Biology Open最新文献

Reproducing intestinal cells in vitro is important in pharmaceutical research and drug development. Caco-2 cells and human iPS cell-derived intestinal epithelial cells are widely used, but few evaluation systems can mimic the complex crypt-villus-like structure. We attempted to generate intestinal cells mimicking the three-dimensional structure from human iPS cells. After inducing the differentiation of iPS cells into intestinal organoids, these were dispersed into single cells and cultured two-dimensionally. An air-liquid interface culture was used, with CHIR99021, forskolin, and A-83-01 used as key compounds. Long-term culture was also performed by adding Wnt3a, Noggin, and RSPO1, which are frequently used in organoid culture. The air-liquid interface culture combined several compounds that successfully induced the formation of a crypt-villus-like structure, which grew rapidly at around day 6. The expression of pharmacokinetic genes such as CYP3A4 was also enhanced. The intestinal stem cells were efficiently maintained by the addition of Wnt3a, Noggin, and RSPO1. We were able to construct a crypt-villus-like structure on cell culture inserts, which is considered a very simple culture platform. This structure had characteristics extremely similar to living intestinal tissues and may have a superior homeostatic mechanism.

Coastal fish populations are threatened by multiple anthropogenic impacts, including the accumulation of industrial contaminants and the increasing frequency of hypoxia. Some populations of the Atlantic killifish (Fundulus heteroclitus), like those in New Bedford Harbor (NBH), Massachusetts, USA, have evolved a resistance to dioxin-like polychlorinated biphenyls (PCBs) that may influence their ability to cope with secondary stressors. To address this question, we compared hepatic gene expression and DNA methylation patterns in response to mild or severe hypoxia in killifish from NBH and Scorton Creek (SC), a reference population from a relatively pristine environment. We hypothesized that NBH fish would show altered responses to hypoxia due to trade-offs linked to toxicant resistance. Our results revealed substantial differences between populations. SC fish demonstrated dose-dependent changes in gene expression in response to hypoxia, while NBH fish exhibited a muted transcriptional response to severe hypoxia. Interestingly, NBH fish showed significant DNA methylation changes in response to hypoxia, while SC fish did not exhibit notable epigenetic alterations. These findings suggest that toxicant-adapted killifish may face trade-offs in their molecular response to environmental stress, potentially impacting their ability to survive severe hypoxia in coastal habitats. Further research is needed to elucidate the functional implications of these epigenetic modifications and their role in adaptive stress responses.

Cell fate decisions during cortical development sculpt the identity of long-range connections that subserve complex behaviors. These decisions are largely dictated by mutually exclusive transcription factors, including CTIP2/Bcl11b for subcerebral projection neurons and BRN1/Pou3f3 for intra-telencephalic projection neurons. We have recently reported that the balance of cortical CTIP2-expressing neurons is altered in a mouse model of DDX3X syndrome, a female-biased neurodevelopmental disorder associated with intellectual disability, autism spectrum disorder, and significant motor challenges. Here, we studied the developmental dynamics of a subpopulation of cortical neurons co-expressing CTIP2 and BRN1. We found that CTIP2+BRN1+ neurons are born during early phases of neurogenesis like other CTIP2+ neurons, peak in expression during perinatal life, and persist in adult brains. We also found that CTIP2+BRN1+ neurons are excessive in number in prenatal and mature cortical motor areas of Ddx3x mutant mice, translating into altered laminar distribution of subcerebral projection neurons extending axons to the brainstem. These findings underscore the critical role of molecular specification during cortical development in health and disease.

Epithelial cell cohesion and barrier function critically depend on α-catenin, an actin-binding protein and essential constituent of cadherin-catenin-based adherens junctions. α-catenin undergoes actomyosin force-dependent unfolding of both actin-binding and middle domains to strongly engage actin filaments and its various effectors; this mechanosensitivity is critical for adherens junction function. We previously showed that α-catenin is highly phosphorylated in an unstructured region that links the mechanosensitive middle and actin-binding domains (known as the P-linker region), but the cellular processes that promote α-catenin phosphorylation have remained elusive. Here, we leverage a previously published phospho-proteomic data set to show that the α-catenin P-linker region is maximally phosphorylated during mitosis. By reconstituting α-catenin CRISPR knockout MDCK cells with wild-type, phospho-mutant and phospho-mimic forms of α-catenin, we show that full phosphorylation restrains mitotic cell rounding in the apical direction, strengthening the interactions between dividing and non-dividing neighbors to limit epithelial barrier leak. As the major scaffold components of adherens junctions, tight junctions and desmosomes are also differentially phosphorylated during mitosis, we reason that epithelial cell division may be a tractable system to understand how junction complexes are coordinately regulated to sustain barrier function under tension-generating morphogenetic processes.

In the developing mouse ventral spinal cord, HES5, a transcription factor downstream of Notch signalling, is expressed as evenly spaced clusters of high HES5-expressing neural progenitor cells along the dorsoventral axis. While Notch signalling requires direct membrane contact for its activation, we have previously shown mathematically that contact needs to extend beyond neighbouring cells for the HES5 pattern to emerge. However, the presence of cellular structures that could enable such long-distance signalling was unclear. Here, we report that cellular protrusions are present all along the apicobasal axis of individual neural progenitor cells. Through live imaging, we show that these protrusions dynamically extend and retract reaching lengths of up to ∼20 µm, enough to extend membrane contact beyond adjacent cells. The Notch ligand DLL1 was found to colocalise with protrusions, further supporting the idea that Notch signalling can be transduced at a distance. The effect of protrusions on the HES5 pattern was tested by reducing the density of protrusions using the CDC42 inhibitor ML141, leading to a tendency to decrease the distance between high HES5 cell clusters. However, this tendency was not significant and leaves an open question about their role in the fine-grained organisation of neurogenesis.

The gut microbiome, which is composed of bacteria, viruses, and fungi, and is involved in multiple essential physiological processes, changes measurably as a person ages, and can be associated with negative health outcomes. Microbiome transplants have been proposed as a method to improve gut function and reduce or reverse multiple disorders, including age-related diseases. Here, we take advantage of the laboratory model organism, Drosophila melanogaster, to test the effects of transplanting the microbiome of a young fly into middle-aged flies, across multiple genetic backgrounds and both sexes, to test whether age-related lifespan could be increased, and late-life physical health declines mitigated. Our results suggest that, overall, microbiome transplants do not improve longevity and may even be detrimental in flies, and the health effects of microbiome transplants were minor, but sex- and genotype-dependent. This discovery supports previous evidence that axenic flies, those with no gut microbiome, live healthier and longer lives than their non-axenic counterparts. The results of this study suggest that, at least for fruit flies, microbiome transplants may not be a viable intervention to improve health and longevity, though more research is still warranted.

Chromosomal aneuploidies are a major cause of developmental failure and pregnancy loss. To investigate the possible consequences of aneuploidy on early embryonic development in vitro, we focused on primed pluripotent stem cells that are relatable to the epiblast of post-implantation embryos in vivo. We used human induced pluripotent stem cells (iPSCs) as an epiblast model and altered chromosome numbers by treating with reversine, a small-molecule inhibitor of monopolar spindle 1 kinase (MSP1) that inactivates the spindle assembly checkpoint, which has been strongly implicated in chromosome mis-segregation and aneuploidy generation. Upon reversine treatment, we obtained cells with varied chromosomal content that retained pluripotency and potential to differentiate into cells of three germ lineages. However, these cells displayed lagging chromosomes, increased micronuclei content, high p53 expression and excessive apoptotic activity. Cell proliferation was not affected. Prolonged in vitro culture of these cells resulted in a selective pool of cells with supernumerary chromosomes, which exhibited cellular hypertrophy, enlarged nuclei, and overproduction of total RNAs and proteins. We conclude that increased DNA damage responses, apoptosis, and improper cellular mass and functions are possible mechanisms that contribute to abnormal epiblast development.

The SUMO-targeted ubiquitin ligase (STUbL) family is involved in multiple cellular processes via a wide range of mechanisms to maintain genome stability. One of the evolutionarily conserved functions of STUbL is to promote changes in the nuclear positioning of DNA lesions, targeting them to the nuclear periphery. In Schizossacharomyces pombe, the STUbL Slx8 is a regulator of SUMOylated proteins and promotes replication stress tolerance by counteracting the toxicity of SUMO conjugates. In order to study the dynamic dialectic between ubiquitinylation and SUMOylation in the nuclear space of the S. pombe genome, we analyzed Slx8 localization. Unexpectedly, we did not detect replication stress-induced Slx8 foci. However, we discovered that Slx8 forms a single nuclear focus, enriched at the nuclear periphery, which marks both clustered centromeres at the spindle pole body and the silent mating-type region. The formation of this single Slx8 focus requires the E3 SUMO ligase Pli1, poly-SUMOylation and the histone methyl transferase Clr4 that is responsible for the heterochromatin histone mark H3-K9 methylation. Finally, we established that Slx8 promotes centromere clustering and gene silencing at heterochromatin domains. Altogether, our data highlight evolutionarily conserved and functional relationships between STUbL and heterochromatin domains to promote gene silencing and nuclear organization.