Biology subjects

Mihajlovic, A. I.

Publications and source records attributed to Mihajlovic, A. I..

4 recordsLinked to original sources

bioRxiv2025

TEAD4 regulates apical domain homeostasis and cell-positioning to maintain the trophectoderm lineage during preimplantation mouse embryo development.

In mammalian preimplantation embryos, different cell lineages occupy specific niches. For example, the outer trophectoderm (TE) comprises a monolayer of epithelialized cells surrounding the inner-cell mass (ICM) and blastocyst cavity. In mice, TEAD4 is known as a transcription factor that regulates TE-specific genes in a polarity-dependent manner during TE specification. Here we show that it also maintains blastocyst TE integrity, as knocking down (KD) Tead4 via clonal siRNA causes abnormal morphology of outer-cell apical domains, which correlates with the atypical contribution of Tead4-KD cell clones to an enlarged ICM throughout blastocyst maturation; with only minimal feedback on established apical polarity. Light-sheet live-cell embryo imaging reveals these cells either actively migrate into the ICM, sometimes involving apical domain abscission, or are positioned post-division, linking disrupted apical morphology to cell repositioning. RNA-Seq data indicate TEAD4 regulates genes related to the cytoskeleton, particularly actin, and cell adhesion, which we propose are required for the appropriate maintenance of the spatial positioning of specified TE cells in the blastocyst. Indeed, knocking down Tead4 in combination with two identified target genes, the atypical GTPases Rnd1 and Rnd3, partially rescues aberrant outer-to-inner cell allocations but does not influence the onset of apical domain morphological abnormalities. These findings indicate that Tead4 and its regulated transcriptome actively contribute to the maintenance of the outer TE lineage until the peri-implantation stage.

developmental biology↗

bioRxiv2023

OOCYTE AND EMBRYO CULTURE UNDER OIL PROFOUNDLY ALTERS EFFECTIVE CONCENTRATIONS OF SMALL MOLECULE INHIBITORS

Culture of oocytes and embryos in media under oil is a cornerstone of fertility treatment, and extensively employed in experimental investigation of early mammalian development. It has been noted anecdotally by some that certain small molecule inhibitors might lose activity in oil-covered culture systems, presumably by drug partitioning into the oil. Here we took a pseudo-pharmacological approach to appraise this formally. Using different culture dish designs with defined media:oil volume ratios, we show that the EC50 of the widely employed microtubule poison nocodazole shifts as a function of the media:oil ratio, such that nocodazole concentrations that prevent cell division in oil-free culture fail to in oil-covered media drops. Relatively subtle changes in culture dish design lead to measurable changes in EC50. This effect is not specific to one type of culture oil, and can be readily observed both in oocyte and embryo culture experiments. We subsequently applied a similar approach to a small panel of widely employed cell cycle-related inhibitors, finding that most lose activity in standard oil-covered oocyte/embryo culture systems. Our data suggest that loss of small molecule activity in oil-covered oocyte and embryo culture is a widespread phenomenon with potentially far-reaching implications for data reproducibility, and we recommend avoiding oil-covered culture for experiments employing inhibitors/drugs wherever possible.

developmental biology↗

bioRxiv2021

Distinct classes of lagging chromosome underpin age-related oocyte aneuploidy in mouse

Chromosome segregation errors that cause oocyte aneuploidy increase in frequency with maternal age and are considered a major contributing factor of age-related fertility decline in females. A common age-associated chromosome segregation phenomenon in oocytes is the lagging anaphase chromosome, but whether anaphase laggards actually missegregate and cause aneuploidy is unclear. Here we show unexpectedly that lagging chromosomes in mouse oocytes comprise two mechanistically distinct classes of motion that we refer to as Class-I and Class-II. We use imaging approaches and mechanistic interventions to dissociate the two classes, and find that whereas Class-II laggards are benign, Class-I laggards can directly cause aneuploidy. Most notably, a controlled prolongation of meiosis-I specifically lessens Class-I lagging to prevent aneuploidy. Our data thus reveal lagging chromosomes to be a cause of age-related aneuploidy in mouse oocytes and suggest that manipulating the cell cycle could increase the yield of useful oocytes in some contexts.

cell biology↗

bioRxiv2019

Human Oocytes Harbouring Damaged DNA Can Complete Meiosis-I

Chromosomal abnormalities such as aneuploidies and DNA damage are considered a major threat to the establishment of healthy eggs and embryos. Recent landmark studies showed that mouse oocytes with damaged DNA can resume meiosis and undergo Germinal Vesicle Breakdown (GVBD), but then arrest in metaphase of meiosis-I in a process involving Spindle Assembly Checkpoint (SAC) signalling. Such a mechanism could help prevent the generation of metaphase-II (Met-II) eggs with damaged DNA. However we report that this is not the case in the human oocyte. DNA damage prevents human oocytes from undergoing GVBD in some cases. Strikingly however, most oocytes harbouring DNA damage progress through meiosis-I and subsequently extrude the first polar body (PB1) to form a metaphase-II egg, revealing the absence of a DNA-damage-induced SAC response. Analysis of the resulting metaphase-II eggs revealed highly disorganised spindles with misaligned and heavily damaged chromosomes. Our results suggest that DNA damage accumulated in meiosis-I, such as could occur during in vitro maturation procedures, does not prevent polar body extrusion and therefore could persist in morphologically normal looking metaphase-II eggs.

developmental biology↗