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Butterfly eyespots represent novel complex traits that display substantial diversity in number and size within and across species. Correlative gene expression studies have implicated a large suite of transcription factors, including Distal-less (Dll), Engrailed (En), and Spalt (Sal), in eyespot development in butterflies, but direct evidence testing the function of any of these proteins is still missing. Here we show that the characteristic two-eyespot pattern of wildtype Bicyclus anynana forewings is correlated with dynamic progression of Dll, En, and Sal expression in larval wings from four spots to two spots, whereas no such decline in gene expression ensues in a four-eyespot mutant. We then conduct transgenic experiments testing whether over-expression of any of these genes in a wild-type genetic background is sufficient to induce eyespot differentiation in these pre-patterned wing compartments. We also produce a Dll-RNAi transgenic line to test how Dll down-regulation affects eyespot development. Finally we test how ectopic expression of these genes during the pupal stages of development alters adults color patters. We show that over-expressing Dll in larvae is sufficient to induce the differentiation of additional eyespots and increase the size of eyespots, whereas down-regulating Dll leads to a decrease in eyespot size. Furthermore, ectopic expression of Dll in the early pupal wing led to the appearance of ectopic patches of black scales. We conclude that Dll is a positive regulator of focal differentiation and eyespot signaling and that this gene is also a possible selector gene for scale melanization in butterflies., (Copyright © 2013 Wiley Periodicals, Inc.)

A transmembrane glycoprotein recently identified on some tumor cells, extracellular matrix metalloproteinase inducer (EMMPRIN), has been shown to induce metalloproteinase (MMP) production by peritumor fibroblasts (PTF). We examined biopsy specimens of normal human oral mucosa and oral squamous cell carcinoma (SCC) for expression of EMMPRIN. In normal mucosa, EMMPRIN was expressed at the cell membrane throughout the epithelium with a slight enhancement along the basal cell layer. In oral SCC, EMMPRIN was expressed at the cell membrane throughout the entire lesion. Immunofluorescence microscopy localized EMMPRIN to the cell membrane in a highly invasive oral SCC cell line in agreement with our in vivo observations. Function-blocking antibodies to EMMPRIN significantly inhibited oral SCC cell migration on tenascin-C (TN-C) and fibronectin as well as invasion through a reconstituted basement membrane (RBM). We previously showed that soluble factors from SCC cells and PTF are required for deposition of a TN-C matrix. To determine whether EMMPRIN may modulate the release or expression of these soluble factors, we again used function-blocking antibodies. Antibodies to EMMPRIN completely inhibited the organization of TN-C matrices and partially reduced the deposition of FN matrices by oral SCC cell /PTF co-cultures. In addition, antibodies to EMMPRIN perturbed the expression of MMP-2. Moreover, antibodies to MMP-2 perturbed oral SCC cell invasion of an RBM by approx. 75%. Our results demonstrate that EMMPRIN is highly expressed in oral SCC, facilitates tumor cell motility, and mediates TN-C matrix deposition. Taken together, these results suggest that EMMPRIN may help regulate oral squamous cell carcinoma invasion.

We have shown that a fibronectin (FN) matrix is required for the organization of tenascin-C (TN-C) matrices by peritumor fibroblasts (PTF) cultured from tissue surrounding oral squamous-cell carcinoma (SCC). In the present study, we detected alternatively spliced FN containing both the EDA and EDB domains decorating the reactive stroma adjacent to the invading tumor nests in oral SCC biopsies. In vitro, PTF cells organized an extensive FN matrix rich in the EDA domain and containing a small amount of EDB. In contrast, normal human fibroblasts deposited a FN matrix which expressed only the EDA domain. PTF-conditioned medium (CM), shown to enhance migration of oral SCC cells on TN-C, was found to enhance their migration on FN and invasion of a reconstituted basement membrane. Addition of antibodies to FN to the PTF-CM inhibited SCC-cell migration on TN-C, and depletion of FN from the PTF-CM abolished its ability to induce migration or invasion by oral SCC cells, suggesting that FN promotes the migration and invasion of oral SCC cells. Western blots of the PTF-CM identified FN containing the EDA but not the EDB domain. When soluble FN was added to the control medium in the lower chamber of the Transwell system, SCC-cell migration increased significantly. These results demonstrate that both the EDA and the EDB domains of FN are expressed in the extracellular matrix of oral SCC in vivo and PTF in vitro and indicate that FN is the probable chemotactic factor in the PTF-conditioned medium.

We previously showed that the extracellular matrix component tenascin-C (TN-C) is upregulated in oral squamous cell carcinoma (SCC) compared with the normal oral mucosa. In this study we examined oral biopsy specimens of mild to moderate dysplasia or carcinoma in situ to study TN-C expression. We found that carcinoma in situ is the stage at which TN-C becomes widely expressed, suggesting it may be involved in the initial stages of tumor progression. To study TN-C matrix production in vitro, we used an invasive oral SCC cell line (HSC-3) and peri-tumor fibroblasts (PTF). Neither cell type organized a TN-C matrix when cultured alone; however, when co-cultured with HSC-3 cells, PTF were able to assemble a TN-C matrix. PTF retained the ability to organize a TN-C matrix when separated from the HSC-3 cells by a semi-permeable membrane, indicating that cell-cell contact is not necessary for TN-C matrix organization and suggesting that soluble factors may be involved. Moreover, PTF were induced to assemble TN-C matrices when grown in medium conditioned by both the PTF and HSC-3 cells. Antibodies to fibronectin (FN) and to the first FN type III repeat blocked both FN and TN-C matrix assembly, indicating that TN-C matrix organization is dependent on an FN template. Antibodies to alpha5, alphav and beta1 integrins also blocked TN-C matrix formation. When seeded onto FN matrices, the co-cultures were unaffected by the anti-integrin and anti-FN antibodies and were able to organize a TN-C matrix. Our results suggest that progression of malignant oral SCC is accompanied by an alteration of the normal ECM to one rich in TN-C, and that the organization of a TN-C matrix is dependent on soluble cues provided by both the SCC cells and the PTF.

In this study we identified tenascin-C (TN-C) and one of its integrin receptors, alpha(v)beta6, in oral squamous-cell carcinoma (SCC) specimens. Neither TN-C nor alpha(v)beta6 are expressed in normal oral mucosa. We also studied 2 human oral squamous-cell carcinoma cell lines: the highly invasive HSC-3 cells, and the poorly invasive SCC-25 cells. We determined that adhesion of these cells to TN-C involves both alpha2 and alpha(v) integrins. Migration on TN-C by oral SCC cells required fibroblast-conditioned medium and did not occur in its absence. This migration was blocked by anti-alpha2 and anti-alpha(v) antibodies and was partially inhibited by antibodies to hepatocyte growth factor, epidermal growth factor and transforming growth factor-beta1. When seeded on TN-C, the poorly invasive SCC-25 cells formed alpha(v)beta6-positive focal contacts; the HSC-3 cells did not. HSC-3, SCC-25 and PTF cells secrete TN-C into the culture medium, as determined by Western blot. However, when HSC-3 cells were inoculated into the floor of the mouth of nude mice, only murine TN-C could be identified in the reactive stroma adjacent to the resulting tumor nests, demonstrating that in vivo, HSC-3 cells do not secrete TN-C. Our results demonstrate that alpha(v)beta6 and tenascin-C are neo-expressed in oral squamous-cell carcinoma, and that the tumor stromal environment is influential in oral SCC behavior.