Papers by Giuseppina Barsacchi
In «The Origin of Species» Charles Darwin (1859) discusses eye evolution in the paragraph «Organs... more In «The Origin of Species» Charles Darwin (1859) discusses eye evolution in the paragraph «Organs of extreme perfection and complication» of Chapter VI, which has the significant title «Difficulties of the Theory». Darwin appears to be fully aware of difficulties on eye evolution: «To suppose that the eye, with all its inimitable contrivances... could have been formed by natural selection, seems, I freely confess, absurd in the highest degree». Indeed, the great variety of eyes in the animal word raises the question on their possible origin, by natural selection, from a simple prototypic ancestral eye. In fact eyes have also been considered an example of functional convergence or parallelism and the view on their possible polyphyletic origin has been authoritatively proposed. The recent emergence of an extensive conservation of the genetic network driving eye development in animals as distant as insects and mammals has brought new life to the debate on the monophyletic versus polyph...
<b>Copyright information:</b>Taken from "A specific box switches the cell fate d... more <b>Copyright information:</b>Taken from "A specific box switches the cell fate determining activity of XOTX2 and XOTX5b in the retina"http://www.neuraldevelopment.com/content/2/1/12Neural Development 2007;2():12-12.Published online 27 Jun 2007PMCID:PMC1929070. Results of lipofection of RPCs with wild-type and , and mutant constructs, showing the overall distribution of retinal cell types in clones lipofected with the different constructs, as indicated (a); PC, photoreceptor cells; HC, horizontal cells; BC, bipolar cells; AC, amacrine cells; GC, ganglion cells. The proportion of each cell type is represented as average ± standard error of the mean. Counted cells are indicated in the histogram (), from 9 retinae for , 10 retinae for , 9 retinae for , and 14 retinae for . Asterisks represent significant differences between constructs and , as calculated by Tukey-Kramer test (*&lt; 0.05, **
<b>Copyright information:</b>Taken from "A specific box switches the cell fate d... more <b>Copyright information:</b>Taken from "A specific box switches the cell fate determining activity of XOTX2 and XOTX5b in the retina"http://www.neuraldevelopment.com/content/2/1/12Neural Development 2007;2():12-12.Published online 27 Jun 2007PMCID:PMC1929070. On the left are schematics of the different constructs; on the right are their sequences in the final region of the homeodomain (HD) and directly downstream of it, with different colors shading the parental sequences of XOTX2 (red), XOTX5b (yellow) and OTD (blue). Lines are introduced for sequence alignment. The divergent region responsible for the different retinal activities of XOTX2 and XOTX5b (RS box) is shown in the blue box.
The International journal of developmental biology, 2000
Vertebrate eye formation is a complex process which involves early specification of the prospecti... more Vertebrate eye formation is a complex process which involves early specification of the prospective eye territory, induction events, patterning along the polarity axes and regional specification, to bring about the proper morphogenetic movements, cell proliferation, cell differentiation and neural connections allowing visual function. The molecular machinery underlying such complex developmental events is presently under an intense research scrutiny and many associated genetic factors have been isolated and characterized. These studies produced striking knowledge in the field, especially with respect to uncovering the role of key genes and their possible evolutionary conservation. Presently, a major task is to define the complex interactions connecting the multiplicity of molecular players that regulate eye development. We recently identified two homeobox genes, Xrx1 and Xvax2, and studied their function by using the Xenopus embryo as a developmental model system. Xrx1 and Xvax2 con...
Development, 1999
The anteriormost part of the neural plate is fated to give rise to the retina and anterior brain ... more The anteriormost part of the neural plate is fated to give rise to the retina and anterior brain regions. In Xenopus, this territory is initially included within the expression domain of the bicoid-class homeobox gene Xotx2 but very soon, at the beginning of neurulation, it becomes devoid of Xotx2 transcripts in spatiotemporal concomitance with the transcriptional activation of the paired-like homeobox gene Xrx1. By use of gain- and loss-of-function approaches, we have studied the role played by Xrx1 in the anterior neural plate and its interactions with other anterior homeobox genes. We find that, at early neurula stage Xrx1 is able to repress Xotx2 expression, thus first defining the retina-diencephalon territory in the anterior neural plate. Overexpression studies indicate that Xrx1 possesses a proliferative activity that is coupled with the specification of anterior fate. Expression of a Xrx1 dominant repressor construct (Xrx1-EnR) results in a severe impairment of eye and anter...
Neural development, Jan 20, 2015
Hedgehog (Hh) signals are instrumental to the dorsoventral patterning of the vertebrate eye, prom... more Hedgehog (Hh) signals are instrumental to the dorsoventral patterning of the vertebrate eye, promoting optic stalk and ventral retinal fates and repressing dorsal retinal identity. There has been limited analysis, however, of the critical window during which Hh molecules control eye polarity and of the temporal changes in the responsiveness of eye cells to these signals. In this study, we used pharmacological and molecular tools to perform stage-specific manipulations of Hh signalling in the developing Xenopus eye. In gain-of-function experiments, most of the eye was sensitive to ventralization when the Hh pathway was activated starting from gastrula/neurula stages. During optic vesicle stages, the dorsal eye became resistant to Hh-dependent ventralization, but this pathway could partially upregulate optic stalk markers within the retina. In loss-of-function assays, inhibition of Hh signalling starting from neurula stages caused expansion of the dorsal retina at the expense of the v...
European Journal of Biochemistry, 1997
To analyse the trans-cleavage activity of the hammerhead ribozyme occurring in the ovary of the n... more To analyse the trans-cleavage activity of the hammerhead ribozyme occurring in the ovary of the newt (Notophthalmus, Triturus) in more detail, six synthetic ribozymes representing natural and modified hammerhead sequences were tested with both short oligoribonucleotides and long transcripts as substrates. The same analysis was also performed with the monomer (330 nucleotides) newt ribozyme and variants thereof. None of the ribozymes comprising the newt natural sequence showed activity under multiple turnover conditions, regardless of sequence changes in stem and loop 11. With excess of ribozyme, the same ribozymes cleaved only to a limited extent a short substrate and extremely poorly a target site embedded within a long transcript. The addition of whole ovary cell extract had little influence on cleavage activity of short substrates. However, sequence changes in stems I and 111 to target different sequences considerably improved cleavage ability of the ribozymes under all conditions used. An RNA secondarystructure folding program showed that ribozymes with the natural newt sequence did not fold in a hammerhead structure whereas those with the changes in stem I and 111 did. These results suggest that the sequence of the stems I and 111 impairs the assembly of the newt ribozyme into a bimolecular hammerhead complex in vitro and that proteins present in the ovaries do not facilitate activity.
The Vertebrate Organizer, 2004
“The complex apparatus of the vertebrate eye originates by a sequence of developmental processes ... more “The complex apparatus of the vertebrate eye originates by a sequence of developmental processes which......are interlocked spatially and temporally”. So wrote Hans Spemann in 1901 (see Hamburger 1988, p. 18), when he introduced his work addressing “......the question whether these developmental processes proceed dependently or independently of each other; that is, whether their spatial and temporal coordination is guaranteed by a causal interaction or by a harmony which dates back to earlier stages, and perhaps to the egg”. A century apart, we are still impressed by Spemann’s ability to sharply define the scientific questions he was approaching experimentally with equal sharpness — well symbolized by his perfectly hand-crafted tools! The question he aimed at was obviously about induction, though this term was still ill-defined at the time. Spemann initially planned to assay induction in various aspects of eye development, a well-known process in 1901: namely, the transformation of the optic vesicle into the optic cup; lens formation; and formation of a transparent cornea. In the end, however, rather than expanding his experimental approaches, he focused in depth on lens formation, thus providing the first experimental evidence on an inductive phenomenon (Spemann 1901). Understanding lens induction paved the way towards understanding the inductive properties of the “Spemann organizer” (Spemann 1938).
The Theory of Evolution and Its Impact, 2011
Evolutionary Developmental Biology (Evo-Devo) deals with the relationships between the individual... more Evolutionary Developmental Biology (Evo-Devo) deals with the relationships between the individual development and the phenotypic changes of the organism during evolution. Major morphological transitions in evolution are presently recognized to be accommodated by a few key developmental genetic changes (part of a “developmental reprogramming”) and “case studies” in snakes, ducks, bats, dolphins, insects, and finches, providing examples of developmental bases of evolutionary change, are presented. On the other hand, the molecular changes occur in an otherwise conserved developmental genetics tool-kit (e.g., the Hox genes for anterior-posterior patterning, the network for eye formation) representing the “deep homology” underlying diversity of forms. Based on a relationship between embryo development and organism evolution, Evo-Devo represents a synthesis between Developmental and Evolutionary Biology.
We found that deprivation of pattern vision in one eye, that leaves luminance detection performan... more We found that deprivation of pattern vision in one eye, that leaves luminance detection performance unaffected, is sufficient to reduce brain-derived neurotrophic factor (but not trkB) messenger RNA in the visual cortex of young and adult rats. Monocular deprivation by means of eyelids' suture was performed during or after the critical period and the cortical amount of brain-derived neurotrophic factor messenger RNA was analysed by in situ hybridization and RNAase protection after 15-30 days of deprivation. A reduction of brain-derived neurotrophic factor messenger RNA was observed in the visual cortex contralateral to the deprived eye in rats monocularly deprived during the critical period. The same reduction was also found in rats monocularly deprived after the end of the critical period, when anatomical or physiological signs of monocular deprivation are absent. The pharmacological blockade of retinal activity equally affected the expression of brain-derived neurotrophic factor messenger RNA in young and adults. Quantitative RNAase protection assays revealed that the cortical level of brain-derived neurotrophic factor messenger RNA was reduced to the same extent when intraocular injections of tetrodotoxin were performed within or after the critical period. A developmental study of brain-derived neurotrophic factor messenger RNA expression in rat visual cortex showed a marked increase around the time of natural eye-opening followed by a plateau from postnatal day 20 until adult age. Messenger RNA for the kinasic domain of brain-derived neurotrophic factor receptor (trkB) was found in the dorsal lateral geniculate nucleus and the visual cortex during development and in adults. Our results suggest that the reduction of brain-derived neurotrophic factor messenger RNA induced by monocular deprivation is related to the absence of pattern vision rather than to the competitive interactions that underlie the effects of monocular deprivation during the critical period.
Developmental Dynamics, 2014
KEY FINDINGS • A screen for genes consistently regulated in Rx1 gain-and loss-of function embryos... more KEY FINDINGS • A screen for genes consistently regulated in Rx1 gain-and loss-of function embryos at early neurula identifies factors mainly involved in transcription regulation, cell migration/adhesion and cell proliferation. • Expression analysis shows that upregulated genes are coexpressed with Rx1, while downregulated genes are not expressed in early retinal precursors.
The Journal of Cell Biology, 1972
The repetitive DNA sequences of the newt, Triturus viridescens, have been studied by nucleic acid... more The repetitive DNA sequences of the newt, Triturus viridescens, have been studied by nucleic acid hybridization procedures. Complementary RNA was synthesized enzymatically from unfractionated newt DNA. This RNA hybridized strongly to the centromeric regions of both somatic and lampbrush chromosomes It also bound to other loci scattered along the lengths of the chromosomes The amplified ribosomal DNA in the multiple oocyte nucleoli was demonstrated by in situ hybridization
The International Journal of Developmental Biology, 2008
Maturation of miRNAs by dicer is required in vertebrates for normal neural development. Here we r... more Maturation of miRNAs by dicer is required in vertebrates for normal neural development. Here we report that dicer inactivation in Xenopus affects cell cycle progression, survival and timing of the generation of retinal cells, resulting in small retinas with lamination defects. In particular, dicer inactivation delays the exit from the cell cycle and the translation of key genes of late neurogenesis, highlighting a crucial role of miRNAs in retinal development.
STEM CELLS, 2013
The molecular mechanisms underlying the acquisition of retinal precursor identity are scarcely de... more The molecular mechanisms underlying the acquisition of retinal precursor identity are scarcely defined. Although the homeobox gene Rx1 (also known as Rax) plays a major role in specifying retinal precursors and maintaining their multipotent state, the involved mechanisms remain to be largely deciphered. Here, following a highthroughput screen for genes regulated by Rx1, we found that this transcription factor specifies the fate of retinal progenitors by repressing genes normally activated in adjacent ectodermal territories. Unexpectedly, we also observed that Rx1, mainly through the activation of the transcriptional repressors TLE2 and Hes4, is necessary and sufficient to inhibit endomesodermal gene expression in retinal precursors of the eye field. In particular, Rx1 knockdown leads retinogenic blastomeres to adopt an endomesodermal fate, indicating a previously undescribed function for Rx1 in preventing the expression of endomesoderm determinants known to inhibit retinal fate. Alt...
Stem Cells, 2009
Driving specific differentiation pathways in multipotent stem cells is a main goal of cell therap... more 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 t...
Protein & Peptide Letters, 2003
An N-terminal polypeptide (XVAX2-P104) of the XVAX2 protein, which is involved in controlling the... more An N-terminal polypeptide (XVAX2-P104) of the XVAX2 protein, which is involved in controlling the dorsoventral patterning of the retina in Xenopus laevis, was expressed and purified as a Histagged fusion protein (pHis-XVAX2-P104), and it was employed to generate an anti-XVAX2 antibody in New Zealand white rabbits. ELISA analysis shows that the titer of the antibody is as high as ~1:100,000. The antibody could specifically recognize the full-length XVAX2 protein in extracts of Xenopus embryos, as determined by Western Blotting.
Proceedings of the National Academy of Sciences, 2009
Cell identity is acquired in different brain structures according to a stereotyped timing schedul... more 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 in...
Proceedings of the National Academy of Sciences, 2010
In the developing central nervous system, the cell cycle clock plays a crucial role in determinin... more In the developing central nervous system, the cell cycle clock plays a crucial role in determining cell fate specification. A second clock, the circadian oscillator, generates daily rhythms of cell cycle progression. Although these two clocks interact, the mechanisms linking circadian cell cycle progression and cell fate determination are still poorly understood. A convenient system to address this issue is the pineal organ of lower vertebrates, which contains only two neuronal types, photoreceptors and projection neurons. In particular, photoreceptors constitute the core of the pineal circadian system, being able to transduce daily light inputs into the rhythmical production of melatonin. However, the genetic program leading to photoreceptor fate largely remains to be deciphered. Here, we report a previously undescribed function for the homeobox gene Bsx in controlling pineal proliferation and photoreceptor fate in Xenopu s. We show that Xenopus Bsx ( Xbsx ) is expressed rhythmical...
Proceedings of the National Academy of Sciences, 1997
Strand-specific transcripts of a satellite DNA of the newts, Notophthalmus and Triturus , are pre... more Strand-specific transcripts of a satellite DNA of the newts, Notophthalmus and Triturus , are present in cells in monomeric and multimeric sizes. These transcripts undergo self-catalyzed, site-specific cleavage in vitro : the reaction requires Mg 2+ and is mediated by a “hammerhead” domain. Transcription of the newt ribozyme appears to be performed by RNA polymerase II under the control of a proximal sequence element and a distal sequence element. In vitro , the newt ribozyme can cleave in trans an RNA substrate, suggesting that in vivo it might be involved in RNA processing events, perhaps as a riboprotein complex. Here we show that the newt ribozyme is in fact present as a riboprotein particle of about 12 S in the oocytes of Triturus. In addition, reconstitution experiments and gel-shift analyses show that a complex is assembled in vitro on the monomeric ribozyme molecules. UV cross-linking studies identify a few polypeptide species, ranging from 31 to 65 kDa, associated to the ne...
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Papers by Giuseppina Barsacchi