Urodele amphibians regenerate appendages through the recruitment of progenitor cells into a blastema that rebuilds the lost tissue. Blastemal formation is accompanied by extensive remodeling of the extracellular matrix. Although this remodeling process is important for appendage regeneration, it is not known whether the remodeled matrix directly influences the generation and behavior of blastemal progenitor cells. By integrating in vivo 3-dimensional spatiotemporal matrix maps with in vitro functional time-lapse imaging, we show that key components of this dynamic matrix, hyaluronic acid, tenascin-C and fibronectin, differentially direct cellular behaviors including DNA synthesis, migration, myotube fragmentation and myoblast fusion. These data indicate that both satellite cells and fragmenting myofibers contribute to the regeneration blastema and that the local extracellular environment provides instructive cues for the regenerative process. The fact that amphibian and mammalian myoblasts exhibit similar responses to various matrices suggests that the ability to sense and respond to regenerative signals is evolutionarily conserved.

Unlike humans, certain adult vertebrates such as newts and zebrafish possess extraordinary abilities to functionally regenerate lost appendages and injured organs, including cardiac muscle. Here, we present new evidence that a remodeled extracellular matrix (ECM) directs cell activities essential for cardiac muscle regeneration. Comprehensive mining of DNA microarrays and Gene Ontology term enrichment analyses for regenerating newt and zebrafish hearts revealed that distinct ECM components and ECM-modifying proteases are among the most significantly enriched genes in response to local injury. In contrast, data analyses for mammalian cardiac injury models indicated that inflammation and metabolic processes are the most significantly activated gene groups. In the regenerating newt heart, we show dynamic spatial and temporal changes in tenascin-C, hyaluronic acid, and fibronectin ECM distribution as early as 3 days postamputation. Linked to distinct matrix remodeling, we demonstrate a myocardium-wide proliferative response and radial migration of progenitor cells. In particular, we report dramatic upregulation of a regeneration-specific matrix in the epicardium that precedes the accumulation and migration of progenitor cells. For the first time, we show that the regenerative ECM component tenascin-C significantly increases newt cardiomyocyte cell cycle reentry in vitro. Thus, the engineering of nature-tested extracellular matrices may provide new strategic opportunities for the enhancement of regenerative responses in mammals.

Ganglioside GM3 inhibits epidermal growth factor (EGF)-dependent cell proliferation in a variety of cell lines. Both in vitro and in vivo, this glycosphingolipid inhibits the kinase activity of the EGF receptor (EGFR). Furthermore, membrane preparations containing EGFR can bind to GM3-coated surfaces. These data suggest that GM3 may interact directly with the EGFR. In this study, the interaction of gangliosides with the extracellular domain (ECD) of the EGFR was investigated. The purified human recombinant ECD from insect cells bound directly to ganglioside GM3. The ganglioside interaction site appears to be distinct from the EGF-binding site. In agreement with previous reports on the effects of specific gangliosides on EGFR kinase activity, the ECD preferentially interacted with GM3. The order of relative binding of other gangliosides investigated was as follows: GM3 > > GM2, GD3, GM4 > GM1, GD1a, GD1b, GT1b, GD2, GQ1b > lactosylceramide. These data suggest that NeuAc-lactose is essential for binding and that any sugar substitution reduces binding. In agreement with the specificity of soluble ECD binding to gangliosides, GM3 specifically inhibited EGFR autophosphorylation. Identification of a ganglioside interaction site on the ECD of the EGFR is consistent with the hypothesis that endogenous GM3 may function as a direct modulator of EGFR activity.

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Recent work on the PDZ-LIM protein family has revealed that it has important activities at the cellular level, mediating signals between the nucleus and the cytoskeleton, with significant impact on organ development. We review and integrate current knowledge about the PDZ-LIM protein family and propose a new functional role, sequestering nuclear factors in the cytoplasm. Characterized by their PDZ and LIM domains, the PDZ-LIM family is comprised of evolutionarily conserved proteins found throughout the animal kingdom, from worms to humans. Combining two functional domains in one protein, PDZ-LIM proteins have wide-ranging and multi-compartmental cell functions during development and homeostasis. In contrast, misregulation can lead to cancer formation and progression. New emerging roles include interactions with integrins, T-box transcription factors, and receptor tyrosine kinases. Facilitating the assembly of protein complexes, PDZ-LIM proteins can act as signal modulators, influence actin dynamics, regulate cell architecture, and control gene transcription.Keywords: actin cytoskeleton; organogenesis; PDZ-LIM; scaffold; T-box Evolutionarily conserved building blocks for a heterogeneous protein familyThe PDZ-LIM family is comprised of proteins with multiple functional domains, and all share a PDZ domain combined with at least one LIM domain. As protein-protein interaction modules, PDZ and LIM domains act as scaffolds, binding to filamentous actin-associated proteins, a range of cytoplasmic signaling molecules, and nuclear proteins, allowing this family to carry out diverse functions during development and adulthood.( Figure 1 shows the architecture of the PDZ-LIM protein subfamilies and their phylogenetic relationship. While both the PDZ and the LIM domains are often found in combination with other peptide motifs or domains, the coevolution of the two invariant functional domains, PDZ and LIM, indicate high functional relevance and, thus, is of particular interest and the focus of this review. All family members associate with the actin cytoskeleton, and this property, organizing protein complexes at the cytoskeleton, may serve a range of unexpected, yet important biological roles. PDZ domainFound in bacteria, yeast, and throughout the animal kingdom, the PDZ domain is one of the most common protein-protein binding domains. (17,19) It is characterized by a highly conserved sequence of 80-90 amino acids consisting of

Tbx5 is involved in congenital heart disease, however, the mechanisms leading to organ malformation are greatly unknown. We hypothesized a model by which the Tbx5 binding protein Pdlim7 controls nuclear/cytoplasmic shuttling and function of the transcription factor. Using the zebrafish, we present in vivo significance for an essential role of Tbx5/Pdlim7 protein interaction in the regulation of cardiac formation. Knock-down of Pdlim7 results in a non-looped heart, strikingly reminiscent of the tbx5 heartstrings mutant phenotype. However, while misregulation of Pdlim7 and Tbx5 produce similar aberrant cardiac morphology, molecular and histological analysis uncovered that the Pdlim7 and Tbx5 cardiac phenotypes are due to opposite effects on valve development. Loss of Pdlim7 function causes no valve tissue to develop while lack of Tbx5 results in increased valve tissue. These opposing defects are evident before valve formation and are the result of distinct gene misregulation during specification of the atrio-ventricular (AV) boundary. We show that Pdlim7/Tbx5 interactions affect the expression of Tbx5 target genes nppa and tbx2b at the AV boundary, and their domains of misexpression directly correlate with the identified valve defects. These studies demonstrate that controlling the correct balance of Tbx5 activity is crucial for the specification of the AV boundary and valve formation.

The limb- and heart-specific Tbx5 transcription factor coexpresses with and directly binds to the novel PDZ-LIM domain protein, LMP4. LMP4 is distributed in the cytoplasm associated with the actin cytoskeleton. In the presence of LMP4, Tbx5 shuttles dynamically between the nucleus and cytoplasm and, in a complex with LMP4, localizes to actin filaments. Nuclear and cytoplasmic Tbx5 distribution in developing chicken wings suggests the functional significance of the LMP4–Tbx5 interaction. In primary epicardial cells, we demonstrate that Tbx5 protein subcellular relocalization can be stimulated by external signals that induce cell differentiation. To test whether the relocalization from nuclear to cytoplasmic sites interferes with downstream gene expression, we used limb-specific Fgf10 and heart-specific Anf promoter-luciferase reporters and demonstrate that LMP4 acts as a repressor of Tbx5 activity. These studies reveal a previously unknown mechanism for Tbx transcription factor regulation in vertebrate limb and heart development and provide a better understanding of the molecular basis of hand/heart birth defects associated with Tbx5 mutations.