Tissue Geometry Determines Sites of Mammary Branching Morphogenesis in Organotypic Cultures

Abstract

The treelike structures of many organs, including the mammary gland, are generated by branching morphogenesis, a reiterative process of branch initiation and invasion from a preexisting epithelium. Using a micropatterning approach to control the initial three-dimensional structure of mouse mammary epithelial tubules in culture, combined with an algorithm to quantify the extent of branching, we found that the geometry of tubules dictates the position of branches. We predicted numerically and confirm experimentally that branches initiate at sites with a local minimum in the concentration of autocrine inhibitory morphogens, such as transforming growth factor-β. These results reveal that tissue geometry can control organ morphogenesis by defining the local cellular microenvironment, a finding that has relevance to control of invasion and metastasis.A burst of dichotomous and lateral branching at puberty transforms the mammary epithelial tubule rudiment present at birth into a fully elaborated ductal tree in the female adult. The overall process of branching morphogenesis is regulated globally by a number of cues, including growth factors, extracellular matrix (ECM) molecules, proteases, and morphogens (1-4). These global cues must be integrated locally within the context of the tissue to determine where branches are initiated; thus, a subgroup of epithelial cells is instructed to form a branch or to bifurcate, whereas neighboring cells are not (5). Current techniques to study this process, which is common to many organs including the lung, kidney, and salivary gland, do not allow for a precise quantitative understanding of how spatial positioning is determined. Given that the mammary ductal network branches out from preexisting epithelial tubules, we hypothesized that the position of cells within a tubule might provide contextual information to instruct branch site initiation.To define the role of positional context, we developed a three-dimensional (3D) micropatterned assay for mammary epithelial branching morphogenesis that allowed us to mimic the mammary rudiment by controlling the initial geometry of tubules and to quantify the positions at which they branched. We engineered epithelial tubules of defined geometry by embedding functionally normal mouse mammary epithelial (EpH4) cells in cavities of collagen gel generated by molding unpolymerized collagen I around a patterned elastomeric stamp (Fig. 1A) (6). Embedded epithelial cells formed hollow tubules (7) conforming to the size and shape of the collagen cavities (Fig. 1, B NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript its magnitude and position statistically, we stained nuclei and stacked multiple fluorescent images in registration such that the stacked image revealed the average spatial distribution of cells within, and branching from, the tubules (Fig. 1D). Stacked images were depicted as frequency maps ( Fig. 1E) (8).The tubules remained quiescent ( Fig. 1E) until induced to undergo branching morphogenesis by addition o...

A spectrophotometer-based diffusivity assay reveals that diffusion hindrance of small molecules in extracellular matrix gels used in 3D cultures is dominated by viscous effects

“…To assess such suitable timewindows, we used a simple model to predict t 50 values. Noteworthy, our predictions were consistent with experimental time-windows reported in 3D culture studies (Table 3) [5,18,43,44]. However, our predictions are likely to be an underestimation for soluble signaling M a n u s c r i p t 19 molecules, since many ECM components contain binding domains for signaling factors [7,8].…”

“…A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t [18] dense COLI n.a. HGF (~80 kDa) ≥ 2 day I 1 day (early branching) [44] dense FIB n.a. vEGF, bFGF, HGF (~20-80 kDa) ~ 3-5 day I 7 day (robust branching) [5] I values based on a 2 mm gel thickness in Fig.…”

Microstructured extracellular matrices in tissue engineering and development

“…1a). 28,30,57 Manual assembly of a flat ECM gel over the patterned one yielded cell-filled cavities that were completely surrounded by ECM. Similar approaches used thin needles as templates to mold ECM, thereby forming open or blindended channels in one step.…”

“…3a), new branches only arise from stereotyped locations defined by the initial tissue geometry. 30 This geometric signal is transmitted to the tissue in the form of concentration gradients of morphogens 30,35 as well as gradients of mechanical stress 11,12 which induce patterns of gene expression within the tissue. 22 It will be interesting to determine whether the signals induced by microstructured ECM are shared by the development of all branching organs.…”

Mechanotransduction and Growth Factor Signalling to Engineer Cellular Microenvironments

“…Multicellular geometry can also feedback to regulate patterns of cell growth, with regions of high traction stress favouring growth and branching morphogenesis (Figure 5c). [72,73] Cells on a surface can sense and react to radii of curvature much larger than a single cell and regions with high local concave (convex) curvature favour (inhibit) tissue growth (Figure 5d). [74,75] On the other end of the size scale, surface nano-and microtopography influence size and shape of cell-matrix adhesion points and thus also play an important role in cell mechanotransduction, as well as in stem cell self-renewal and multipotency.…”