Cell death and cell proliferation are basic cellular processes that need to be precisely controlled during embryonic development. The developing vertebrate limb illustrates particularly well how correct morphogenesis depends on the appropriate spatial and temporal balance between cell death and cell proliferation. Precise knowledge of the patterns of cell proliferation and cell death during limb development is required to understand how their modifications may contribute to the generation of the great diversity of limb phenotypes that result from spontaneous mutations or induced genetic manipulations. We have performed a comprehensive analysis of the patterns of cell death, assayed by terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick end-labeling (TUNEL), and cell proliferation, assayed by anti-phosphorylated histone H3 immunohistochemistry, in consecutive sections of forelimbs and hindlimbs covering an extensive period of chick and mouse limb development. Our results confirm and expand previous reports and show common and specific areas of cell death for each species. Mitotic cells were found scattered in a uniform distribution across the early limb bud, with the exception of the areas of cell death in which mitotic cells were scarce. At later stages, mitotic cells were seen more abundantly in the digital tips. The aim of the present study was to satisfy the need for organized data sets describing these processes, which will allow the side-by-side comparison between the two major model organisms of limb development, i.e., the mouse and the chick.

Summary. Using a histochemical technique, alkaline phosphatase has been shown to appear in the stroma of the mouse uterus during implantation and after initiation of the oil-induced deciduoma.In sections of pregnant uteri the enzyme first appeared on the 5th day after mating as a small crescentic area in the stroma on the antimesometrial side of the uterus close to the implanting blastocyst.In pseudopregnant mice after instillation of oil into the uterine lumen (on the 4th day) a similar crescentic area of alkaline phosphatase activity appeared around the uterine lumen on the antimesometrial side on the 5th day.The epithelium in both cases is intact on the 5th day but degeneration occurs on the antimesometrial side on the 6th day. This suggests that epithelial degeneration is an inherent property of the uterus during the period of implantation, and is not directly caused by the activities of the giant cells of the trophoblast.There was no alkaline phosphatase activity in the uterine stroma of pseudopregnant untreated mice on the 4th, 5ht and 6th days after sterile mating and the epithelium showed no evidence of degeneration. Further, injection of oil into the uteri of ovariectomized mice failed to cause epithelial degeneration or result in alkaline phosphatase activity in the stroma.The bearing of these results on the role of the blastocyst in implantation is discussed.

The origin of birds and avian flight from within the archosaurian radiation has been among the most contentious issues in paleobiology. Although there is general agreement that birds are related to theropod dinosaurs at some level, debate centers on whether birds are derived directly from highly derived theropods, the current dogma, or from an earlier common ancestor lacking suites of derived anatomical characters. Recent discoveries from the Early Cretaceous of China have highlighted the debate, with claims of the discovery of all stages of feather evolution and ancestral birds (theropod dinosaurs), although the deposits are at least 25 million years younger than those containing the earliest known bird Archaeopteryx. In the first part of the study we examine the fossil evidence relating to alleged feather progenitors, commonly referred to as protofeathers, in these putative ancestors of birds. Our findings show no evidence for the existence of protofeathers and consequently no evidence in support of the follicular theory of the morphogenesis of the feather. Rather, based on histological studies of the integument of modern reptiles, which show complex patterns of the collagen fibers of the dermis, we conclude that "protofeathers" are probably the remains of collagenous fiber "meshworks" that reinforced the dinosaur integument. These "meshworks" of the skin frequently formed aberrant patterns resembling feathers as a consequence of decomposition. Our findings also draw support from new paleontological evidence. We describe integumental structures, very similar to "protofeathers," preserved within the rib area of a Psittacosaurus specimen from Nanjing, China, an ornithopod dinosaur unconnected with the ancestry of birds. These integumental structures show a strong resemblance to the collagenous fiber systems in the dermis of many animals. We also report the presence of scales in the forearm of the theropod ornithomimid (bird mimic) dinosaur, Pelecanimimus, from Spain. In the second part of the study we examine evidence relating to the most critical character thought to link birds to derived theropods, a tridactyl hand composed of digits 1-2-3. We maintain the evidence supports interpretation of bird wing digit identity as 2,3,4, which appears different from that in theropod dinosaurs. The phylogenetic significance of Chinese microraptors is also discussed, with respect to bird origins and flight origins. We suggest that a possible solution to the disparate data is that Aves plus bird-like maniraptoran theropods (e.g., microraptors and others) may be a separate clade, distinctive from the main lineage of Theropoda, a remnant of the early avian radiation, exhibiting all stages of flight and flightlessness.

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The aim of the present report is to provide a detailed description of the morphogenesis and initial differentiation of the long tendons of the chick foot, the long autopodial tendons (LAT), from day 6 to day 11 of development. The fine structure of the developing LAT was studied by light and transmission electron microscopy. The characterization by immunofluorescent techniques of the extracellular matrix was performed using laser scanning confocal (tenascin, elastin, fibrillin, emilin, collagen type I, II, III, IV and VI) or routine fluorescence (tenascin, 13F4) microscopy. In addition, cell proliferation in pretendinous blastemas was analyzed by the detection of BrdU incorporation by immunofluorescence. The light microscopic analysis permitted the identification of different stages during LAT morphogenesis. The first stage is the formation of a thick ectoderm-mesenchyme interface along the digital rays, followed by the differentiation of the "mesenchyme lamina", an extracellular matrix tendon precursor, and ending with the formation and differentiation of the cellular condensation that forms the tendon blastema around this lamina. The immunofluorescence study revealed the presence and arrangement of the different molecules analyzed. Tenascin and collagen type VI are precocious markers of the developing tendons and remain present during the whole process of tendon formation. Collagen type I becomes mainly restricted to the developing tendons from day 7.5. Collagens type II and IV are never detected in the developing tendons, while a faint labeling for collagen type III is first detected at day 7. The analysis of the distribution of the elastic matrix components in the developing tendons is a major contribution of our study. Elastin was detected in the periphery of the tendons from day 8 and also in fibrils anchoring the tendons to the skeletal elements. At the same stage, emilin strongly stains the core of the tendon rods, while fibrillin is detected a little later. Our study indicates the existence of an ectoderm-mesoderm interaction at the first stage of the tendon formation. In addition, our results show the different spatial and temporal pattern of distribution of extracellular matrix molecules in developing tendons. Of special importance are the findings concerning the tendinous elastic matrix and its possible role in tendon maturation and stabilization.

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Carbon particles and isotopic quail grafts were used as markers to study the salient features of the fate map of the chick forelimb between stages 20 and 27. The grafting technique confirmed the reliability of the carbon method: they both revealed striking asymmetries in which apical mesodermal tissue was progressively displaced in a proximal direction (as would be expected on the basis of growth by net apical addition of tissue) but also in a preaxial direction, while postaxial tissue became elongated in the direction of limb outgrowth. Ectoderm showed a similar preaxial-postaxial asymmetry but became displaced from initially underlying mesoderm. In marked contrast to mesoderm, distal ectoderm remained at a constant distance from the apical ectodermal ridge (or became incorporated into it), thus implying that the ectodermal sheet is anchored distally and grows by uniform stretching proximally. Within the ectoderm itself, the outer peridermal layer is displaced distally relative to the underlying epidermal basal layer. Peripheral mesoderm showed patterns of displacement which were intermediate between those of ectoderm and chondrogenic core mesoderm. It is argued that such morphogenetic phenomena are integral components of developmental mechanisms of significance in the control of pattern generation. Implications of the interpretation and use of the fate map in relation to theories of limb development, particularly those based on mechanisms defined in terms of limb axes, are reviewed.