Novel Notch alleles reveal a Deltex-dependent pathway repressing neural fate.

BACKGROUND: The Notch receptor triggers a wide range of cell fate choices in higher organisms. In Drosophila, segregation of neural from epidermal lineages results from competition among equivalent cells. These cells express achaete/scute genes, which confer neural potential. During lateral inhibition, a single neural precursor is selected, and neighboring cells are forced to adopt an epidermal fate. Lateral inhibition relies on proteolytic cleavage of Notch induced by the ligand Delta and translocation of the Notch intracellular domain (NICD) to the nuclei of inhibited cells. The activated NICD, interacting with Suppressor of Hairless [Su(H)], stimulates genes of the E(spl) complex, which in turn repress the proneural genes achaete/scute. RESULTS: Here, we describe new alleles of Notch that specifically display loss of microchaetae sensory precursors. This phenotype arises from a repression of neural fate, by a Notch signaling distinct from that involved in lateral inhibition. We show that the loss of sensory organs associated with this phenotype results from a constitutive activation of a Deltex-dependent Notch-signaling event. These novel Notch alleles encode truncated receptors lacking the carboxy terminus of the NICD, which is the binding site for the repressor Dishevelled (Dsh). Dsh is known to be involved in crosstalk between Wingless and Notch pathways. CONCLUSIONS: Our results reveal an antineural activity of Notch distinct from lateral inhibition mediated by Su(H). This activity, mediated by Deltex (Dx), represses neural fate and is antagonized by elements of the Wingless (Wg)-signaling cascade to allow alternative cell fate choices.

Interactions between chip and the achaete/scute-daughterless heterodimers are required for pannier-driven proneural patterning.

The GATA factor Pannier activates the achaete-scute (ASC) proneural complex through enhancer binding and provides positional information for sensory bristle patterning in Drosophila. Chip was previously identified as a cofactor of the dorsal selector Apterous, and we show here that both Apterous and Chip also regulate ASC expression. Chip cooperates with Pannier in bridging the GATA factor with the HLH Ac/Sc and Daughterless proteins to allow enhancer-promoter interactions, leading to activation of the proneural genes, whereas Apterous antagonizes Pannier function. Within the Pannier domain of expression, Pannier and Apterous may compete for binding to their common Chip cofactor, and the accurate stoichiometry between these three proteins is essential for both proneural prepattern and compartmentalization of the thorax.

Different contributions of pannier and wingless to the patterning of the dorsal mesothorax of Drosophila.

In Drosophila, the GATA family transcription factor Pannier and the Wnt secreted protein Wingless are known to be important for the patterning of the notum, a part of the dorsal mesothorax of the fly. Thus, both proteins are necessary for the development of the dorsocentral mechanosensory bristles, although their roles in this process have not been clarified. Here, we show that Pannier directly activates the proneural genes achaete and scute by binding to the enhancer responsible for the expression of these genes in the dorsocentral proneural cluster. Moreover, the boundary of the expression domain of Pannier appears to delimit the proneural cluster laterally, while antagonism of Pannier function by the Zn-finger protein U-shaped sets its limit dorsally. So, Pannier and U-shaped provide positional information for the patterning of the dorsocentral cluster. In contrast and contrary to previous suggestions, Wingless does not play a similar role, since the levels and vectorial orientation of its concentration gradient in the dorsocentral area can be greatly modified without affecting the position of the dorsocentral cluster. Thus, Wingless has only a permissive role on dorsocentral achaete-scute expression. We also provide evidence indicating that Pannier and U-shaped are main effectors of the regulation of wingless expression in the presumptive notum.

u-shaped encodes a zinc finger protein that regulates the proneural genes achaete and scute during the formation of bristles in Drosophila.

The pattern of the large sensory bristles on the notum of Drosophila arises as a consequence of the expression of the achaete and scute genes. The gene u-shaped encodes a novel zinc finger that acts as a transregulator of achaete and scute in the dorsal region of the notum. Viable hypomorphic u-shaped mutants display additional dorsocentral and scutellar bristles that result from overexpression of achaete and scute. In contrast, overexpression of u-shaped causes a loss of achaete-scute expression and consequently a loss of dorsal bristles. The effects on the dorsocentral bristles appear to be mediated through the enhancer sequences that regulate achaete and scute at this site. The effects of u-shaped mutants are similar to those of a class of dominant alleles of the gene pannier with which they display allele-specific interactions, suggesting that the products of both genes cooperate in the regulation of achaete and scute. A study of the sites at which the dorsocentral bristles arise in mosaic u-shaped nota, suggests that the levels of the u-shaped protein are crucial for the precise positioning of the precursors of these bristles.

Transcriptional activity of pannier is regulated negatively by heterodimerization of the GATA DNA-binding domain with a cofactor encoded by the u-shaped gene of Drosophila.

The genes pannier (pnr) and u-shaped (ush) are required for the regulation of achaete-scute during establishment of the bristle pattern in Drosophila. pnr encodes a protein belonging to the GATA family of transcription factors, whereas ush encodes a novel zinc finger protein. Genetic interactions between dominant pnr mutants bearing lesions situated in the amino-terminal zinc finger of the GATA domain and ush mutants have been described. We show here that both wild-type Pannier and the dominant mutant form activate transcription from the heterologous alpha globin promoter when transfected into chicken embryonic fibroblasts. Furthermore, Pnr and Ush are found to heterodimerize through the amino-terminal zinc finger of Pnr and when associated with Ush, the transcriptional activity of Pnr is lost. In contrast, the mutant pnr protein with lesions in this finger associates only poorly with Ush and activates transcription even when cotransfected with Ush. These interactions have been investigated in vivo by overexpression of the mutant and wild-type proteins. The results suggest an antagonistic effect of Ush on Pnr function and reveal a new mode of regulation of GATA factors during development.

A genetic analysis of pannier, a gene necessary for viability of dorsal tissues and bristle positioning in Drosophila.

A genetic and phenotypic analysis of the gene pannier is described. Animals mutant for strong alleles die as embryos in which the cells of the amnioserosa are prematurely lost. This leads to a dorsal cuticular hole. The dorsal-most cells of the imagos are also affected: viable mutants exhibit a cleft along the dorsal midline. pannier mRNA accumulates specifically in the dorsal-most regions of the embryo and the imaginal discs. Viable mutants and mutant combinations also affect the thoracic and head bristle patterns in a complex fashion. Only those bristles within the area of expression of pannier are affected. A large number of alleles have been studied and reveal that pannier may have opposing effects on the expression of achaete and scute leading to a loss or a gain of bristles.

pannier, a negative regulator of achaete and scute in Drosophila, encodes a zinc finger protein with homology to the vertebrate transcription factor GATA-1.

The gene pannier acts as a repressor of achaete and scute, two transcription factors expressed in discrete subsets of cells at the sites where neural precursors develop. Molecular analysis of mutant alleles revealed the presence of two functional domains within the pannier protein: a zinc finger domain showing homology to the GATA-1 family of vertebrate transcription factors and a domain comprising two putative amphipathic helices. Mutants associated with lesions in the zinc finger domain display an overexpression of achaete and scute and the development of extra neural precursors. Mutant proteins in which the domain including the putative helices is deleted act as hyperactive repressor molecules causing a loss of achaete/scute expression and a loss of neural precursors. Other results suggest that the activity of pannier may be modulated by association with position-specific factors.

Sgs-3 chromatin structure and trans-activators: developmental and ecdysone induction of a glue enhancer-binding factor, GEBF-I, in Drosophila larvae.

The transcription of the Drosophila melanogaster 68C salivary gland glue gene Sgs-3 involves the interaction of a distal and a proximal regulatory region. These are marked in vivo by a specific chromatin structure which is established sequentially during development, starting early in embryogenesis. The distal region is characterized by a stage- and tissue-specific DNase I hypersensitive site. A stage- and tissue-specific factor, GEBF-I, binds in this region and is missing in 2B5 mutant larvae which lack Sgs-3 transcripts. This binding involves the simultaneous interaction with two distinct DNA sequences which induces conformational changes in the protein. Salivary glands acquire competence to respond to ecdysone in the mid-third larval instar, whereafter the hormone rapidly induces both the GEBF-I protein and Sgs-3 transcription.

Analysis of a DNase I-hypersensitive site in transgenic Drosophila reveals a key regulatory element of Sgs3.

We have undertaken chromatin studies on transformed Drosophila strains carrying DNA sequences modified in the region of the DNase I (EC 3.1.4.5)-hypersensitive sites -750 and -600 base pairs upstream from the Sgs3 start site. Although both sites are developmentally specific, modifications in the -750 site have little or no effect on Sgs3-encoded transcript levels, whereas either deletion or replacement of sequences at the -600 site causes an important reduction in transcript levels. The element associated with the -600 site enhances Sgs3 transcription when displaced with respect to the start site. This combined approach has defined sequence elements necessary both for normal transcript levels as well as the chromatin structure characteristic of Sgs3 activity in vivo.

Changes in the chromatin structure of Drosophila glue genes accompany developmental cessation of transcription in wild type and transformed strains.

Three Drosophila salivary gland glue genes show a dramatic transition in their DNAse I hypersensitive sites during the short period between the late third instar and the white prepupa, which correlates with the cessation of their transcription. In culture cells, where the genes are inactive, there is a chromatin configuration similar to that of prepupal salivary glands. In two transformed fly strains where the sgs3 gene is active at new chromosomal sites, including one in which 2.6 kb of sgs3 upstream sequences have been inverted, the same DNAase I hypersensitive sites and developmental transitions are seen over the same DNA regions. These results, together with the analysis of transformants carrying rearranged sgs3 genes, suggest that there is at least one distal DNAase I hypersensitive site associated with an element of regulation which may be exchanged between sgs genes.