Auswahl der wissenschaftlichen Literatur zum Thema „Homeobox protein engrailed2“

The Engrailed-1 gene, En1, a murine homologue of the Drosophila homeobox gene engrailed (en), is required for midbrain and cerebellum development and dorsal/ventral patterning of the limbs. In Drosophila, en is involved in regulating a number of key patterning processes including segmentation of the epidermis. An important question is whether, during evolution, the biochemical properties of En proteins have been conserved, revealing a common underlying molecular mechanism to their diverse developmental activities. To address this question, we have replaced the coding sequences of En1 with Drosophila en. Mice expressing Drosophila en in place of En1 have a near complete rescue of the lethal En1 mutant brain defect and most skeletal abnormalities. In contrast, expression of Drosophila en in the embryonic limbs of En1 mutants does not lead to repression of Wnt7a in the embryonic ventral ectoderm or full rescue of the embryonic dorsal/ventral patterning defects. Furthermore, neither En2 nor en rescue the postnatal limb abnormalities that develop in rare En1 null mutants that survive. These studies demonstrate that the biochemical activity utilized in mouse to mediate brain development has been retained by Engrailed proteins across the phyla, and indicate that during evolution vertebrate En proteins have acquired two unique functions during embryonic and postnatal limb development and that only En1 can function postnatally.

By a complex and little understood mechanism, segment polarity genes control patterning in each segment of the Drosophila embryo. During this process, cell to cell communication plays a pivotal role and is under direct control of the products of segment polarity genes. Many of the cloned segment polarity genes have been found to be highly conserved in evolution, providing a model system for cellular interactions in other organisms. In Drosophila, two of these genes, engrailed and wingless, are expressed on either side of the parasegment border, wingless encodes a secreted molecule and engrailed a nuclear protein with a homeobox. Maintenance of engrailed expression is dependent on wingless and vice versa. To investigate the role of other segment polarity genes in the mutual control between these two genes, we have examined wingless and engrailed protein distribution in embryos mutant for each of the segment polarity genes. In embryos mutant for armadillo, dishevelled and porcupine, the changes in engrailed expression are identical to those in wingless mutant embryos, suggesting that their gene products act in the wingless pathway. In embryos mutant for hedgehog, fused, cubitus interruptus Dominant and gooseberry, expression of engrailed is affected to varying degrees. However wingless expression in the latter group decays in a similar way earlier than engrailed expression, indicating that these gene products might function in the maintenance of wingless expression. Using double mutant embryos, epistatic relationships between some segment polarity genes have been established. We present a model showing a current view of segment polarity gene interactions.

We have isolated and characterized cDNAs corresponding to the Xenopus En-2 gene. Comparison of amino acid sequences between the entire Xenopus En-2 and the Drosophila engrailed proteins confirms conservation of sequences inside as well as proximal to the homeobox and reveals a region of similarity towards the N terminus. Two transcripts encode the Xenopus En-2 protein. Both transcripts are regulated temporally in an identical fashion and are likely to be transcribed from two copies of the En-2 gene. We have also analyzed the distribution of the protein in the head tissue and in the dissected brain of tailbud stage embryos. In addition to the main band of expression at the midbrain-hindbrain boundary, we show that the protein is expressed in three novel areas: the mandibular arch, the optic tectum and the region of anterior pituitary.

Phylogenetic analyses imply that multiple engrailed-family gene duplications occurred during hexapod evolution, a view supported by previous reports of only a single engrailed-family gene in members of the grasshopper genus Schistocerca and in the beetle Tribolium castaneum . Here, we report the cloning of a second engrailed-family gene from Schistocerca gregaria and present evidence for two engrailed-family genes from four additional hexapod species. We also report the existence of a second engrailed-family gene in the Tribolium genome. We suggest that the engrailed and invected genes of Drosophila melanogaster have existed as a conserved gene cassette throughout holometabolous insect evolution. In total 11 phylogenetically diverse hexapod orders are now known to contain species that possess two engrailed-family paralogues, with in each case only one paralogue encoding the RS-motif, a characteristic feature of holometabolous insect invected proteins. We propose that the homeoboxes of hexapod engrailed-family paralogues are evolving in a concerted fashion, resulting in gene trees that overestimate the frequency of gene duplication. We present new phylogenetic analyses using non-homeodomain amino acid sequence that support this view. The S. gregaria engrailed-family paralogues provide strong evidence that concerted evolution might in part be explained by recurrent gene conversion. Finally, we hypothesize that the RS-motif is part of a serine-rich domain targeted for phosphorylation.

The hedgehog gene product, secreted from engrailed-expressing neuroectoderm, is required for the formation of post-S1 neuroblasts in rows 2, 5 and 6. The hedgehog protein functions not only as a paracrine but also as an autocrine factor and its transient action on the neuroectoderm 1–2 hours (at 18 degrees C) prior to neuroblast delamination is necessary and sufficient to form normal neuroblasts. In contrast to epidermal development, hedgehog expression required for neuroblast formation is regulated by neither engrailed nor wingless. hedgehog and wingless bestow composite positional cues on the neuroectodermal regions for S2-S4 neuroblasts at virtually the same time and, consequently, post-S1 neuroblasts in different rows can acquire different positional values along the anterior-posterior axis. The average number of proneural cells for each of three eagle-positive S4-S5 neuroblasts was found to be 5–9, the same for S1 NBs. As with wingless (Chu-LaGraff et al., Neuron 15, 1041–1051, 1995), huckebein expression in putative proneural regions for certain post-S1 neuroblasts is under the control of hedgehog. hedgehog and wingless are involved in separate, parallel pathways and loss of either is compensated for by the other in NB 7–3 formation. NBs 6–4 and 7–3, arising from the engrailed domain, were also found to be specified by the differential expression of two homeobox genes, gooseberry-distal and engrailed.

The PRH (proline-rich homeodomain) [also known as Hex (haematopoietically expressed homeobox)] protein is a transcription factor that functions as an important regulator of vertebrate development and many other processes in the adult including haematopoiesis. The Groucho/TLE (transducin-like enhancer) family of co-repressor proteins also regulate development and modulate the activity of many DNA-binding transcription factors during a range of diverse cellular processes including haematopoiesis. We have shown previously that PRH is a repressor of transcription in haematopoietic cells and that an Eh-1 (Engrailed homology) motif present within the N-terminal transcription repression domain of PRH mediates binding to Groucho/TLE proteins and enables co-repression. In the present study we demonstrate that PRH regulates the nuclear retention of TLE proteins during cellular fractionation. We show that transcriptional repression and the nuclear retention of TLE proteins requires PRH to bind to both TLE and DNA. In addition, we characterize a trans-dominant-negative PRH protein that inhibits wild-type PRH activity by sequestering TLE proteins to specific subnuclear domains. These results demonstrate that transcriptional repression by PRH is dependent on TLE availability and suggest that subnuclear localization of TLE plays an important role in transcriptional repression by PRH.

Significant progress has been made towards understanding how pattern formation occurs in the imaginal discs that give rise to the limbs of Drosophila melanogaster. Here, we examine the process of regional specification that occurs in the eye-antennal discs, which form the head of the adult fruitfly. We demonstrate genetically that there is a graded requirement for the activity of the orthodenticle homeobox gene in forming specific structures of the developing head. Consistent with this result, we show that OTD protein is expressed in a graded fashion across the disc primordia of these structures and that different threshold levels of OTD are required for the formation of specific subdomains of the head. Finally, we provide evidence suggesting that otd acts through the segment polarity gene engrailed to specify medial head development.

Recovery from acute renal failure (ARF) requires the replacement of injured cells with new cells that restore tubule epithelial integrity. We described recently the expression of a wide range of nephrogenic proteins in tubular cells after ARF induced by ischemia-reperfusion (I/R) (Villanueva S, Cespedes C, and Vio CP. Am J Physiol Regul Integr Comp Physiol 290: R861–R870, 2006). These markers, namely, Vimentin, neural cell adhesion molecules (Ncam), basic fibroblast growth factor (bFGF), paired homeobox-2 (Pax-2), bone morphogene protein-7 (BMP-7), Noggin, Lim-1, Engrailed, Smad, phospho-Smad, hypoxia-induced factor-1α (HIF-1α), VEGF, and Tie-2, are expressed in a time frame similar to that observed in normal kidney development. bFGF participates in early kidney development as a morphogen involved in mesenchyme/epithelial transition, and it is reexpressed in the recovery phase of ARF. To test the hypothesis that bFGF can accelerate the regeneration after renal damage, we used recombinant bFGF and studied the expression pattern of the above described morphogens in ARF. Male Sprague-Dawley rats were subjected to 30 min of renal ischemic injury and were injected with bFGF 30 μg/kg followed by reperfusion. Rats were killed and the expression of nephrogenic proteins were analyzed by immunohistochemistry and Western blot analysis. In the animals subjected to I/R treated with bFGF, we observed a 12- to 24-h earlier and more abundant reexpression of the proteins Ncam, bFGF, Pax-2, BMP-7, Noggin, Lim-1, Engrailed, VEGF, and Tie-2 than the I/R untreated rats. In addition, we observed a reduction in renal damage markers ED-1 and α-smooth muscle actin. These results indicate that bFGF can participate in the regeneration process and suggest that the treatment with bFGF can induce an earlier regeneration process after ischemic acute renal failure.

beta-catenin plays an integral role in cell-cell adhesion by linking the cadherin complex of the adherens junction to the underlying actin cytoskeleton. In addition, beta-catenin transduces intracellular signals within the Wnt developmental pathway that are crucial to the proper establishment of embryonic axes and pattern formation of early mesoderm and ectoderm. For example, in the context of a defined dorsal ‘organizer’ region of early Xenopus embryos, beta-catenin enters the nucleus and associates with transcription factors of the HMG (High Mobility Group) Lef/Tcf protein family. Consequently, genes such as siamois, a homeobox gene contributing to the specification of the dorsoanterior axis, are activated. To further examine the role that beta-catenin plays in Wnt signaling, we generated a chimeric protein, beta-Engrailed (beta-Eng), in which the C-terminal trans-activation domain of beta-catenin is replaced with the transcriptional repression domain of Drosophila Engrailed. Dorsal overexpression of this mRNA in early Xenopus embryos leads to suppression of organizer-specific molecular markers such as siamois, Xnr-3 and goosecoid, corresponding with the dramatic morphological ventralization of embryos. Ventralized embryos further exhibit reduced activity of the Wnt pathway, as indicated by the loss of the notochord/organizer marker, chordin. Importantly, beta-Eng associates and functions normally with the known components of the cadherin complex, providing the experimental opportunity to repress beta-catenin's signaling function apart from its role in cadherin-mediated cell-cell adhesion.

Meis-family homeobox proteins have been shown to regulate cell fate specification in vertebrate and invertebrate embryos. Ectopic expression of RNA encoding the Xenopus Meis3 (XMeis3) protein caused anterior neural truncations with a concomitant expansion of hindbrain and spinal cord markers in Xenopus embryos. In naïve animal cap explants, XMeis3 activated expression of posterior neural markers in the absence of pan-neural markers. Supporting its role as a neural caudalizer, XMeis3 is expressed in the hindbrain and spinal cord. We show that XMeis3 acts like a transcriptional activator, and its caudalizing effects can be mimicked by injecting RNA encoding a VP16-XMeis3 fusion protein. To address the role of endogenous XMeis3 protein in neural patterning, XMeis3 activity was antagonized by injecting RNA encoding an Engrailed-XMeis3 antimorph fusion protein or XMeis3 antisense morpholino oligonucleotides. In these embryos, anterior neural structures were expanded and posterior neural tissues from the midbrain-hindbrain junction through the hindbrain were perturbed. In neuralized animal cap explants, XMeis3-antimorph protein modified caudalization by basic fibroblast growth factor and Wnt3a. XMeis3-antimorph protein did not inhibit caudalization per se, but re-directed posterior neural marker expression to more anterior levels; it reduced expression of spinal cord and hindbrain markers, yet increased expression of the more rostral En2 marker. These results provide evidence that XMeis3 protein in the hindbrain is required to modify anterior neural-inducing activity, thus, enabling the transformation of these cells to posterior fates.

Les homéoprotéines constituent une large famille de facteurs de transcription régulant de nombreux processus neuro-développementaux, dont certains de manière paracrine. Les mécanismes non-conventionnels de transfert intercellulaire restent mal connus. Dans cette thèse, nous cherchons à caractériser ces mécanismes et leur potentiels régulateurs dans le cas de l’homéoprotéine Engrailed2 (EN2). Dans un premier temps, nous avons développé des méthodes quantitatives pour mesurer les deux étapes du transfert d’EN2. Cette stratégie nous a permis de démontrer pour la première fois, la translocation d’EN2 à travers la membrane plasmique. Ensuite, nous avons pu identifier certains acteurs indispensables de cette translocation : les phosphoinositides PIP2, le cholestérol et les protéoglycans. Grâce à ces travaux, nous avons maintenant une meilleure compréhension de ces processus de translocation membranaire. Ensuite, nous avons testé l’hypothèse d’une régulation par H2O2 du transfert d’EN2, démontré l’existence d’une boucle de contrôle entre H2O2 et EN2 et illustré l’importance de cette régulation au cours du développement du cerveau du zebrafish. Enfin, nous avons étendu nos outils de quantification et notre hypothèse de travail au trafic d’un morphogène plus conventionnel : Sonic Hedgehog (Shh). Malgré des mécanismes de transfert plus complexes (que pour EN2), nous avons également mis en évidence une boucle de contrôle entre les signalisations Shh et redox. Si cette régulation redox du transfert des homéoprotéines et morphogènes est conservée pour différentes protéines, ces résultats proposent des pistes pour comprendre les relations entre morphogénèse et métabolisme cellulaire
Homeoproteins (HP) constitute a large family of transcription factors endowed with both autocrine and paracrine activities. Homeoprotein paracrine action controls patterning processes, including axonal guidance and boundary formation. Internalization and secretion, the steps of intercellular transfer, rely on unconventional mechanisms, which still need to be fully characterized. We have deciphered the mechanism of HP transfer, responsible for their paracrine activity, using Engrailed (EN2) as a paradigm. First, I have developed tools to quantify EN2 uptake and secretion. This original strategy allowed us to demonstrate EN2 bidirectional transfer through direct plasma membrane translocation. Then, I identified the molecular requirements for EN2 transfer, highlighting the pivotal role of PIP2, cholesterol, and proteoglycans. This work illustrates how soluble protein are able to cross the plasma membrane, giving new clues to the study of cell-penetrating peptides derived from HP but also to other unconventionnally secreted proteins. Next, I have addressed the contribution of redox signaling in EN2 transfer, and demonstrated that EN2 and H2O2 act in synergy to shape the optic tectum in the zebrafish. Finally, I have extended this study to conventional morphogens and showed that H2O2 regulates the traffic of Sonic Hedgehog. If this regulation of protein trafficking can be generalized to other HPs and morphogens remains unknown, but if so, it would provide an understanding for how tissue morphogenesis and cell metabolism influence each other during development