Tulane University Cell and Molecular Biology

Dr. Muneoka has been a member of the Cell and Molecular Biology faculty since 1986 and served as Chair of the department from 1993 to 2005.

Dr. Muneoka's primary research interests are in limb development and regeneration, pattern formation, wound healing, cell migration, and growth control. His research is presently funded by grants from the National Institutes of Health.

Dr. Muneoka teaches CELL 413, Embryology, and CELL 608, Advanced Developmental and Cell Biology II. Follow the links on the left slide of the page for more information about Dr. Muneoka's research and teaching.

2004 marks my 25th year carrying out research in the field of vertebrate limb formation. During that time I have utilized a number of experimental models including the regenerating salamander limb, the regenerating mouse digit, and the developing limbs of salamanders, frogs, chicks and mice. I have remained both productive and actively funded during this time. My studies have primarily focused on how cells communicate with one another to establish the 3-dimensional pattern of the adult limb. I have been lucky enough to be a participant in a field that has undergone an amazing transformation to become the best understood developing vertebrate organ, and to witness how our understanding of this organ has gained prominence in many aspects of applied science ranging from teratology to ecology.

The limb is formed by a series of interactions that occur between a specialized group of ectodermal cells at the tip of the limb bud that forms a structure called the apical ectodermal ridge (AER), and the mesenchymal cells that underlie the AER. The apical ectoderm produces factors that are necessary for distal outgrowth by the mesenchyme. Mesenchymal cells interact with one another and with the AER to establish spatially distinct patterns of gene expression followed by the differentiation of specific structures. The primary focus of my research is to understand how cells become distinct from one another. In the 1980’s my cell lineage work on developing and regenerating amphibian limbs demonstrated similarities between development and regeneration, and also established the over-contribution of fibroblasts in the regeneration response. To begin to address regeneration in higher vertebrates, I pioneered in utero surgical techniques that make it possible to carry out regeneration studies on the developing mammalian limb. In the early 1990’s I participated with Susan Bryant’s lab in a study that demonstrated retinoic acid acted to induce a mesenchymal signaling center in the limb bud called the Zone of Polarizing Activity (ZPA). This finding has now been demonstrated with loss of function studies and with more sophisticated molecular probes with the same conclusion.

Muneoka, K. and Bryant, S.V. (1982). Evidence that patterning mechanisms in developing and regenerating limbs are the same. Nature 298, 369-371. pdf

Muneoka, K., and Bryant, S. V. (1984). Cellular contribution to supernumerary limbs resulting from the interaction between developing and regenerating tissues in the axolotl. Develop. Biol. 105, 179-187. pdf

Muneoka, K., Fox, W.F. and Bryant, S.V. (1986). Cellular contribution from dermis and cartilage to the regenerating limb blastema in axolotls. Develop. Biol. 116, 256-260. pdf

Muneoka, K., Wanek, N. and Bryant, S.V. (1986). Mouse embryos develop normally exo utero. J. Exp. Zool. 239, 289-293. pdf

Wanek, N., Gardiner, D.M., Muneoka, K. and Bryant, S.V. (1991). Retinoic acid changes anterior cells into ZPA cells in the chick wing bud. Nature, 350, 81-83.

Studies from my lab established an initial link between the production of fibroblast growth factors (FGF) by the AER and the signaling ability of the ZPA. Signaling by the ZPA is now known to be mediated by a secreted factor called sonic hedgehog (Shh) and it is now well-established that FGFs modulate Shh production. Since that time we have continued to focus on FGF signaling and our studies have focused on how FGFs modify positional characteristics of non-ZPA cells and how FGFs modulate limb outgrowth. We have shown that FGFs can induce a regenerative response from the non-regenerating chick limb bud and we have gone on to show that FGF-4 functions as a chemotactic factor produced by the AER to regulate cell movements important for limb morphogenesis. We have also shown that limb bud cells possess differential cell adhesive properties that influence cell behavior during limb morphogenesis. The idea that cell motility and differential cell adhesion plays a critical role in limb morphogenesis leading to outgrowth is novel for the field and we have proposed a dynamic model for this process.

FGFs are secreted factors that interact with specific cell surface receptors to elicit a response. One of the feastures of the FGF family is that they have an affinity for heparan sulfate moieties present in the extracellular matrix (ECM). We have investigated the role of ECM during limb development utilizing the ZPA signaling assay in the chick limb bud. The ZPA signaling assay is based on the stimulation of supernumerary digits following tissue grafts into the anterior limb bud. Historically, the ZPA is identified and characterized based on its ability to induce supernumerary digits and more recently, there is evidence that the production of SHH by ZPA cells is responsible for this induction. Using this assay we have discovered that ECM preparations from a number of cell lines have the ability to induce supernumerary digits in the absence of ectopic SHH. Remarkably, we find that stripping the ECM preparation of bound factors enhances signaling levels suggesting that this activity involved the inactivation of inhibitory activity in the limb bud. These studies are truly unique in that they represent the first experimental evidence that ECM components are playing a key role in modulating cell-cell signaling during limb development.

Anderson, R., Landry, M. and Muneoka, K. (1993). Maintenance of ZPA signaling in cultured mouse limb bud cells. Development 117, 1421-1433. pdf

Anderson, R., Landry, M., Reginelli, A., Taylor, G., Achkar, C., Gudas, L., and Muneoka, K. (1994). Conversion of anterior limb bud cells to ZPA signaling cells in vitro and in vivo. Develop. Biol. 164, 241-257.

Taylor, G.P., Anderson, R., Reginelli, A.D. and Muneoka, K. (1994). FGF-2 induces regeneration of the chick limb bud. Develop. Biol. 163, 282-284. pdf

Li, S. and Muneoka, K. (1999). Cell migration and chick limb development: chemotactic action of FGF-4 and the AER. Develop. Biol., 211, 335-347. pdf

Schaller, S and Muneoka, K. (2001). Inhibition of polarizing activity in the anterior limb bud is regulated by extracellular factors. Develop. Biol., 240, 443-456. pdf

Omi, M., Anderson, R. & Muneoka, K. (2002). Differential cell affinity and sorting of anterior and posterior cells during outgrowth of recombinant avian limb buds. Develop. Biol. 250, 292-304. pdf

Ngo-Muller, V., Li, S., Schaller, S.A., Han M., Farrington, J., Omi, M., Anderson, R., & Muneoka, K. (2004). FGF4 and Skeletal Morphogenesis. In: The Skeleton, Biochemical, Genetic, and Molecular Interactions in Development and Homeostasis (E.J. Massaro, J.M. Rogers, Eds), Humana Press, Totowa, New Jersey. pp 131-145. pdf

Our FGF studies have been extended to the developing mouse limb where we have discovered that FGF-4 modulates cell movements during digit morphogenesis. In this case, we find that FGF4 induces expansion of the digit tip leading to digit bifurcation. FGFs are known to regulate the expression of the Msx homeobox-containing genes (Msx1 and Msx2) during limb outgrowth, and we show that Msx1 gene expression is expanded in association with digit tip bifurcation. We had previously shown that digit tip regeneration in fetal and neonatal stages correlated with the expression domain of Msx1 in the nail region. In recent studies we have found that mutant mice that lack the Msx1 gene are defective in their ability to regenerate the digit tip. This is the first demonstration of a gene in mammals that is necessary to mount a digit tip regeneration response. Our studies also show that during digit formation the expression of the Bmp4 gene is dependent on Msx1 and Msx2 expression, and that adding BMP4 to amputated Msx1 mutant digits rescues the regeneration response. Finally, we have shown that inhibiting the activity of BMPs during digit tip regeneration in wildtype mice results in the inhibition of digit tip regeneration. In sum, our studies identify a signaling cascade that plays a regulatory role in a regeneration response in mammals. For the regenerating digit, this is the first regulatory signal that has been identified. Ongoing studies in the lab are following up on this important discovery and additional regulatory signals are being identified.

Muneoka, K. and Sassoon, D. (1992). Regeneration in developing vertebrate limbs. Develop. Biol. 152, 37-49. pdf

Reginelli, A.D., Wang, Y., Sassoon, D., and Muneoka, K. (1995). Digit tip regeneration correlates with regions of msx1 (formerly Hox7) expression in fetal and newborn mice. Development 121, 1065-1076. pdf

Muller, T.L., Ngo-Muller, V., Reginelli, A., Taylor, G., Anderson, R. and Muneoka, K. (1999). Regeneration in higher vertebrates: Limb buds and digit tips. Sem. Cell & Develop. Biol., 10, 405-413. pdf

Ngo-Muller, V. and Muneoka, K. (2000). Influence of FGF4 on digit morphogenesis during limb development in the mouse. Develop. Biol., 219, 224-236. pdf

Han, M., Yang, X., Farrington, J.E. & Muneoka, K. (2003). Digit regeneration is regulated by Msx1 and BMP4 in fetal mice. Development 130, 5123-5132. pdf

“BMP and FGF signaling in mammalian digit regeneration”, Ken Muneoka, P.I., National Institute of Child Health and Human Development, R01 HD043277, 2/1/04 – 1/31/09.

There are few documented instances in which human organs are not able to respond to injury by complete and perfect replacement of the damaged parts. Under the appropriate conditions the adult digit tip is one such organ. The digit is composed of a diverse number of distinct cell types making up the various tissues of the digit (e.g. epidermis, nail, nailbed, dermis, adipose, bone), and in response to amputation, a perfect replica of the tip of the digit including the nail and fingerprint regenerates. This regeneration response is level-specific and only occurs following amputations at the level of the nailbed. An additional aspect of digit regeneration is that wound healing occurs without the deposition of scar tissue, whereas wound healing of amputations just proximal to the nailbed (regeneration-incompetent) results in scar tissue formation. Thus, digit amputation in mammals represents a model system to study both organ regeneration and scar-free wound healing. It is therefore surprising that there has been little interest in studying this remarkable phenomenon, despite the fact that the most frequent body part injured is the hand and that in the US approximately 19,000 experience loss of a digit in any one year.

The mouse digit offers the best model for mammalian regeneration studies and recent findings provide evidence that the activity of the homeobox-containing gene Msx1, and BMP signaling is required for a successful regeneration response. These data come from in vitro and in vivo studies using the Msx1 mutant digit that displays a fetal digit regeneration defect. In an in vitro model the Msx1 regeneration defect is rescued by application of BMP4, and treatment of regenerating wildtype digits with the BMP binding protein, NOGGIN, represses the endogenous regeneration response. Our studies provide evidence that Bmp4 expression is under the control of the Msx homeobox-containing genes (Msx1 and Msx2). Studies proposed in this grant will investigate the role of BMP signaling in the control of fetal digit regeneration with the aim of inducing an enhanced response from proximal (non-regenerating) amputations. We also explore regulatory elements of this signaling pathway both downstream of BMP and upstream of Msx1. Finally, we will study the role of the wound epidermis in this regenerative response.