Maria A. Ros

Pitx2 Participates in the Late Phase of the Pathway Controlling Left-Right Asymmetry

Pitx2, a member of the bicoid-related family of homeobox-containing genes, is asymmetrically expressed in the left lateral plate mesoderm and derived tissues during chick and mouse development. Modifications of Pitx2 pattern of expression in the iv mouse mutation correlate with the situs alterations characteristic of the mutation. Misexpression experiments demonstrate that Shh and nodal positively regulate Pitx2 expression. Our results are compatible with a Pitx2 function in the late phase of the gene cascade controlling laterality.

Limb-patterning activity and restricted posterior localization of the amino-terminal product of Sonic hedgehog cleavage

BACKGROUND Sonic hedgehog (Shh), a vertebrate homolog of the Drosophila segment polarity gene hedgehog (hh), has been implicated in patterning of the developing chick limb. Such a role is suggested by the restricted expression of Shh along the posterior limb bud margin, and by the observation that heterologous cells expressing Shh have limb-polarizing activity resembling that of cells from the polarizing region of the posterior limb bud margin. It has not been demonstrated, however, that the Sonic hedgehog protein (SHH) alone is sufficient for limb patterning. SHH has been shown to undergo autoproteolytic cleavage in vitro, yielding two smaller products. It is of interest, therefore, to determine whether processing of SHH occurs in the developing limb and how such processing influences the function of SHH. RESULTS We demonstrate that SHH is proteolytically processed in developing chick limbs. Grafts of cells expressing SHH protein variants that correspond to individual cleavage products demonstrate that the ability to induce patterned gene expression and to impose morphological pattern upon the limb bud is limited to the amino-terminal product (SHH-N) of SHH proteolytic cleavage. We also demonstrate that bacterially synthesized and purified SHH-N, released from implanted beads, is sufficient for limb-patterning activity. Finally, we show that the endogenous amino-terminal cleavage product is tightly localized to the posterior margin of the limb bud. CONCLUSIONS Our data show that, of the two cleavage products resulting from SHH autoproteolysis, SHH-N expressed in grafted heterologous cells or supplied in purified form is sufficient to impose pattern upon the developing limb. Moreover, the restricted localization of the endogenous amino-terminal SHH cleavage product to the posterior border of the chick limb bud makes it unlikely that its patterning activity results from it being distributed in a broad gradient across the antero-posterior axis. More consistent with the observed localization is a model in which the amino-terminal SHH cleavage product exerts its patterning effects by local induction in or near the polarizing region, initiating a cascade of gene expression that ultimately extends across the developing limb.

Hox genes regulate digit patterning by controlling the wavelength of a turing-type mechanism

Digit Determination Pentadactyly has been an early and rapid innovation of tetrapods. Sheth et al. (p. 1476) report that a dramatic reduction in distally expressed Hox genes, in the absence of a functional morphogen signaling pathway, results in extreme polydactyly in mice. Mutant digits exhibited patterns reminiscent of the endoskeleton of fins, suggesting that an ancestral patterning mechanism has been deeply conserved in evolution. Mouse genetics and computer modeling suggest that a reaction-diffusion mechanism defines digit pattern. The formation of repetitive structures (such as stripes) in nature is often consistent with a reaction-diffusion mechanism, or Turing model, of self-organizing systems. We used mouse genetics to analyze how digit patterning (an iterative digit/nondigit pattern) is generated. We showed that the progressive reduction in Hoxa13 and Hoxd11-Hoxd13 genes (hereafter referred to as distal Hox genes) from the Gli3-null background results in progressively more severe polydactyly, displaying thinner and densely packed digits. Combined with computer modeling, our results argue for a Turing-type mechanism underlying digit patterning, in which the dose of distal Hox genes modulates the digit period or wavelength. The phenotypic similarity with fish-fin endoskeleton patterns suggests that the pentadactyl state has been achieved through modification of an ancestral Turing-type mechanism.

Initiation of proximal-distal patterning in the vertebrate limb by signals and growth.

Growth of limb cells in culture conditions with subsequent in vivo transplantation allows the dissection of limb patterning. Two broad classes of models have been proposed to explain the patterning of the proximal-distal axis of the vertebrate limb (from the shoulder to the digit tips). Differentiating between them, we demonstrate that early limb mesenchyme in the chick is initially maintained in a state capable of generating all limb segments through exposure to a combination of proximal and distal signals. As the limb bud grows, the proximal limb is established through continued exposure to flank-derived signal(s), whereas the developmental program determining the medial and distal segments is initiated in domains that grow beyond proximal influence. In addition, the system we have developed, combining in vitro and in vivo culture, opens the door to a new level of analysis of patterning mechanisms in the limb.

The chick oligozeugodactyly (ozd) mutant lacks sonic hedgehog function in the limb.

We have analyzed a new limb mutant in the chicken that we name oligozeugodactyly (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 the ozd limbs develop in the absence of Shh expression, while all other organs express Shh and develop normally. Neither Ptc1 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 of Hoxd11-13 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 to ozd limbs; however, retinoic acid fails to induce Shh in ozd limb 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.

Diffusible Signals, Not Autonomous Mechanisms, Determine the Main Proximodistal Limb Subdivision

Cells compare proximal and distal signals to set their identity along the vertebrate limb. Vertebrate limbs develop three main proximodistal (PD) segments (upper arm, forearm, and hand) in a proximal-to-distal sequence. Despite extensive research into limb development, whether PD specification occurs through nonautonomous or autonomous mechanisms is not resolved. Heterotopic transplantation of intact and recombinant chicken limb buds identifies signals in the embryo trunk that proximalize distal limb cells to generate a complete PD axis. In these transplants, retinoic acid induces proximalization, which is counteracted by fibroblast growth factors from the distal limb bud; these related actions suggest that the first limb-bud PD regionalization results from the balance between proximal and distal signals. The plasticity of limb progenitor cell identity in response to diffusible signals provides a unifying view of PD patterning during vertebrate limb development and regeneration.

Birth and death of cells in limb development: A mapping study

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. Developmental Dynamics 235:2521–2537, 2006.

The Apical Ectodermal Ridge: morphological aspects and signaling pathways

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.

A BMP- Shh negative-feedback loop restricts Shh expression during limb development

Normal patterning of tissues and organs requires the tight restriction of signaling molecules to well-defined organizing centers. In the limb bud, one of the main signaling centers is the zone of polarizing activity (ZPA) that controls growth and patterning through the production of sonic hedgehog (SHH). The appropriate temporal and spatial expression of Shh is crucial for normal limb bud patterning, because modifications, even if subtle, have important phenotypic consequences. However, although there is a lot of information about the factors that activate and maintain Shh expression, much less is known about the mechanisms that restrict its expression to the ZPA. In this study, we show that BMP activity negatively regulates Shh transcription and that a BMP-Shh negative-feedback loop serves to confine Shh expression. BMP-dependent downregulation of Shh is achieved by interfering with the FGF and Wnt signaling activities that maintain Shh expression. We also show that FGF induction of Shh requires protein synthesis and is mediated by the ERK1/2 MAPK transduction pathway. BMP gene expression in the posterior limb bud mesoderm is positively regulated by FGF signaling and finely regulated by an auto-regulatory loop. Our study emphasizes the intricacy of the crosstalk between the major signaling pathways in the posterior limb bud.

IMMUNOHISTOLOGICAL AND ULTRASTRUCTURAL STUDY OF THE DEVELOPING TENDONS OF THE AVIAN FOOT

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 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.