Biomedical subjects

Gabriele Amore

Publications and source records attributed to Gabriele Amore.

6 recordsLinked to original sources

PubMed2006

cis-Regulatory control of cyclophilin, a member of the ETS-DRI skeletogenic gene battery in the sea urchin embryo.

The Strongylocentrotus purpuratus cyclophilin1 gene (Sp-cyp1) is expressed exclusively in skeletogenic mesenchyme cells of the embryo, beginning in the micromere lineage of the early blastula stage and continuing after gastrulation during the syncytial deposition of the skeleton. This gene encodes a protein which is a member of the peptidyl-prolyl cis-trans isomerase (PPIase) family. Sp-cyp1 is among the differentiation genes activated in the skeletogenic territory as a terminal function of the endomesodermal gene regulatory network. Network perturbation analysis had predicted the skeletogenic regulators Ets1 and Deadringer (Dri) to be its driver inputs. Here, we show that a 218-bp cis-regulatory DNA fragment recapitulates skeletogenic Sp-cyp1 expression; that elimination of either Ets1 or Dri inputs severely depresses the activity of expression constructs containing this DNA fragment; and that Ets1 and Dri target sites within the 218 bp fragment are required for normal expression. This indicates that the predicted inputs are direct. Other studies indicate that the same inputs are evidently necessary for expression of several other skeletogenic differentiation genes, and these genes probably constitute a skeletogenic gene battery, defined by its Ets plus Dri regulatory inputs.

Amino Acid Sequence↗

PubMed2004

SpHnf6, a transcription factor that executes multiple functions in sea urchin embryogenesis.

The Strongylocentrotus purpuratus hnf6 (Sphnf6) gene encodes a new member of the ONECUT family of transcription factors. The expression of hnf6 in the developing embryo is triphasic, and loss-of-function analysis shows that the Hnf6 protein is a transcription factor that has multiple distinct roles in sea urchin development. hnf6 is expressed maternally, and before gastrulation its transcripts are distributed globally. Early in development, its expression is required for the activation of PMC differentiation genes such as sm50, pm27, and msp130, but not for the activation of any known PMC regulatory genes, for example, alx, ets1, pmar1, or tbrain. Micromere transplantation experiments show that the gene is not involved in early micromere signaling. Early hnf6 expression is also required for expression of the mesodermal regulator gatac. The second known role of hnf6 is its participation after gastrulation in the oral ectoderm gene regulatory network (GRN), in which its expression is essential for the maintenance of the state of oral ectoderm specification. The third role is in the neurogenic ciliated band, which is foreshadowed exactly by a trapezoidal band of hnf6 expression at the border of the oral ectoderm and where it continues to be expressed through the end of embryogenesis. Neither oral ectoderm regulatory functions nor ciliated band formation occur normally in the absence of hnf6 expression.

Amino Acid Sequence↗

PubMed2004

Expression of an NK2 homeodomain gene in the apical ectoderm defines a new territory in the early sea urchin embryo.

We have identified an NK2 family homeodomain transcription factor, SpNK2.1, in the sea urchin Strongylocentrotus purpuratus whose transcripts are initially detected within the apical plate ectoderm of the hatching blastula and are confined to the apical organ at least through 2 weeks of development. Protein localization studies demonstrate that SpNK2.1 is restricted to the apical plate epithelium, but is excluded from the nucleus of serotonergic neurons. The expression profile of SpNK2.1 is dictated via two separate regulatory systems. Initially, SpNK2.1 is restricted to the apical pole domain by beta-catenin-dependent processes operating along the animal-vegetal axis, as evidenced by an expansion of SpNK2.1 expression upon cadherin overexpression. Starting at gastrulation, expression in the apical plate is maintained by SpDri, the sea urchin orthologue of dead ringer. Abrogation of SpDri results in the downregulation of SpNK2.1 after gastrulation, but SpDri is not necessary for the initial activation of SpNK2.1. Loss of function experiments using SpNK2.1-specific morpholino antisense oligonucleotides and SpNK2.1 overexpression experiments do not disrupt embryonic development and have no effect upon the development of neuronal components of the apical organ. Nonetheless, SpNK2.1 defines a new early territory of the sea urchin embryo.

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PubMed2003

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.

Amino Acid Sequence↗

PubMed2002

A provisional regulatory gene network for specification of endomesoderm in the sea urchin embryo.

We present the current form of a provisional DNA sequence-based regulatory gene network that explains in outline how endomesodermal specification in the sea urchin embryo is controlled. The model of the network is in a continuous process of revision and growth as new genes are added and new experimental results become available; see http://www.its.caltech.edu/~mirsky/endomeso.htm (End-mes Gene Network Update) for the latest version. The network contains over 40 genes at present, many newly uncovered in the course of this work, and most encoding DNA-binding transcriptional regulatory factors. The architecture of the network was approached initially by construction of a logic model that integrated the extensive experimental evidence now available on endomesoderm specification. The internal linkages between genes in the network have been determined functionally, by measurement of the effects of regulatory perturbations on the expression of all relevant genes in the network. Five kinds of perturbation have been applied: (1) use of morpholino antisense oligonucleotides targeted to many of the key regulatory genes in the network; (2) transformation of other regulatory factors into dominant repressors by construction of Engrailed repressor domain fusions; (3) ectopic expression of given regulatory factors, from genetic expression constructs and from injected mRNAs; (4) blockade of the beta-catenin/Tcf pathway by introduction of mRNA encoding the intracellular domain of cadherin; and (5) blockade of the Notch signaling pathway by introduction of mRNA encoding the extracellular domain of the Notch receptor. The network model predicts the cis-regulatory inputs that link each gene into the network. Therefore, its architecture is testable by cis-regulatory analysis. Strongylocentrotus purpuratus and Lytechinus variegatus genomic BAC recombinants that include a large number of the genes in the network have been sequenced and annotated. Tests of the cis-regulatory predictions of the model are greatly facilitated by interspecific computational sequence comparison, which affords a rapid identification of likely cis-regulatory elements in advance of experimental analysis. The network specifies genomically encoded regulatory processes between early cleavage and gastrula stages. These control the specification of the micromere lineage and of the initial veg(2) endomesodermal domain; the blastula-stage separation of the central veg(2) mesodermal domain (i.e., the secondary mesenchyme progenitor field) from the peripheral veg(2) endodermal domain; the stabilization of specification state within these domains; and activation of some downstream differentiation genes. Each of the temporal-spatial phases of specification is represented in a subelement of the network model, that treats regulatory events within the relevant embryonic nuclei at particular stages.

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PubMed2002

A genomic regulatory network for development.

Development of the body plan is controlled by large networks of regulatory genes. A gene regulatory network that controls the specification of endoderm and mesoderm in the sea urchin embryo is summarized here. The network was derived from large-scale perturbation analyses, in combination with computational methodologies, genomic data, cis-regulatory analysis, and molecular embryology. The network contains over 40 genes at present, and each node can be directly verified at the DNA sequence level by cis-regulatory analysis. Its architecture reveals specific and general aspects of development, such as how given cells generate their ordained fates in the embryo and why the process moves inexorably forward in developmental time.

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