CentTracker: a trainable, machine-learning–based tool for large-scale analyses of Caenorhabditis elegans germline stem cell mitosis

Abstract

Investigating the complex interactions between stem cells and their native environment requires an efficient means to image them in situ. Caenorhabditis elegans germline stem cells (GSCs) are distinctly accessible for intravital imaging; however, long-term image acquisition and analysis of dividing GSCs can be technically challenging. Here we present a systematic investigation into the technical factors impacting GSC physiology during live imaging and provide an optimized method for monitoring GSC mitosis under minimally disruptive conditions. We describe CentTracker, an automated and generalizable image analysis tool that uses machine learning to pair mitotic centrosomes and which can extract a variety of mitotic parameters rapidly from large-scale datasets. We employ CentTracker to assess a range of mitotic features in a large GSC data set. We observe spatial clustering of mitoses within the germline tissue, but no evidence that subpopulations with distinct mitotic profiles exist within the stem cell pool. We further find biases in GSC spindle orientation relative to the germline's distal-proximal axis, and thus the niche. The technical and analytical tools provided herein pave the way for large-scale screening studies of multiple mitotic processes in GSCs dividing in situ, in an intact tissue, in a living animal, under seemingly physiological conditions.

Non-autonomous insulin signaling delays mitotic progression inC. elegansgermline stem and progenitor cells

“…In addition to providing a measure of mitotic duration, spindle pole tracking allows us to extract a range of mitotic features related to spindle assembly and/or chromosome alignment and segregation ( S2A Fig ; (20)). When we compared mitotic features across all genotypes, we did not observe any obvious trends that might explain changes in the duration of mitosis ( S2B Fig ).…”

“…GSPC mitosis can be observed directly by live-cell imaging in whole-mount animals (18–20), making them an excellent model system for studying stem and progenitor cells mitosis in vivo . We have shown previously that caloric restriction leads to checkpoint-dependent mitotic delays in GSPCs and may perturb spindle assembly (18), supporting the idea that mitosis in vivo is influenced by the physiological status of the whole animal.…”

The initial expansion of the C. elegans syncytial germ line is coupled to incomplete primordial germ cell cytokinesis

“…To gain further insight into syncytial expansion during germ cell division, we employed live confocal imaging of animals coexpressing FP-tagged ANI-1 and a membrane marker to monitor actomyosin dynamics during PGC division. Because live imaging of PGCs requires that animals are removed from food, a condition that causes germ cells to rapidly exit mitotic proliferation (Zellag et al, 2021), we tracked PGC cytokinesis in cells that had already entered mitosis and formed a metaphase plate (Fig. 3C; Movie 4).…”

Uncoupling cell division and cytokinesis during germline development in metazoans

“…However, not all syncytial germ lines exhibit strict synchronous development. For example, in the syncytial C. elegans adult gonad, mitotic divisions are only loosely clustered within the pool of mitotic germ cells, and adjacent mitotic cells divide asynchronously ( Maciejowski et al, 2006 ; Gerhold et al, 2015 ; Zellag et al, 2021 ). Similarly, the border between the mitotic and meiotic regions of the adult C. elegans gonad is not sharp and mitotic and meiotic cells are interspersed ( Hansen et al, 2004 ; Crittenden et al, 2006 ).…”