Transcriptional profiling is a powerful approach for understanding development and disease. Current cell type-specific RNA purification methods have limitations, including cell dissociation trauma or inability to identify all RNA species. Here, we describe ''mouse thiouracil (TU) tagging,'' a genetic and chemical intersectional method for covalent labeling and purification of cell type-specific RNA in vivo. Cre-induced expression of uracil phosphoribosyltransferase (UPRT) provides spatial specificity; injection of 4-thiouracil (4TU) provides temporal specificity. Only UPRT + cells exposed to 4TU produce thio-RNA, which is then purified for RNA sequencing (RNA-seq). This method can purify transcripts from spatially complex and rare (<5%) cells, such as Tie2:Cre + brain endothelia/microglia (76% validated by expression pattern), or temporally dynamic transcripts, such as those acutely induced by lipopolysaccharide (LPS) injection. Moreover, generating chimeric mice via UPRT + bone marrow transplants identifies immune versus niche spleen RNA. TU tagging provides a novel method for identifying actively transcribed genes in specific cells at specific times within intact mice.

Transmission electron microscopy (TEM) is widely used as an imaging modality to provide high-resolution details of subcellular components within cells and tissues. Mitochondria and endoplasmic reticulum (ER) are organelles of particular interest to those investigating metabolic disorders. A straightforward method for quantifying and characterizing particular aspects of these organelles would be a useful tool. In this protocol, we outline how to accurately assess the morphology of these important subcellular structures using open source software ImageJ, originally developed by the National Institutes of Health (NIH). Specifically, we detail how to obtain mitochondrial length, width, area, and circularity, in addition to assessing cristae morphology and measuring mito/endoplasmic reticulum (ER) interactions. These procedures provide useful tools for quantifying and characterizing key features of sub-cellular morphology, leading to accurate and reproducible measurements and visualizations of mitochondria and ER.

High-resolution 3D images of organelles are of paramount importance in cellular biology. Although light microscopy and transmission electron microscopy (TEM) have provided the standard for imaging cellular structures, they cannot provide 3D images. However, recent technological advances such as serial block-face scanning electron microscopy (SBF-SEM) and focused ion beam scanning electron microscopy (FIB-SEM) provide the tools to create 3D images for the ultrastructural analysis of organelles. Here, we describe a standardized protocol using the visualization software, Amira, to quantify organelle morphologies in 3D, thereby providing accurate and reproducible measurements of these cellular substructures. We demonstrate applications of SBF-SEM and Amira to quantify mitochondria and endoplasmic reticulum (ER) structures.

We identify problematic areas throughout the Science, Technology, Engineering and Mathematics (STEM) pipeline that perpetuate racial disparities in academia. Distinct ways to curtail these disparities include early exposure and access to resources, supportive mentoring networks and comprehensive training programs specifically for racially minoritized students and trainees at each career stage. These actions will revitalize the STEM pipeline. ll

Genome-wide transcriptional changes in development provide important insight into mechanisms underlying growth, differentiation, and patterning. However, such large-scale developmental studies have been limited to a few representatives of Ecdysozoans and Chordates. Here, we characterize transcriptomes of embryonic, larval, and metamorphic development in the marine mollusc Aplysia californica and reveal novel molecular components associated with life history transitions. Specifically, we identify more than 20 signal peptides, putative hormones, and transcription factors in association with early development and metamorphic stages—many of which seem to be evolutionarily conserved elements of signal transduction pathways. We also characterize genes related to biomineralization—a critical process of molluscan development. In summary, our experiment provides the first large-scale survey of gene expression in mollusc development, and complements previous studies on the regulatory mechanisms underlying body plan patterning and the formation of larval and juvenile structures. This study serves as a resource for further functional annotation of transcripts and genes in Aplysia, specifically and molluscs in general. A comparison of the Aplysia developmental transcriptome with similar studies in the zebra fish Danio rerio, the fruit fly Drosophila melanogaster, the nematode Caenorhabditis elegans, and other studies on molluscs suggests an overall highly divergent pattern of gene regulatory mechanisms that are likely a consequence of the different developmental modes of these organisms.

Endometrial or uterine glands secrete substances essential for uterine receptivity to the embryo, implantation, conceptus survival, and growth. Adenogenesis is the process of gland formation within the stroma of the uterus. In the mouse, uterine gland formation initiates at postnatal day (P) 5. Uterine gland morphology is poorly understood because it is primarily based on two-dimensional (2D) histology. To more fully describe uterine gland morphogenesis, we generated three-dimensional (3D) models of postnatal uterine glands from P0 to P21, based on volumetric imaging using light sheet microscopy. At birth (P0), there were no glands. At P8, we found bud- and teardrop-shaped epithelial invaginations. By P11, the forming glands were elongated epithelial tubes. By P21, the elongated tubes had a sinuous morphology. These morphologies are homogeneously distributed along the anterior-posterior axis of the uterus. To facilitate uterine gland analyses, we propose a novel 3D staging system of uterine gland morphology during development in the prepubertal mouse. We define five uterine gland stages: Stage 1: bud; Stage 2: teardrop; Stage 3: elongated; Stage 4: sinuous; and Stage 5: primary branches. This staging system provides a standardized key to assess and quantify prepubertal uterine gland morphology that can be used for studies of uterine gland development and pathology. In addition, our studies suggest that gland formation initiation occurs during P8 and P11. However, between P11 and P21 gland formation initiation stops and all glands elongate and become sinuous. We also found that the mesometrial epithelium develops a unique morphology we term the uterine rail.

Mentoring is a developmental experience intended to increase the willingness to learn and establish credibility while building positive relationships through networking. In this commentary, we focus on intentional mentoring for underrepresented mentees, including individuals that belong to minority racial, ethnic, and gender identity groups in Science, Technology, Engineering, Mathematics, and Medicine (STEMM) fields. Intentional mentoring is the superpower action necessary for developing harmony and comprehending the purpose and value of the mentor/mentee relationship. Regardless of a mentor's career stage, we believe the strategies discussed may be used to create a supportive and constructive mentorship environment; thereby improving the retention rates of underrepresented mentees within the scientific community.

The Portuguese man of war, Physalia physalis, is one of the most conspicuous, but poorly understood members of the pleuston, a community of organisms that occupy a habitat at the sea-air interface. Physalia physalis is a siphonophore that uses a gas-filled float as a sail to catch the wind. The development, morphology, and colony organization of P. physalis is very different from all other siphonophores. Here, we look at live and fixed larval and juvenile specimens, and use optical projection tomography to build on existing knowledge about the morphology and development of this species. We also propose a framework for homologizing the axes with other siphonophores, and also suggest that the tentacle bearing zooids should be called tentacular palpons. Previous descriptions of P. physalis larvae, especially descriptions of budding order, were often framed with the mature colony in mind. However, we use the simpler organization of larvae and the juvenile specimens to inform our understanding of the morphology, budding order, and colony organization in the mature specimen. Finally, we review what is known about the ecology and lifecycle of P. physalis.