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L T Pecorino

Publications and source records attributed to L T Pecorino.

7 recordsLinked to original sources
PubMed1996

Activation of a single retinoic acid receptor isoform mediates proximodistal respecification.

BACKGROUND: The regenerating limb of urodele amphibians is an important system for evaluating the effects of retinoic acid (RA) on pattern formation. Regeneration proceeds by local formation of the blastema, a mesenchymal growth zone which normally only gives rise to structures distal to its level of origin. RA can respecify proximodistal identity in amphibian limb regeneration, and this activity of RA on the blastema is observed in two contexts. First, exposure to RA proximalizes a distal blastema resulting in duplication of structures proximal to the level of amputation. Second, after transplantation of a distal blastema to a proximal stump, the transplanted cells normally make only a minor contribution to the intercalary regenerate, but if transplanted cells are exposed to RA they occupy positions proximal to their level of origin and contribute to the regeneration of the intermediate tissue. Multiple isoforms of RA receptors (RARs) are expressed in the newt limb and are thought to mediate the respecification of positional identity. RESULTS: To identify which receptor(s) mediates proximodistal respecification, we have used the biolistics (particle bombardment) technique to transfect the blastemal mesenchyme with plasmids encoding chimeric proteins containing partial amino-acid sequences of the various newt RAR isoforms fused to a partial sequence of the thyroid hormone (3,5, 3'-triiodothyronine; T3) receptor. We then used T3 treatment to selectively activate individual RAR isoforms in vivo. By analyzing the distributions of transfected cells in regenerates derived from distal-to-proximal transplantation we find that activation of a single RAR isoform, delta 2, specifically mediates proximalization. CONCLUSIONS: These results demonstrate that the ability of RA to respecify proximodistal identity is mediated by a specific RAR isoform, delta 2. Activation of the RA pathway in individual cells indicates that positional respecification can be cell-autonomous. RA can respecify axial identity in several contexts in vertebrate development, but this is the first case where the RAR mediating respecification has been identified.

Animals↗
PubMed1996

Semi-automated positional analysis using laser scanning microscopy of cells transfected in a regenerating newt limb.

Limb regeneration in urodele amphibians such as the newt is a key system for investigating the positional identity of cells. The regenerate arises locally from blastemal cells, mesenchymal progenitors that normally give rise to structures distal to the amputation plane but which can be respecified (proximalized) by treatment with retinoic acid (RA) such that proximal structures are formed. To establish an assay for positional identity, cells of distal and RA-treated distal blastemas are labeled by transfection with an alkaline phosphatase marker gene using particle bombardment (biolistics). After grafting the distal blastema to a proximal stump, a context known as intercalary regeneration, the proximodistal distribution of labeled cells in the resulting regenerate is an index of positional identity. We use enzyme-labeled fluorescence (ELF) in conjunction with laser scanning microscopy to detect transfected cells within a section of the entire regenerate. A semi-automated analysis of the positional distribution of marked cells along the proximal-distal axis demonstrates that cells from both distal and RA-treated blastemas contribute to the regenerate. This procedure provides an efficient and accurate tool for positional analysis of transfected cells, and should be applicable for studying genes that play a role in specifying cell position during morphogenesis.

Alkaline Phosphatase↗
PubMed1994

Isoform-specific induction of a retinoid-responsive antigen after biolistic transfection of chimaeric retinoic acid/thyroid hormone receptors into a regenerating limb.

Retinoic acid (RA) induces secretory differentiation in the wound epidermis of a regenerating amphibian limb. We investigated the role of individual RA receptor (RAR) types in the newt wound epidermis by introducing chimaeric RA/thyroid hormone (T3) receptors (chi alpha 1 and chi delta 1) that can be activated by T3. A biolistic particle delivery system was employed to transfect cells in the wound epidermis of a regenerating limb and approximately 10% of the cells in targeted surface areas expressed marker genes. Both chi alpha 1 and chi delta 1 were comparable in their ability to stimulate transcription of a synthetic reporter construct through a RA response element after activation with T3 in situ. This activation was also comparable to that obtained by the endogenous complement of RARs in the RA-treated, transfected wound epidermis. The RA-inducible WE3 antigen, a marker for secretory differentiation, which distinguishes the wound epidermis from normal skin (Tassava, R. A., Johnson-Wint, B. and Gross, J. 1986, J. Exp. Zool. 239, 229-240), was used to assess the functional role of chi alpha 1 and chi delta 1. Chimaeric receptors were transfected with an alkaline phosphatase marker gene, activated with T3, and the expression of both the marker and WE3 was analyzed by double-label immunofluorescence. Newt limbs transfected with chi delta 1 showed many double-labelled cells dependent on the presence of T3, whereas contralateral limbs transfected with an alkaline phosphatase marker lacking chimaeric receptor sequences did not.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

In vitro analysis of the tissue plasminogen activator promoter reveals a GC box-binding activity present in murine brain but undetectable in kidney and liver.

Tissue plasminogen activator (t-PA) mRNA levels are high in murine brain, lower in kidney, and undetectable in liver. Differences in t-PA mRNA levels are regulated in part at the transcriptional level. Brain, kidney, and liver nuclear extracts direct regulated transcription from the murine t-PA promoter in a manner that reflects the relative levels of t-PA gene expression in these tissues in vivo. Analysis of mutants has defined two GC box motifs as important elements for regulated transcription in vitro. Upon investigation of protein-DNA binding, we detected an activity in brain extracts which was not detected in kidney or liver extracts. An Sp1-like factor also binds to this region in all three tissue types. DNA interference experiments show that the brain-enriched binding activity and the Sp1-like factor contact the same GC-rich sequences. These studies provide additional evidence that brain-enriched DNA-binding activities can interact with sequences also recognized by ubiquitous transcription factors.

Animals↗
PubMed1990

Transcription factor Sp1 is important for retinoic acid-induced expression of the tissue plasminogen activator gene during F9 teratocarcinoma cell differentiation.

The induced differentiation of F9 cells by retinoic acid (RA) and cyclic AMP (cAMP) activated transcription of the tissue plasminogen activator (t-PA) gene. This differentiation-responsive regulation of the t-PA promoter was also observed in transient assays. Multiple sequence elements within 243 bp of t-PA DNA contributed to the high level of transcription in retinoic acid- and cyclic AMP-differentiated cells. To investigate the factors involved in controlling t-PA transcription upon differentiation, we used F9 cell extracts to examine proteins that bind two proximal promoter elements. These elements (boxes 4 and 5) are homologous to GC boxes that are known binding sites for transcription factor Sp1. Mobility shift assays in the presence and absence of anti-Sp1 antibodies demonstrated that the proteins which bound to this region were immunologically related to human Sp1. The proteins also had a DNA-binding specificity similar to that of a truncated form of Sp1. Mutations of the GC motif within boxes 4 and 5 that interfered with Sp1 binding reduced in parallel the binding of the F9 cellular factors and lowered transcription in vitro as well as in vivo. Although this proximal region of the t-PA promoter was active in vivo only in differentiated cells, the Sp1-like binding proteins were present in equal concentrations and had similar properties in extracts of both stem and differentiated cells. These data suggest that other cellular elements participate with this Sp1-like factor in controlling differentiation-specific expression.

Animals↗
PubMed1988

Anti-sense inhibition of tissue plasminogen activator production in differentiated F9 teratocarcinoma cells.

F9 teratocarcinoma cells secrete the serine protease, tissue plasminogen activator (t-PA), upon differentiation induced in vitro by retinoic acid (RA) or RA and dibutyryl cAMP (RA/dbcAMP). A recombinant plasmid capable of directing the production of t-PA anti-sense RNA was constructed and transfected into F9 stem cells in an attempt to create a hypomorphic phenotype for t-PA synthesis. Several colonies were isolated which contained anti-sense RNA and which showed greater than a 50% reduction in t-PA activity upon differentiation. One such colony, 3b4, exhibited a 75% reduction in t-PA activity and was analyzed further. Large quantities of t-PA anti-sense transcript were expressed in the stem cells which are characterized by the absence of t-PA gene expression. In the induced cells, which normally express t-PA, the amount of detectable anti-sense transcript was significantly decreased. The amount of t-PA mRNA in differentiated cells containing t-PA anti-sense RNA was comparable to that in differentiated control cells. Subcellular localization of the mRNA in induced 3b4 cells appeared to be the same as induced control cells. Expression of collagen type IV, another marker of differentiation, was also monitored and was unaffected by the presence of t-PA anti-sense RNA in RA/dbcAMP-treated cells. The inhibition of differentiation-specific gene expression by anti-sense RNA may be useful for further studies of developmentally regulated genes.

Animals↗