Gabriele Amore

Spdeadringer, a sea urchin embryo gene required separately in skeletogenic and oral ectoderm gene regulatory networks

The Spdeadringer (Spdri) gene encodes an ARID-class transcription factor not previously known in sea urchin embryos. We show that Spdri is a key player in two separate developmental gene regulatory networks (GRNs). Spdri is expressed in a biphasic manner, first, after 12 h and until ingression in the skeletogenic descendants of the large micromeres; second, after about 20 h in the oral ectoderm, where its transcripts remain present at 30-50 mRNA molecules/cell far into development. In both territories, the periods of Spdri expression follow prior territorial specification events. The functional significance of each phase of expression was assessed by determining the effect of an alphaSpdri morpholino antisense oligonucleotide (MASO) on expression of 17 different mesodermal genes, 8 different oral ectoderm genes, and 18 other genes expressed specifically during endomesoderm specification. These effects were measured by quantitative PCR, supplemented by whole-mount in situ hybridization and morphological observations. Spdri is shown to act in the micromere descendants in the pathways that result in the expression of batteries of terminal skeletogenic genes. But, in the oral ectoderm, the same gene participates in the central GRN controlling oral ectoderm identity. Spdri is linked in the oral ectoderm GRN with several other genes encoding transcriptional regulators that are expressed specifically in various regions of the oral ectoderm. If its expression is blocked by treatment with alphaSpdri MASO, oral-specific features disappear and expression of the aboral ectoderm marker spec1 encompasses the whole of the ectoderm. In addition to disappearance of the oral ectoderm, morphological consequences of alphaSpdri MASO treatment include failure of spiculogenesis and of correct primary mesenchyme cell (pmc) patterning in the postgastrular embryo, and also failure of gastrulation. To further analyze these phenotypes, chimeric embryos were constructed consisting of two labeled micromeres combined with micromereless 4th cleavage host embryos; either the micromeres or the hosts contained alphaSpdri MASO. These experiments showed that, while Spdri expression is required autonomously for expression of skeletogenic genes prior to ingression, complete skeletogenesis also requires the expression of oral ectoderm patterning information. Presentation of this information on the oral side of the blastocoel in turn depends on Spdri expression in the oral ectoderm. Failure of gastrulation is not due to indirect interference with endomesodermal specification per se, since all endomesodermal genes tested function normally in alphaSpdri MASO embryos. Part of its cause is interference by alphaSpdri MASO with a late signaling function on the part of the micromere descendants that is needed to complete clearance of the Soxb1 repressor of gastrulation from the prospective endoderm, but in addition there is a nonautonomous oral ectoderm effect.

Presence of Candida zemplinina in Sicilian Musts and Selection of a Strain for Wine Mixed Fermentations

The purpose of this work was to investigate the presence of C. zemplinina yeasts in Sicilian musts and grapes and to identify strains of oenological interest. We report on the taxonomical reclassification of Candida yeast isolates from Sicilian musts and on the selection of one strain of oenological interest (Cz3), based on mixed micro-fermentation experiments in sterile Nero d’Avola musts. Our results show that Candida zemplinina is abundant in Sicilian grapes and musts, and that the Cz3 strain is suitable for Candida zemplinina/Saccharomyces cerevisiae mixed fermentations. The higher glycerol content and the lower ethanol level stood out as the most promising features of the wines obtained upon sequential inoculation of the Cz3 and (S. cerevisiae) NDA21 yeast starters. We therefore have isolated a Sicilian Cz strain endowed with very promising features for the future development of mixed fermentation protocols.

Biodiversity of indigenous Saccharomyces populations from old wineries of south-eastern Sicily (Italy): preservation and economic potential.

In recent years, the preservation of biodiversity has become an important issue. Despite much public discussion, however, current practices in the food industry seldom take account of its potential economic importance: on the contrary, the introduction of industrialized agriculture practices over large areas has often resulted in a dramatic reduction in biodiversity. In this paper, we report on the remarkable degree of biodiversity in the wine yeast populations naturally present in a small area of Sicily (Italy) where traditional (non-industrial) winery practices are still in place. Out of more than 900 Saccharomyces yeast isolates recovered from late spontaneous fermentations, we detected at least 209 strains. Most interestingly, when evaluated at the fermentation and technological level, a number of isolates were found to be superior to industrial yeast strains. Out of a selected group, isolates from two strains were used for experimental fermentations in a winery environment and the quality of the wines produced was assessed at the technological, quality and sensory levels. Given that the characteristics of the wines produced were found to be industrially appealing, the study demonstrated the economic potential of preserving the patrimony of Sicilian yeast biodiversity and highlighted the importance of maintaining traditional wine making practices.

A Hh-driven gene network controls specification, pattern and size of the Drosophila simple eyes

During development, extracellular signaling molecules interact with intracellular gene networks to control the specification, pattern and size of organs. One such signaling molecule is Hedgehog (Hh). Hh is known to act as a morphogen, instructing different fates depending on the distance to its source. However, how Hh, when signaling across a cell field, impacts organ-specific transcriptional networks is still poorly understood. Here, we investigate this issue during the development of the Drosophila ocellar complex. The development of this sensory structure, which is composed of three simple eyes (or ocelli) located at the vertices of a triangular patch of cuticle on the dorsal head, depends on Hh signaling and on the definition of three domains: two areas of eya and so expression – the prospective anterior and posterior ocelli – and the intervening interocellar domain. Our results highlight the role of the homeodomain transcription factor engrailed (en) both as a target and as a transcriptional repressor of hh signaling in the prospective interocellar region. Furthermore, we identify a requirement for the Notch pathway in the establishment of en maintenance in a Hh-independent manner. Therefore, hh signals transiently during the specification of the interocellar domain, with en being required here for hh signaling attenuation. Computational analysis further suggests that this network design confers robustness to signaling noise and constrains phenotypic variation. In summary, using genetics and modeling we have expanded the ocellar gene network to explain how the interaction between the Hh gradient and this gene network results in the generation of stable mutually exclusive gene expression domains. In addition, we discuss some general implications our model may have in some Hh-driven gene networks.

Built shallow to maintain homeostasis and persistent infection: insight into the transcriptional regulatory network of the gastric human pathogen Helicobacter pylori.

Transcriptional regulatory networks (TRNs) transduce environmental signals into coordinated output expression of the genome. Accordingly, they are central for the adaptation of bacteria to their living environments and in host–pathogen interactions. Few attempts have been made to describe a TRN for a human pathogen, because even in model organisms, such as Escherichia coli, the analysis is hindered by the large number of transcription factors involved. In light of the paucity of regulators, the gastric human pathogen Helicobacter pylori represents a very appealing system for understanding how bacterial TRNs are wired up to support infection in the host. Herein, we review and analyze the available molecular and “-omic” data in a coherent ensemble, including protein–DNA and protein–protein interactions relevant for transcriptional control of pathogenic responses. The analysis covers ∼80% of the annotated H. pylori regulators, and provides to our knowledge the first in-depth description of a TRN for an important pathogen. The emerging picture indicates a shallow TRN, made of four main modules (origons) that process the physiological responses needed to colonize the gastric niche. Specific network motifs confer distinct transcriptional response dynamics to the TRN, while long regulatory cascades are absent. Rather than having a plethora of specialized regulators, the TRN of H. pylori appears to transduce separate environmental inputs by using different combinations of a small set of regulators.

Regulation of ocellar specification and size by twin of eyeless and homothorax

The retinal determination gene network (RDGN) constitutes a paradigm of a gene network controlling organ specification and growth. In this study, we probed the RDGN in the Drosophila ocelli, a set of simple eyes located on the flys dorsal head, by studying the expression, regulation, and function of toy, hth, eya, and so, members of the Pax6, Meis, Eya, and Six gene families. Our results highlight the role of the pax6 gene toy, together with the hh signaling pathway, in the initiation of eya and so expression; the engagement of eya and so in a feedback loop necessary for their full expression; and the interplay between hh signaling and hth as a mechanism of organ size control, as general regulatory steps in the specification of visual organs. Developmental Dynamics, 2011.

Highly conserved elements discovered in vertebrates are present in non-syntenic loci of tunicates, act as enhancers and can be transcribed during development

Co-option of cis-regulatory modules has been suggested as a mechanism for the evolution of expression sites during development. However, the extent and mechanisms involved in mobilization of cis-regulatory modules remains elusive. To trace the history of non-coding elements, which may represent candidate ancestral cis-regulatory modules affirmed during chordate evolution, we have searched for conserved elements in tunicate and vertebrate (Olfactores) genomes. We identified, for the first time, 183 non-coding sequences that are highly conserved between the two groups. Our results show that all but one element are conserved in non-syntenic regions between vertebrate and tunicate genomes, while being syntenic among vertebrates. Nevertheless, in all the groups, they are significantly associated with transcription factors showing specific functions fundamental to animal development, such as multicellular organism development and sequence-specific DNA binding. The majority of these regions map onto ultraconserved elements and we demonstrate that they can act as functional enhancers within the organism of origin, as well as in cross-transgenesis experiments, and that they are transcribed in extant species of Olfactores. We refer to the elements as ‘Olfactores conserved non-coding elements’.

Candida zemplinina for production of wines with less alcohol and more glycerol

We developed a new protocol for winery mixed fermentations, using the selected Candida zemplinina yeast strain Cz3. The results of a two-year study, in which red musts (Merlot in 2010; Merlot, Nero d’Avola and Frappato in 2011) were inoculated with Cz3, is discussed. These wines were compared with wines obtained by inoculation with commercial Saccharomyces cerevisiae yeast strains (NDA21 and AR06 in 2010; NDA21 in 2011), or with those obtained by spontaneous fermentation (only in 2011). The inoculation of Cz3 always resulted in a two-phase fermentation: the first phase was driven by the C. zemplinina strain, while the second was dominated by the indigenous S. cerevisiae yeasts coming from the grapes and/or the winery. In both years the Cz3 wines contained less alcohol and more glycerol than those made with the commercial yeast strains or those obtained by spontaneous fermentation. Triangle tests showed that a sensorial difference between wines could only be achieved through the utilisation of Cz3.

Ca2+ influx-linked protein kinase C activity regulates the β-catenin localization, micromere induction signalling and the oral–aboral axis formation in early sea urchin embryos

Summary Sea urchin embryos initiate cell specifications at the 16-cell stage by forming the mesomeres, macromeres and micromeres according to the relative position of the cells in the animal–vegetal axis. The most vegetal cells, micromeres, autonomously differentiate into skeletons and induce the neighbouring macromere cells to become mesoendoderm in the β-catenin-dependent Wnt8 signalling pathway. Although the underlying molecular mechanism for this progression is largely unknown, we have previously reported that the initial events might be triggered by the Ca2+ influxes through the egg-originated L-type Ca2+ channels distributed asymmetrically along the animal–vegetal axis and through the stretch-dependent Ca2+channels expressed specifically in the micromere at the 4th cleavage. In this communication, we have examined whether one of the earliest Ca2+ targets, protein kinase C (PKC), plays a role in cell specification upstream of β-catenin. To this end, we surveyed the expression pattern of β-catenin in early embryos in the presence or absence of the specific peptide inhibitor of Hemicentrotus pulcherrimus PKC (HpPKC-I). Unlike previous knowledge, we have found that the initial nuclear entrance of β-catenin does not take place in the micromeres, but in the macromeres at the 16-cell stage. Using the HpPKC-I, we have demonstrated further that PKC not only determines cell-specific nucleation of β-catenin, but also regulates a variety of cell specification events in the early sea urchin embryos by modulating the cell adhesion structures, actin dynamics, intracellular Ca2+ signalling, and the expression of key transcription factors.

Compositional genome contexts affect gene expression control in sea urchin embryo.

Gene expression is widely perceived as exclusively controlled by the information contained in cis-regulatory regions. These are built in a modular way, each module being a cluster of binding sites for the transcription factors that control the level, the location and the time at which gene transcription takes place. On the other hand, results from our laboratory have shown that gene expression is affected by the compositional properties (GC levels) of the isochores in which genes are embedded, i.e. the genome context. To clarify how compositional genomic properties affect the way cis-regulatory information is utilized, we have changed the genome context of a GFP-reporter gene containing the complete cis-regulatory region of the gene spdeadringer (spdri), expressed during sea urchin embryogenesis. We have observed that GC levels higher or lower than those found in the natural genome context can alter the reporter expression pattern. We explain this as the result of an interference with the functionality of specific modules in the genes cis-regulatory region. From these observations we derive the notion that the compositional properties of the genome context can affect cis-regulatory control of gene expression. Therefore although the way a gene works depends on the information contained in its cis-regulatory region, availability of such information depends on the compositional properties of the genomic context.