Maria Pannese

Xotx genes in the developing brain of Xenopus laevis

The vertebrate Otx gene family is related to otd, a gene contributing to head development in Drosophila. We previously reported on the expression of Xotx2 gene, homologous to the murine Otx2 gene, during early Xenopus development. In the present paper we report an extensive analysis of the expression pattern of Xotx2 during later stages of development and also the cloning and developmental expression of two additional Otx Xenopus genes, Xotx1 and Xotx4. These latter two genes bear a good degree of homology to murine Otx1, higher for Xotx1 than for Xotx4. Both these genes are expressed in the forebrain and midbrain regions and their developmental patterns of expression are very similar, although not perfectly superimposable. Spatial and temporal expression patterns of the three Xotx genes suggest that they may be involved in the early subdivision of the rostral brain, providing antero-posterior positional information within the most anterior districts of the neuraxis. The three Xotx genes are expressed in all the developing sense organs of the head, eyes, olfactory system and otic vesicles. By in situ hybridization the earliest detectable expression is found in anterior mesendoderm for Xotx2, and in presumptive anterior neuroectoderm for Xotx1 and Xotx4. In addition, we examined whether Xotx1 is expressed in exogastrulae, finding that Xotx1 expression can be activated in the apparent absence of vertical signals of neural induction.

The Xenopus Emx genes identify presumptive dorsal telencephalon and are induced by head organizer signals.

We have isolated and studied the expression pattern of Xemx1 and Xemx2 genes in Xenopus laevis. Xemx genes are the homologues of mouse Emx genes, related to Drosophila empty spiracles. They are expressed in selected regions of the developing brain, particularly in the telencephalon, and, outside the brain, in the otic vesicles, olfactory placodes, visceral arches and the developing excretory system. We also report on experiments concerning the tissue and molecular signals responsible for their activation in competent ectoderm. Xemx genes are activated in ectoderm conjugated with head organizer tissue, but not with tail organizer tissue. Furthermore, they are not activated in animal cap either by noggin or by Xnr3, thus suggesting that a different inducer or the integration of several signals may be responsible for their activation.

Anf: A novel class of vertebrate homeobox genes expressed at the anterior end of the main embryonic axis

Five novel genes homologous to the homeobox-containing genes Xanf-1 and Xanf-2 of Xenopus and Hesx-1/Rpx of mouse have been identified as a result of a PCR survey of cDNA in sturgeon, zebrafish, newt, chicken and human. Comparative analysis of the homeodomain primary structure of these genes revealed that they belong to a novel class of homeobox genes, which we name Anf. All genes of this class investigated so far have similar patterns of expression during early embryogenesis, characterized by maximal transcript levels being present at the anterior extremity of the main embryonic body axis. The data obtained also suggest that, despite considerable high structural divergence between their homeodomains, all known Anf genes may be orthologues, and thus represent one of the most quickly evolving classes of vertebrate homeobox genes.

Calponin modulates the exclusion of Otx-expressing cells from convergence extension movements

Otx2, a vertebrate homologue of the Drosophila orthodenticle gene, coordinates two processes in early embryonic development. Not only does it specify cell fate in the anterior regions of the embryo, it also prevents the cells that express it from participating in the convergence extension movements that shape the rest of the body axis. Here we show that, in Xenopus, this latter function is mediated by XclpH3, transcription of which is directly stimulated by Xotx2. XclpH3 is a Xenopus homologue of the mammalian calponin gene, the product of which binds both actin and myosin and prevents the generation of contractile force by actin filaments.

Wnt Signaling Has Opposing Roles in the Developing and the Adult Brain That Are Modulated by Hipk1

The canonical Wnt/Wingless pathway is implicated in regulating cell proliferation and cell differentiation of neural stem/progenitor cells. Depending on the context, β-Catenin, a key mediator of the Wnt signaling pathway, may regulate either cell proliferation or differentiation. Here, we show that β-Catenin signaling regulates the differentiation of neural stem/progenitor cells in the presence of the β-Catenin interactor Homeodomain interacting protein kinase-1 gene (Hipk1). On one hand, Hipk1 is expressed at low levels during the entire embryonic forebrain development, allowing β-Catenin to foster proliferation and to inhibit differentiation of neural stem/progenitor cells. On the other hand, Hipk1 expression dramatically increases in neural stem/progenitor cells, residing within the subventricular zone (SVZ), at the time when the canonical Wnt signaling induces cell differentiation. Analysis of mouse brains electroporated with Hipk1, and the active form of β-Catenin reveals that coexpression of both genes induces proliferating neural stem/progenitor cells to escape the cell cycle. Moreover, in SVZ derive neurospheres cultures, the overexpression of both genes increases the expression of the cell-cycle inhibitor P16Ink4. Therefore, our data confirm that the β-Catenin signaling plays a dual role in controlling cell proliferation/differentiation in the brain and indicate that Hipk1 is the crucial interactor able to revert the outcome of β-Catenin signaling in neural stem/progenitor cells of adult germinal niches.

Xotx1 maternal transcripts are vegetally localized in Xenopus laevis oocytes.

Xotx1 is a Xenopus homeobox gene related to the Drosophila gene orthodenticle (otd). We previously reported that Xotx1 transcripts are already present in unfertilized egg. Here we report that maternal Xotx1 mRNA is vegetally localized during oogenesis. In stage II oocytes Xotx1 transcripts are localized within the mitochondrial cloud, in a perinuclear position; later on, they are translocated to the vegetal cortex within the mitochondrial cloud. We also observed that in stage III oocytes the expression domain of Wnt11 is contained within the one of Xotx1 while, at stage IV, the Xotx1 expression domain is contained within the one of Vg1.

Evolution of Emx genes and brain development in vertebrates

Emx1 and Emx2 genes are known to be involved in mammalian forebrain development. In order to investigate the evolution of the Emx gene family in vertebrates, a phylogenetic analysis was carried out on the Emx genes sequenced in man, mice, frogs, coelacanths and zebrafish. The results demonstrated the existence of two clades (Emx1 and Emx2 ), each grouping one of the two genes of the investigated taxa. The only exception was the zebrafish Emx1–like gene which turned out to be a sister group to both the Emx1 and Emx2 clusters. Such striking sequence divergence observed for the zebrafish Emx1–like gene could indicate that it is not orthologous to the other Emx1 genes, and therefore, in vertebrates there must be three Emx genes. Alternatively, if the zebrafish emx1 gene is orthologous to the tetrapod one, it must have undergone to strong diversifying selection.

Growth defects and impaired cognitive-behavioral abilities in mice with knockout for Eif4h, a gene located in the mouse homolog of the Williams-Beuren syndrome critical region

Protein synthesis is a tightly regulated, energy-consuming process. The control of mRNA translation into protein is fundamentally important for the fine-tuning of gene expression; additionally, precise translational control plays a critical role in many cellular processes, including development, cellular growth, proliferation, differentiation, synaptic plasticity, memory, and learning. Eukaryotic translation initiation factor 4h (Eif4h) encodes a protein involved in the process of protein synthesis, at the level of initiation phase. Its human homolog, WBSCR1, maps on 7q11.23, inside the 1.6 Mb region that is commonly deleted in patients affected by the Williams-Beuren syndrome, which is a complex neurodevelopmental disorder characterized by cardiovascular defects, cerebral dysplasias and a peculiar cognitive-behavioral profile. In this study, we generated knockout mice deficient in Eif4h. These mice displayed growth retardation with a significant reduction of body weight that began from the first week of postnatal development. Neuroanatomical profiling results generated by magnetic resonance imaging analysis revealed a smaller brain volume in null mice compared with controls as well as altered brain morphology, where anterior and posterior brain regions were differentially affected. The inactivation of Eif4h also led to a reduction in both the number and complexity of neurons. Behavioral studies revealed severe impairments of fear-related associative learning and memory formation. These alterations suggest that Eif4h might contribute to certain deficits associated with Williams-Beuren syndrome.

Key contribution of eIF4H-mediated translational control in tumor promotion

Dysregulated expression of translation initiation factors has been associated with carcinogenesis, but underlying mechanisms remains to be fully understood. Here we show that eIF4H (eukaryotic translation initiation factor 4H), an activator of the RNA helicase eIF4A, is overexpressed in lung carcinomas and predictive of response to chemotherapy. In lung cancer cells, depletion of eIF4H enhances sensitization to chemotherapy, decreases cell migration and inhibits tumor growth in vivo, in association with reduced translation of mRNA encoding cell-proliferation (c-Myc, cyclin D1) angiogenic (FGF-2) and anti-apoptotic factors (CIAP-1, BCL-xL). Conversely, each isoform of eIF4H acts as an oncogene in NIH3T3 cells by stimulating transformation, invasion, tumor growth and resistance to drug-induced apoptosis together with increased translation of IRES-containing or structured 5′UTR mRNAs. These results demonstrate that eIF4H plays a crucial role in translational control and can promote cellular transformation by preferentially regulating the translation of potent growth and survival factor mRNAs, indicating that eIF4H is a promising new molecular target for cancer therapy.

The Targeted Delivery of Interleukin 4 Inhibits Development of Endometriotic Lesions in a Mouse Model

Endometriosis is caused by the displacement of endometrium outside the uterus contributing heavily to infertility and debilitating pelvic pain. Ectopic adhesion and growth are believed to occur under the influence of a favorable hormonal environment and immunological factors. The objective of this study is to analyze the effect of a targeted therapy with an antibody-based pharmacodelivery of interleukin 4 (F8-IL4) in a mouse model of experimentally induced endometriosis. Endometriosis-like lesions were induced in Balb/c mice. The animals were treated intravenously with F8-IL4 or with untargeted IL4 (KSF-IL4). Twelve days after disease induction, the lesions were isolated. A significant reduction in the number of total lesions/mouse and in the total volume of lesions/mouse was observed in mice treated with F8-IL4 compared to controls (P = .029 and P = .006, respectively), while no difference was found between KSF-IL4-treated mice and their controls. Gene expression was evaluated by quantitative real-time polymerase chain reaction. Expression of genes involved in cell adhesion, extracellular matrix invasion, and neovascularization was significantly downregulated in F8-IL4-treated mice compared to their controls (integrin β1: P = .02; metalloproteinase [MMP] 3: P = .02; MMP9: P = .04; vascular endothelial growth factor: P = .04). Gene expression of inflammatory cytokines (tumor necrosis factor α, IL1β, IL1α, and IL6) did not vary in the ectopic lesions isolated from F8-IL4-treated mice compared to their controls. Immunohistochemistry demonstrated a significantly reduced expression of E-cadherin and β-catenin in the lesions of mice treated with F8-IL4. Our results show that the antibody-mediated targeted delivery of IL4 inhibits the development of endometriosis in a syngeneic mouse model by likely impairing adhesion, invasion, and vascularization of the ectopic endometrium.