Defects and rescue of the minor salivary glands in Eda pathway mutants

Despite their importance to oral health, the mechanisms of minor salivary gland (SG) development are largely unexplored. Here we present in vivo and in vitro analyses of developing minor SGs in wild type and mutant mice. Eda, Shh and Fgf signalling pathway genes are expressed in these glands from an early stage of development. Developing minor SGs are absent in Eda pathway mutant embryos, and these mice exhibit a dysplastic circumvallate papilla with disrupted Shh expression. Supplementation of Eda pathway mutant minor SG explants with recombinant EDA rescues minor SG induction. Supplementation with Fgf8 or Shh, previously reported targets of Eda signalling, leads to induction of gland like structures in a few cases, but these fail to develop into minor SGs.     

Stimulation of ectodermal organ development by Ectodysplasin-A1

Organs developing as ectodermal appendages share similar early morphogenesis and molecular mechanisms. Ectodysplasin, a signaling molecule belonging to the tumor necrosis factor family, and its receptor Edar are required for normal development of several ectodermal organs in humans and mice. We have overexpressed two splice forms of ectodysplasin, Eda-A1 and Eda-A2, binding to Edar and another TNF receptor, Xedar, respectively, under the keratin 14 (K14) promoter in the ectoderm of transgenic mice. Eda-A2 overexpression did not cause a detectable phenotype. On the contrary, overexpression of Eda-A1 resulted in alterations in a variety of ectodermal organs, most notably in extra organs. Hair development was initiated continuously from E14 until birth, and in addition, the transgenic mice had supernumerary teeth and mammary glands, phenotypes not reported previously in transgenic mice. Also, hair composition and structure was abnormal, and the cycling of hairs was altered so that the growth phase (anagen) was prolonged. Both hairs and nails grew longer than normal. Molar teeth were of abnormal shape, and enamel formation was severely disturbed in incisors. Furthermore, sweat gland function was stimulated and sebaceous glands were enlarged. We conclude that ectodysplasin-Edar signaling has several roles in ectodermal organ development controlling their initiation, as well as morphogenesis and differentiation.     

Endostatin suppresses colorectal tumor-induced lymphangiogenesis by inhibiting expression of fibronectin extra domain A and integrin α9

Endostatin is a natural occurring anti-angiogenic peptide and has been shown to inhibit tumor lymphangiogenesis by suppressing the expression of tumor-stimulating growth factors. We have previously shown that fibronectin alternative extra domain A (EDA) facilitates lymphangiogenesis of colorectal tumors. Since it is known that EDA interacts with integrin α9 in the lymphatic endothelial cells (LECs), we hypothesized that endostatin may target EDA-integrin α9 pathway to inhibit colorectal tumor-induced lymphangiogenesis. To test this hypothesis, we examined the effect of endostatin on EDA secreted by SW480 colorectal cancer cells and treated human LECs with different doses of endostatin in the presence of conditional medium from SW480 cells. We found that endostatin significantly reduced EDA secretion by SW480 cells and the expression of integrin α9 in LECs. Immunofluorescence studies showed that EDA and integrin α9 colocalized on the cell membrane of LECs and these colocalizations were dramatically reduced by endostatin. Co-immunoprecipitation studies demonstrated that EDA interacted with integrin α9 in LECs, and showed that endostatin treatment inhibited the formation of EDA-integrin α9 complex in LECs. Furthermore, we found that the arrangement and polarity of LEC cytoskeletons were destroyed by endostatin substantially, leading to a reduced formation of tube-like structures of LECs and a suppressed chemotaxis of LECs toward SW480 cells. Consistently, EDA and integrin α9 expressions as well as lymphangiogenesis were significantly suppressed by endostatin in colorectal cancer xenografts. In conclusion, our results suggest that endostatin reduces colorectal tumor-induced lymphangiogenesis, at least in part, by inhibiting EDA-integrin α9 pathway.     

MULTIPLE EVOLUTIONARY PATHWAYS TO DECREASED LATERAL PLATE COVERAGE IN FRESHWATER THREESPINE STICKLEBACKS

Adaptation to novel environments can be based either on standing genetic variation or variation attributable to new mutations. When standing genetic variation for a functional adaptation is lacking, and variation due to new mutations is not yet available, adaptation is possible only through alternative functional solutions. Reduction in the number of bony lateral plates as an adaptation to freshwater colonization by marine threespine sticklebacks (Gasterosteus aculeatus) has occurred in numerous independent cases through allelic substitution in the ectodysplasin‐a (Eda) gene. Studying the phenotypic and genetic variation in plate number and size in five marine and six freshwater threespine stickleback populations, we found that when variation in Eda was limiting (i.e., alleles associated with the low‐plate morph were missing or in extremely low frequency), plate number reduction did not take place in freshwater populations, but reduced lateral plate coverage was achieved by a reduction in the size of lateral plates. Our results suggest that this phenotypically and genetically discrete "small‐plated" threespine stickleback—which is the dominant form in three northern European freshwater populations—may be functionally equivalent to the low‐plated morph and hence, serve as an example of convergent evolution toward functional similarity in the face of genetic constraints.     

Phosphorus limitation does not drive loss of bony lateral plates in freshwater stickleback (Gasterosteus aculeatus)

Connecting the selective forces that drive the evolution of phenotypes to their underlying genotypes is key to understanding adaptation, but such connections are rarely tested experimentally. Threespine stickleback (Gasterosteus aculeatus) are a powerful model for such tests because genotypes that underlie putatively adaptive traits have been identified. For example, a regulatory mutation in the Ectodysplasin (Eda) gene causes a reduction in the number of bony armor plates, which occurs rapidly and repeatedly when marine sticklebacks invade freshwater. However, the source of selection on plate loss in freshwater is unknown. Here, we tested whether dietary reduction of phosphorus can account for selection on plate loss due to a growth advantage of low-plated fish in freshwater. We crossed marine fish heterozygous for the 16 kilobase freshwater Eda haplotype and compared the growth of offspring with different genotypes under contrasting levels of dietary phosphorus in both saltwater and freshwater. Eda genotype was not associated with growth differences in any treatment, or with mechanisms that could mitigate the impacts of phosphorus limitation, such as differential phosphorus deposition, phosphorus excretion, or intestine length. This study highlights the importance of experimentally testing the putative selective forces acting on phenotypes and their underlying genotypes in the wild.     

Ectodysplasin regulates pattern formation in the mammalian hair coat

In mammalian skin, hair follicles develop at regular intervals and with site-specific morphologies. This process generates distinct patterns of hair, but the mechanisms that establish these patterns remain largely unknown. Here we present evidence of follicular patterning by ectodysplasin-A1 (Eda-A1), a signaling protein necessary for the proper development of hair and other appendages. In transgenic mice, Eda-A1 was targeted to the epithelial compartment of the developing skin. At periodic locations, multiple hair follicles were induced side by side, without any interfollicular space. These follicles grew into the dermis as a fusion and subsequently branched to create discrete stalks and hair bulbs. Thus, at sites where interfollicular skin normally forms, hair follicles developed instead. This result shows that Eda-A1 can regulate basic developmental decisions, as cells were switched from interfollicular to follicular fates. Given these effects, it is likely that Eda-A1 is among the key regulators of pattern formation in the skin.