Biology subjects

Drutovic, D.

Publications and source records attributed to Drutovic, D..

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↗

bioRxiv2024

Spatio-temporal requirements of Aurora kinase A in mouse oocytes meiotic spindle building

Meiotic spindles are critical to ensure proper chromosome segregation during gamete formation. Oocytes lack centrosomes and use alternative microtubule nucleation pathways for spindle building. However, how these mechanisms are regulated is still unknown. Aurora kinase A (AURKA) is necessary and sufficient for oocyte meiosis in mouse because Aurka KO oocytes arrest in meiosis I [1] and AURKA compensates for loss of Aurkb/Aurkc [2]. AURKA is required early in pro-metaphase I to trigger microtubule organizing center fragmentation, a step necessary to effectively build a bipolar spindle. Moreover, in double Aurkb/Aurkc knockouts, AURKA localizes to spindles and chromatin to support meiosis. Although these mouse models were useful for foundational studies, we were unable to resolve AURKA spatial and temporal functions. Here we provide high-resolution analyses of AURKA requirements during multiple steps of meiotic spindle building and identify the subcellular populations that carry out these functions. By combining mouse genetics and pharmacological approaches we show that AURKA is specifically required in early spindle building and later for spindle stability, whereas AURKC is specifically required in late pro-metaphase. Through expression of targeted AURKA constructs expressed in triple Aurora kinase knockout oocytes and high-resolution live imaging, we demonstrate that the spindle pole population of AURKA is the predominate pool that controls meiotic spindle building and stability.

cell biology↗

bioRxiv2022

CHK1-CDC25A-CDK1 regulate cell cycle progression in early mouse embryos to protect genome integrity

After fertilization, remodeling of the oocyte and sperm genomes is essential to convert these highly differentiated non-dividing transcriptionally quiescent cells into early cleavage-stage transcriptionally active totipotent blastomeres. This developmental transition is accompanied by cell cycle adaptation such as lengthening or shortening of the gap phases G1 and G2. However, regulation of these cell cycle changes is poorly understood, especially in mammals. Checkpoint kinase 1 (CHK1) is a protein kinase that regulates cell cycle progression in somatic cells. Here, we show that CHK1 regulates cell cycle progression in early mouse embryos by restraining CDK1 kinase activity due to CDC25A phosphatase degradation. CHK1 kinase also ensures the long G2 phase needed for genome activation and reprogramming gene expression in 2-cell stage mouse embryos. Last, Chk1 depletion leads to DNA damage and chromosome segregation errors that result in aneuploidy and infertility.

developmental biology↗

bioRxiv2021

Aurora kinase A is essential for meiosis in mouse oocytes

The Aurora protein kinases are well-established regulators of spindle building and chromosome segregation in mitotic and meiotic cells. In mouse oocytes, there is significant Aurora kinase A (AURKA) compensatory abilities when the other Aurora kinase homologs are deleted. Whether the other homologs, AURKB or AURKC can compensate for loss of AURKA is not known. Using a conditional mouse oocyte knockout model, we demonstrate that this compensation is not reciprocal because female oocyte-specific knockout mice are sterile and their oocytes fail to complete meiosis I. In determining the AURKA-specific functions, we demonstrate that its first meiotic requirement is to activate Polo-like kinase 1 at microtubule organizing centers (MTOCs; meiotic spindle poles). This activation induces fragmentation of the MTOCs, a step essential for building a bipolar spindle. The next step that requires AURKA is building the liquid-like spindle domain that involves TACC3. Finally, we find that AURKA is also required for anaphase I onset to trigger cohesin cleavage in an APC/C independent manner. We conclude that AURKA has multiple functions essential to completing MI that are distinct from AURKB and AURKC. Author SummaryFemale gametes, oocytes, are uniquely prone to chromosome segregation errors in meiosis I that are associated with early miscarriages. The Aurora protein kinases are essential to control chromosome segregation in all cell types. During mitosis, Aurora kinase A (AURKA) regulates the building of the spindle, the machinery responsible for pulling chromosomes apart. Here, we use a genetic approach to demonstrate that AURKA is essential for meiosis I in mouse oocytes. AURKA is required at multiple steps in meiosis I, first to trigger fragmentation of protein structures that make up the two ends of the meiotic spindle, later to regulate building of a specialized phase-separated spindle domain, and finally to trigger efficient cleavage of cohesin, the molecular glue that holds chromosomes together until anaphase onset. These findings are the first demonstration of distinct Aurora kinase function that cannot be compensated for by the other two homologs. Therefore, this mouse model is excellent tool for pinpointing specific Aurora kinase functions and identifying AURKA target proteins critical for chromosome segregation in meiosis I.

cell biology↗