Dynamic regulation of ribosome levels and translation during development.

Single-cell sequencing technologies have led to a revolution in our knowledge of the diversity of cell types, connections between biological levels of organization, and relationships between genotype and phenotype. These advances have mainly come from using model organisms; however, using single-cell sequencing in non-model organisms could enable investigations of questions inaccessible with typical model organisms. This primer describes a general workflow for single-cell sequencing studies and considerations for using non-model organisms (limited to multicellular animals). Importantly, single-cell sequencing, when further applied in non-model organisms, will allow for a deeper understanding of the mechanisms between genotype and phenotype and the basis for biological variation.

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A Primer for Single-Cell Sequencing in Non-Model Organisms

Background: The establishment of male or female identity (sex determination) is essential for creating the anatomical, physiological, and behavioral differences between 2 sexes of the same species (sexual dimorphism). In many organisms, including mammals and Drosophila, sex is determined by inheritance of sex chromosomes, while in other animals, sex is determined by environmental factors. Arguably the most important consequence of sex determination is the production of healthy gametes necessary for reproduction: female oocytes and male spermatids. Summary: The generation of sperm and oocytes requires cooperation between 2 different cell types within the gonad: germ cells and somatic cells. Defects in sex determination in either the somatic gonad or germline lead to disorders of sexual development and infertility. In Drosophila, the gene Sex lethal (Sxl) is the key determinant of sex in both the soma and the germline. However, how Sxl controls sex determination is much more well understood in the soma than the germline. Key Mesage: This review will focus on Sxl in the germline, how it is activated specifically in female germ cells, and how it regulates germline sex determination and sexual development.

https://www.karger.com/Article/Pdf/521235

The Regulation of Germline Sex Determination in Drosophila by Sex lethal

The origin of functional new genes is a basic biological process that has significant contribution to organismal diversity. Previous studies in both Drosophila and mammals showed that new genes tend to be expressed in testes and avoid the X chromosome, presumably because of meiotic sex chromosome inactivation (MSCI). Here, we analyze the published single-cell transcriptome data of Drosophila adult testis and find an enrichment of male germline mitotic genes, but an underrepresentation of meiotic genes on the X chromosome. This can be attributed to an excess of autosomal meiotic genes that were derived from their X-linked mitotic progenitors, which provides direct cell-level evidence for MSCI in Drosophila. We reveal that new genes, particularly those produced by retrotransposition, tend to exhibit an expression shift toward late spermatogenesis compared with their parental copies, probably due to the more intensive sperm competition or sexual conflict. Our results dissect the complex factors including age, the origination mechanisms and the chromosomal locations that influence the new gene origination and evolution in testes, and identify new gene cases that show divergent cell-level expression patterns from their progenitors for future functional studies.

On the Origin and Evolution of Drosophila New Genes during Spermatogenesis.

Many animal species employ sperm nuclear basic proteins (SNBPs) or protamines to package sperm genomes tightly. SNBPs vary across animal lineages and evolve rapidly in mammals. We used a phylogenomic approach to investigate SNBP diversification in Drosophila species. We found that most SNBP genes in Drosophila melanogaster evolve under positive selection except for genes essential for male fertility. Unexpectedly, evolutionarily young SNBP genes are more likely to be critical for fertility than ancient, conserved SNBP genes. For example, CG30056 is dispensable for male fertility despite being one of three SNBP genes universally retained in Drosophila species. We found 19 independent SNBP gene amplification events that occurred preferentially on sex chromosomes. Conversely, the montium group of Drosophila species lost otherwise-conserved SNBP genes, coincident with an X-Y chromosomal fusion. Furthermore, SNBP genes that became linked to sex chromosomes via chromosomal fusions were more likely to degenerate or relocate back to autosomes. We hypothesize that autosomal SNBP genes suppress meiotic drive, whereas sex-chromosomal SNBP expansions lead to meiotic drive. X-Y fusions in the montium group render autosomal SNBPs dispensable by making X-versus-Y meiotic drive obsolete or costly. Thus, genetic conflicts between sex chromosomes may drive SNBP rapid evolution during spermatogenesis in Drosophila species.In sperm, DNA is packaged more tightly than in other cells thanks to small proteins called ‘sperm nuclear basic proteins’ (SNBPs), also called protamines in mammals. SNBPs are important for sperm to develop properly and correctly perform their role during fertilization. Although the evolution of SNBPs has been studied in mammals, these proteins have not been as thoroughly examined in invertebrates. Chang et al. took advantage of the availability of high-quality sequences for the genomes of 78 species of Drosophila flies to investigate the evolution of the genes that code for SNBPs in these flies. The results showed that, just like in mammals, in Drosophila the protein sequences of SNBPs evolve rapidly. However, unlike mammals, Chang et al. also found that Drosophila species frequently gained and lost genes coding for SNBPs. Interestingly, the ‘older’ genes (genes that appeared earlier in evolution) that code for SNBPs are not essential for reproduction in the fruit fly Drosophila melanogaster. This is an unexpected finding because older genes usually have essential roles for survival and reproduction, which require them to be passed on to the next generation and remain in the genome. In contrast, younger SNBP genes that had appeared more recently and were not shared between different species of Drosophila were often essential for fertility. These results, combined with other observations about where SNBP genes are located in the genome, led Chang et al. to hypothesize that SNBPs present in sex chromosomes act as ‘meiotic drivers’ while those on other chromosomes (known as autosomes) suppress meiotic drive. In other words, SNBP genes present in the sex chromosomes may be responsible for killing sister sperm cells that do not carry those genes, while SNBP genes that are not located on sex chromosomes may suppress this activity. This is of particular interest because it indicates that SNBPs are involved in genetic conflicts between the two sex chromosomes: sperm that carry SNBPs on the X chromosome may kill sperm with a Y chromosome, and vice versa. The results of Chang et al. shed light on the mysterious evolution of SNBPs in Drosophila flies. Although previous hypotheses regarding the rapid evolution of SNBPs evolution have focused on their role in genome packaging, this new analysis suggests that much of the evolutionary change is likely driven by genetic conflicts between sex chromosomes. 

Expansion and loss of sperm nuclear basic protein genes in Drosophila correspond with genetic conflicts between sex chromosomes

Dosage compensation equalizes X-linked expression between XY males and XX females. In male fruit flies, expression levels of the X-chromosome are increased approximately two-fold to compensate for their single X chromosome. In testis, dosage compensation is thought to cease during meiosis; however, the timing and degree of the resulting transcriptional suppression is difficult to separate from global meiotic downregulation of each chromosome. To address this, we analyzed testis single-cell RNA-sequencing (scRNA-seq) data from two Drosophila melanogaster strains. We found evidence that the X chromosome is equally transcriptionally active as autosomes in somatic and pre-meiotic cells, and less transcriptionally active than autosomes in meiotic and post-meiotic cells. In cells experiencing dosage compensation, close proximity to MSL (male-specific lethal) chromatin entry sites (CES) correlates with increased X chromosome transcription. We found low or undetectable levels of germline expression of most msl genes, mle, roX1 and roX2 via scRNA-seq and RNA-FISH, and no evidence of germline nuclear roX1/2 localization. Our results suggest that, although dosage compensation occurs in somatic and pre-meiotic germ cells in Drosophila testis, there might be non-canonical factors involved in the dosage compensation mechanism. The single-cell expression patterns and enrichment statistics of detected genes can be explored interactively in our database: https://zhao.labapps.rockefeller.edu/gene-expr/.

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Single-cell RNA-sequencing reveals pre-meiotic X-chromosome dosage compensation in Drosophila testis

Dosage compensation (DC) is a mechanism by which X chromosome transcription is equalized in the somatic cells of both males and females In male fruit flies, expression levels of the X-chromosome are increased two-fold to compensate for their single X chromosome In testis, dosage compensation is thought to cease during meiosis, however, the timing and degree of the resulting transcriptional suppression is difficult to separate from global meiotic downregulation of each chromosome To address this, we analyzed testis single-cell RNA-sequencing (scRNA-seq) data from two Drosophila melanogaster strains We found evidence that the X chromosome is equally transcriptionally active as autosomes in somatic and pre-meiotic cells, and less transcriptionally active than autosomes in meiotic and post-meiotic cells In cells experiencing dosage compensation, close proximity to MSL (male-specific lethal) chromatin entry sites (CES) correlates with increased X chromosome transcription We found low or undetectable level of germline expression of most msl genes, mle, roX1 and roX2 via sequencing or RNA-FISH, and no evidence of germline nuclear roX1/2 localization Our results suggest that, although DC occurs in somatic and premeiotic germ cells in Drosophila testis, there might be non-canonical factors involved in the dosage compensation The single-cell expression patterns and enrichment statistics of detected genes can be explored interactively in our database: https://zhaolabappsrockefelleredu/gene-expr/

https://biorxiv.org/content/10.1101/2021.02.05.429952v1.full.pdf

Zhantao Shao

Papers

Single-cell RNA-sequencing reveals pre-meiotic X-chromosome dosage compensation in Drosophila testis

Single-cell RNA-sequencing reveals pre-meiotic X-chromosome dosage compensation in Drosophila testis