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We developed a three-dimensional assay prepared from primary breast cancer tissue and quantified tumor response to tamoxifen therapy. Freshly harvested breast cancer biopsies obtained at the time of curative surgical resection were fragmented and embedded into collagen I cushions. Changes in proliferation, apoptosis and tumor volume in response to tamoxifen treatment were quantified using image analysis software and optical projection tomography. Individual and collective invasion of epithelial cells into the surrounding collagen I was observed over the course of the experiment using phase contrast light microscopy and histopathological methods. Addition of tamoxifen to preparations derived from ER+ tumors demonstrated a range of response as measured by proliferative and apoptotic markers. In keeping with published data, tamoxifen reduced the percentage of apoptotic cells expressing cleaved caspase-3 (p = 0.02, Poisson regression analysis). Tamoxifen also reduced residual epithelial volume in ER+ tumors (p = 0.001, Mann-Whitney test), but not in ER low/- tumors (p = 0.78). Changes in tumor volume, as measured by optical projection tomography, allowed stratification into responsive and non-responsive tumors. The model mirrors observations of breast cancer response and histopathological changes to tamoxifen in neo-adjuvant trials. This assay provides a method of screening a battery of therapeutics against individual cancers, informing subsequent design of neo-adjuvant trials., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

We describe lens defects in heterozygous small eye mice, and autonomous deficiencies of Pax6(+/-) cells in the developing lens of Pax6(+/+) <--> Pax6(+/-) chimeras. Two separate defects of the lens were identified by analyzing the distribution of heterozygous cells in chimeras: Pax6(+/-) cells are less readily incorporated into the lens placode than wild type, and those that are incorporated into the lens are not maintained efficiently in the proliferating lens epithelium. The lens of chimeric eyes is, therefore, predominantly wild type from embryonic day 16.5 onwards, whereas heterozygous cells contribute normally to all other eye tissues. Eye size and defects of the iris and cornea are corrected in fetal and adult chimeras with up to 80% mutant cells. Therefore, these aspects of the phenotype may be secondary consequences of primary defects in the lens, which has clinical relevance for the human aniridia (PAX6(+/-)) phenotype.

Mouse mesenchyme was grafted into chick embryos to investigate the control of mesenchymal expression of Msx-1 in the developing limb and face. In situ hybridization, using species-specific probes, allows a comparison between Msx-1 expression in the graft and the host tissue. The results show that Msx-1 expression in both limb-to-limb and face-to-face grafts corresponds closely with the level of Msx-1 expression in the surrounding chick mesenchyme. Cells in grafts that end up within the host domain of Msx-1 express the gene irrespective of whether they were from normally expressing, or non-expressing, regions. Therefore Msx-1 expression in both the developing limb and the developing face appears to be position-dependent. Mesenchyme from each of the three major facial primordia behaved in the same way when grafted to the chick maxillary primordium. Reciprocal grafts between face and limb gave a different result: Msx-1 expression was activated when facial mesenchyme was grafted to the limb but not when limb mesenchyme was grafted to the face. This suggests either that there are quantitative or qualitative differences in two local signalling systems or that additional factors determine the responsiveness of the mesenchyme cells.

We have isolated, sequenced and examined the expression pattern of two tandemly arranged homeobox-containing genes from the chicken. The predicted amino acid sequences of the homeodomain and the adjacent carboxyterminal portion of the protein of the first gene is virtually identical (99%) to that of murine homeobox 2.1 and hence we refer to it as Ghox 2.1 (Gallus homeobox). The closest mouse homologue of the second homeodomain is Hox 2.2 (95% identical within the homeobox), and hence referred to as Ghox 2.2. Northern analysis of embryonic RNA reveals major transcripts of 2 kb for Ghox 2.1 and 1.7 kb for Ghox 2.2. To investigate the transcript pattern, embryos of various stages were dissected into heads, trunks and limb buds and the RNA was analysed by Northern blotting and RNase protection assays. Ghox 2.1 transcripts are present in all three regions. Ghox 2.2 RNA is found in trunks and limb buds, but it is strikingly absent from the developing head. In situ hybridization with 35S-labelled antisense riboprobes derived from Ghox 2.1 demonstrates that this gene is expressed at high levels in spinal chord, myelencephalon and mesonephros. Dorsal root ganglia and the lung rudiment also contain Ghox 2.1 message, but in somewhat lower amounts. Mid- and forebrain, the heart, presomitic mesenchyme and notochord do not contain detectable levels of Ghox 2.1 mRNA. Of particular interest is the expression of Ghox 2.1 in a well-defined patch of mesenchymal tissue situated in an anterioproximal region of the limb bud.

The development of the chick face involves outgrowth of buds of tissue, accompanied by the differentiation of cartilage and bone in spatially defined patterns. To investigate the role of epithelial-mesenchymal interactions in facial morphogenesis, small fragments of facial tissue have been grafted to host chick wing buds to continue their development in isolation. Fragments of the frontonasal mass give rise to typical upper-beak-like structures: a long central rod of cartilage, the prenasal cartilage and an egg tooth. Meckel's cartilage, characteristic of the lower beak, develops from fragments of the mandible. Removal of the ectoderm prior to grafting leads to truncated development. In fragments of frontonasal mass mesenchyme only a small spur of cartilage differentiates and there is no outgrowth. The mandible is less affected; a rod of cartilage still forms but the amount of outgrowth is reduced. Retinoid treatment of chick embryos specifically affects the development of the upper beak and outgrowth and cartilage differentiation in the frontonasal mass are inhibited. The mandibles, however, are unaffected and develop normally. In order to investigate whether the epithelium or the mesenchyme of the frontonasal mass is the target of retinoid action, recombinations of retinoid-treated and untreated facial tissue have been grafted to host wing buds. Recombinations of retinoid-treated frontonasal mass ectoderm with untreated mesenchyme develop normally whereas recombinations of untreated ectoderm with retinoid-treated mesenchyme lead to truncations. The amount of outgrowth in fragments of mandibular tissue is slightly reduced when either the ectoderm or the mesenchyme has been treated with retinoids. These recombination experiments demonstrate that the mesenchyme of the frontonasal mass is the target of retinoid action. This suggests that retinoids interfere with the reciprocal epithelial-mesenchymal interactions necessary for outgrowth and normal upper beak development.

It has long been proposed that concentration gradients of morphogens provide cues to specify cell fate in embryonic fields. Recent work in a variety of vertebrate systems give bona fide evidence that retinoic acid, the biologically active form of vitamin A, is a candidate for such a morphogen. In the developing chick wing, for example, locally applied retinoic acid triggers striking changes in the pattern along the anteroposterior axis. Instead of giving rise to a wing with the normal 234 digit pattern, wing buds treated with retinoic acid develop a 432234 mirror-image symmetrical digit pattern. For this review, we focus on three aspects of limb morphogenesis. (1) We summarize the experimental evidence supporting the notion that retinoic acid is a candidate morphogen. (2) Limb buds contain high levels of cellular retinoic-acid-binding protein (CRABP). Using order of magnitude calculations, we evaluate how the concentration of CRABP might affect the occupancy state of the retinoic acid receptor. (3) We discuss the spatio-temporal expression pattern of homeobox-containing genes in the developing limb and speculate about the possibility that retinoic acid influences the pattern of expression of homeobox genes.

Retinoic acid was locally applied to presumptive limb regions of chick embryos to find out the earliest time at which the limb pattern can be reprogrammed. When beads soaked in retinoic acid were placed in the appropriate positions in embryos at stage 10 or older, duplicated or reduced leg patterns resulted. To pin point the time at which the cells in the limb rudiment respond to the retinoid, beads were removed at various times and the lengths of exposure required to reprogramme limb development found. The early limb rudiments require longer exposures to give duplications than late rudiments. The effective treatment periods last at least until stage 17 when the limb bud and apical ectodermal ridge develop. In contrast, the length of exposure to reduce the limb is constant at early stages. Retinoids first start acting to produce duplicated structures between stages 10 and 13. Therefore, retinoids appear to begin to reprogramme the cells as soon as they are determined to give rise to a limb.

Pattern formation is the developmental process that leads to the spatial ordering of cell differentiation. We have explored the problem of pattern formation in the development of the face of chick embryos. At early stages, the developing face consists of a series of small buds of tissue, the facial primordia that encircle the primitive mouth. The concepts of positional information provide a framework for considering how the patterns of differentiated cells are generated in the face. We suggest that the cranial neural crest cells must first be informed to which facial primordium they belong and then of their position within that primordium. The cells of the early primordia appear indistinguishable. However, when the mesenchyme cells are placed in high-density culture, cartilage differentiates. The extent and pattern of cartilage differentiation is characteristic for the cell population of each facial primordium. Myogenic cells also differentiate in the cultures, but the proportion of myogenic cells is independent of the extent of chondrogenesis. Within the facial primordia, a set of epithelial-mesenchymal interactions appears to be required for outgrowth and pattern formation along the proximodistal axis of the chick beaks. In culture, face epithelium locally inhibits cartilage differentiation and suggests that another set of epithelial-mesenchymal interactions may be involved in cell patterning. The mechanisms involved in specifying the mediolateral axis of the face, for example, the midpoint of the upper beak, are not known. Vitamin A derivatives, collectively known as retinoids, affect the development of the face of chick embryos and lead to a specific facial defect. Upper beak development is inhibited but the lower beak develops normally. The response to retinoids could be related to the specification of cells to belong to the facial primordium that will form the upper beak. Alternatively, retinoids may interfere with positional cues that operate to inform cells of their position within that primordium.