Genetic and Mechanical Regulation of Intestinal Smooth Muscle Development.

The gastrointestinal tract is enveloped by concentric and orthogonally aligned layers of smooth muscle; however, an understanding of the mechanisms by which these muscles become patterned and aligned in the embryo has been lacking. We find that Hedgehog acts through Bmp to delineate the position of the circumferentially oriented inner muscle layer, whereas localized Bmp inhibition is critical for allowing formation of the later-forming, longitudinally oriented outer layer. Because the layers form at different developmental stages, the muscle cells are exposed to unique mechanical stimuli that direct their alignments. Differential growth within the early gut tube generates residual strains that orient the first layer circumferentially, and when formed, the spontaneous contractions of this layer align the second layer longitudinally. Our data link morphogen-based patterning to mechanically controlled smooth muscle cell alignment and provide a mechanistic context for potentially understanding smooth muscle organization in a wide variety of tubular organs.

The developmental mechanics of divergent buckling patterns in the chick gut.

Tissue buckling is an increasingly appreciated mode of morphogenesis in the embryo, but it is often unclear how geometric and material parameters are molecularly determined in native developmental contexts to generate diverse functional patterns. Here, we study the link between differential mechanical properties and the morphogenesis of distinct anteroposterior compartments in the intestinal tract-the esophagus, small intestine, and large intestine. These regions originate from a simple, common tube but adopt unique forms. Using measured data from the developing chick gut coupled with a minimal theory and simulations of differential growth, we investigate divergent lumen morphologies along the entire early gut and demonstrate that spatiotemporal geometries, moduli, and growth rates control the segment-specific patterns of mucosal buckling. Primary buckling into wrinkles, folds, and creases along the gut, as well as secondary buckling phenomena, including period-doubling in the foregut and multiscale creasing-wrinkling in the hindgut, are captured and well explained by mechanical models. This study advances our existing knowledge of how identity leads to form in these regions, laying the foundation for future work uncovering the relationship between molecules and mechanics in gut morphological regionalization.

Characterization of the development of the high-acuity area of the chick retina.

The fovea is a small region within the central retina that is responsible for our high acuity daylight vision. Chickens also have a high acuity area (HAA), and are one of the few species that enables studies of the mechanisms of HAA development, due to accessible embryonic tissue and methods to readily perturb gene expression. To enable such studies, we characterized the development of the chick HAA using single molecule fluorescent in situ hybridization (smFISH), along with more classical methods. We found that Fgf8 provides a molecular marker for the HAA throughout development and into adult stages, allowing studies of the cellular composition of this area over time. The radial dimension of the ganglion cell layer (GCL) was seen to be the greatest at the HAA throughout development, beginning during the period of neurogenesis, suggesting that genesis, rather than cell death, creates a higher level of retinal ganglion cells (RGCs) in this area. In contrast, the HAA acquired its characteristic high density of cone photoreceptors post-hatching, which is well after the period of neurogenesis. We also confirmed that rod photoreceptors are not present in the HAA. Analyses of cell death in the developing photoreceptor layer, where rods would reside, did not show apoptotic cells, suggesting that lack of genesis, rather than death, created the "rod-free zone" (RFZ). Quantification of each cone photoreceptor subtype showed an ordered mosaic of most cone subtypes. The changes in cellular densities and cell subtypes between the developing and mature HAA provide some answers to the overarching strategy used by the retina to create this area and provide a framework for future studies of the mechanisms underlying its formation.

Integrity of Narrow Epithelial Tubes in the C. elegans Excretory System Requires a Transient Luminal Matrix.

Most epithelial cells secrete a glycoprotein-rich apical extracellular matrix that can have diverse but still poorly understood roles in development and physiology. Zona Pellucida (ZP) domain glycoproteins are common constituents of these matrices, and their loss in humans is associated with a number of diseases. Understanding of the functions, organization and regulation of apical matrices has been hampered by difficulties in imaging them both in vivo and ex vivo. We identified the PAN-Apple, mucin and ZP domain glycoprotein LET-653 as an early and transient apical matrix component that shapes developing epithelia in C. elegans. LET-653 has modest effects on shaping of the vulva and epidermis, but is essential to prevent lumen fragmentation in the very narrow, unicellular excretory duct tube. We were able to image the transient LET-653 matrix by both live confocal imaging and transmission electron microscopy. Structure/function and fluorescence recovery after photobleaching studies revealed that LET-653 exists in two separate luminal matrix pools, a loose fibrillar matrix in the central core of the lumen, to which it binds dynamically via its PAN domains, and an apical-membrane-associated matrix, to which it binds stably via its ZP domain. The PAN domains are both necessary and sufficient to confer a cyclic pattern of duct lumen localization that precedes each molt, while the ZP domain is required for lumen integrity. Ectopic expression of full-length LET-653, but not the PAN domains alone, could expand lumen diameter in the developing gut tube, where LET-653 is not normally expressed. Together, these data support a model in which the PAN domains regulate the ability of the LET-653 ZP domain to interact with other factors at the apical membrane, and this ZP domain interaction promotes expansion and maintenance of lumen diameter. These data identify a transient apical matrix component present prior to cuticle secretion in C. elegans, demonstrate critical roles for this matrix component in supporting lumen integrity within narrow bore tubes such as those found in the mammalian microvasculature, and reveal functional importance of the evolutionarily conserved ZP domain in this tube protecting activity.