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INTRODUCTION: Acute renal failure (ARF) after cardiac surgery is a risk factor associated with mortality and use of resources. Some studies have reported beneficial effects of pulsatile flow on cardiopulmonary bypass (CPB) on renal function. The aim of this study is to describe the echographic morphology of the renal arterial wave modifying the parameters of pulsatile CPB. MATERIAL AND METHOD: Descriptive study was performed on 10 patients without previous AFR and undergoing cardiac surgery with CPB. Pre-, intra- and post-surgery renal ultrasound was performed. During pulsatile CPB, the amplitude and the baseline flow were modified. Recordings of pulsed Doppler in intrarenal arteries were obtained by measuring maximum systolic velocity, minimum diastolic velocity, resistance index (RI) and acceleration time (AT). RESULTS: Statistical differences were found in ultrasounds pre-CPB between A50F50 modality (P=.013), A50F30 (P=.013) and A60F50 (P=.003). No statistically significance was found with A30F30 modality (P=.125). CONCLUSIONS: The decrease in the amplitude and the baseline flow of pulsatility during CPB shows a renal ultrasound morphology that is more similar to the physiological one. Subsequent studies using these characteristics during pulsatile CPB could thus show perfusion over the ARF that occurs after cardiac surgery.

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Externally the vertebrate body plan presents a bilateral symmetry in relation to the midline. However, inside the body the distribution of the visceral organs follows a very particular pattern that is not symmetrical in relation to the midline. The last 10 years have seen remarkable advances in our understanding of how the internal asymmetries typical of the vertebrate body are established and controlled. The use of different development models has permitted to uncover fascinating ways of creating asymmetry, like the activity of the nodal cilia. A host of studies has also unravelled the involvement of many genes in the left right patterning pathway. Based on this knowledge the genetic basis of human laterality defects are beginning to be revealed. It is a major challenge now to understand how all these genes control left right development as well as the complex set of interactions established between them.

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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.

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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.

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The homeobox gene Pitx2 has been characterized as a mediator of left-right signaling in heart, gut, and lung morphogenesis. However, the relationship between the developmental role of Pitx2 and its expression pattern at the organ level has not been explored. In this study we focus on the role of Pitx2 in heart morphogenesis. Chicken Pitx2 transcripts are present in the left portion of the cardiac crescent and in the left side of the heart tube. Through looping Pitx2 is present in the left atrium, in the ventral portion of the ventricles and in the left-ventral part of the outflow tract. Mouse Pitx2 shows a similar developmental profile of expression. To test whether Pitx2 represents a lineage marker we have tagged the left portion of the chicken cardiac tube with fluorescent DiD. Labeled cells were found at HH16 in the left atrium and in the ventral region of the ventricles and the outflow tract. In the iv/iv mouse model of cardiac heterotaxia Pitx2 was abnormally expressed in the atrial and in the ventricular chambers. Furthermore, altered Pitx2 expression correlated with the occurrence of DORV. Our data reveal the existence of molecular isomerism not only in the atrial, but also in the ventricular compartment of the heart.

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Vertebrate limbs develop in a temporal proximodistal sequence, with proximal regions specified and generated earlier than distal ones. Whereas considerable information is available on the mechanisms promoting limb growth, those involved in determining the proximodistal identity of limb parts remain largely unknown. We show here that retinoic acid (RA) is an upstream activator of the proximal determinant genes Meis1 and Meis2. RA promotes proximalization of limb cells and endogenous RA signaling is required to maintain the proximal Meis domain in the limb. RA synthesis and signaling range, which initially span the entire lateral plate mesoderm, become restricted to proximal limb domains by the apical ectodermal ridge (AER) activity following limb initiation. We identify fibroblast growth factor (FGF) as the main molecule responsible for this AER activity and propose a model integrating the role of FGF in limb cell proliferation, with a specific function in promoting distalization through inhibition of RA production and signaling.

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dHAND is a basic helix-loop-helix (bHLH) transcription factor essential for cardiovascular development. Here we analyze its pattern of expression and functional role during chick limb development. dHAND expression was observed in the lateral plate mesoderm prior to emergence of the limb buds. Coincident with limb initiation, expression of dHAND became restricted to the posterior half of the limb bud. Experimental procedures that caused mirror-image duplications of the limb resulted in mirror-image duplications of the pattern of dHAND expression along the anterior-posterior axis. Retroviral overexpression of dHAND in the limb bud produced preaxial polydactyly, corresponding to mild polarizing activity at the anterior border. At the molecular level, misexpression of dHAND caused ectopic activation of members of the Sonic hedgehog (Shh) pathway, including Gli and Patched, in the anterior limb bud. A subset of infected embryos displayed ectopic anterior activation of Shh. Other factors implicated in anterior-posterior polarization of the bud such as the most 5' Hoxd genes and Bmp2 were also ectopically activated at the anterior border. Our results indicate a role for dHAND in the establishment of anterior-posterior polarization of the limb bud.

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Recombinant limbs were performed by ensembling dissociated-reaggregated wing bud mesoderm inside an ectodermal hull. The zone of polarizing activity was excluded from the mesoderm used to perform the recombinant limbs (non-polarized recombinants), and grafted when desired (polarized recombinants). Reorganization of patterning progressively occurred in the newly formed progress zone under the influence of the apical ectodermal ridge (AER), explaining the proximo-distal gradient of morphogenesis observed in developed recombinant limbs. The AER, without the influence of the polarizing region (ZPA), was sufficient to direct outgrowth and appropriate proximo-distal patterning, as observed in the expression of the Hoxa-11 and Hoxa-13 genes. The development of the recombinant limbs coursed with symmetric AER and downregulation of Bmp expression in the mesoderm supporting a negative effect of Bmp signaling upon the apical ridge. The recombinant ectoderm maintained previously established compartments of gene expressions and organized a correct dorso-ventral patterning in the recombinant progress zone. Finally, the ZPA effect was only detected on Bmp expression and pattern formation along the antero-posterior axis.

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The recombinant limb is a model system that has proved fruitful for analyzing epithelial-mesenchymal interactions and understanding the functional properties of the components of the limb bud. Here we present an overview of some of the insights obtained through the use of this technique. Among these are the understanding that fore or hind limb identity is inherent to the limb bud mesoderm, that the apical ectodermal ridge (AER) is a permissive signaling center and that the limb bud ectoderm plays a central role in the control of dorsoventral polarity. Recombinant limb studies have also allowed the identification of the affected tissue component in several limb mutants. More recently this model has been applied to the study of regulation of gene expressions related to patterning. In this report we use recombinant limbs to analyze pattering of the Pax3 expressing limb muscle cell lineage in the early stages of limb development. In recombinant limbs made without the zone of polarizing activity (ZPA), myoblasts appear intermingled with other mesodermal cells at the beginning of the recombinant limb development. Rapidly thereafter, the muscle precursors segregate and organize around the central forming chondrogenic core of the recombinant. Although this segregation is reminiscent of that occurring during normal development, the myoblasts in the recombinant fail to proliferate appropriately and also fail to migrate distally. Consequently, the muscle pattern in the recombinant limb is defective indicating that normal patterning cues are absent. However, recombinant limbs polarized with a ZPA exhibited a larger mass of muscle cells and a more normal morphogenesis, supporting a role for this signaling center in limb muscle development. Finally, we have ruled out host somite contributions to recombinant limbs by grafting chick recombinant limbs to quail hosts. This initial report demonstrates the value of the recombinant limb model system for dissecting the environmental cues required for normal muscle limb patterning.

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Eph receptors and their ligands, the ephrins, have been implicated in early patterning and axon guidance in vertebrate embryos. Members of these families play pivotal roles in the formation of topographic maps in the central nervous system, the formation of brain commissures, and in the guidance of neural crest cells and motor axons through the anterior half of the somites. Here, we report a highly dynamic expression pattern of the chick EphA7 gene in the developing limb. Expression is detected in discrete domains of the dorsal mesenchyme from 3 days of incubation. The expressing cells are adjacent to the routes where axons grow to innervate the limb at several key points: the region of plexus formation, the bifurcation between dorsal and ventral fascicles, and the pathway followed by axons innervating the dorsal muscle mass. These results suggested a role for EphA7 in cell-cell contact-mediated signalling in dorsal limb patterning and/or axon guidance. We carried out experimental manipulations in the chick embryo wing bud to alter the dorsoventral patterning of the limb. The analyses of EphA7 expression and innervation in the operated wings indicate that a signal emanating from the dorsal ectoderm regulates EphA7 in such a way that, in its absence, the wing bud lacks EphA7 expression and shows innervation defects at the regions where the gene was downregulated. EphA7 downregulation in the dorsal mesenchyme after dorsal ectoderm removal is more rapid than that of Lmx-1, the gene known to mediate dorsalisation in response to the ectodermal signal. These results add a new gene to the dorsalisation signalling pathway in the limb. Moreover, they implicate the Eph receptor family in the patterning and innervation of the developing limb, extending its role in axon pathfinding to the distal periphery.

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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.

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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.

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The great advances made over the last few years in the identification of signalling molecules that pattern the limb bud along the three axes make the limb an excellent model system with which to study developmental mechanisms in vertebrates. The understanding of the signalling networks and their mutual interactions during limb development requires the characterisation of the corresponding downstream genes. In this study we report the expression pattern of Slug, a zinc-finger-containing gene of the snail family, during the development of the limb, and its regulation by distinct axial signalling systems. Slug expression is highly dynamic, and at different stages of limb development can be correlated with the zone of polarizing activity, the progress zone and the interdigital areas. We show that the maintenance of its expression is dependent on signals from the apical ectodermal ridge and independent of Sonic Hedgehog. We also report that, in the interdigit, apoptotic cells lie outside of the domains of Slug expression. The correlation of Slug expression with areas of undifferentiated mesenchyme at stages of tissue differentiation is consistent with its role in early development, in maintaining the mesenchymal phenotype and repressing differentiation processes. We suggest that Slug is involved in the epithelial-mesenchymal interactions that lead to the maintenance of the progress zone.

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Bone Morphogenetic Protein 2 (BMP-2) and Osteogenic Protein 1 (OP-1, also termed BMP-7) are members of the transforming growth factor beta superfamily. In the present study, we have analyzed the effects of administering them locally at different stages and locations of the chick limb bud using heparin beads as carriers. Our results show that these BMPs are potent apoptotic signals for the undifferentiated limb mesoderm but not for the ectoderm or the differentiating chondrogenic cells. In addition, they promote intense radial growth of the differentiating cartilages and disturb the formation of joints accompanied by alterations in the pattern of Indian hedgehog and ck-erg expression. Interestingly, the effects of these two BMPs on joint formation were found to be different. While the predominant effect of BMP-2 is alteration in joint shape, OP-1 is a potent inhibitory factor for joint formation. In situ hybridizations to check whether this finding was indicative of specific roles for these BMPs in the formation of joints revealed a distinct and complementary pattern of expression of these genes during the formation of the skeleton of the digits. While Op-1 exhibited an intense expression in the perichondrium of the developing cartilages with characteristic interruptions in the zones of joint formation, Bmp-2 expression was a positive marker for the articular interspaces. These data suggest that, in addition to the proposed role for BMP-2 and OP-1 in the establishment of the anteroposterior axis of the limb, they may also play direct roles in limb morphogenesis: (i) in regulating the amount and spatial distribution of the undifferentiated prechondrogenic mesenchyme and (ii) in controlling the location of the joints and the diaphyses of the cartilaginous primordia of the long bones once the chondrogenic aggregates are established.

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There is evidence that the interdigital mesoderm may be in an undifferentiated state. For example, under experimental manipulation in vivo it may be diverted from cell death to digit formation. In the present work we wanted to analyze the maximum morphogenetic potential of the interdigital cells. To do this we made recombinant limbs of three types, the first using dissociated-reaggregated leg interdigital mesoderm, the second using the same tissue but without dissociation and the third adding a piece of polarizing region to the dissociated interdigit. In all three the massive cell death of the interdigit failed to occur. The first type of recombinant formed a small nodule of cartilage while the other two formed a well-developed digit. Our data indicate that the maximum morphogenetic potential of the interdigital tissue appears constrained to form digits and that dissociation of the tissue decreased this ability; polarizing region restores the ability of dissociated cell recombinants to form a digit. We also analyzed in these recombinants the expression of a battery of genes implicated in interdigital cell death or in digital morphogenesis. The pattern of expression of each gene analyzed was identical in the three types of recombinant limbs. The expression of Msx1 and Msx2 genes was maintained under the ridge indicating a good interaction between the interdigital cells, both dissociated and undissociated, and the apical ridge. The expression of Hoxd-12, Hoxd-13 and Hoxa-13 genes was maintained in the recombinants, indicating that these cells carry information about their autopodial origin, and this correlates well with their distal restricted morphogenetic potential. Finally, the patterns of expression of the Bmp-2, Bmp-4 and Bmp-7 genes indicated that they are independently regulated in the recombinants and that Bmp-4 and Bmp-7 have wider expression domains than the areas of cell death that were only detected under the regressing apical ridge during day 3 of the experiment.

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