bioRxiv Science⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/693853

Efficient generation of zebrafish maternal-zygotic mutants through transplantation of ectopically induced and Cas9/gRNA targeted PGCs

Sun, Y.·Zhang, F.·Li, X.·He, M.·Ye, D.·Xiong, F.·Amin, G.·Zhu, Z.
View original source ↗Export metadata ↓

Abstract

The CRISPR/Cas9 technology has been widely utilized for knocking out genes involved in various biological processes in zebrafish. Despite this technology is efficient for generating different mutations, one of the main drawbacks is low survival rates during embryogenesis when knocking out some embryonic lethal genes. To overcome this problem, we developed a novel strategy using a combination of CRISPR/Cas9 mediated gene knockout with primordial germ cells (PGCs) transplantation to facilitate and speed up the process of zebrafish mutant generation, particularly for embryonic lethal genes. First, we optimized the procedure for gRNA targeted PGCs transplantation (PGCT), by increasing the efficiencies of genome mutation in PGCs and induction of PGCs fates in donor embryos for PGCT. Second, the combined CRISPR/Cas9 with PGCT was utilized for generation of maternal zygotic (MZ) mutants of tcf7l1a (essential gene for head development), pou5f3 (essential gene for zygotic genome activation) and chd (essential gene for dorsal development) at F1 generation with high efficiency. Finally, we revealed some novel phenotypes in the maternal zygotic mutant of tcf7l1a and chd, while MZtcf7l1a showed elevated neural crest development, and MZchd have stronger ventralization than its zygotic counterparts. Therefore, this study presents an efficient and powerful method for generating MZ mutants of embryonic lethal genes in zebrafish.

Source connections

Explore related subjects

developmental biologyPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Sun, Y., Zhang, F., Li, X., He, M., Ye, D., Xiong, F., Amin, G., Zhu, Z.. 2019-07-05. Efficient generation of zebrafish maternal-zygotic mutants through transplantation of ectopically induced and Cas9/gRNA targeted PGCs. https://doi.org/10.1101/693853

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Neogenin-1 marks myeloid-primed fetal hematopoietic stem cells that undergo progressive lineage-restriction with age

During aging, hematopoietic stem cells (HSCs) increasingly shift from balanced to myeloid-biased differentiation, resulting in reduced lymphoid output and impaired adaptive immunity. The question of whether this lineage bias is established in a subset of HSCs during early development or primarily emerges with aging warrants further investigation. Here, we investigate whether myeloid-biased HSCs (my-HSCs) are established at the fetal liver stage by specifically examining Neogenin-1 (NEO1), a previously defined marker of my-HSCs. We identify two distinct populations of Hoxb5+ HSCs in the fetal liver: NEO1+ and NEO1-, with NEO1+ HSCs exhibiting transcriptional and functional characteristics consistent with my-HSCs. With age, my-HSC-associated transcriptional programs become increasingly reinforced across the Hoxb5+ pHSC compartment, with NEO1+ cells showing early enrichment of this program and both NEO1+ and NEO1- cells acquiring broader myeloid-biased features in aging. These findings suggest that lineage programming can begin early in development and is further shaped by age-related changes, potentially contributing to the functional decline observed in the aging hematopoietic system.

developmental biology↗
bioRxiv2026

Distinct roles for partially redundant transcription factors in Caenorhabditis elegans mesoderm lineage development

Developmental transcription factors often have overlapping functions, making it difficult to define the distinct roles of individual factors during lineage specification. We investigated the partially redundant transcription factors TBX-35 and CEH-51 in the Caenorhabditis elegans embryonic MS mesodermal lineage using 4D lineage tracing, reporter imaging, genetics, and single-cell RNA sequencing. In tbx-35 mutants, MS descendants showed progressively slower cell cycles and a division pattern that increasingly resembled the cousin C lineage. Fate-regulator expression also shifted toward C-like features, including ectopic pal-1 and expanded HLH-1 expression, although mutant cells did not simply adopt normal C-lineage positions. Loss of tbx-35 also impaired a later MS-dependent Notch induction in the AB lineage while leaving an earlier induction intact. CEH-51 showed a different pattern of activity whereby its protein became enriched in anterior MS daughters, and ceh-51 mutants produced later, more restricted lineage defects that were strongest in descendants of cells with higher CEH-51 levels. Single-cell profiling identified overlapping but nonidentical sets of genes dependent on the two factors. TBX-35-dependent changes were strongest at earlier stages, whereas CEH-51-dependent genes became more prominent later and were enriched in anterior MS sublineages. Finally, temperature-shift experiments determined that the severity and onset of tbx-35 mutant phenotypes depend on the maternal temperature environment and cannot be explained by differences in residual CEH-51 expression. These findings reveal that TBX-35 and CEH-51 contribute differently across the MS lineage and that reliable mesoderm development is supported by overlapping zygotic and maternal regulatory inputs.

developmental biology↗
bioRxiv2026

Dynamic microtubules drive yolk-cytoplasm segregation in the syncytial Drosophila embryo

Yolk-cytoplasm segregation is among the earliest spatial organization events in the developing embryo of many oviparous animals. The segregation process is intimately linked to early embryonic cleavage and pattern formation, and exhibits a wide range of spatial and temporal diversity. However, the underlying cytoskeletal mechanism remains largely unknown, except for a small number of species. Using quantitative live imaging, we investigated yolk segregation in the Drosophila embryo during the syncytial nuclear cycles 11-14. We find that the yolk vesicles move progressively inward in spatial and temporal coordination with the inward expanding microtubule networks that are nucleated from centrosomes positioned at the cortex, whereas cortical actin meshwork remains spatially restricted. Using the gnu RNAi embryo to decouple nuclear migration and division from cytoskeletal dynamics, we establish causality with targeted pharmacological disruption and find that microtubule dynamics is required for yolk segregation, while depolymerization of actin has no discernible effect. In support of a mechanism of growth-propelled passive displacement, microtubule plus end comets come in apparent contact with yolk vesicles, and injected, inert microbeads are displaced towards the embryo center presumably by the same pushing force. These findings identify microtubule polymerization as a predominant driver of yolk-cytoplasm segregation in Drosophila and suggest that diverse cytoskeletal mechanisms evolved to accomplish this crucial reorganization process

developmental biology↗