Circumventing Zygotic Lethality to Generate Maternal Mutants in Zebrafish

Maternal gene products accumulated during oogenesis are essential for supporting early developmental processes in both invertebrates and vertebrates. Therefore, understanding their regulatory functions should provide insights into the maternal control of embryogenesis. The CRISPR/Cas9 genome editing technology has provided a powerful tool for creating genetic mutations to study gene functions and developing disease models to identify new therapeutics. However, many maternal genes are also essential after zygotic genome activation; as a result, loss of their zygotic functions often leads to lethality or sterility, thus preventing the generation of maternal mutants by classical crossing between zygotic homozygous mutant adult animals. Although several approaches, such as the rescue of mutant phenotypes through an injection of the wild-type mRNA, germ-line replacement, and the generation of genetically mosaic females, have been developed to overcome this difficulty, they are often technically challenging and time-consuming or inappropriate for many genes that are essential for late developmental events or for germ-line formation. Recently, a method based on the oocyte transgenic expression of CRISPR/Cas9 and guide RNAs has been designed to eliminate maternal gene products in zebrafish. This approach introduces several tandem guide RNA expression cassettes and a GFP reporter into transgenic embryos expressing Cas9 to create biallelic mutations and inactivate genes of interest specifically in the developing oocytes. It is particularly accessible and allows for the elimination of maternal gene products in one fish generation. By further improving its efficiency, this method can be used for the systematic characterization of maternal-effect genes.

Three classes of epigenomic regulators converge to hyperactivate the essential maternal gene deadhead within a heterochromatin mini-domain

The formation of a diploid zygote is a highly complex cellular process that is entirely controlled by maternal gene products stored in the egg cytoplasm. This highly specialized transcriptional program is tightly controlled at the chromatin level in the female germline. As an extreme case in point, the massive and specific ovarian expression of the essential thioredoxin Deadhead (DHD) is critically regulated in Drosophila by the histone demethylase Lid and its partner, the histone deacetylase complex Sin3A/Rpd3, via yet unknown mechanisms. Here, we identified Snr1 and Mod(mdg4) as essential for dhd expression and investigated how these epigenomic effectors act with Lid and Sin3A to hyperactivate dhd. Using Cut&Run chromatin profiling with a dedicated data analysis procedure, we found that dhd is intriguingly embedded in an H3K27me3/H3K9me3-enriched mini-domain flanked by DNA regulatory elements, including a dhd promoter-proximal element essential for its expression. Surprisingly, Lid, Sin3a, Snr1 and Mod(mdg4) impact H3K27me3 and this regulatory element in distinct manners. However, we show that these effectors activate dhd independently of H3K27me3/H3K9me3, and that dhd remains silent in the absence of these marks. Together, our study demonstrates an atypical and critical role for chromatin regulators Lid, Sin3A, Snr1 and Mod(mdg4) to trigger tissue-specific hyperactivation within a unique heterochromatin mini-domain.

Dynamics of maternal gene expression in Rhodnius prolixus

The study of the developmental processes in Rhodnius prolixus has recently advanced with the sequencing of the genome. In this work, we study maternal gene expression driving oogenesis and early embryogenesis in R. prolixus. We analyze the transcriptional profile of mRNAs to establish the genes expressed across the ovary, unfertilized eggs and different embryonic stages of R. prolixus until the formation of the germ band anlage (0, 12, 24, and 48 hours post egg laying). We identified 81 putative maternal and ovary-related genes and validated their expression by qRT-PCR. Consistent with a role in oogenesis and early development of R. prolixus, we show that parental RNAi against Rp-BicD results in embryos that did not show any distinguishable embryonic structure. In this framework we propose three hierarchies of maternal genes that affect early and late oogenesis, and embryonic patterning.

Rapid generation of maternal mutants via oocyte transgenic expression of CRISPR-Cas9 and sgRNAs in zebrafish

Maternal products are exclusive factors to drive oogenesis and early embryonic development. As disrupting maternal gene functions is either time-consuming or technically challenging, early developmental programs regulated by maternal factors remain mostly elusive. We provide a transgenic approach to inactivate maternal genes in zebrafish primary oocytes. By introducing three tandem single guide RNA (sgRNA) expression cassettes and a green fluorescent protein (GFP) reporter into Tg(zpc:zcas9) embryos, we efficiently obtained maternal nanog and ctnnb2 mutants among GFP-positive F1 offspring. Notably, most of these maternal mutants displayed either sgRNA site–spanning genomic deletions or unintended large deletions extending distantly from the sgRNA targets, suggesting a prominent deletion-prone tendency of genome editing in the oocyte. Thus, our method allows maternal gene knockout in the absence of viable and fertile homozygous mutant adults. This approach is particularly time-saving and can be applied for functional screening of maternal factors and generating genomic deletions in zebrafish.

Three classes of epigenomic regulators converge to hyperactivate the essential maternal gene deadhead within a heterochromatin mini-domain.

The formation of a diploid zygote is a highly complex cellular process that is entirely controlled by maternal gene products stored in the egg cytoplasm. This highly specialized transcriptional program is tightly controlled at the chromatin level in the female germline. As an extreme case in point, the massive and specific ovarian expression of the essential thioredoxin Deadhead (DHD) is critically regulated in Drosophila by the histone demethylase Lid and its partner, the histone deacetylase complex scaffold Sin3A, via yet unknown mechanisms. Here, we identified the Brahma chromatin remodeler sub-unit Snr1 and the insulator component Mod(mdg4) as essential for dhd expression and investigated how these epigenomic effectors act with Lid and Sin3A to hyperactivate dhd . Using Cut&Run chromatin profiling with a dedicated data analysis procedure, we found that dhd is intriguingly embedded in an H3K27me3/H3K9me3-enriched mini-domain flanked by DNA regulatory elements, including a dhd promoter-proximal element essential for its expression. Surprisingly, Lid, Sin3A, Snr1 and Mod(mdg4) impact H3K27me3 and this regulatory element in distinct manners. However, we show that these effectors activate dhd independently of H3K27me3/H3K9me3, and that these marks are not required to repress dhd . Together, our study demonstrates an atypical and critical role for chromatin regulators Lid, Sin3A, Snr1 and Mod(mdg4) to trigger tissue-specific hyperactivation within a unique heterochromatin mini-domain.

Axis formation in annual killifish: Nodal coordinates morphogenesis in absence of Huluwa prepatterning

Axis formation in fish and amphibians is initiated by a prepattern of maternal gene products in the blastula. The embryogenesis of annual killifish challenges prepatterning models because blastomeres disperse and then re-aggregate to form the germ layers and body axes. This dispersion-aggregation process prompts the question how axis determinants such as Huluwa and germ layer inducers such as Nodal function in annual killifish. Here we show in Nothobranchius furzeri that huluwa, the factor thought to break symmetry by stabilizing β-catenin, is a non-functional pseudogene. Nuclear β-catenin is not selectively stabilized on one side of the blastula but accumulates in cells forming the incipient aggregate. Inhibition of Nodal signaling blocks aggregation and disrupts coordinated cell migration, establishing a novel role for this signaling pathway. These results reveal a surprising departure from classic mechanisms of axis formation: canonical Huluwa-mediated prepatterning is dispensable and Nodal coordinates morphogenesis.

Polyploidization, hybridization, and maternal and paternal lineages in Cyprinids (Teleostei: Cypriniformes)

Background The reconstruction of phylogenetic relationships for allopolyploids using genomic data is challenging because hybridization and polyploidy can blur history. In cyprinids, one allopolyploidization event involves goldfish and common carp, yet little is known about the origins of other cyprinid polyploid lineages, including their maternal and paternal ancestral lineages. Results Herein we employ 10 HOX genes, genomic and transcriptomic data of representative species from seven subfamilies in Cyprinidae to investigate the origins of polyploid lineages. Analyses of the nuclear and mitochondria genomes reveal that the Schizothoracinae and Cyprininae share the same maternal common ancestor, and identify the candidate genes for identifying maternal gene-copies from Cyprininae. Gene-trees show a close relationship between Sinocyclocheilus grahami and Cyprininae, and indicate that they share the same whole genome duplication (WGD) event about 12.10 ~ 14.03 Ma. Another allopolyploidization event involves Torinae; one duplication clustering with Schizothoracinae and Schizopygopsinae identifies them as paternal siblings. Further, Labeoninae has a history of recurrent hybridization, which is supported by both gene-trees from genomes and HOX genes. Conclusions Collectively, the diverse WGD history makes Cyprinidae a candidate system for investigating the origin of hybridization and polyploidization in vertebrates. Further investigations should enlarge the representatives, combine morphological traits, and reconstruct gene-trees based on whole-genome markers.

Middle eastern genetic legacy in the paternal and maternal gene pools of Chuetas

Chuetas are a group of descendants of Majorcan Crypto-Jews (Balearic Islands, Spain) who were socially stigmatized and segregated by their Majorcan neighbours until recently; generating a community that, although after the seventeenth century no longer contained Judaic religious elements, maintained strong group cohesion, Jewishness consciousness, and endogamy. Collective memory fixed 15 surnames as a most important defining element of Chueta families. Previous studies demonstrated Chuetas were a differentiated population, with a considerable proportion of their original genetic make-up. Genetic data of Y-chromosome polymorphism and mtDNA control region showed, in Chuetas’ paternal lineages, high prevalence of haplogroups J2-M172 (33%) and J1-M267 (18%). In maternal lineages, the Chuetas hallmark is the presence of a new sub-branching of the rare haplogroup R0a2m as their modal haplogroup (21%). Genetic diversity in both Y-chromosome and mtDNA indicates the Chueta community has managed to avoid the expected heterogeneity decrease in their gene pool after centuries of isolation and inbreeding. Moreover, the composition of their uniparentally transmitted lineages demonstrates a remarkable signature of Middle Eastern ancestry—despite some degree of host admixture—confirming Chuetas have retained over the centuries a considerable degree of ancestral genetic signature along with the cultural memory of their Jewish origin.

Differences in maternal gene expression in Cesarean section delivery compared with vaginal delivery

Cesarean section (CS) is recognized as being a shared environmental risk factor associated with chronic immune disease. A study of maternal gene expression changes between different delivery modes can add to our understanding of how CS contributes to disease patterns later in life. We evaluated the association of delivery mode with postpartum gene expression using a cross-sectional study of 324 mothers who delivered full-term (≥ 37 weeks) singletons. Of these, 181 mothers had a vaginal delivery and 143 had a CS delivery (60 with and 83 without labor). Antimicrobial peptides (AMP) were upregulated in vaginal delivery compared to CS with or without labor. Peptidase inhibitor 3 (PI3), a gene in the antimicrobial peptide pathway and known to be involved in antimicrobial and anti-inflammatory activities, showed a twofold increase in vaginal delivery compared to CS with or without labor (adjusted p-value 1.57 × 10–11 and 3.70 × 10–13, respectively). This study evaluates differences in gene expression by delivery mode and provides evidence of antimicrobial peptide upregulation in vaginal delivery compared to CS with or without labor. Further exploration is needed to determine if AMP upregulation provides protection against CS-associated diseases later in life.