The bHLH transcription factor dHAND is required for establishment of SHH signaling by the limb bud organizer in posterior mesenchyme, a step crucial to development of vertebrate paired appendages. We show that the transcriptional repressor GLI3 restricts dHAND expression to posterior mesenchyme prior to activation of SHH signaling in mouse limb buds. dHAND, in turn, excludes anterior genes such as Gli3 and Alx4 from posterior mesenchyme. Furthermore, genetic interaction of GLI3 and dHAND directs establishment of the SHH/ FGF signaling feedback loop by restricting the BMP antagonist GREMLIN posteriorly. These interactions polarize the nascent limb bud mesenchyme prior to SHH signaling.

Growth of limb cells in culture conditions with subsequent in vivo transplantation allows the dissection of limb patterning.

We have analyzed a new limb mutant in the chicken that we nameoligozeugodactyly (ozd). The limbs of this mutant have a longitudinal postaxial defect, lacking the posterior element in the zeugopod(ulna/fibula) and all digits except digit 1 in the leg. Classical recombination experiments show that the limb mesoderm is the defective tissue layer in ozd limb buds. Molecular analysis revealed that theozd limbs develop in the absence of Shh expression, while all other organs express Shh and develop normally. NeitherPtc1 nor Gli1 are detectable in mutant limb buds. However,Bmp2 and dHAND are expressed in the posterior wing and leg bud mesoderm, although at lower levels than in normal embryos. Activation ofHoxd11-13 occurs normally in ozd limbs but progressively declines with time. Phase III of expression is more affected than phase II,and expression is more severely affected in the more 5′ genes. Interestingly, re-expression of Hoxd13 occurs at late stages in the distal mesoderm of ozd leg buds, correlating with formation of digit 1. Fgf8 and Fgf4 expression are initiated normally in the mutant AER but their expression is progressively downregulated in the anterior AER. Recombinant Shh protein or ZPA grafts restore normal pattern toozd limbs; however, retinoic acid fails to induce Shh in ozdlimb mesoderm. We conclude that Shh function is required for limb development distal to the elbow/knee joints, similar to the Shh-/-mouse. Accordingly we classify the limb skeletal elements as Shh dependent or independent, with the ulna/fibula and digits other than digit 1 in the leg being Shh dependent. Finally we propose that the ozd mutation is most likely a defect in a regulatory element that controls limb-specific expression of Shh.

The Apical Ectodermal Ridge (AER) is one of the main signaling centers during limb development. It controls outgrowth and patterning in the proximo-distal axis. In the last few years a considerable amount of new data regarding the cellular and molecular mechanisms underlying AER function and structure has been obtained. In this review, we describe and discuss current knowledge of the regulatory networks which control the induction, maturation and regression of the AER, as well as the link between dorso-ventral patterning and the formation and position of the AER. Our aim is to integrate both recent and old knowledge to produce a wider picture of the AER which enhances our understanding of this relevant structure.

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.

Congenital malformations of the limbs are among the most frequent congenital anomalies found in humans, and they preferentially affect the distal part--the hand or foot. The presence of extra digits, a condition called polydactyly, is the most common limb deformity of the human hand and is the consequence of disturbances in the normal program of limb development. However, despite the extensive use of the developing limb as a classical developmental model, the cellular and genetic mechanisms that control the number and identity of the digits are not completely understood. The aim of this review is to introduce the reader to the current state of knowledge in limb development and to provide the necessary background for an understanding of how deviations from the normal developmental program may lead to polydactyly.

The ventricular fiber architecture has been studied anatomically and histologically in 25 human hearts. Special attention has been paid to the form of insertion of the myocardial fibers and to the existence or nonexistence of an independent system of fibers for each ventricle. Three different myocardial layers--superficial (subepicardial), middle, and deep (subendocardial)--have been distinguished according to the fiber direction through the ventricular mass. All the fibers are common for both ventricles with the exception of a thick middle layer peculiar to the left ventricle. The tendinous cords, the central fibrous body, and the arterial fibrous rings are the major zones of insertion of myocardial fibers. On the basis of our results we have attempted to correlate the ventricular architecture with the valvular dynamics.

With rapid progress in understanding the genes that control limb development and patterning interest is becoming focused on the factors that permit the emergence of the limb bud. The current hypothesis is that FGF-8 from the mesonephros induces limb initiation. To test this, the inductive interaction between the Wolffian duct and intermediate mesoderm was blocked rostral to the limb field, preventing mesonephric differentiation while maintaining the integrity of the limb field. The experimental outcome was monitored by following expression of cSim1 and Lmx1, molecular markers for the duct and the mesonephros, respectively. Evidence is presented that the intermediate mesoderm undergoes apoptosis when the inductive interaction with the Wolffian duct is blocked. fgf-8 expression was undetectable in the mesonephric area of embryos with confirmed absence of mesonephros; nevertheless, limb buds formed and limb development was normal. The mesonephros in general, and specifically its fgf-8 expression, was shown to be unnecessary for limb initiation and development; the hypothesis linking the mesonephros and limb development is not supported. Further studies of axial influences on limb initiation should now concentrate on medial structures such as Hensen's node and paraxial mesoderm; the alternative that no axial influences are required should also be examined.