Andrew Lassar - US grants

9317533 Lassar The regulation of cell type determination is a crucial aspect of early embryogenesis in all multicellular organisms. A variety of approaches taken by several laboratories has led to the identification of a family of structurally related skeletal muscle transcriptional regulators (the myogenic HLH proteins) which play a crucial role in skeletal muscle determination and differentiation. The myogenic HLH regulators can activate muscle gene expression in various cellular contexts implying that no tissue specific factors other than this family of regulatory proteins are needed to initiate muscle differentiation in the mesodermal and non-mesodermal cells tested. During vertebrate development skeletal muscle precursors are first observed in the somite, an axial structure which gives rise to dermis, skeletal muscle, cartilage and bone. The earliest expression of the myogenic bHLH regulators occurs in the myotomal cells of the somite, which give rise to all skeletal muscle in vertebrates. The expression of the myogenic bHLH genes during cell type specification of skeletal muscle suggests that activation of these regulators may in fact commit cells to the skeletal muscle cell lineage. There are a number of unanswered questions regarding specification of somitic cells into the muscle lineage: 1) When are the somitic cells giving rise to the myogenic lineage first specified to this fate, 2) what triggers the final commitment and 3) which signals are involved in this process? Experiments have demonstrated that early somite cells of chicken or mouse, when explanted in tissue culture, are not able to differentiate into muscle unless they are co-cultured with either neural tube or overlying epithelial cells. Thus neural tube (and some types of epithelia) are inducing the pluripotent early somite cells to differentiate into muscle. Dr. Lassar has recently demonstrated that neural tube dependent induction of somitic skeletal muscle differentiation is ac companied by activation of the myogenic HLH regulators. Thus a factor(s) from the neural tube induces skeletal muscle differentiation in somitic cells via activation of the myogenic HLH regulatory family. By employing an in vitro induction assay, he will attempt to characterize and molecularly clone the neural factor(s) responsible for inducing chick and /or mouse early somite cells to enter the skeletal muscle cell lineage. The goal of this work is to understand both the inductive events as well as eventually identify the intracellular mediators necessary for activation of the myogenic bHLH regulators. ***

DESCRIPTION (adapted from the applicant's abstract): The broad aim of this project is to understand how the cardiac myocyte differentiation program is activated during vertebrate development. In previous work the principal investigator has identified a cardiac-inducing activity in the anterior lateral plate endoderm of the gastrula-stage avian embryo, which can induce cells from the posterior primitive streak (which normally gives rise to extra embryonic tissues and to blood) to give rise to cardiac myocytes. Recently, the principal investigator has extended these studies to demonstrate that a combination of at least two signals is necessary to induce cardiac myocyte determination. During mid-primitive streak stage in chick embryos, cells which contact anterior endoderm seem to lie in a developmental field in which cells display a potential to become heart. BMP-2 and/or BMP-4 then appear to be capable of inducing cells within this field to initiate the cardiac differentiation program. One of the first cardiac markers that the principal investigator has found to be induced in primitive streak cells exposed to heart inducing signals is the homeobox-containing gene, Nkx-2.5. In Drosophila, the homologous gene tinman is expressed in the heart and is required for heart formation. Work from the principal investigator's lab suggests that a combination of at least two signals, a cardiac promoting signal from the anterior endoderm plus a BMP-2/-4 signal, are together necessary to initiate expression of the Nkx-2.5 gene. The goal of this proposal is to identify the sequence elements in the Nkx-2.5 gene that are responsive to these signaling molecules and the transcription factors that induce cardiac-specific expression of this gene. Aim 1. Map the region of the chick Nkx-2.5 gene that responds to cardiac inducing signals in an in vitro cardiac induction assay. Aim 2. Identify the transcription factors that bind to the cardiac responsive sequences in the chick Nkx-2.5 gene. Aim 3. Determine if a chick Mad1 family member expressed in the precardiac mesoderm can mediate the cardiac inducing properties of BMP-2/-4. Aim 4. Identify transcription factors that mediate activation of Nkx-2.5 in response to BMP signals by screening for proteins that interact with chick Mad1 in precardiac mesoderm.

DESCRIPTION: The central goal of this proposal is to understand how signals from surrounding tissues activate somite myogenesis. Although it is clear that expression of either MyoD or Myf-5 is essential for the formation of skeletal muscle, it has hitherto been unclear how these genes are activated during development. The PI has begun to address what regulatory molecules mediate the induction of MyoD and Myf-5 by signals from either the axial tissues (i.e., the neural tube/notochord) or the overlying ectoderm. He has found that the muscle promoting signals from the axial tissues can be mimicked by in vitro supplied Wnt and Shh signals. Furthermore, he has found that signals from either the overlying ectoderm or in vitro supplied Wnt and Shh signals can induce somitic expression of the paired box transcription factors, Pax-3 and Pax-7, concomitant with expression of Myf-5 and prior to that of MyoD. Moreover, infection of somites in vitro with a retrovirus encoding Pax-3 (which is normally expressed in the dermomyotome) is sufficient to induce expression of MyoD, Myf-5 and myogenin in paraxial mesoderm in the absence of inducing tissues. This finding has led the PI to speculate that the muscle-promoting signals from the axial tissues, the overlying ectoderm or in vitro supplied Wnt and Shh signals may activate somitic myogenesis via a Pax-3 dependent pathway. The focus of this proposal is to elucidate how Pax-3 activates myogenic bHLH gene expression in the somite. Pax-3 is the first identified transcription factor shown to be capable of activating the expression of MyoD and Myf-5 in somitic tissue. The major goals of this grant are focused on determining how Pax-3 activates myogenic bHLH gene expression and how this activity of Pax-3 is modulated. The goals are: 1. Determine if Pax-7 and/or an alternatively spliced isoform of Pax-3 activate somitic myogenesis. 2. Determine if MyoD and Myf-5 expression are directly or indirectly activated by Pax-3. 3. Identify somitic genes whose expression is positively or negatively regulated by Pax-3. 4. Determine how the muscle promoting activity of endogenous Pax-3 is blocked in the neural tube.

(Adapted from the Applicant's Abstract) The objective of the Morphology Core is to provide both a state-of-the-art histology service and image analysis facility for documentation of gene expression in manipulated mouse and avian embryos.

(Adapted from the Applicant's Abstract) The broad aim of this project is to understand how the cardiac myocyte differentiation program is activated during vertebrate development. In previous work the investigators have identified a cardiac-inducing activity in the anterior lateral plate endoderm of the gastrula stage avian embryos, which can induce cells from the posterior primitive streak (which normally gives rise to extra- embryonic tissues and to blood) to give rise to cardiac myocytes. Recently, the investigators have extended these studies to demonstrate that in addition to a cardiac inducing signal from the anterior endoderm, Bone Morphogenetic Proteins (BMPs) expressed in both endoderm and ectoderm also play an important role in heart induction. The result suggest that there are at least two stages in vertebrate cardiac myocyte determination Cells emerging from the primitive streak are still plastic with respect to their ability to form heart. Those mesodermal cells which contact anterior endoderm seem to lie in a developmental field in which cells display a potential to become heart. BMP-2 developmental field in which cells display a potential to become heart. BMP-2 and/or BMP-4 then appear to be capable of inducing cells within this field to initiated the cardiac differentiation program. Thus, a combination of two distinct signals is necessary for the induction of cardiac myocytes: a BMP signal present in the lateral regions of the embryo collaborates with an as yet to be identified signal secreted by the anterior endoderm to induce heart formation. The goal of this proposal will be to identify the signal secreted by cells in the anterior endoderm that induces the formation of cardiac myocytes in collaboration with a BMP signal. By employing subtractive hybridization technology the investigations have recently identified a signaling molecule, termed "crescent", which is a putative Wnt inhibitor, and is specifically expressed in the anterior endoderm at the time when cardiac myocytes are being induced in the chick embryos. During early chick development, crescent is expressed in a domain that precisely overlaps the location where the endodermal heart inducing activity lies. In this proposal the investigators intend to firstly investigate where crescent is the heart inducing activity in the anterior endoderm. If such is the case, the investigators will focus on understanding how this such is the case, the investigators will focus on understanding on understanding how this signaling molecule collaborates with BMP signals to induce heart formation in the various assay systems that the investigators have developed, they will employ an alternative approach to isolate the heart inducing activity in the anterior endoderm. Specific Aim 1 will investigate whether crescent, a putative anti-Wnt molecule secreted by the anterior endoderm collaborates with BMP signals to induce the formation of cardiac myocytes. Specific Aim 2 if crescent can induce the formation of cardiac myocytes in collaboration with a BMP signal, the investigators will determine whether crescent displays an anti-Wnt activity in this induction. Specific Aim 3 will determine whether mice genetically engineered to lack crescent display a defect in heart formation. Specific Aim 4 if the investigators find that crescent fails to display any heart inducing activity either in vitro or in vivo, they will develop develop a functional screen to identify the cardiac inducing molecule secreted by the anterior endoderm.

DESCRIPTION (Taken from the applicants abstract): This is a proposal to convene an international meeting on "Molecular Biology of Muscle Development an, Disease" to be held at the Asilomar Conference Center in Pacific Grove, California from May 21-26, 2000. The conference focuses on the induction and regulation of myogenesis in both vertebrates and invertebrates, nerve muscle interactions, and diseases of skeletal muscle. The field of skeletal muscle molecular biology has undergone enormous advances within the past several years, including the identification of transcription factors and signaling molecules that control the formation of skeletal muscle during early development, the signals that pattern this tissue into different types of musculature, the factors that control the proliferation and differentiation of skeletal muscle, the signals that regulate synapse-specific gene expression and control the formation of the neuromuscular junction. Furthermore, the last decade has seen significant advances in our understanding the molecular biology of a number of muscle pathologies, including: Duchene muscular dystrophy, Limb-Girdle muscular dystrophy, Freidrich's Ataxia, Amyotrophic Lateral Sclerosis, Myotonic Dystrophy, and proximal spinal muscular atrophy. This conference will include presentations by the world's experts in each of these fields. This conference is unique in the muscle biology community at bringing together investigators studying the biology and embryology of muscle development in both vertebrate and invertebrate systems with those studying muscle pathology in disease. This conference has a 20 year history and has previously been held as a Keystone symposium, convening every three years. They have changed the venue this coming year to Asilomar, and anticipate that as in past conferences they will attract between 400-500 participants from around the globe.

DESCRIPTION (provided by applicant): The broad aim of this project is to elucidate the transcription factors that control chondrocyte differentiation during vertebrate development. Prior work in my lab has established that Sonic Hedgehog (Shh) and Wnt signals pattern somitic cell fate. We have recently found that one of the genes induced by Shh, Nkx3.2, confers competence for subsequent BMP signals to induce chondrogenesis. Activation of somitic chondrogenesis by Nkx3.2 requires BMP signaling and correlates with the ability of this regulator to induce the expression of another chondrocyte transcription factor, Sox-9. We have found that the transcriptional repressor activity of Nkx3.2 requires interaction of Nkx3.2 with both BMP-dependent Smads and histone deacetylases and is necessary for Nkx3.2 to induce both somitic chondrogenesis and Sox-9 gene expression. Forced expression of Sox-9 in somites can similarly induce competence for subsequent BMP signals to induce chondrogenesis. Together, these findings suggest that Nkx3.2 induces a prochondrogenic state in somites by repressing the transcription of an inhibitor of Sox-9 expression. Later in development Nkx3.2 is specifically expressed in immature cartilage in the developing limb bud and is excluded from regions of chondrocyte hypertrophy. Forced expression of Nkx3.2 throughout the limb bud blocks chondrocyte maturation and chondrocyte hypertrophy. The transcriptional repressor activity of Nkx3.2 is necessary for this transcription factor to block cartilage maturation, and suggests that Nkx3.2 blocks the transcription of a factor(s) required for cartilage maturation. Our findings indicate that Nkx3.2 plays a role in the generation and maturation of cartilage cells. In this proposal I outline a series of experiments to address the following specific aims. (1) Determine how BMP-dependent SMADs promote chondrogenesis. (2) Determine if GATA genes repress a chondrogenic response to BMP signaling (3) Determine how Nkx3.2 represses chondrocyte maturation. (4) Determine if other Nkx family members expressed in the developing bones act together with Nkx3.2 to repress chondrocyte maturation.

DESCRIPTION (provided by applicant): The broad aim of this project is to elucidate the regulatory circuits that pattern paraxial mesodermal cell fates in both the somites and in the cranial paraxial mesoderm. Prior studies have indicated that Shh and Wnt signals from the axial tissues and from the surface ectoderm, work in combination with BMP signals from the lateral plate mesoderm to pattern different cell types in the somites. Signals that induce dermomyotomal and myotomal gene expression simultaneously repress the expression of sclerotomal markers. Conversely, signals that induce sclerotomal gene expression simultaneously repress the expression of dermomyotomal/myotomal markers. In contrast to somitic myogenesis, the source of signals that induce skeletal muscle formation in the head remains obscure. While Wnt and Hedgehog signals induce myogenesis in somites these signals have the opposite effect on head mesoderm, where they block the formation of skeletal muscle. Whereas trunk myogenesis is induced by signals from the axial tissues, head myogenesis is blocked by cues from the dorsal neural tube. We have found that the timing and kinetics of head myogenesis are correlated with an inductive signal(s) secreted by the cranial neural crest (CNC), that both isolated CNC cells or medium conditioned by such cells can directly induce skeletal myogenesis in head mesoderm, and that BMP-antagonists expressed in the cranial neural crest can mimic the muscle inducing activity of this tissue. In this proposal I outline the following specific aims. (1). Determine if Pax-3, and its related gene Pax-7, are necessary and sufficient to both induce dorsal somitic cell fates and block the induction of sclerotomal gene expression in the dorsal regions of the somite. (2) Determine if Nkx3.2, Nkx3.1 and Sox-9 are necessary and sufficient to induce both ventral somitic cell fates and block the induction of dermomyotomal/myotomal gene expression in the ventral regions of the somite. (3) Determine if dermomyotomal gene expression requires both Wnt and notch signals. (4) Determine if signals from the cranial neural crest are necessary for the formation of cranial muscle, and determine how Wnt signals block the formation of head muscle.