Merlin, the Drosophila homologue of neurofibromatosis-2, is specifically required in posterior follicle cells for axis formation in the oocyte.

In Drosophila, the formation of the embryonic axes is initiated by Gurken, a transforming growth factor alpha signal from the oocyte to the posterior follicle cells, and an unknown polarising signal back to the oocyte. We report that Drosophila Merlin is specifically required only within the posterior follicle cells to initiate axis formation. Merlin mutants show defects in nuclear migration and mRNA localisation in the oocyte. Merlin is not required to specify posterior follicle cell identity in response to the Gurken signal from the oocyte, but is required for the unknown polarising signal back to the oocyte. Merlin is also required non-autonomously, only in follicle cells that have received the Gurken signal, to maintain cell polarity and limit proliferation, but is not required in embryos and larvae. These results are consistent with the fact that human Merlin is encoded by the gene for the tumour suppressor neurofibromatosis-2 and is a member of the Ezrin-Radixin-Moesin family of proteins that link actin to transmembrane proteins. We propose that Merlin acts in response to the Gurken signal by apically targeting the signal that initiates axis specification in the oocyte.

Small bristles, the Drosophila ortholog of NXF-1, is essential for mRNA export throughout development.

We identified a temperature-sensitive allele of small bristles (sbr), the Drosophila ortholog of human TAP/NXF-1 and yeast Mex67, in a screen for mutants defective in mRNA export. We show that sbr is essential for the nuclear export of all mRNAs tested in a wide range of tissues and times in development. High resolution and sensitive in situ hybridization detect the rapid accumulation of individual mRNA species in sbr mutant nuclei in particles that are distinct from nascent transcript foci and resemble wild-type export intermediates. The particles become more numerous and intense with increasing time at the restrictive temperature and are exported very rapidly after shifting back to the permissive temperature. The mRNA export block is not due indirectly to a defect in splicing, nuclear protein import, or aberrant nuclear ultrastructure, suggesting that in sbr mutants, mRNA is competent for export but fails to dock or translocate through NPCs. We conclude that NXF-1 is an essential ubiquitous export factor for all mRNAs throughout development in higher eukaryotes.

Squid hnRNP protein promotes apical cytoplasmic transport and localization of Drosophila pair-rule transcripts.

Drosophila melanogaster pair-rule segmentation gene transcripts localize apically of nuclei in blastoderm embryos. This might occur by asymmetric (vectorial) export from one side of the nucleus or by transport within the cytoplasm. We have followed fluorescently labeled pair-rule transcripts postinjection into Drosophila embryos. Naked, microinjected fushi tarazu (ftz) transcripts do not localize in blastoderm embryos, indicating that cytoplasmic mechanisms alone are insufficient for apical targeting. However, prior exposure of ftz to Drosophila or human embryonic nuclear extract leads to rapid, specific, microtubule-dependent transport, arguing against vectorial export. We present evidence that ftz transcript localization involves the Squid (Hrp40) hnRNP protein and that the activity of hnRNP proteins in promoting transcript localization is evolutionarily conserved. We propose that cytoplasmic localization machineries recognize transcripts in the context of nuclear partner proteins.

Asymmetric localization of Drosophila pair-rule transcripts from displaced nuclei: evidence for directional nuclear export.

Drosophila pair-rule transcripts accumulate exclusively apical of the layer of peripheral nuclei in syncytial blastoderm stage embryos. Here, we use aneuploid embryos to test zygotic gene requirements for pair-rule transcript localization. As apical localization is maintained in all genotypes tested, the required components must be maternally encoded. In aneuploid embryos with multiple layers or cortical nuclei, pair-rule transcripts lie apical of both superficial and internalized nuclei. In the latter case, the transcripts are 'pseudo-apical', i.e. apical of the nuclei from which they derive, but basal of superficial nuclei. We show that internalized nuclei maintain their apico-basal nuclear orientation, and that they lack the apical cytoskeletal assemblies which lie adjacent to superficial nuclei. These results support a mechanism of localizing pair-rule transcripts by directional (vectorial) nuclear export.

Redundant domains contribute to the transcriptional activity of the thyroid transcription factor 1.

The thyroid transcription factor 1 (TTF-1) is a homeodomain-containing protein implicated in the activation of thyroid-specific gene expression. Here we report that TTF-1 is capable of activating transcription from thyroglobulin and, to a lesser extent, thyroperoxidase gene promoters in nonthyroid cells. Full transcriptional activation of the thyroglobulin promoter by TTF-1 requires the presence of at least two TTF-1 binding sites. TTF-1 activates transcription via two functionally redundant transcriptional activation domains that as suggested by competition experiments, could use a common intermediary factor.

Degradation of maternal string mRNA is controlled by proteins encoded on maternally contributed transcripts.

In Drosophila, maternal string mRNAs are stable for the first few hours of development, but undergo specific timed degradation at the cellularisation stage. To determine whether the proteins that control this degradation are maternally or zygotically transcribed, we have used in situ hybridisation to determine the fate of maternal string transcripts in mutant embryos which lack individual chromosome arms. Our data indicate that maternal string mRNA persists for the normal period in all these mutants. Using alpha-amanitin to inhibit zygotic transcription we have shown that degradation of maternal mRNA is unaffected. Therefore, the proteins required to activate the degradation of string mRNA are encoded on a maternally contributed mRNA. We discuss possible models to explain the degradation pathway.

Multiple mechanisms of interference between transformation and differentiation in thyroid cells.

Transformation of the thyroid cell line FRTL-5 results in loss or reduction of differentiation as measured by the expression of thyroglobulin and thyroperoxidase, two proteins whose genes are exclusively expressed in thyroid follicular cells. The biochemical mechanisms leading to this phenomenon were investigated in three cell lines obtained by transformation of FRTL-5 cells with Ki-ras, Ha-ras, and polyomavirus middle-T oncogenes. With the ras oncogenes, transformation leads to undetectable expression of the thyroglobulin and thyroperoxidase genes. However, the mechanisms responsible for the extinction of the differentiated phenotype seem to be different for the two ras oncogenes. In Ki-ras-transformed cells, the mRNA encoding TTF-1, a transcription factor controlling thyroglobulin and thyroperoxidase gene expression, is severely reduced. On the contrary, nearly wild-type levels of TTF-1 mRNA are detected in Ha-ras-transformed cells. Furthermore, overexpression of TTF-1 can activate transcription of the thyroglobulin promoter in Ki-ras-transformed cells, whereas it has no effect on thyroglobulin transcription in the Ha-ras-transformed line. Expression of polyoma middle-T antigen in thyroid cells leads to only a reduction of differentiation and does not severely affect either the activity or the amount of TTF-1. Another thyroid cell-specific transcription factor, TTF-2, is more sensitive to transformation, since it disappears in all three transformed lines, and probably contributes to the reduced expression of the differentiated phenotype.

Pax-8, a paired domain-containing protein, binds to a sequence overlapping the recognition site of a homeodomain and activates transcription from two thyroid-specific promoters.

The Pax-8 gene, a member of the murine family of paired box-containing genes (Pax genes), is expressed in adult thyroid and in cultured thyroid cell lines. The Pax-8 protein binds, through its paired domain, to the promoters of thyroglobulin and thyroperoxidase, genes that are exclusively expressed in the thyroid. In both promoters, the binding site of Pax-8 overlaps with that of TTF-1, a homeodomain-containing protein involved in the activation of thyroid-specific transcription. Pax-8 activates transcription from cotransfected thyroperoxidase and thyroglobulin promoters, indicating that it may be involved in the establishment, control, or maintenance of the thyroid-differentiated phenotype. Thus, the promoters of thyroglobulin and thyroperoxidase represent the first identified natural targets for transcriptional activation by a paired domain-containing protein.

Functional role of TTF-1 binding sites in bovine thyroglobulin promoter.

We have studied the binding of purified TTF-1 on the bovine thyroglobulin gene promoter. DNase I footprinting experiments revealed three binding sites which corresponded in location to the A, B and C sites found in the rat thyroglobulin promoter. Mutants in the A and C regions showing reduced binding of TTF-1, also exhibited largely decreased promoter activity in transient expression experiments in primary-cultured dog thyrocytes. Two mutants in the B site that exhibited a reduced capacity to bind TTF-1 also displayed a drastically affected transcriptional activity in transient assays. As in the rat, sites A and C only are critical for promoter activity, these results suggest that full occupancy of the B site is required for thyroglobulin promoter activity in the cow only.

Cell-type-specific expression of the rat thyroperoxidase promoter indicates common mechanisms for thyroid-specific gene expression.

A 420-bp fragment from the 5' end of the rat thyroperoxidase (TPO) gene was fused to a luciferase reporter and shown to direct cell-type-specific expression when transfected into rat thyroid FRTL-5 cells. Analysis of this DNA fragment revealed four regions of the promoter which interact with DNA-binding proteins present in FRTL-5 cells. Mutation of the DNA sequence within any of these regions reduced TPO promoter activity. The trans-acting factors binding to these sequences were compared with thyroid transcription factor 1 (TTF-1) and TTF-2, previously identified as transcriptional activators of another thyroid-specific gene, the thyroglobulin (Tg) gene. Purified TTF-1 binds to three regions of TPO which are protected by FRTL-5 proteins. Two of the binding sites overlap with recognition sites for other DNA-binding proteins. One TTF-1 site can also bind a protein (UFB) present in the nuclei of both expressing and nonexpressing cells. TTF-1 binding to the proximal region overlaps with that for a novel protein present in FRTL-5 cells which can also recognize the promoter-proximal region of Tg. Using a combination of techniques, the factor binding to the fourth TPO promoter site was shown to be TTF-2. We conclude, therefore, that the FRTL-5-specific expression of two thyroid restricted genes, encoding TPO and Tg, relies on a combination of the same trans-acting factors present in thyroid cells.