Two-step nuclear centring by competing microtubule- and actin-based mechanisms in 2-cell mouse embryos.

Microtubules typically promote nuclear centring during early embryonic divisions in centrosome-containing vertebrates. In acentrosomal mouse zygotes, microtubules also centre male and female pronuclei prior to the first mitosis, this time in concert with actin. How nuclear centring is brought about in subsequent acentrosomal embryonic divisions has not been studied. Here, using time-lapse imaging in mouse embryos, we find that although nuclei are delivered to the cell centre upon completion of the first mitotic anaphase, the majority do not remain stationary and instead travel all the way to the cortex in a microtubule-dependent manner. High cytoplasmic viscosity in 2-cell embryos is associated with non-diffusive mechanisms involving actin for subsequent nuclear centring when microtubules again exert a negative influence. Thus, following the first mitotic division, pro-centring actin-dependent mechanisms work against microtubule-dependent de-centring forces. Disrupting the equilibrium of this tug-of-war compromises nuclear centring and symmetry of the subsequent division potentially risking embryonic development. This circuitous centring process exposes an embryonic vulnerability imposed by microtubule-dependent de-centring forces.

A surge in cytoplasmic viscosity triggers nuclear remodeling required for Dux silencing and pre-implantation embryo development.

Embryonic genome activation (EGA) marks the transition from dependence on maternal transcripts to an embryonic transcriptional program. The precise temporal regulation of gene expression, specifically the silencing of the Dux/murine endogenous retrovirus type L (MERVL) program during late 2-cell interphase, is crucial for developmental progression in mouse embryos. How this finely tuned regulation is achieved within this specific window is poorly understood. Here, using particle-tracking microrheology throughout the mouse oocyte-to-embryo transition, we identify a surge in cytoplasmic viscosity specific to late 2-cell interphase brought about by high microtubule and endomembrane density. Importantly, preventing the rise in 2-cell viscosity severely impairs nuclear reorganization, resulting in a persistently open chromatin configuration and failure to silence Dux/MERVL. This, in turn, derails embryo development beyond the 2- and 4-cell stages. Our findings reveal a mechanical role of the cytoplasm in regulating Dux/MERVL repression via nuclear remodeling during a temporally confined period in late 2-cell interphase.

Isolation and in vitro Culture of Mouse Oocytes.

Females are endowed at birth with a fixed reserve of oocytes, which declines both in quantity and quality with advancing age. Understanding the molecular mechanisms regulating oocyte quality is crucial for improving the chances of pregnancy success in fertility clinics. In vitro culture systems enable researchers to analyse important molecular and genetic regulators of oocyte maturation and fertilisation. Here, we describe in detail a highly reproducible technique for the isolation and culture of fully grown mouse oocytes. We include the considerations and precautionary measures required for minimising the detrimental effects of in vitro culture conditions. This technique forms the starting point for a wide range of experimental approaches such as post-transcriptional gene silencing, immunocytochemistry, Western blotting, high-resolution 4D time-lapse imaging, and in vitro fertilization, which are instrumental in dissecting the molecular determinants of oocyte quality. Hence, this protocol serves as a useful, practical guide for any oocyte researcher beginning experiments aimed at investigating important oocyte molecular factors. Graphic abstract: A step-by-step protocol for the isolation and in vitro culture of oocytes from mice.

DRAXIN regulates interhemispheric fissure remodelling to influence the extent of corpus callosum formation.

Corpus callosum dysgenesis (CCD) is a congenital disorder that incorporates either partial or complete absence of the largest cerebral commissure. Remodelling of the interhemispheric fissure (IHF) provides a substrate for callosal axons to cross between hemispheres, and its failure is the main cause of complete CCD. However, it is unclear whether defects in this process could give rise to the heterogeneity of expressivity and phenotypes seen in human cases of CCD. We identify incomplete IHF remodelling as the key structural correlate for the range of callosal abnormalities in inbred and outcrossed BTBR mouse strains, as well as in humans with partial CCD. We identify an eight base-pair deletion in Draxin and misregulated astroglial and leptomeningeal proliferation as genetic and cellular factors for variable IHF remodelling and CCD in BTBR strains. These findings support a model where genetic events determine corpus callosum structure by influencing leptomeningeal-astroglial interactions at the IHF.

DCC regulates astroglial development essential for telencephalic morphogenesis and corpus callosum formation.

The forebrain hemispheres are predominantly separated during embryogenesis by the interhemispheric fissure (IHF). Radial astroglia remodel the IHF to form a continuous substrate between the hemispheres for midline crossing of the corpus callosum (CC) and hippocampal commissure (HC). Deleted in colorectal carcinoma (DCC) and netrin 1 (NTN1) are molecules that have an evolutionarily conserved function in commissural axon guidance. The CC and HC are absent in Dcc and Ntn1 knockout mice, while other commissures are only partially affected, suggesting an additional aetiology in forebrain commissure formation. Here, we find that these molecules play a critical role in regulating astroglial development and IHF remodelling during CC and HC formation. Human subjects with DCC mutations display disrupted IHF remodelling associated with CC and HC malformations. Thus, axon guidance molecules such as DCC and NTN1 first regulate the formation of a midline substrate for dorsal commissures prior to their role in regulating axonal growth and guidance across it.

Gene regulation could be attributed to TCF3 and other key transcription factors in the muscle of pubertal heifers.

Puberty is a whole-body event, driven by the hypothalamic integration of peripheral signals such as leptin or IGF-1. In the process of puberty, reproductive development is simultaneous to growth, including muscle growth. To enhance our understanding of muscle function related to puberty, we performed transcriptome analyses of muscle samples from six pre- and six post-pubertal Brahman heifers (Bos indicus). Our aims were to perform differential expression analyses and co-expression analyses to derive a regulatory gene network associate with puberty. As a result, we identified 431 differentially expressed (DEx) transcripts (genes and non-coding RNAs) when comparing pre- to post-pubertal average gene expression. The DEx transcripts were compared with all expressed transcripts in our samples (over 14,000 transcripts) for functional enrichment analyses. The DEx transcripts were associated with "extracellular region," "inflammatory response" and "hormone activity" (adjusted p < .05). Inflammatory response for muscle regeneration is a necessary aspect of muscle growth, which is accelerated during puberty. The term "hormone activity" may signal genes that respond to progesterone signalling in the muscle, as the presence of this hormone is an important difference between pre- and post-pubertal heifers in our experimental design. The DEx transcript with the highest average expression difference was a mitochondrial gene, ENSBTAG00000043574 that might be another important link between energy metabolism and puberty. In the derived co-expression gene network, we identified six hub genes: CDC5L, MYC, TCF3, RUNX2, ATF2 and CREB1. In the same network, 48 key regulators of DEx transcripts were identified, using a regulatory impact factor metric. The hub gene TCF3 was also a key regulator. The majority of the key regulators (22 genes) are members of the zinc finger family, which has been implicated in bovine puberty in other tissues. In conclusion, we described how puberty may affect muscle gene expression in cattle.

Combined allelic dosage of Nfia and Nfib regulates cortical development.

BACKGROUND: Nuclear factor I family members nuclear factor I A and nuclear factor I B play important roles during cerebral cortical development. Nuclear factor I A and nuclear factor I B regulate similar biological processes, as their expression patterns, regulation of target genes and individual knockout phenotypes overlap. We hypothesised that the combined allelic loss of Nfia and Nfib would culminate in more severe defects in the cerebral cortex than loss of a single member. METHODS: We combined immunofluorescence, co-immunoprecipitation, gene expression analysis and immunohistochemistry on knockout mouse models to investigate whether nuclear factor I A and nuclear factor I B function similarly and whether increasing allelic loss of Nfia and Nfib caused a more severe phenotype. RESULTS: We determined that the biological functions of nuclear factor I A and nuclear factor I B overlap during early cortical development. These proteins are co-expressed and can form heterodimers in vivo. Differentially regulated genes that are shared between Nfia and Nfib knockout mice are highly enriched for nuclear factor I binding sites in their promoters and are associated with neurodevelopment. We found that compound heterozygous deletion of both genes resulted in a cortical phenotype similar to that of single homozygous Nfia or Nfib knockout embryos. This was characterised by retention of the interhemispheric fissure, dysgenesis of the corpus callosum and a malformed dentate gyrus. Double homozygous knockout of Nfia and Nfib resulted in a more severe phenotype, with increased ventricular enlargement and decreased numbers of differentiated glia and neurons. CONCLUSION: In the developing cerebral cortex, nuclear factor I A and nuclear factor I B share similar biological functions and function additively, as the combined allelic loss of these genes directly correlates with the severity of the developmental brain phenotype.