Research Summary
Lee Niswander is interested in the genetic, cellular, and molecular processes that regulate development of the vertebrate embryonic neural tube, limb, and lung.
Our research is focused on the genetic and cellular mechanisms that control embryonic development, particularly the processes required for closure of the neural tube and for development of the vertebrate limb and lung. Our approaches include a forward genetic screen in mice to identify mutations that affect embryonic development, live imaging of the developing neural tube and lung, and identification of the key kinases and phosphatases that control lung branching morphogenesis.
ENU Mutagenesis Screen for Genes Required for Mouse Development
A forward genetic screen is an ideal and unbiased approach for identifying key genes that regulate specific developmental processes. Using an ENU (ethylnitrosourea) mutagenesis screen in mice, we have identified a number of recessive mutations that disrupt specific aspects of embryonic development, particularly closure of the neural tube and development of the limb and lung.
Movie 1: Time-lapse video microscopy of embryonic mouse lung explant cultures provides mechanistic insights into the dynamics of lung branching morphogenesis. Embryonic day 11 lung expressing GFP (green fluorescent protein) in the epithelium driven by the surfactant-C promoter was cultured on transwell filter support for 24 hours. Images were taken every 10 minutes on a Zeiss LSM 510 confocal microscope. The movie suggests a period of bud enlargement, then bud flattening, followed by bifurcation in which epithelial cells in the middle remain in place and cells on the two sides of the initial bud undergo outgrowth to form two new buds. Scale bar, 2 mm.
Culture and images produced by Carsten Schnatwinkel. Mouse line courtesy of Jeffrey Whitsett and Brigid Hogan.
Neural Development
The neural tube is the embryonic precursor of the central nervous system. Closure of the neural tube is a complex event requiring the coordination of many genes that control a variety of developmental processes, including pattern formation, proliferation, cell interactions, and morphogenetic processes to roll the flat neural ectoderm into a closed tube. Failure to close the neural tube in the brain or spinal cord leads to exencephaly or spina bifida, respectively. Although neural tube defects (NTDs) are the second most common human birth defect, the underlying genetic basis of NTDs in humans is poorly understood.
Genetics of neural tube closure. To advance an understanding of the causes of birth defects of the brain and spinal cord in humans, we are using the mouse as a model to identify the genes involved in closure of the neural tube, followed by a comprehensive cellular and molecular characterization of their mechanism of action. From the ENU mutagenesis screen we have identified a large number of mouse lines with NTDs. Our approach is to identify the genes by genetic mapping and to characterize the phenotypes to elucidate the developmental processes that are affected. Moreover, we are using these mouse models to understand the mechanism behind the suppression of NTDs by folic acid and to determine whether other dietary supplementations are capable of rescuing the folic acidresistant NTDs.
Imaging of neural tube closure. Neurulation is a dynamic process in which the flat neural plate rolls up into a tube and separates from the overlying ectoderm. Neurulation has largely been studied in static images of fixed tissue, which does not reveal the dynamics and complexity of the cell behaviors. We are undertaking real-time imaging of the embryonic neural tube to define the cellular events that take place during neural closure. Knowledge of these events is then used to identify the cellular basis for the defects observed in the mouse mutants derived from the ENU mutagenesis screen.
Movie 2: Time-lapse video microscopy of embryonic mouse lung explant cultures. Embryonic day 11 lung expressing GFP under the b-actin promoter (created by Andras Nagy) highlights highly motile cells in the epithelium and mesenchyme. The lung was cultured for 24 hours. Images were taken every 4 minutes on a Zeiss LSM 510 confocal microscope. Bright-field, GFP fluorescence, and merged images are shown. The cell rearrangements that occur in both the epithelium and mesenchyme during branching morphogenesis are highly dynamic. Note the mesenchymal cells, which move around the epithelium and contact the neighboring epithelial branch. Those cells appear to (a) apply forces to support the bifurcation event and (b) communicate with the neighboring epithelial branch.
Culture and images produced by Carsten Schnatwinkel.
Limb Development
Key signals that control limb growth and patterning have been identified in the past 10 years, yet surprisingly little is known about how these regulators function, what their upstream activators and downstream targets are, or how patterning information is translated into skeletal elements of appropriate size and shape.
From the mutagenesis screen we have identified limb mutants that cause polydactyly (extra digits), soft tissue syndactyly (the webbing between the digits does not undergo regression), and other skeletal defects, including shortening or loss of specific limb elements and aberrant cartilage formation. We are also exploring genetic interactions between various mutant lines to determine whether they regulate similar functions or act within a genetic pathway. Moreover, we are performing real-time imaging of limb mesenchyme as it undergoes cartilage formation to define the cell behaviors involved as these undifferentiated cells undergo the early stages of chondrocyte differentiation. Limb mesenchyme from mutants with an abnormal number of digits and aberrant chondrogenesis will be dynamically imaged to identify the cell behaviors that are disrupted. These studies are an unbiased means to identify key developmental regulators and will contribute significantly to a greater understanding of the genetic and cellular control of vertebrate limb development.
Lung Development and Disease
The lung and other highly branched organs such as the kidney and lacrimal gland develop from a simple epithelial bud into a complex three dimensionally patterned functional organ. This happens through a process called branching morphogenesis. We wish to elucidate the fundamental processes underlying the development of the lung, as well as the pathogenesis of lung disease. To do so we use a combination of forward and reverse genetics in mice and molecular manipulations in organ cultures to identify key regulators of branching morphogenesis. Through the mouse ENU mutagenesis screen we have identified novel regulators of lung development. In addition, we are identifying the phosphoregulators (kinases and phosphatases) that control the branching pattern, the position and shape of the buds, and tubule size. Furthermore, we have developed dynamic imaging of lung branching morphogenesis to provide insight into the cell biological processes that drive lung branching. We are now coupling our forward and reverse genetic mutants with live imaging to decipher how molecular networks influence cell behaviors. Another long-term goal is to elucidate the function of developmentally important genes in the repair of lung tissue following injury or disease.
This work is supported in part by the National Institutes of Health, the Sandler Program for Asthma Research, and the Pediatrics Department at the University of Colorado Denver.
As of May 30, 2012
