila melanogaster.Here,we implement the SMALT system in zebrafish,recording a median of 14 substitution mutations on a one-kilobase-pair barcoding sequence for one-day post-fertilization embryos.Leveraging this system,we reconstruct four cell lineage trees for zebrafish fin cells,encompassing both original and regenerated fin.Each tree consists of hundreds of internal nodes with a median bootstrap support of 99%.Analysis of the obtained cell lineage trees reveals that regenerated fin cells mainly originate from cells in the same part of the fins.Through multiple times sampling germ cells from the same individual,we show the stability of the germ cell pool and the early separation of germ cell and somatic cell progenitors.Our system offers the potential for reconstructing high-quality cell phylogenies across diverse tissues,providing valuable insights into development and disease in zebrafish.

A theoretical exploration of the origin and early evolution of a pandemic

A virus that can cause a global pandemic must be highly adaptive to human conditions.Such adaptation is not likely to have emerged suddenly but,instead,may have evolved step by step with each step favored by natural selection.It is thus necessary to develop a theory about the origin in order to guide the search.Here,we propose such a model whereby evolution occurs in both the virus and the hosts(where the evolution is somatic;i.e.,in the immune system).The hosts comprise three groups–the wild animal hosts,the nearby human population,and farther-away human populations.The theory suggests that the conditions under which the pandemic has initially evolved are:(i)an abundance of wild animals in the place of origin(PL_(0));(ii)a nearby human population of low density;(iii)frequent and long-term animal-human contacts to permit step-by-step evolution;and(iv)a level of herd immunity in the animal and human hosts.In this model,the evolving virus may have regularly spread out of PL_(0) although such invasions often fail,leaving sporadic cases of early infections.The place of the first epidemic(PL_(1)),where humans are immunologically naïve to the virus,is likely a distance away from PL_(0).Finally,this current model is only a first attempt and more theoretical models can be expected to guide the search for the origin of SARS-CoV-2.

The evolution of sex chromosome dosage compensation in animals

The evolution of heteromorphic sex chromosomes shall lead to gene expression dosage problems,as in at least one of the sexes,the sex-linked gene dose has been reduced by half.It has been proposed that the transcriptional output of the whole X or Z chromosome should be doubled for complete dosage compensation in heterogametic sex.However,owing to the variability of the existing methods to determine the transcriptional differences between sex chromosomes and autosomes(S:A ratios)in different studies,we collected more than 500 public RNA-Seq data set from multiple tissues and species in major clades and proposed a unified computational framework for unbiased and comparable measurement of the S:A ratios of multiple species.We also tested the evolution of dosage compensation more directly by assessing changes in the expression levels of the current sex-linked genes relative to those of the ancestral sex-linked genes.We found that in mammals and birds,the S:A ratio is approximately 0.5,whereas in insects,fishes,and flatworms,the S:A ratio is approximately 1.0.Further analysis showed that the fraction of dosage-sensitive housekeeping genes on the X/Z chromosome is significantly correlated with the S:A ratio.In addition,the degree of degeneration of the Y chromosome may be responsible for the change in the S:A ratio in mammals without a dosage compensation mechanism.Our observations offer unequivocal support for the sex chromosome insensitivity hypothesis in animals and suggest that dosage sensitivity states of sex chromosomes are a major factor underlying different evolutionary strategies of dosage compensation.

The expression tractability of biological traits shaped by natural selection

Understanding how gene expression is translated to phenotype is central to modern molecular biology,and the success is contingent on the intrinsic tractability of the specific traits under examination.However, an a priori estimate of trait tractability from the perspective of gene expression is unavailable.Motivated by the concept of entropy in a thermodynamic system, we here propose such an estimate(S_T)by gauging the number(N) of expression states that underlie the same trait abnormality, with large S_T corresponding to large N. By analyzing over 200 yeast morphological traits, we show that S_T predicts the tractability of an expression-trait relationship. We further show that S_T is ultimately determined by natural selection, which builds co-regulated gene modules to minimize possible expression states.

Development of a high-fidelity Cas9-dependent adenine base editor (ABE) system for genome editing with high-fidelity Cas9 variants

One of the main reasons that hinders the application of base editors in the clinic is the trade-off between editing efficiency and editing fidelity.The off-target base editing includes Cas9-independent and Cas9-dependent manners.1 The Cas9-independent off-target results from the intrinsic deamination activity of the deaminase domain of a base editor,which affects random RNA or DNA molecules.

Defining endogenous barcoding sites for CRISPR/Cas9-based cell lineage tracing in zebrafish

There is a growing interest in developing experimental methods for tracking the developmental cell lineages of a complex organism.The recently developed CRISPR/Cas9-based barcoding method is,although highly promising,difficult to scale up because it relies on exogenous barcoding sequences that are engineered into the genome.In this study,we characterized 78 high-quality endogenous sites in the zebrafish genome that can be used as CRISPR/Cas9-based barcoding sites.The 78 sites are all highly expressed in most of the cell types according to single-cell RNA sequencing(scRNA-seq)data.Hence,the barcoding information of the 78 endogenous sites is recovered by the available scRNA-seq platforms,enabling simultaneous characterization of cell type and cell lineage information.

An instantaneous coalescent method insensitive to population structure

Conventional coalescent inferences of population history make the critical assumption that the population under examination is panmictic.However,most populations are structured.This complicates the prevailing coalescent analyses and sometimes leads to inaccurate estimates.To develop a coalescent method unhampered by population structure,we perform two analyses.First,we demonstrate that the coalescent probability of two randomly sampled alleles from the immediate preceding generation(one generation back)is independent of population structure.Second,motivated by this finding,we propose a new coalescent method:i-coalescent analysis.The i-coalescent analysis computes the instantaneous coalescent rate by using a phylogenetic tree of sampled alleles.Using simulated data,we broadly demonstrate the capability of i-coalescent analysis to accurately reconstruct population size dynamics of highly structured populations,although we find this method often requires larger sample sizes for structured populations than for panmictic populations.Overall,our results indicate i-coalescent analysis to be a useful tool,especially for the inference of population histories with intractable structure such as the developmental history of cell populations in the organs of complex organisms.