Pak2 reduction induces a failure of early embryonic development in mice

Background The quality of the early embryo is vital to embryonic development and implantation. As a highly conserved serine/threonine kinase, p21-activated kinase 2 (Pak2) participates in diverse biologic processes, especially in cytoskeleton remodeling and cell apoptosis. In mice, Pak2 knock out and endothelial depletion of Pak2 showed embryonic lethality. However, the role of Pak2 in preimplantation embryos remains unelucidated. Methods In the present work, Pak2 was reduced using a specific small interfering RNA in early mouse embryos, validating the unique roles of Pak2 in spindle assembly and DNA repair during mice early embryonic development. We also employed immunoblotting, immunostaining, in vitro fertilization (IVF) and image quantification analyses to test the Pak2 knockdown on the embryonic development progression, spindle assembly, chromosome alignment, oxidative stress, DNA lesions and blastocyst cell apoptosis. Areas in chromatin with γH2AX were detected by immunofluorescence microscopy and serve as a biomarker of DNA damages. Results We found that Pak2 knockdown significantly reduced blastocyst formation of early embryos. In addition, Pak2 reduction led to dramatically increased abnormal spindle assembly and chromosomal aberrations in the embryos. We noted the overproduction of reactive oxygen species (ROS) with Pak2 knockdown in embryos. In response to DNA double strand breaks (DSBs), the histone protein H2AX is specifically phosphorylated at serine139 to generate γH2AX, which is used to quantitative DSBs. In this research, Pak2 knockdown also resulted in the accumulation of phosphorylated γH2AX, indicative of increased embryonic DNA damage. Commensurate with this, a significantly augmented rate of blastocyst cell apoptosis was detected in Pak2-KD embryos compared to their controls. Conclusions Collectively, our data suggest that Pak2 may serve as an important regulator of spindle assembly and DNA repair, and thus participate in the development of early mouse embryos.

Vangl2-environment interaction causes severe neural tube defects, without abnormal neuroepithelial convergent extension

Planar cell polarity (PCP) signalling is vital for initiation of mouse neurulation, with diminished convergent extension (CE) cell movements leading to craniorachischisis, a severe neural tube defect (NTD). Some humans with NTDs also have PCP gene mutations but these are heterozygous, not homozygous as in mice. Other genetic or environmental factors may interact with partial loss of PCP function in human NTDs. We found that reduced sulfation of glycosaminoglycans interacts with heterozygosity for the Lp allele of Vangl2 (a core PCP gene), to cause craniorachischisis in cultured mouse embryos, with rescue by exogenous sulphate. We hypothesised this glycosaminoglycan-PCP interaction may regulate CE but, surprisingly, DiO labeling of the embryonic node demonstrates no abnormality of midline axial extension in sulfation-depleted Lp/+ embryos. Positive-control Lp/Lp embryos show severe CE defects. Abnormalities were detected in the size and shape of somites that flank the closing neural tube in sulfation-depleted Lp/+ embryos. We conclude that failure of closure initiation can arise by a mechanism other than faulty neuroepithelial CE, with possible involvement of matrix-mediated somite expansion, adjacent to the closing neural tube.

Over 400 million years of cooperation: Untangling the chondrocranium-dermatocranium connection

The cranial endo- and dermal skeletons, which comprise the vertebrate skull, evolved independently and form separately during embryogenesis. In mammals, the mostly cartilaginous cranial endoskeleton forms prior to the bony dermatocranium. Many features of the chondrocranium are transient, undergoing endochondral ossification or disappearing, so its role in skull morphogenesis is not understood The fibroblast growth factor (FGF) and receptor (FGFR) signaling pathway contributes significantly to the regulation of osteochondroprogenitor cell function. Mutations in FGFR genes are associated with diseases that impact the skull including dwarfing chondrodyplasia and craniosynostosis syndromes. We investigate the developing chondrocranium and dermatocranium using a mouse model for craniosynostosis carrying a gain of function mutation in Fgfr2 to assess development of these cranial skeleton systems. Dermatocrania and chondrocrania of Fgfr2cC342Y/+ mice and their Fgfr2c+/+ littermates were quantified in 3D from microcomputed tomography images of mouse embryos. Chondrocrania of embryonic mice carrying the Fgfr2 mutation are larger than their Fgfr2c+/+ littermates and include novel extensions of cartilage over the lateral and dorsal aspect of the brain. Like the forming chondrocranium, the embryonic dermatocranium is larger in Fgfr2cC342Y/+ mice throughout embryogenesis but after disappearance of much of the chondrocranium, the dermatocranium becomes progressively smaller relative to Fgfr2c+/+ littermates during postnatal growth. Results reveal the direct effects of this Fgfr2c mutation on embryonic cranial cartilage, the impact of chondrocranial structure on developing dermatocranial elements, the importance of the chondrocranium in decoding the impact of specific genetic variants on head morphogenesis, and the potential for harnessing these effects as therapeutic targets.

A 37 kb region upstream of Brachyury comprising a notochord enhancer is essential for notochord and tail development

The node-streak border region comprising notochord progenitor cells (NPCs) at the posterior node and neuro-mesodermal progenitor cells (NMPs) in the adjacent epiblast is the prime organizing center for axial elongation in mouse embryos. The T-box transcription factor Brachyury (T) is essential for both, formation of the notochord and maintenance of NMPs, and thus is a key regulator of trunk and tail development. The T promoter controlling T expression in NMPs and nascent mesoderm has been characterized in detail. However, control elements for T expression in the notochord have not been identified yet. We have generated a series of deletion alleles by CRISPR/Cas9 genome editing in mESCs, and analyzed their effects in mutant mouse embryos. We identified a 37 kb region upstream of T essential for notochord function and tailbud outgrowth. Within that region we discovered a T binding enhancer required for notochord cell specification and differentiation. Our data reveal a complex regulatory landscape controlling cell type-specific expression and function of T in NMP/nascent mesoderm and node/notochord allowing proper trunk and tail development.

Artefacts in Volume Data Generated with High Resolution Episcopic Microscopy (HREM)

High resolution episcopic microscopy (HREM) produces digital volume data by physically sectioning histologically processed specimens, while capturing images of the subsequently exposed block faces. Our study aims to systematically define the spectrum of typical artefacts inherent to HREM data and to research their effect on the interpretation of the phenotype of wildtype and mutant mouse embryos. A total of 607 (198 wildtypes, 409 mutants) HREM data sets of mouse embryos harvested at embryonic day (E) 14.5 were systematically and comprehensively examined. The specimens had been processed according to essentially identical protocols. Each data set comprised 2000 to 4000 single digital images. Voxel dimensions were 3 × 3 × 3 µm3. Using 3D volume models and virtual resections, we identified a number of characteristic artefacts and grouped them according to their most likely causality. Furthermore, we highlight those that affect the interpretation of embryo data and provide examples for artefacts mimicking tissue defects and structural pathologies. Our results aid in optimizing specimen preparation and data generation, are vital for the correct interpretation of HREM data and allow distinguishing tissue defects and pathologies from harmless artificial alterations. In particular, they enable correct diagnosis of pathologies in mouse embryos serving as models for deciphering the mechanisms of developmental disorders.

Iron overload in endometriosis peritoneal fluid induces early embryo ferroptosis mediated by HMOX1

Endometriosis is one of the most common disorders that causes infertility in women. Iron is overloaded in endometriosis peritoneal fluid (PF), with harmful effects on early embryo development. However, the mechanism by which endometriosis peritoneal fluid affects embryonic development remains unclear. Hence, this study investigated the effect of iron overload on mouse embryos and elucidated the molecular mechanism. Iron overload in endometriosis PF disrupted blastocyst formation, decreased GPX4 expression and induced lipid peroxidation, suggesting that iron overload causes embryotoxicity and induces ferroptosis. Moreover, mitochondrial damage occurs in iron overload-treated embryos, presenting as decreased ATP levels, increased ROS levels and MMP hyperpolarization. The cytotoxicity of iron overload is attenuated by the ferroptosis inhibitor Fer-1. Furthermore, Smart-seq analysis revealed that HMOX1 is upregulated in embryo ferroptosis and that HMOX1 suppresses ferroptosis by maintaining mitochondrial function. This study provides new insight into the mechanism of endometriosis infertility and a potential target for future endometriosis infertility treatment efforts.