Nanog, Pou5f1 and SoxB1 activate zygotic gene expression during the maternal-to-zygotic transition.

After fertilization, maternal factors direct development and trigger zygotic genome activation (ZGA) at the maternal-to-zygotic transition (MZT). In zebrafish, ZGA is required for gastrulation and clearance of maternal messenger RNAs, which is in part regulated by the conserved microRNA miR-430. However, the factors that activate the zygotic program in vertebrates are unknown. Here we show that Nanog, Pou5f1 (also called Oct4) and SoxB1 regulate zygotic gene activation in zebrafish. We identified several hundred genes directly activated by maternal factors, constituting the first wave of zygotic transcription. Ribosome profiling revealed that nanog, sox19b and pou5f1 are the most highly translated transcription factors pre-MZT. Combined loss of these factors resulted in developmental arrest before gastrulation and a failure to activate >75% of zygotic genes, including miR-430. Our results demonstrate that maternal Nanog, Pou5f1 and SoxB1 are required to initiate the zygotic developmental program and induce clearance of the maternal program by activating miR-430 expression.

Identification of small ORFs in vertebrates using ribosome footprinting and evolutionary conservation.

Identification of the coding elements in the genome is a fundamental step to understanding the building blocks of living systems. Short peptides (< 100 aa) have emerged as important regulators of development and physiology, but their identification has been limited by their size. We have leveraged the periodicity of ribosome movement on the mRNA to define actively translated ORFs by ribosome footprinting. This approach identifies several hundred translated small ORFs in zebrafish and human. Computational prediction of small ORFs from codon conservation patterns corroborates and extends these findings and identifies conserved sequences in zebrafish and human, suggesting functional peptide products (micropeptides). These results identify micropeptide-encoding genes in vertebrates, providing an entry point to define their function in vivo.

Skin Microbiome Variation with Cancer Progression in Human Cutaneous Squamous Cell Carcinoma.

The skin microbiome plays a critical role in skin homeostasis and disorders. UVR is the major cause of nonmelanoma skin cancer, but other risk factors, including immune suppression, chronic inflammation, and antibiotic usage, suggest the microbiome as an additional, unexplored risk factor and potential disease biomarker. The overarching goal was to study the skin microbiome in squamous cell carcinoma (SCC) and premalignant actinic keratosis compared with that in healthy skin to identify skin cancer‒associated changes in the skin microbiome. We performed a high-resolution analysis of shotgun metagenomes of actinic keratosis and SCC in healthy skin, revealing the microbial community shifts specific to actinic keratosis and SCC. Most prominently, the relative abundance of pathobiont Staphylococcus aureus was increased at the expense of commensal Cutibacterium acnes in SCC compared with that in healthy skin, and enrichment of functional pathways in SCC reflected this shift. Notably, C. acnes associated with lesional versus healthy skin differed at the strain level, suggesting the specific functional changes associated with its depletion in SCC. Our study revealed a transitional microbial dysbiosis from healthy skin to actinic keratosis to SCC, supporting further investigation of the skin microbiome for use as a biomarker and providing hypotheses for studies investigating how these microbes might influence skin cancer progression.

Spindle misorientation in tumors from APC(min/+) mice.

The adenomatous polyposis coli (APC) tumor suppressor gene is mutated in the majority of colon cancers, and its mutation may initiate cancer by multiple mechanisms. Recently, abnormal mitotic spindle orientation was shown in normal-appearing tissues from mice heterozygous for APC mutation. To determine the effect of APC mutation on spindle orientation in tumors, and to better understand its mechanism, we measured mitotic spindle orientation in intestinal tumors from APC mutant mice, with three-dimensionally reconstructed confocal stacks of microtubule immunofluorescence images. We found spindle angles were increased in crypts heterozygous for the APC(min) mutation, and further increased in tumors. Astral microtubules of these spindles were clearly evident, suggesting astral microtubule loss is not a major mechanism of spindle misorientation in intestinal cells lacking wild-type APC. beta-Catenin staining was markedly abnormal in crypts and tumors from the mutant mice, suggesting a possible role in spindle orientation. Spindle angles in colon tumors with wild-type APC were equivalent to those in wild-type mice, showing that spindle misorientation is specific to APC mutation and not a general feature of tumors. We suggest spindle misorientation may contribute to the loss of normal tissue organization during tumor formation and could offer new insights into early carcinogenic events.

Planar spindle orientation and asymmetric cytokinesis in the mouse small intestine.

A major feature of epithelial cell polarity is regulated positioning of the mitotic spindle within the cell. Spindles in cells of symmetrically expanding tissues are predicted to align parallel to the tissue plane. Direct measurement of this alignment has been difficult in mammalian tissues. Here, we analyzed the position of spindles in intact mouse intestinal epithelium using microtubule immunofluorescence and three-dimensional confocal imaging. Mitotic cells were identified in the proliferative zone of intestinal crypts. Spindle angle relative to the apical cell surface was determined either by direct measurement from confocal images or with a computational algorithm. Angles averaged within 10 degrees of parallel to the apical surface in metaphase and anaphase cells, consistent with robust planar spindle positioning, whereas spindles in prometaphase cells showed much greater angle variability. Interestingly, cytokinetic furrows appeared to extend from the basal cell surface toward the apical surface. This type of image analysis may be useful for studying the regulation of spindle position during tissue remodeling and tumor formation.