Burrus

1. To elucidate the role of Wnt proteins in myogenesis.

The development of a vertebrate organism from a single fertilized egg is a complex series of events involving both cell proliferation and specialization. Many of these events have been shown to be mediated by intercellular signals. One such family of signaling factors is the Wnt gene family, which is comprised of at least 20 related vertebrate genes (Adamson et al., 1994; Bouillet et al., 1996; Christian et al., 1991; Christiansen et al., 1995; Gavin et al., 1990; Hume and Dodd, 1993; Kispert et al., 1996; Ku and Melton, 1993; Nusse and Varmus, 1982; Roelink and Nusse, 1991; Roelink et al., 1990; Sidow, 1992; Tanda et al., 1995; Wainwright et al., 1988; Wolda and Moon, 1992). Wnt genes encode cysteine-rich polypeptides possessing 50-60% amino acid identity which are thought to be secreted signaling molecules involved in cell growth, survival, and/or differentiation (McMahon et al., 1992; Nusse and Varmus, 1992). My laboratory is particularly interested in the role(s) of these signals in directing pluripotential chick somite cells to become skeletal muscle. Although several Wnt family members have been shown to be sufficient and necessary for the induction of myogenesis in vertebrates (Leyns et al., 1997; Münsterberg et al., 1995; Stern et al., 1995; Wang et al., 1997), we are lacking important information about how Wnt proteins act at a cellular level. Despite the identification of the Wnt-1 cDNA more than thirteen years ago (Fung et al., 1985), studies pertaining to the cellular roles of Wnt proteins have been severely hampered by the lack of soluble active ligand. For reasons that are poorly understood, Wnt genes are extremely refractory to overexpression. Michelle Baranski is working on generating a COS cell expression system to generate Wnt fusion proteins which can easily be purified and used as immunogens for the generation of anti-Wnt monoclonal antibodies. By comparing the biochemical properties of recombinant Wnt proteins and endogenous Wnt proteins, we hope to identify the differences that will help us pinpoint the reason that it has been so difficult to generate active soluble recombinant Wnt protein. Furthermore, Wnt protein purified from the COS cell expression system and/or from chick embryos (using immunoaffinity columns) will be used to directly assess the role of Wnt proteins in myogenic specification. The development of these key reagents will open the door to numerous future studies pertaining to both development and cancer. This project is funded by a grant from NIH (MBRS).

2. To understand the role of Frzb in myogenic specification and differentiation.

Recently, a secreted molecule with Wnt-binding activity has been identified (Hoang et al, 1996; Shirozu et al, 1996; Finch et al, 1997; Leyns et al, 1997; Rattner et al, 1997; Wang et al, 1997). This molecule (Frzb) shares a region of homology with the extracellular domain of the candidate Wnt receptor, Frizzled. This sequence homology has led researchers to speculate that Frzb acts as an antagonist of Wnt activity by binding to secreted Wnt proteins in the extracellular space and making them unavailable to bind and activate Wnt receptors. In support of this hypothesis, it has been shown that segmental plate and early somite explants cannot specify skeletal muscle lineages in response to Wnts alone while more mature somites can. Interestingly, mouse Frzb-1 is expressed in newly formed somites, suggesting the exciting possibility that it may function as a negative regulator of Wnt activity in immature somites (Leyns et al, 1997). In order to determine whether Frzb-1 may play a similar role in chick embryos, Eli Berdougo and James Sandler, former members of my laboratory at SFSU, have isolated the chick homologue of Frzb-1. Our analysis of its expression pattern suggests that it may play a role in regulating neural crest development (a project that Tina Cheng and Steve Wang are now pursuing), but that it is not likely to play a role in inhibiting early myogenic specification. Thus, we sought to isolate other chick Frzb family members in hopes of identifying one with an expression pattern similar to that of mouse Frzb-1. Karianne Terry, one of my undergraduate students, has isolated two additional Frzb homologues from the chick, sfrp-1 and sfrp-2, and we are currently analyzing their expression patterns. Once we have isolated all of the chick Frzb homologues, we will be able to carry out experiments which are designed to answer some important questions in the field of muscle biology. First, is there a Frzb family member that is expressed in early chick somites? And if so, does this Frzb participate in somite patterning by mediating Wnt activity in somites? This work is currently funded by an NIH-AREA grant.

Last modified July 10, 2002


		Dr. Laura Burrus
			lburrus@sfsu.edu
	http://userwww.sfsu.edu/~lburrus/
1. To elucidate the role of Wnt proteins in myogenesis. The development of a vertebrate organism from a single fertilized egg is a complex series of events involving both cell proliferation and specialization. Many of these events have been shown to be mediated by intercellular signals. One such family of signaling factors is the Wnt gene family, which is comprised of at least 20 related vertebrate genes (Adamson et al., 1994; Bouillet et al., 1996; Christian et al., 1991; Christiansen et al., 1995; Gavin et al., 1990; Hume and Dodd, 1993; Kispert et al., 1996; Ku and Melton, 1993; Nusse and Varmus, 1982; Roelink and Nusse, 1991; Roelink et al., 1990; Sidow, 1992; Tanda et al., 1995; Wainwright et al., 1988; Wolda and Moon, 1992). Wnt genes encode cysteine-rich polypeptides possessing 50-60% amino acid identity which are thought to be secreted signaling molecules involved in cell growth, survival, and/or differentiation (McMahon et al., 1992; Nusse and Varmus, 1992). My laboratory is particularly interested in the role(s) of these signals in directing pluripotential chick somite cells to become skeletal muscle. Although several Wnt family members have been shown to be sufficient and necessary for the induction of myogenesis in vertebrates (Leyns et al., 1997; Münsterberg et al., 1995; Stern et al., 1995; Wang et al., 1997), we are lacking important information about how Wnt proteins act at a cellular level. Despite the identification of the Wnt-1 cDNA more than thirteen years ago (Fung et al., 1985), studies pertaining to the cellular roles of Wnt proteins have been severely hampered by the lack of soluble active ligand. For reasons that are poorly understood, Wnt genes are extremely refractory to overexpression. Michelle Baranski is working on generating a COS cell expression system to generate Wnt fusion proteins which can easily be purified and used as immunogens for the generation of anti-Wnt monoclonal antibodies. By comparing the biochemical properties of recombinant Wnt proteins and endogenous Wnt proteins, we hope to identify the differences that will help us pinpoint the reason that it has been so difficult to generate active soluble recombinant Wnt protein. Furthermore, Wnt protein purified from the COS cell expression system and/or from chick embryos (using immunoaffinity columns) will be used to directly assess the role of Wnt proteins in myogenic specification. The development of these key reagents will open the door to numerous future studies pertaining to both development and cancer. This project is funded by a grant from NIH (MBRS). 2. To understand the role of Frzb in myogenic specification and differentiation. Recently, a secreted molecule with Wnt-binding activity has been identified (Hoang et al, 1996; Shirozu et al, 1996; Finch et al, 1997; Leyns et al, 1997; Rattner et al, 1997; Wang et al, 1997). This molecule (Frzb) shares a region of homology with the extracellular domain of the candidate Wnt receptor, Frizzled. This sequence homology has led researchers to speculate that Frzb acts as an antagonist of Wnt activity by binding to secreted Wnt proteins in the extracellular space and making them unavailable to bind and activate Wnt receptors. In support of this hypothesis, it has been shown that segmental plate and early somite explants cannot specify skeletal muscle lineages in response to Wnts alone while more mature somites can. Interestingly, mouse Frzb-1 is expressed in newly formed somites, suggesting the exciting possibility that it may function as a negative regulator of Wnt activity in immature somites (Leyns et al, 1997). In order to determine whether Frzb-1 may play a similar role in chick embryos, Eli Berdougo and James Sandler, former members of my laboratory at SFSU, have isolated the chick homologue of Frzb-1. Our analysis of its expression pattern suggests that it may play a role in regulating neural crest development (a project that Tina Cheng and Steve Wang are now pursuing), but that it is not likely to play a role in inhibiting early myogenic specification. Thus, we sought to isolate other chick Frzb family members in hopes of identifying one with an expression pattern similar to that of mouse Frzb-1. Karianne Terry, one of my undergraduate students, has isolated two additional Frzb homologues from the chick, sfrp-1 and sfrp-2, and we are currently analyzing their expression patterns. Once we have isolated all of the chick Frzb homologues, we will be able to carry out experiments which are designed to answer some important questions in the field of muscle biology. First, is there a Frzb family member that is expressed in early chick somites? And if so, does this Frzb participate in somite patterning by mediating Wnt activity in somites? This work is currently funded by an NIH-AREA grant. Last modified July 10, 2002