Mammary mechanobiology - investigating roles for mechanically activated ion channels in lactation and involution.

The ability of a mother to produce a nutritionally complete neonatal food source has provided a powerful evolutionary advantage to mammals. Milk production by mammary epithelial cells is adaptive, its release is exquisitely timed, and its own glandular stagnation with the permanent cessation of suckling triggers the cell death and tissue remodeling that enables female mammals to nurse successive progeny. Chemical and mechanical signals both play a role in this process. However, despite this duality of input, much remains unknown about the nature and function of mechanical forces in this organ. Here, we characterize the force landscape in the functionally mature gland and the capacity of luminal and basal cells to experience and exert force. We explore molecular instruments for force-sensing, in particular channel-mediated mechanotransduction, revealing increased expression of Piezo1 in mammary tissue in lactation and confirming functional expression in luminal cells. We also reveal, however, that lactation and involution proceed normally in mice with luminal-specific Piezo1 deletion. These findings support a multifaceted system of chemical and mechanical sensing in the mammary gland, and a protective redundancy that ensures continued lactational competence and offspring survival.

Glial Cell Expansion Coincides with Neural Circuit Formation in the Developing Auditory Brainstem.

Neural circuit formation involves maturation of neuronal, glial and vascular cells, as well as cell proliferation and cell death. A fundamental understanding of cellular mechanisms is enhanced by quantification of cell types during key events in synapse formation and pruning and possessing qualified genetic tools for cell type-specific manipulation. Acquiring this information in turn requires validated cell markers and genetic tools. We quantified changing proportions of neurons, astrocytes, oligodendrocytes, and microglia in the medial nucleus of the trapezoid body (MNTB) during neural circuit development. Cell type-specific markers, light microscopy and 3D virtual reality software, the latter developed in our laboratory, were used to count cells within distinct cell populations at postnatal days (P)3 and P6, bracketing the period of nerve terminal growth and pruning in this system. These data revealed a change from roughly equal numbers of neurons and glia at P3 to a 1.5:1 ratio of glia to neurons at P6. PCNA and PH3 labeling revealed that proliferation of oligodendrocytes contributed to the increase in glial cell number during this timeframe. We next evaluated Cre driver lines for selectivity in labeling cell populations. En1-Cre was specific for MNTB neurons. PDGFRα-Cre and Aldh1L1-Cre, thought to be mostly specific for oligodendrocyte lineage cells and astrocytes, respectively, both labeled significant numbers of neurons, oligodendrocytes, and astrocytes and are non-specific genetic tools in this neural system.

Rapid and Semi-automated Extraction of Neuronal Cell Bodies and Nuclei from Electron Microscopy Image Stacks.

Connectomics-the study of how neurons wire together in the brain-is at the forefront of modern neuroscience research. However, many connectomics studies are limited by the time and precision needed to correctly segment large volumes of electron microscopy (EM) image data. We present here a semi-automated segmentation pipeline using freely available software that can significantly decrease segmentation time for extracting both nuclei and cell bodies from EM image volumes.