John A. Klingensmith, Ph.D. - US grants

DESCRIPTION (Adapted from the Investigator's Abstract): Craniofacial development is a uniquely complex morphogenetic process in vertebrate ontogeny, creating evolutionary plasticity but also developmental vulnerability. The head and face are derived from many tissue precursors, which require a precise orchestration of pattern formation, cell migration, proliferation, apoptosis, and inductive interactions to achieve a functional end. Many of these events are mediated by secreted cytokines, whose activity must be precisely regulated to preclude inappropriate cellular responses. Bone Morphogenetic Proteins (BMPs) are a family of secreted ligands which have potent effects on many aspects of craniofacial development, particularly the closely related proteins BMP2 and BMP4. Their activity is thought to be important in the growth or patterning of such diverse tissues as the brain, the skull, the pituitary gland, the teeth, and the precursors of the face. Research from Drosophila and Xenopus indicate that BMP2/4 signal transduction is regulated in large part by antagonistic proteins such as Chordin (Chd) and Noggin (Nog). In frogs, Chd and Nog promote anterior development, and Chd is essential for normal head development in zebrafish. Preliminary work described in this proposal shows that these genes are required for development of the mammalian head. Lack of Chd in an inbred genetic background results in a group of craniofacial skeletal and soft-tissue defects involving neural crest derivatives, similar to those seen in certain human syndromes. Chd and Nog together are required early for head development, but are also involved specifically in development of the forebrain, mouth, nose, mandible, numerous bones of the skull, and other craniofacial tissues. The major aims of this proposal are: 1) to characterize the spatiotemporal expression patterns of Chd and Nog to clarify their roles in craniofacial development; 2) to determine the critical sites and times of action for Chd and Nog in head induction using embryonic stem cell chimeras and tissue recombinants; 3) to determine the functions of these genes in growth and patterning of craniofacial tissues; and 4) to assess whether ectopic BMP signaling reproduces the craniofacial defects of the mutants.

[unreadable] DESCRIPTION (provided by applicant): The outflow tract of the embryonic heart contributes to the formation of the aortic and pulmonary artery. Defects in outflow tract development result in severe congenital heart defects such as persistent truncus arteriosus and double outlet right ventricle. Similarly, atrio-ventricular (A-V) cushions contribute to the formation of the mitral and tricuspid valves as well as to the septa of the adult heart. Abnormalities in A-V cushion development can result in severe congenital heart defects such as A-V canal or tricuspid and mitral valve atresias. Understanding how the outflow tract forms and the A-V cushions develop is therefore critical to understanding the development of many congenital heart defects. Classic studies have demonstrated the requirement of cardiac neural crest cells (CNCC) in the septation of the single outflow tract into the aortic and pulmonary arteries. More recent studies have provided new insight into the development of the myocardium of the outflow tract of the heart. These studies demonstrate the existence of heart precursor cells that are added to the outflow tract and right ventricle. Our preliminary data suggests that the Shh signaling pathway is critical for these "anterior heart field" (AHF) cells as well as for migratory CNCC during outflow tract formation and septation. In addition, Shh appears to be required for A-V cushion formation and therefore valve development. Loss of the Shh signal results in a single outflow tract (pulmonary artery atresia) and a single A-V valve. We propose that the cardiac defects seen in Shh mutants are due to abnormal development of both CNCC and the AHF during outflow tract development as well as abnormal endocardial cushion formation during valve formation. We propose examining the cell autonomous requirement for hedgehog signaling within AHF and CNCC in the coordinated development of the outflow tract using a Cre/LoxP approach. Similarly we will test the cell autonomous requirement of hedgehog signaling during A-V valve formation using genetic manipulations in the mouse. [unreadable] [unreadable] [unreadable]

An exciting finding of recent research in developmental biology is that defective development of the rostral foregut endoderm (FGE) can result in both intrinsic and extrinsic malformations of direct relevance to major human birth defects. An important group of foregut defects is tracheoesophageal fistula, where the trachea and esophagus fail to be formed correctly from the early endodermal tube. The FGE is also the source of critical signals that regulate craniofacial development; for example, defective foregut signaling can lead to severe mandibular hypoplasia (underdevelopment of the lower jaw). Despite their pragmatic importance, the mechanisms controlling these intrinsic and extrinsic aspects of foregut development remain largely unknown. The long-term objective of our work is to understand how intercellular signaling directs the development of rostral tissues in the mouse embryo. Our previous work showed that loss of the BMP antagonists Noggin and Chordin results in both tracheo-esophageal fistula and mandibular hypoplasia. These BMP antagonists are expressed in the anterior primitive streak, the source of the FGE. Later, they are expressed in the axial midline, including floorplate, notochord and dorsal FGE. Based on our current data, our central hypothesis is that ongoing axial midline BMP antagonism is an active regulator of an endodermal signaling network essential for foregut and craniofacial development. However, there is also evidence consistent with the alternative hypothesis: That in either case the relevant requirement for BMP antagonism is during and immediately after gastrulation for normal formation of the early foregut endoderm, and thus indirectly for the foregut's subsequent intrinsic and extrinsic developmental roles. Resolving these questions will provide key insight into the essential roles of BMP antagonism and the mechanisms of these birth defects in general. Accordingly, we directly test both our central and alternative hypotheses, by means of the following specific aims: 1. Determine the tissues in which BMP antagonist expression is required for formation of the trachea and esophagus; 2. Determine the spatiotemporal requirement for Noggin and Chordin in mandibular outgrowth; and 3. Determine the interactions of BMP antagonism with the foregut endodermal signaling network.

DESCRIPTION (provided by applicant): This application addresses broad Challenge Area (15) Translational Science, and the specific Challenge Topic, 15-NS-106: Identifying mechanisms that underlie nervous system development and function. This Challenge solicits mechanistic studies that elucidate principles of nervous system formation, as well as analyses of how normal mechanisms are perturbed in neurological disease. Spina bifida is one of the most common structural malformations in man;despite its high mortality and morbidity, the etiological causes of spina bifida remain poorly understood. We propose a mechanistic analysis of the causes of lumbar spina bifida in Noggin mutant mice. In contrast to virtually all other mouse strains with neural tube defects, the spina bifida phenotype in Noggin does not occur through a failure of the dorsal neural folds to close into a tube. Rather, a day or so after closure the lumbar spinal cord reopens dorsally in isolated regions along the midline. This results in a lumbar spina bifida phenotype. This mutant represents a novel mouse model for an unexplored mechanism of spina bifida that is likely to be more relevant to pathogenesis of some human cases of spina bifida than models in which the neural tube fails to close. We use tissue-specific gene ablation to determine the individual tissue requirements Noggin for maintenance of neurulation. Our preliminary data indicate that Noggin promotes adhesion of neural tube cells to cell adhesion molecules such as N-cadherin. Our overall hypothesis is that Noggin is required in the closed seam along the dorsal neural tube for maintenance of closure. We test this as well as alternative models, and evaluate downstream molecular and cellular pathways. PUBLIC HEALTH RELEVANCE: This Challenge Grant application addresses mechanisms of spina bifida using the mouse model system. Using a genetic approach, complemented with molecular and cellular assays, we dissect the developmental cause(s) of the fully penetrant spina bifida phenotype in mouse noggin mutants. Spina bifida is poorly understood in humans, yet affects affects approximately 2,000 of the approximately 4 million babies born each year in the US. Although usually not fatal in live-born infants if repaired surgically, lifetime complications typically occur, at an average cost of over $1 million per case.

DESCRIPTION (provided by Principal Investigator): Proper development of embryonic foregut derivatives is essential for mammalian survival at birth. The rostral endodermal foregut tube divides into ventral respiratory (larynx and trachea) and dorsal alimentary (esophagus and stomach) components. Remarkably little is known about the underlying mechanisms, despite their pragmatic importance. For example, defects in foregut compartmentalization result in common birth defects involving abnormal communications between these systems, known clinically as tracheoesophageal fistulas (TEF) and laryngo-esophageal clefts. Sonic hedgehog (Shh) is essential for generation of distinct laryngeal/tracheal and esophageal tubes, as the Shh-null mouse foregut is uncompartmentalized. Our preliminary data indicate that later Shh expression in the developing trachea and esophagus is critical for the stratification of foregut epithelia and development of specialized foregut mesoderm. Our overall hypothesis is that shh signaling acts as a master regulator of foregut development, initially to control tissue remodeling essential for formation of esophagus, trachea and larynx from a common precursor, and subsequently to pattern the endodermal and mesodermal tissues of these nascent organs. We propose a series of tissue-specific and temporal genetic manipulations of Shh expression and reception in the mouse embryo, coupled with histological and molecular analyses, to investigate the roles of Shh signaling in foregut development. Aim 1 determines the role of Shh signaling in normal separation of trachea from esophagus, the morphogenic process by which this occurs, and how the disruption of Shh signaling results in TEF. Aim 2 in turn addresses the separation of larynx from esophagus, with special attention to the role of neural crest-derived laryngeal cartilages in this process. Aim 3 elucidates the role of Shh in the different patterns of stratification and differentiation within the endoderm. In Aim 4, we determine the spatiotemporal functions of Shh signaling in differentiation of specialized foregut mesoderm, including the tracheal cartilage rings, trachealis smooth muscle, and esophageal smooth muscle. Altogether, these studies will provide important insight into the roles of Shh signaling in normal development and congenital defects of the trachea, esophagus, and larynx. PUBLIC HEALTH RELEVANCE: Proper development of embryonic foregut derivatives is essential for mammalian survival at birth. The rostral endodermal foregut tube divides into ventral respiratory (larynx and trachea) and dorsal alimentary (esophagus and stomach) components. These must then develop into functional organs. Little is known about the underlying mechanisms, despite their pragmatic importance. This proposal is for a research project to dissect the roles of the Sonic hedgehog intercellular signaling pathway in mouse foregut development. We elucidate critical roles in the initial compartmentalization of the common foregut tube, and in the organ-specific differentiation of endoderm and mesoderm. Our results are of direct relevance to the mechanisms of human foregut birth defects and potentially to the pathogenesis of esophageal cancer.