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The use of dielectrophoresis for the construction of artificial skin tissue with skin cells in follicle-like 3D cell aggregates in well-defined patterns is demonstrated. To analyse the patterns produced and to study their development after their formation a Virtual Instrument (VI) system was developed using the LabVIEW IMAQ Vision Development Module. A series of programming functions (algorithms) was used to isolate the features on the image (in our case; the patterned aggregates) and separate them from all other unwanted regions on the image. The image was subsequently converted into a binary version, covering only the desired microarray regions which could then be analysed by computer for automatic object measurements. The analysis utilized the simple and easy-to-use User-Specified Multi-Regions Masking (MRM) technique, which allows one to concentrate the analysis on the desired regions specified in the mask. This simplified the algorithms for the analysis of images of cell arrays having similar geometrical properties. By having a collection of scripts containing masks of different patterns, it was possible to quickly and efficiently develop sets of custom virtual instruments for the offline or online analysis of images of cell arrays in the database.

22 will be presented as scheduled, but will not be published due to lack of license agreement between authors and publisher. doi:10.1016/j.ydbio.2009.05.029 Program/Abstract # 23 Pathways controlling cell fate decisions in the early mouse embryo Elizabeth J. Robertson, Sebastian Arnold, Mathias Groszer, Elizabeth Bikoff Department of Pathology, Univ of Oxford, Oxford, UK Wellcome Trust Centre Human Genetics, Univ of Oxford, UK TGFβ pathways are instrumental in patterning the somatic lineages of the early mouse embryo, as well as for formation of the germ line. Previous experiments argue that graded Nodal activities control the very earliest cell fate decisions during axis patterning, and are essential for correct mesodermal patterning and specification of axial mesendoderm and definitive endoderm. During gastrulation high levels of nodal induce endoderm progenitors, whereas lower levels specify mesoderm. In addition Nodal directly acts to maintain undifferentiated trophoblast stem cells within the extraembryonic ectoderm and thereby maintain the expression of Bmp4 required for germ cell specification. Our recent studies show that the Tbox transcription factor Eomesodermin (Eomes), acting downstream of nodal signalling, plays multiple roles in the developing embryo. Eomes activities in the trophectoderm are required for maintaining trophoblast stem cells, while in the epiblast Eomes has two roles namely to promote nascent mesoderm to undergo EMT during gastrulation, and for specification of the definitive endoderm lineage. Interestingly Eomes is also transiently expressed in the subventricular zone of the developing cortex. Sox1.Cre mediated deletion causes microcephaly and severe behavioural defects. This can be attributed to a reduction in the expansion of the SVZ progenitor cells leading to a disturbance in the formation of upper cortical neurons. Thus Eomes has emerged as a key regulator of multiple processes in the mouse embryo. doi:10.1016/j.ydbio.2009.05.030 Program/Abstract # 24 Localized Xenopus Trim36 regulates cortical rotation and dorsal axis formation Douglas W. Houston, Tawny N. Cuykendall Department of Biology, The University of Iowa, Iowa City, IA, USA The activation of Wnt/beta-catenin signaling on the future dorsal side of the blastula is necessary and sufficient for axis formation in Xenopus and other vertebrates. Wnt signaling is initiated by dorsal enrichment of vegetally localized molecules following rotation of the egg cortex after fertilization. Both localized wnt11 mRNA and protein inhibitors of beta-catenin degradation, Dishevelled and GBP, have been implicated, but the mechanisms activating Wnt signaling in axis formation still remain elusive. Because vegetally localized RNAs are important for this process, we have conducted a microarray screen to identify novel mRNAs localized to the vegetal cortex. We present evidence that a localized mRNA encoding a Tripartite Motif Protein (Trim), Trim36, plays a critical role in Xenopus axis formation. Maternal antisense inhibition of Trim36 resulted in ventralized embryos, with reductions in dorsal beta-catenin accumulation and Wnt target gene expression. We further present experiments to identify the extent that Trim36 interacts with Wnt/beta-catenin signaling and cortical rotation mechanisms. doi:10.1016/j.ydbio.2009.05.031 Abstracts / Developmental Biology 331 (2009) 390–391 391

The hair follicles and the interfollicular epidermis of intact mature skin are maintained by distinct stem cell populations. Upon wounding, however, emigration of hair follicle keratinocytes to the interfollicular epidermis plays a role in acute stages of healing. In addition to this repair function, rapidly cycling cells of the upper hair follicle have been observed transiting to the interfollicular epidermis in neonatal skin. Here we report that an absence of hair follicle development leads to shortening and kinking of the mouse tail. These skeletal defects are reduced by stimulating keratinocyte proliferation, suggesting that they arise from impaired epidermal expansion. We confirm that rapidly cycling cells of the hair follicle emigrate to the interfollicular epidermis of the neonatal tail. These results suggest that an absence of hair follicles results in impaired skin growth that is unable to keep pace with the rapidly elongating axial skeleton of the tail. Thus, in addition to their role in wound repair, hair follicles can make a significant contribution to lateral expansion of the interfollicular epidermis in the absence of trauma.

Hair follicles have been observed to provide a major cellular contribution to epidermal healing, with emigration of stem-derived cells from the follicles aiding in wound reepithelialization. However, the functional requirements for this hair follicle input are unknown. Here we have characterized the keratinocyte stem cell status of mutant mice that lack all hair follicle development on their tail, and analyzed the consequent alterations in epidermal wound healing rate and mechanisms. In analyzing stem cell behavior in embryonic skin we found that clonogenic keratinocytes are relatively frequent in the ectoderm prior to hair follicle formation. However, their frequency in the interfollicular epidermis drops sharply by birth, at which time the majority of stem cells are present within the hair follicles. We find that in the absence of hair follicles cutaneous wounds heal with an acute delay in reepithelialization. This delay is followed by expansion of the region of activated epidermis, beyond that seen in normal haired skin, followed by appropriate wound closure. JID Journal Club article: for questions, answers, and open discussion about this article please go to http://network.nature.com/group/jidclub.

The 14-3-3 sigma (Stratifin; Sfn) is a cell cycle regulator intimately involved in the program of epithelial keratinization. 14-3-3 sigma is unique in that it is expressed primarily in epithelial cells and is frequently silenced in epithelial cancers. Despite its well-documented role as a cell cycle regulator and as a tumor suppressor, the function of 14-3-3 sigma in the intricate balance of proliferation and differentiation in epithelial development is poorly understood. A mutation in 14-3-3 sigma was found to be responsible for the repeated epilation (Er) phenotype. It has previously been shown that Sfn(+/Er) mice are characterized by repeated hair loss and regrowth, whereas Sfn(Er/Er) mice die at birth displaying severe oral fusions and limb abnormalities as a result of defects in keratinizing epithelia. Here we show that mice heterozygous for the 14-3-3 sigma mutation have severe defects in hair shaft differentiation, resulting in destruction of the hair shaft during morphogenesis. Furthermore, we report that the interfollicular epidermis and sebaceous glands are hyperproliferative, coincident with expanded nuclear Yap1 (Yes-associated protein 1)-a critical modulator of epidermal stem cell proliferation. We also report that hair follicle stem cells in the bulge cycle abnormally, raising important questions as to the role of 14-3-3 sigma in the bulge.

Mutations in members of the ectodysplasin (TNF-related) signalling pathway, EDA, EDAR, and EDARADD in mice and humans produce an ectodermal dysplasia phenotype that includes missing teeth and smaller teeth with reduced cusps. Using the keratin 14 promoter to target expression of an activated form of Edar in transgenic mice, we show that expression of this transgene is able to rescue the tooth phenotype in Tabby (Eda) and Sleek (Edar) mutant mice. High levels of expression of the transgene in wild-type mice result in molar teeth with extra cusps, and in some cases supernumerary teeth, the opposite of the mutant phenotype. The level of activation of Edar thus determines cusp number and tooth number during tooth development.

The development of several types of skin appendages is guided by prenatal ectodysplasin signaling. In this issue, Cui et al. report on the dose and duration of ectodysplasin signaling required for the maintenance and morphogenesis of different types of appendages. They report that achievement of an intimate arrangement between epithelial and mesenchymal cell populations correlates with the acquisition of autonomy from ectodysplasin stimulation.

Background Scaleless (sc/sc) chickens carry a single recessive mutation that causes a lack of almost all body feathers, as well as foot scales and spurs, due to a failure of skin patterning during embryogenesis. This spontaneous mutant line, first described in the 1950s, has been used extensively to explore the tissue interactions involved in ectodermal appendage formation in embryonic skin. Moreover, the trait is potentially useful in tropical agriculture due to the ability of featherless chickens to tolerate heat, which is at present a major constraint to efficient poultry meat production in hot climates. In the interests of enhancing our understanding of feather placode development, and to provide the poultry industry with a strategy to breed heat-tolerant meat-type chickens (broilers), we mapped and identified the sc mutation. Results Through a cost-effective and labour-efficient SNP array mapping approach using DNA from sc/sc and sc/+ blood sample pools, we map the sc trait to chromosome 4 and show that a nonsense mutation in FGF20 is completely associated with the sc/sc phenotype. This mutation, common to all sc/sc individuals and absent from wild type, is predicted to lead to loss of a highly conserved region of the FGF20 protein important for FGF signalling. In situ hybridisation and quantitative RT-PCR studies reveal that FGF20 is epidermally expressed during the early stages of feather placode patterning. In addition, we describe a dCAPS genotyping assay based on the mutation, developed to facilitate discrimination between wild type and sc alleles. Conclusions This work represents the first loss of function genetic evidence supporting a role for FGF ligand signalling in feather development, and suggests FGF20 as a novel central player in the development of vertebrate skin appendages, including hair follicles and exocrine glands. In addition, this is to our knowledge the first report describing the use of the chicken SNP array to map genes based on genotyping of DNA samples from pooled whole blood. The identification of the sc mutation has important implications for the future breeding of this potentially useful trait for the poultry industry, and our genotyping assay can facilitate its rapid introgression into production lines.