Sun Lab Research Description

Goals of Our Research

Molecular Genetics of Limb Formation

Molecular Genetics of Lung Formation and Maintenance

Mechanisms of Morphological Diversification through Evolution

Goals of Our Research

1. To understand how the vertebrate organs achieve their size, pattern and cellular functions. We focus on the limb and the lung. Knowledge gained will contribute to our understanding of birth defects.

2. To use vertebrate embryos as simple settings to explore similar events in adult organ maintenance. Knowledge gained will contribute to our understanding of stem cell biology and the etiology of diseases such as cancer and asthma.

3. To understand how the lung in diverse vertebrate species exhibits different morphologies. Knowledge gained will contribute to our understanding of molecular evolution.

Molecular Genetics of Limb Formation

Tetrapod limbs initiate as limb buds that protrude from the sides of the main body axis (Fig. 1a). Each limb bud is comprised of two cell populations: a layer of ectoderm-derived epithelial cells encasing mesoderm-derived mesenchymal cells (Fig. 1b). In a young limb bud, the mesenchymal cells appear homogeneous in character. As development proceeds, this cell population will grow and differentiate into skeletal elements and connective tissues of the adult limb, while other components, the muscles and endothelial cells, migrate in from outside the limb bud.

   The end product of limb development is an adult limb with distinct structures along all three of its axes: the proximal-distal (P-D, upper arm to fingertip ) axis, the anterior-posterior (A-P, thumb to pinky) axis and dorsal-ventral (D-V, back of hand to palm) axis (Fig. 1c,d). The distinct size and shape of skeletal elements along each of these axes are produced in a stereotypical manner, suggestive of precise genetic control.

Figure 1. Limb morphology and axes. a-c, Sanning EM images of whole mouse embryo (a) or limb buds (b, c) at Embryonic day (E)10.5. Forelimb (FL) and hindlimb (HL) buds are labeled. Dashed line in a indicates the plane of section used to generate the image in b. Arrowheads in c point to the thickened apical ectodermal ridge which forms at the dorsal- ventral boundary of the limb bud. d, Anewborn mouse forelimb skeleton with the shoulder girdle attached to the three segments of the limb: the sylopod (S), zeugopod (Z) and autopod (A). The arrows on the upper right corner indicate two of the three axes of the limb. Other abbreviations: an, anterior; di, distal; do, dorsal; ect, ectoderm; mes, mesoderm; po, posterior; pr, proximal; ve, ventral. SEM images are from http://www.med.unc.edu/embryo_images/.

   Embryo manipulations and genetic studies have established that members of the Fibroblast Growth Factor (FGF) family of signaling molecules play a central role in limb formation. In particular, two of the family members, Fgf4 and Fgf8, are required in combination for the formation of the entire limb (Fig. 2). However, the mechanism of their function as well as the fundamental mechanism of limb patterning remain in dispute. Our recent genetic study of the Fgf mutants led to data that challenge the Progress Zone model, a prominent model in developmental biology that provides a mechanism for generating limb patterns. Our current work focuses on testing alternative models of limb development, addressing the precise role of FGF in limb formation and identifying the mediators of FGF function. An understanding of normal development will shed light on the causes of limb-related birth defects.

Molecular Genetics of Lung Formation and Maintenance

At birth, the fetal lung is highly efficient gas exchange machinery that is vital for survival.The sophisticated design of the lung is achieved during gestation through numerous coordinated developmental steps, including budding, branching, patterning and differentiation (Fig. 3). The preciseness of cellular programming is maintained in the adult. Deviations from this blueprint lead to debilitating lung diseases such as cancer and asthma.

    Lung branching is mediated by reciprocal interactions between the epithelium and the overlying mesenchyme. A model postulates that the feedback interaction between signaling molecules such as FGFs, Sonic Hedgehog (SHH) and Bone Morophogenetic Protein 4 (BMP4) are important for this stereotyped branching. We are interested in the precise mechanism of this interaction and the involvement of other molecules, such as transcription factors, in this process. Our investigation uses a combination of genetic, genomic and organ culture approaches.

Figure 3. Embryonic lung morphology. Lung organogenesis starts early in gestation (5 weeks in human, E9.5 in mouse) and is completed shortly after birth in three stages: (1) Initiation: the lung originates as a ventral outpouching of the foregut epithelia, separates from the prospective esophagus and develops into two primary lung buds. (2) Branching and Patterning: the lung buds grow and branch repeatedly to form an elaborate respiratory tree with bronchi, bronchioles and terminal air sacs. The core epithelium (blue) and the surrounding mesenchyme (white) are specified, but are not yet terminally differentiated at this stage. (3) Differentiation: functional cell types form to facilitate lung inflation and air-blood exchange, among other roles.

    Many recent studies present collective evidence that there are “tissue stem cells” in the lung: cells in a particular region of the lung which, upon stimulation (e.g. assault by toxic agents), can re-enter the cell cycle and differentiate to replace the damaged cells locally. Whether this happens through activation of epithelial progenitor cells that lie “dormant” in the adult lung or through reprogramming of already differentiated cells is under intense debate, and may vary depending on cell type. Nevertheless, the cellular events that occur during regeneration are similar to those executed during lung formation. This justifies the use of the embryonic lung as a simple setting to understand stem cell activation and maintenance.

    A view into lung diseases such as cancer and asthma at a cellular level reveals that they are caused by defects in processes that are similar to those carried out during lung organogenesis. For example, epithelial lung cancer is a result of aberrant cell proliferation and/or differentiation programs. On the molecular level, there are also emerging similarities between organogenesis and cancer. It has been shown that Epidermal Growth Factor (EGF) signaling, which is involved in branching morphogenesis, is upregulated in 30% of non-small cell lung cancers, one of the two major categories of lung cancer. Many of the airway remodeling events in asthma are reiterations of cellular differentiation programs executed/carried out during lung development. Recent evidence suggests that airway remodeling is not only a result of the inflammation but is also an essential determinant of asthma severity and airway dysfunction. Thus, understanding cellular events during lung formation can directly impact risk assessment, early detection, prognosis and therapy for lung cancer and asthma.

Mechanisms of Morphological Diversification through Evolution

An additional aspect of the complexity of the lung is evidenced by the amount of structural diversity observed among vertebrate lungs (Fig.3). This is in contrast to many other organs, which exhibit a high degree of conservation across species. Such diversity in lung is a result of functional adaptations to accommodate diverse body sizes, habitats and metabolic requirements. Despite significant investigation into mammalian lung development, the events leading to the rich variance in lung structures among all vertebrate species arenot well understood. By studying the molecular characteristics of embryonic lungs from different species, we hope to contribute to the understanding of the genetic basis of adaptation.

Figure 4. Diverse morphology of mammalian versus avian adult lungs. Latex castings of (left) an adult pig (representing mammalian) lung and (right) chicken (representing avian) lung are shown. The castings outline the epithelial structures. The mature mammalian lung is contrived of multiple bifurcations of the primary branches, forming a “respiratory tree.”The branches terminate in blind-ended air sacs known as alveoli, which are the functional units for gas exchange. The mature mammalian lung is elastic and expandable.Breathing takes place following movement of the diaphragm. The chick lung develops through branching and anastomosis (a process in which the secondary and tertiary bronchi join), constructing a continuous, closed loop structure.The chick lung is rigid and inexpansible; air sacs branch off of the lungs and serve as bellows to push air throughout the organ. This highly efficient cross-current system is believed to have evolved as a consequence of the high metabolic demands required for flight. Photos from “Functional Morphology of the Vertebrate Respiratory Systems”, by J.N. Maina, Science Publishers, Inc.