Translational control in cortical development.

Differentiation of specific neuronal types in the nervous system is worked out through a complex series of gene regulation events. Within the mammalian neocortex, the appropriate expression of key transcription factors allocates neurons to different cortical layers according to an inside-out model and endows them with specific properties. Precise timing is required to ensure the proper sequential appearance of key transcription factors that dictate the identity of neurons within the different cortical layers. Recent evidence suggests that aspects of this time-controlled regulation of gene products rely on post-transcriptional control, and point at micro-RNAs (miRs) and RNA-binding proteins as important players in cortical development. Being able to simultaneously target many different mRNAs, these players may be involved in controlling the global expression of gene products in progenitors and post-mitotic cells, in a gene expression framework where parallel to transcriptional gene regulation, a further level of control is provided to refine and coordinate the appearance of the final protein products. miRs and RNA-binding proteins (RBPs), by delaying protein appearance, may play heterochronic effects that have recently been shown to be relevant for the full differentiation of cortical neurons and for their projection abilities. Such heterochronies may be the base for evolutionary novelties that have enriched the spectrum of cortical cell types within the mammalian clade.

A eutherian-specific microRNA controls the translation of Satb2 in a model of cortical differentiation.

Cerebral cortical development is controlled by key transcription factors that specify the neuronal identities in the different layers. The mechanisms controlling their expression in distinct cells are only partially known. We investigated the expression and stability of Tbr1, Bcl11b, Fezf2, Satb2, and Cux1 mRNAs in single developing mouse cortical cells. We observe that Satb2 mRNA appears much earlier than its protein and in a set of cells broader than expected, suggesting an initial inhibition of its translation, subsequently released during development. Mechanistically, Satb2 3'UTR modulates protein translation of GFP reporters during mouse corticogenesis. We select miR-541, a eutherian-specific miRNA, and miR-92a/b as the best candidates responsible for SATB2 inhibition, being strongly expressed in early and reduced in late progenitor cells. Their inactivation triggers robust and premature SATB2 translation in both mouse and human cortical cells. Our findings indicate RNA interference as a major mechanism in timing cortical cell identities.

Protopine/Gemcitabine Combination Induces Cytotoxic or Cytoprotective Effects in Cell Type-Specific and Dose-Dependent Manner on Human Cancer and Normal Cells.

The natural alkaloid protopine (PRO) exhibits pharmacological properties including anticancer activity. We investigated the effects of PRO, alone and in combination with the chemotherapeutic gemcitabine (GEM), on human tumor cell lines and non-tumor human dermal fibroblasts (HDFs). We found that treatments with different PRO/GEM combinations were cytotoxic or cytoprotective, depending on concentration and cell type. PRO/GEM decreased viability in pancreatic cancer MIA PaCa-2 and PANC-1 cells, while it rescued the GEM-induced viability decline in HDFs and in tumor MCF-7 cells. Moreover, PRO/GEM decreased G1, S and G2/M phases, concomitantly with an increase of subG1 phase in MIA PaCa-2 and PANC-1 cells. Differently, PRO/GEM restored the normal progression of the cell cycle, altered by GEM, and decreased cell death in HDFs. PRO alone increased mitochondrial reactive oxygen species (ROS) in MIA PaCa-2, PANC-1 cells and HDFs, while PRO/GEM increased both intracellular and mitochondrial ROS in the three cell lines. These results indicate that specific combinations of PRO/GEM may be used to induce cytotoxic effects in pancreatic tumor MIA PaCa-2 and PANC-1 cells, but have cytoprotective or no effects in HDFs.

HMGA Genes and Proteins in Development and Evolution.

HMGA (high mobility group A) (HMGA1 and HMGA2) are small non-histone proteins that can bind DNA and modify chromatin state, thus modulating the accessibility of regulatory factors to the DNA and contributing to the overall panorama of gene expression tuning. In general, they are abundantly expressed during embryogenesis, but are downregulated in the adult differentiated tissues. In the present review, we summarize some aspects of their role during development, also dealing with relevant studies that have shed light on their functioning in cell biology and with emerging possible involvement of HMGA1 and HMGA2 in evolutionary biology.

Cell-specific pattern of berberine pleiotropic effects on different human cell lines.

The natural alkaloid berberine has several pharmacological properties and recently received attention as a potential anticancer agent. In this work, we investigated the molecular mechanisms underlying the anti-tumor effect of berberine on glioblastoma U343 and pancreatic carcinoma MIA PaCa-2 cells. Human dermal fibroblasts (HDF) were used as non-cancer cells. We show that berberine differentially affects cell viability, displaying a higher cytotoxicity on the two cancer cell lines than on HDF. Berberine also affects cell cycle progression, senescence, caspase-3 activity, autophagy and migration in a cell-specific manner. In particular, in HDF it induces cell cycle arrest in G2 and senescence, but not autophagy; in the U343 cells, berberine leads to cell cycle arrest in G2 and induces both senescence and autophagy; in MIA PaCa-2 cells, the alkaloid induces arrest in G1, senescence, autophagy, it increases caspase-3 activity and impairs migration/invasion. As demonstrated by decreased citrate synthase activity, the three cell lines show mitochondrial dysfunction following berberine exposure. Finally, we observed that berberine modulates the expression profile of genes involved in different pathways of tumorigenesis in a cell line-specific manner. These findings have valuable implications for understanding the complex functional interactions between berberine and specific cell types.

The HMGA gene family in chordates: evolutionary perspectives from amphioxus.

High mobility group A proteins of vertebrates, HMGA1 and 2, are chromatin architectural factors involved in development, cell differentiation, and neoplastic transformation. Here, we characterize an amphioxus HMGA gene ortholog and analyze its expression. As a basal chordate, amphioxus is well placed to provide insights into the evolution of the HMGA gene family, particularly in the transition from invertebrates to vertebrates. Our phylogenetic analysis supports the basal position of amphioxus, echinoderm, and hemichordate HMGA sequences to those of vertebrate HMGA1 and HMGA2. Consistent with this, the genomic landscape around amphioxus HMGA shares features with both. Whole mount in situ hybridization shows that amphioxus HMGA mRNA is detectable from neurula stage onwards in both nervous and non-nervous tissues. This correlates with protein expression monitored immunocytochemically using antibodies against human HMGA2 protein, revealing especially high levels of expression in cells of the lamellar body, the amphioxus homolog of the pineal, suggesting that the gene may have, among its many functions, an evolutionarily conserved role in photoreceptor differentiation.

Hmga2 is required for neural crest cell specification in Xenopus laevis.

HMGA proteins are small nuclear proteins that bind DNA by conserved AT-hook motifs, modify chromatin architecture and assist in gene expression. Two HMGAs (HMGA1 and HMGA2), encoded by distinct genes, exist in mammals and are highly expressed during embryogenesis or reactivated in tumour progression. We here addressed the in vivo role of Xenopus hmga2 in the neural crest cells (NCCs). We show that hmga2 is required for normal NCC specification and development. hmga2 knockdown leads to severe disruption of major skeletal derivatives of anterior NCCs. We show that, within the NCC genetic network, hmga2 acts downstream of msx1, and is required for msx1, pax3 and snail2 activities, thus participating at different levels of the network. Because of hmga2 early effects in NCC specification, the subsequent epithelial-mesenchymal transition (EMT) and migration of NCCs towards the branchial pouches are also compromised. Strictly paralleling results on embryos, interfering with Hmga2 in a breast cancer cell model for EMT leads to molecular effects largely consistent with those observed on NCCs. These data indicate that Hmga2 is recruited in key molecular events that are shared by both NCCs and tumour cells.

Noggin-Mediated Retinal Induction Reveals a Novel Interplay Between Bone Morphogenetic Protein Inhibition, Transforming Growth Factor ß, and Sonic Hedgehog Signaling.

It has long been known that the depletion of bone morphogenetic protein (BMP) is one of the key factors necessary for the development of anterior neuroectodermal structures. However, the precise molecular mechanisms that underlie forebrain regionalization are still not completely understood. Here, we show that Noggin1 is involved in the regionalization of anterior neural structures in a dose-dependent manner. Low doses of Noggin1 expand prosencephalic territories, while higher doses specify diencephalic and retinal regions at the expense of telencephalic areas. A similar dose-dependent mechanism determines the ability of Noggin1 to convert pluripotent cells in prosencephalic or diencephalic/retinal precursors, as shown by transplant experiments and molecular analyses. At a molecular level, the strong inhibition of BMP signaling exerted by high doses of Noggin1 reinforces the Nodal/transforming growth factor (TGF)ß signaling pathway, leading to activation of Gli1 and Gli2 and subsequent activation of Sonic Hedgehog (SHH) signaling. We propose a new role for Noggin1 in determining specific anterior neural structures by the modulation of TGFß and SHH signaling.

Identification and evolution of molecular domains involved in differentiating the cement gland-promoting activity of Otx proteins in Xenopus laevis.

Otx genes are a class of vertebrate homeobox genes, homologous to the orthodenticle gene of Drosophila melanogaster, that play a crucial role in anterior embryo patterning and sensory organ formation. In the frog, Xenopus laevis, at least three members of this class have been isolated: otx1, otx2 and otx5 (crx); they are involved in regulating both shared and differential processes during frog development. In particular, while otx2 and otx5 are both capable to promote cement gland (CG) formation, otx1 is not. We performed a molecular dissection of Otx5 and Otx1 proteins to characterize the functional parts of the proteins that make them differently able to promote CG formation. We show that a CG promoting domain (CGPD) is localized at the Otx5 C-terminus, and is bipartite: CGPD1 (aa210-255) is the most effective domain, while CGPD2 (aa177-209) has a lower activity. A histidine stretch disrupts CGPD1 continuity in Otx1 determining its loss of CG promoting activity; this histidine-rich region acts as an actively CG repressing domain. Another Otx1 specific domain, a serine-rich stretch, may also be involved in repressing Otx1 potential to trigger CG formation, though at a much lower level. This is the first evidence that these domains, specific of the Otx1 orthology group, play a role during development in differentiating Otx1 action compared to other Otx family members. We discuss the potential implications of their appearance in light of the evolution of Otx functional activities.

Expression and functional characterization of Xhmg-at-hook genes in Xenopus laevis.

High Mobility Group A proteins (HMGA1 and HMGA2) are architectural nuclear factors involved in development, cell differentiation, and cancer formation and progression. Here we report the cloning, developmental expression and functional analysis of a new multi-AT-hook factor in Xenopus laevis (XHMG-AT-hook) that exists in three different isoforms. Xhmg-at-hook1 and 3 isoforms, but not isoform 2, are expressed throughout the entire development of Xenopus, both in the maternal and zygotic phase. Localized transcripts are present in the animal pole in the early maternal phase; during the zygotic phase, mRNA can be detected in the developing central nervous system (CNS), including the eye, and in the neural crest. We show evidence that XHMG-AT-hook proteins differ from typical HMGA proteins in terms of their properties in DNA binding and in protein/protein interaction. Finally, we provide evidence that they are involved in early CNS development and in neural crest differentiation.

MicroRNAs couple cell fate and developmental timing in retina.

Cell identity is acquired in different brain structures according to a stereotyped timing schedule, by accommodating the proliferation of multipotent progenitor cells and the generation of distinct types of mature nerve cells at precise times. However, the molecular mechanisms coupling the identity of a specific neuron and its birth date are poorly understood. In the neural retina, only late progenitor cells that divide slowly can become bipolar neurons, by the activation of otx2 and vsx1 genes. In Xenopus, we found that Xotx2 and Xvsx1 translation is inhibited in early progenitor cells that divide rapidly by a set of cell cycle-related microRNAs (miRNAs). Through expression and functional screenings, we selected 4 miRNAs--mir-129, mir-155, mir-214, and mir-222--that are highly expressed at early developmental stages in the embryonic retina and bind to the 3' UTR of Xotx2 and Xvsx1 mRNAs inhibiting their translation. The functional inactivation of these miRNAs in vivo releases the inhibition, supporting the generation of additional bipolar cells. We propose a model in which the proliferation rate and the age of a retinal progenitor are linked to each other and determine the progenitor fate through the activity of a set of miRNAs.

Noggin elicits retinal fate in Xenopus animal cap embryonic stem cells.

Driving specific differentiation pathways in multipotent stem cells is a main goal of cell therapy. Here we exploited the differentiating potential of Xenopus animal cap embryonic stem (ACES) cells to investigate the factors necessary to drive multipotent stem cells toward retinal fates. ACES cells are multipotent, and can be diverged from their default ectodermal fate to give rise to cell types from all three germ layers. We found that a single secreted molecule, Noggin, is sufficient to elicit retinal fates in ACES cells. Reverse-transcription polymerase chain reaction, immunohistochemistry, and in situ hybridization experiments showed that high doses of Noggin are able to support the expression of terminal differentiation markers of the neural retina in ACES cells in vitro. Following in vivo transplantation, ACES cells expressing high Noggin doses form eyes, both in the presumptive eye field region and in ectopic posterior locations. The eyes originating from the transplants in the eye field region are functionally equivalent to normal eyes, as seen by electrophysiology and c-fos expression in response to light. Our data show that in Xenopus embryos, proper doses of a single molecule, Noggin, can drive ACES cells toward retinal cell differentiation without additional cues. This makes Xenopus ACES cells a suitable model system to direct differentiation of stem cells toward retinal fates and encourages further studies on the role of Noggin in the retinal differentiation of mammalian stem cells.

A specific box switches the cell fate determining activity of XOTX2 and XOTX5b in the Xenopus retina.

BACKGROUND: Otx genes, orthologues of the Drosophila orthodenticle gene (otd), play crucial roles in vertebrate brain development. In the Xenopus eye, Xotx2 and Xotx5b promote bipolar and photoreceptor cell fates, respectively. The molecular basis of their differential action is not completely understood, though the carboxyl termini of the two proteins seem to be crucial. To define the molecular domains that make the action of these proteins so different, and to determine whether their retinal abilities are shared by Drosophila OTD, we performed an in vivo molecular dissection of their activity by transfecting retinal progenitors with several wild-type, deletion and chimeric constructs of Xotx2, Xotx5b and otd. RESULTS: We identified a small 8-10 amino acid divergent region, directly downstream of the homeodomain, that is crucial for the respective activities of XOTX2 and XOTX5b. In lipofection experiments, the exchange of this 'specificity box' completely switches the retinal activity of XOTX5b into that of XOTX2 and vice versa. Moreover, the insertion of this box into Drosophila OTD, which has no effect on retinal cell fate, endows it with the specific activity of either XOTX protein. Significantly, in cell transfection experiments, the diverse ability of XOTX2 and XOTX5b to synergize with NRL, a cofactor essential for vertebrate rod development, to transactivate the rhodopsin promoter is also switched depending on the box. We also show by GST-pull down that XOTX2 and XOTX5b differentially interact with NRL, though this property is not strictly dependent on the box. CONCLUSION: Our data provide molecular evidence on how closely related homeodomain gene products can differentiate their functions to regulate distinct cell fates. A small 'specificity box' is both necessary and sufficient to confer on XOTX2 and XOTX5b their distinct activities in the developing frog retina and to convert the neutral orthologous OTD protein of Drosophila into a positive and specific XOTX-like retinal regulator. Relatively little is known of what gives developmental specificity to homeodomain regulators. We propose that this box is a major domain of XOTX proteins that provides them with the appropriate developmental specificity in retinal histogenesis.

Identification and developmental expression of Xenopus hmga2beta.

HMGA proteins are "architectural modifiers" of the chromatin, characterized by three conserved "AT-hook" motifs, with which they bind AT-rich regions of the DNA, to assist in gene transcription. We report the identification and developmental expression of Xenopus laevis hmga2beta (Xlhmga2beta). We provide evidence of two forms of hmga2 (Xlhmga2alpha and Xlhmga2beta) and of a splicing variant for Xlhmga2beta with an additional AT-hook. By comparing X. laevis and X. tropicalis hmga2 DNA sequences to those of other organisms we show a high conservation of the Xlhmga2beta variant. By RT-PCR, Xlhmga2beta transcripts are first detected before the midblastula transition (MBT), and then become more abundant. By in situ hybridization, localized transcripts are first detected at neurula stages, in the presumptive central nervous system (CNS). At tailbud and tadpole stages, Xlhmga2beta mRNA is detected in the CNS, in the otic vesicles, in neural crest cell derivatives, in the notochord, and in the medio-lateral mesoderm.

Timing the generation of distinct retinal cells by homeobox proteins.

The reason why different types of vertebrate nerve cells are generated in a particular sequence is still poorly understood. In the vertebrate retina, homeobox genes play a crucial role in establishing different cell identities. Here we provide evidence of a cellular clock that sequentially activates distinct homeobox genes in embryonic retinal cells, linking the identity of a retinal cell to its time of generation. By in situ expression analysis, we found that the three Xenopus homeobox genes Xotx5b, Xvsx1, and Xotx2 are initially transcribed but not translated in early retinal progenitors. Their translation requires cell cycle progression and is sequentially activated in photoreceptors (Xotx5b) and bipolar cells (Xvsx1 and Xotx2). Furthermore, by in vivo lipofection of "sensors" in which green fluorescent protein translation is under control of the 3' untranslated region (UTR), we found that the 3' UTRs of Xotx5b, Xvsx1, and Xotx2 are sufficient to drive a spatiotemporal pattern of translation matching that of the corresponding proteins and consistent with the time of generation of photoreceptors (Xotx5b) and bipolar cells (Xvsx1 and Xotx2). The block of cell cycle progression of single early retinal progenitors impairs their differentiation as photoreceptors and bipolar cells, but is rescued by the lipofection of Xotx5b and Xvsx1 coding sequences, respectively. This is the first evidence to our knowledge that vertebrate homeobox proteins can work as effectors of a cellular clock to establish distinct cell identities.

Xenopus Xotx2 and Drosophila otd share similar activities in anterior patterning of the frog embryo.

Despite the obvious anatomical differences between the fly and the vertebrate body plans, several genes involved in their development are largely conserved. In this work we provide evidence that overexpression of the Drosophila orthodenticle (otd) gene in Xenopus laevis has a similar effect to that of its homolog Xotx2. Injections of otd mRNA in whole embryos lead to posterior truncations and to induction of ectopic cement glands, similar to Xotx2 injections. In animal cap assays, otd, like Xotx2, is able to activate the cement gland marker XAG and to suppress the expression of the epidermal marker XK81. Finally, as assayed by Einsteck transplantation assays, otd, like Xotx2, is able to respecify a tail/trunk organizer to a head organizer. In this work we also show that Xotx2 and otd share molecular functions that regulate early regional specification of the Xenopus anterior neural plate. Gain-of-function experiment targeting low doses of either otd or Xotx2 mRNAs in the neural plate promote reduction of Xrx1 and Xbf1 expression domain; no changes are observed for the anterior mesodermal marker Xgsc, the dorsal diencephalic marker Xbh1, and the midbrain/hindbrain marker Xen2. otd/Xotx2 inhibition activity of Xrx1 and Xbf1 expression is consistent with the strong inhibition of Xfgf8 expression in the anterior neural ridge observed upon otd/Xotx2 mRNA injection.

The homeobox gene Xbh1 cooperates with proneural genes to specify ganglion cell fate within the Xenopus neural retina.

Recent studies on vertebrate eye development have focused on the molecular mechanisms of specification of different retinal cell types during development. Only a limited number of genes involved in this process has been identified. In Drosophila, BarH genes are necessary for the correct specification of R1/R6 eye photoreceptors. Vertebrate Bar homologues have been identified and are expressed in vertebrate retinal ganglion cells during differentiation; however, their retinal function has not yet been addressed. In this study, we report on the role of the Xenopus Bar homologue Xbh1 in retinal ganglion cell development and its interaction with the proneural genes Xath5 and Xath3, whose ability to promote ganglion cell fate has been demonstrated. We show that XHB1 plays a crucial role in retinal cell determination, acting as a switch towards ganglion cell fate. Detailed expression analysis, animal cap assays and in vivo lipofection assays, indicate that Xbh1 acts as a late transcriptional repressor downstream of the atonal genes Xath3 and Xath5. However, the action of Xbh1 on ganglion cell development is different and more specific than that of the Xath genes, and accounts for only a part of their activities during retinogenesis.

My perpetual cycle: from student to researcher to teacher to student ...

This contribution stems from the personal experience of the author regarding how he became acquainted with embryology and how he finally entered the field of developmental biology. It reports his feelings as a student of the Histology and Embryology course as it was taught in the late 1970s, and his present efforts in teaching developmental biology to university students. In the Developmental Biology course at Pisa University today, students are taught the tissue, molecular and genetic mechanisms that regulate development of several model systems. Drosophila is introduced at the beginning, because of the great knowledge that it has brought to the unraveling of the molecular aspects of development and because it allows several basic concepts to be introduced, and vertebrate systems follow. Other topics include the classic experiments on amphibian systems, which are explained in the light of recent molecular advances, as well as the genetically more versatile vertebrate systems such as the mouse.

XOtx5b and XOtx2 regulate photoreceptor and bipolar fates in the Xenopus retina.

Photoreceptor and bipolar cells are molecularly related cell types in the vertebrate retina. XOtx5b is expressed in both photoreceptors and bipolars, while a closely related member of the same family of transcription factors, XOtx2, is expressed in bipolar cells only. Lipofection of retinal precursors with XOtx5b biases them toward photoreceptor fates whereas a similar experiment with XOtx2 promotes bipolar cell fates. Domain swap experiments show that the ability to specify different cell fates is largely contained in the divergent sequence C-terminal to the homeodomain, while the more homologous N-terminal and homeodomain regions of both genes, when fused to VP16 activators, promote only photoreceptor fates. XOtx5b is closely related to Crx and like Crx it drives expression from an opsin reporter in vivo. XOtx2 suppresses this XOtx5b-driven reporter activity providing a possible explanation for why bipolars do not express opsin. Similarly, co-lipofection of XOtx2 with XOtx5b overrides the latter's ability to promote photoreceptor fates and the combination drives bipolar fates. The results suggest that the shared and divergent parts of these homologous genes may be involved in specifying the shared and distinct characters of related cell types in the vertebrate retina.

Induction and patterning of the telencephalon in Xenopus laevis.

We report an analysis of the tissue and molecular interplay involved in the early specification of the forebrain, and in particular telencephalic, regions of the Xenopus embryo. In dissection/recombination experiments, different parts of the organizer region were explanted at gastrula stage and tested for their inducing/patterning activities on either naive ectoderm or on midgastrula stage dorsal ectoderm. We show that the anterior dorsal mesendoderm of the organizer region has a weak neural inducing activity compared with the presumptive anterior notochord, but is able to pattern either neuralized stage 10.5 dorsal ectoderm or animal caps injected with BMP inhibitors to a dorsal telencephalic fate. Furthermore, we found that a subset of this tissue, the anterior dorsal endoderm, still retains this patterning activity. At least part of the dorsal telencephalic inducing activities may be reproduced by the anterior endoderm secreted molecule cerberus, but not by simple BMP inhibition, and requires the N-terminal region of cerberus that includes its Wnt-binding domain. Furthermore, we show that FGF action is both necessary and sufficient for ventral forebrain marker expression in neuralized animal caps, and possibly also required for dorsal telencephalic specification. Therefore, integration of organizer secreted molecules and of FGF, may account for patterning of the more rostral part of Xenopus CNS.