Research: M. Larsen

Research Statement, Mindy Larsen

My lab is interested in deciphering the molecular mechanisms controlling branching morphogenesis, which is a process required for the development of many mammalian organs, including the lung, kidney, prostate, mammary glands, and salivary glands. Branching requires coordination of many cellular processes such as proliferation, cytoskeletal contraction, and basement membrane remodeling. These cellular processes result in cleft formation and bud outgrowth, which together comprise the process of branching morphogenesis (Figure 1). Understanding the control and coordination of these cellular processes may lead to a better understanding of how adult tissues can be repaired or regenerated.

Figure 1. Branching morphogenesis in the salivary gland and other organs requires coordination of multiple signaling pathways initiated by transmembrane signaling proteins, including integrins and growth factor receptors (GFR), which lead to the cellular processes of proliferation, cytoskeletal contraction, and basement membrane remodeling. These cellular processes result in cleft formation and bud outgrowth that together comprise branching morphogenesis. From Larsen, et al. 2006. Current Opinion in Cell Biology. 2006. 18:463-471.

We recently demonstrated that cell migration is involved in salivary gland branching morphogenesis. We use embryonic salivary glands as a model system for the study of branching morphogenesis. In embryonic salivary glands grown as organ cultures, we can monitor changes in temporal-spatial distribution of targets using time-lapse confocal microscopy. By tracking cells in embryonic day 13 (E13) salivary glands that were labeled with green fluorescent protein (GFP), we observed that early embryonic epithelial cells undergo substantial, rapid cell migration during the early stages of branching morphogenesis (Figure 2). This cell migration is independent of cell division and does not occur later in development when the epithelial cells have started to differentiate and become polarized. A current question under investigation is: how are cell movements controlled during branching morphogenesis? The Rho family of small GTPases regulates the actin cytoskeleton to induce changes in cell shape and in cell motility and are candidate molecules to serve this function. A current project is to investigate a role for Rho proteins in regulating actin rearrangements during branching morphogenesis.

Figure 2. Still frames from time-lapse image analysis. A. E13 salivary glands were labeled with green fluorescent protein (GFP, green) and Alexa-Fluor-647-fibronectin (pseudocolored red), and images were captured at multiple time-points, shown are images captured at 0:00 and 4:06 hrs. B. Tracking of individual cell movements through all frames of the time-lapse showed that cells are migrating during branching morphogenesis. An example cell track is displayed on a single XY frame (bottom right), a XZ projection (top) and a YZ projection (left). Bars, 50 µm. Modified from Larsen, et. al. 2006. Journal of Cell Science, 119: 3376-3384.

The extracellular matrix plays a critical function during branching morphogenesis. We previously found that the extracellular matrix protein, fibronectin, is required for branching morphogenesis (Figure 1). It is expressed in the cleft, a structure in the basement membrane where branching initiates. Knockdown of fibronectin mRNA with siRNAs or inhibition of protein function with inhibitory antibodies prevented morphogenesis. Pulse-chase labeling of exogenously-added, fluorescently labeled fibronectin (Alexa-Fluor-647-fibronectin), indicated that fibronectin is assembled inward into the gland, providing a driving force for formation of clefts and, thus, progression of branching morphogenesis. Current projects are focused on the upstream signal driving expression of fibronectin and on the downstream targets of fibronectin signaling through its integrin receptors.

We use cell, molecular, biochemical, and imaging techniques to address developmental questions. To profile gene expression during development in vivo and in 3D cell and organ culture models, we use the SAGE gene profiling technique as well as real-time PCR and in situ analysis. Protein expression is evaluated by Western analysis, and confocal imaging. We manipulate gene expression and protein function using small inhibitory RNAS (siRNAs), inhibitory antibodies, and pharmacological inhibitors. To study branching morphogenesis in real time, we image the 3D salivary gland organ culture system using both standard light microscopy and live time-lapse confocal microscopy.