Immunoprecipitation mapping of the TRX-associated chromosome elements in the fork head gene promoter in the Drosophila melanogaster salivary gland cells

Using the method of immunoprecipitation of the in vivo crosslinked and sheared by sonication chromatin, mapping of potential trithorax-associated regulatory elements within the extended (9 kb) promoter region of the fork head gene (fkh) in the Drosophila melanogaster salivary gland cells was performed. Relative homogeneity of the salivary gland cells, along with the parallel use of the antibodies to different domains of the same trithorax protein (TRX), and the introduction of cross-hybridization steps for additional specific enrichment of initial DNA libraries, provided improvement of the method effectiveness and identification of one major and two less expressed potential TRX-binding sites.     

Regulatory elements controlling expression of the Drosophila homeotic gene fork head.

The region-specific homeotic gene fork head (fkh) is expressed and required in a variety of tissues of the developing Drosophila embryo. In order to identify the cis regulatory elements directing the complex spatio-temporal expression pattern of fkh, we have studied the subpatterns directed by defined fragments of fkh genomic DNA. These experiments enabled us to distinguish separate regulatory elements specific for the different expression domains of fkh. In addition, our analysis revealed several unexpected features such as the redundancy of regulatory elements and the overlap of regulatory elements with the transcribed regions of other genes. Moreover, the separation of normally contiguous elements effecting expression in the posterior terminal fkh domain appears to lead to novel expression domains which do not correspond to known developmental units in the embryo.     

Organogenesis in Drosophila melanogaster: embryonic salivary gland determination is controlled by homeotic and dorsoventral patterning genes.

We have investigated Drosophila salivary gland determination by examining the effects of mutations in pattern forming genes on the salivary gland primordium. We find that the anterior-posterior extent of the primordium, a placode of columnar epithelial cells derived from parasegment 2, is established by the positive action of the homeotic gene Sex combs reduced (Scr). Embryos mutant for Scr lack a detectable placode, while ectopic Scr expression leads to the formation of ectopic salivary glands. In contrast, the dorsal-ventral extent of the placode is regulated negatively. Functions dependent on the decapentaplegic product place a dorsal limit on the placode, while dorsal-dependent genes act to limit the placode ventrally. We propose a model in which these pattern forming genes act early to determine the salivary gland anlage by regulating the expression of salivary gland determining genes, which in turn control genes that are involved in salivary gland morphogenesis.     

The Drosophila fork head factor directly controls larval salivary gland-specific expression of the glue protein gene Sgs3.

The Drosophila Fork head protein participates in salivary gland formation, since salivary glands are missing in fork head embryos. Here we show that the fork head encoded protein binds to an upstream regulatory region of the larval salivary gland glue protein gene Sgs3. Mobility shift assay in the presence of an anti-Fork head antibody demonstrated that the Fork head factor interacts with the TGTTTGC box shown to be involved in tissue-specific Sgs3 expression. Experiments employing a set of oligonucleotide competitors revealed that Fork head binding was prevented by the same single base substitutions that were previously shown to interfere with the TGTTTGC element function in vivo. Furthermore, the anti-Fork head antibody bound to >60 sites of polytene chromosomes, including the puffs of all Sgs genes and Fork head protein was detected in the nuclei of salivary glands of larvae of all examined stages. These data provide experimental evidence for the hypothesis that the protein encoded by the fork head gene is required initially for salivary gland formation and is utilized subsequently in the control of larval genes specifically expressed in this organ.     

Analysis of Drosophila salivary gland, epidermis and CNS development suggests an additional function of brinker in anterior-posterior cell fate specification

Salivary glands are simple structured organs which can serve as a model system in the study of organogenesis. Following a large EMS mutagenesis we have identified a number of genes required for normal salivary gland development. Mutations in the locus small salivary glands-1 (ssg-1) lead to a drastic reduction in the size of the salivary glands. The gene ssg-1 was cloned and subsequent sequence and genetic analysis showed identity to the recently published gene brinker. The salivary gland placode in brinker mutants appears reduced along both the anterior-posterior and dorso-ventral axis. Analysis of the brinker cuticle phenotype revealed a similar loss of anterior-posterior as well as lateral cell fates. The abdominal ventral denticle belts show a reduced number of setae in the first denticle row. Furthermore, we observed a preferential loss of lateral neuroblasts in the anterior parasegment. Together, these phenotypes suggest that brinker not only plays a role in dorso-ventral but also in anterior-posterior axis patterning.     

Terminal versus segmental development in the Drosophila embryo: the role of the homeotic gene fork head

Summary Mutations of the homeotic gene fork head (fkh) of Drosophila transform the non-segmented terminal regions of the embryonic ectoderm into segmental derivatives: Pre-oral head structures and the foregut are replaced by post-oral head structures which are occasionally associated with thoracic structures. Posterior tail structures including the hindgut and the Malpighian tubules are replaced by post-oral head structures associated with anterior tail structures. The fkh gene shows no maternal effect and is required only during embryogenesis. The phenotypes of double mutants indicate that fkh acts independently of other homeotic genes (ANT-C, BX-C, spalt) and caudal. In addition, the fkh domains are not expanded in Polycomb (Pc) group mutant embryos. Ectopic expression of the homeotic selector genes of the ANT-C and BX-C in Pc group mutant embryos causes segmental transformations in terminal regions of the embryo only in the absence of fkh gene activity. Thus, fkh is a region-specific homeotic rather than a selector gene, which promotes terminal as opposed to segmental development. Offprint requests to: Institut für Biologie II (Genetik), Universität Tübingen, Auf der Morgenstelle 28, D-7400 Tübingen, Federal Republic of Germany     

Regulation of Six1 expression by evolutionarily conserved enhancers in tetrapods

The Six1 homeobox gene plays critical roles in vertebrate organogenesis. Mice deficient for Six1 show severe defects in organs such as skeletal muscle, kidney, thymus, sensory organs and ganglia derived from cranial placodes, and mutations in human SIX1 cause branchio-oto-renal syndrome, an autosomal dominant developmental disorder characterized by hearing loss and branchial defects. The present study was designed to identify enhancers responsible for the dynamic expression pattern of Six1 during mouse embryogenesis. The results showed distinct enhancer activities of seven conserved non-coding sequences (CNSs) retained in tetrapod Six1 loci. The activities were detected in all cranial placodes (excluding the lens placode), dorsal root ganglia, somites, nephrogenic cord, notochord and cranial mesoderm. The major Six1-expression domains during development were covered by the sum of activities of these enhancers, together with the previously identified enhancer for the pre-placodal region and foregut endoderm. Thus, the eight CNSs identified in a series of our study represent major evolutionarily conserved enhancers responsible for the expression of Six1 in tetrapods. The results also confirmed that chick electroporation is a robust means to decipher regulatory information stored in vertebrate genomes. Mutational analysis of the most conserved placode-specific enhancer, Six1-21, indicated that the enhancer integrates a variety of inputs from Sox, Pax, Fox, Six, Wnt/Lef1 and basic helix-loop-helix proteins. Positive autoregulation of Six1 is achieved through the regulation of Six protein-binding sites. The identified Six1 enhancers provide valuable tools to understand the mechanism of Six1 regulation and to manipulate gene expression in the developing embryo, particularly in the sensory organs.     

Role of the hindbrain in patterning the otic vesicle: a study of the zebrafish vhnf1 mutant

The vertebrate inner ear develops from an ectodermal placode adjacent to rhombomeres 4 to 6 of the segmented hindbrain. The placode then transforms into a vesicle and becomes regionalised along its anteroposterior, dorsoventral and mediolateral axes. To investigate the role of hindbrain signals in instructing otic vesicle regionalisation, we analysed ear development in zebrafish mutants for vhnf1, a gene expressed in the caudal hindbrain during otic induction and regionalisation. We show that, in vhnf1 homozygous embryos, the patterning of the otic vesicle is affected along both the anteroposterior and dorsoventral axes. First, anterior gene expression domains are either expanded along the whole anteroposterior axis of the vesicle or duplicated in the posterior region. Second, the dorsal domain is severely reduced, and cell groups normally located ventrally are shifted dorsally, sometimes forming a single dorsal patch along the whole AP extent of the otic vesicle. Third, and probably as a consequence, the size and organization of the sensory and neurogenic epithelia are disturbed. These results demonstrate that, in zebrafish, signals from the hindbrain control the patterning of the otic vesicle, not only along the anteroposterior axis, but also, as in amniotes, along the dorsoventral axis. They suggest that, despite the evolution of inner ear structure and function, some of the mechanisms underlying the regionalisation of the otic vesicle in fish and amniotes have been conserved.     

Regulation and function of Scr, exd, and hth in the Drosophila salivary gland

Salivary gland formation in the Drosophila embryo is dependent on the homeotic gene Sex combs reduced (Scr). When Scr function is missing, salivary glands do not form, and when SCR is expressed everywhere in the embryo, salivary glands form in new places. Scr is normally expressed in all the cells that form the salivary gland. However, as the salivary gland invaginates, Scr mRNA and protein disappear. Homeotic genes, such as Scr, specify tissue identity by regulating the expression of downstream target genes. For many homeotic proteins, target gene specificity is achieved by cooperatively binding DNA with cofactors. Therefore, it is likely that SCR also requires a cofactor(s) to specifically bind to DNA and regulate salivary gland target gene expression. Here, we show that two homeodomain-containing proteins encoded by the extradenticle (exd) and homothorax (hth) genes are also required for salivary gland formation. exd and hth function at two levels: (1) exd and hth are required to maintain the expression of Scr in the salivary gland primordia prior to invagination and (2) exd and hth are required in parallel with Scr to regulate the expression of downstream salivary gland genes. We also show that Scr regulates the nuclear localization of EXD in the salivary gland primordia through repression of homothorax (hth) expression, linking the regulation of Scr activity to the disappearance of Scr expression in invaginating salivary glands.     

Suppression of neural fate and control of inner ear morphogenesis by Tbx1

Inner ear sensory organs and VIIIth cranial ganglion neurons of the auditory/vestibular pathway derive from an ectodermal placode that invaginates to form an otocyst. We show that in the mouse otocyst epithelium, Tbx1 suppresses neurogenin 1-mediated neural fate determination and is required for induction or proper patterning of gene expression related to sensory organ morphogenesis (Otx1 and Bmp4, respectively). Tbx1 loss-of-function causes dysregulation of neural competence in otocyst regions linked to the formation of either mechanosensory or structural sensory organ epithelia. Subsequently, VIIIth ganglion rudiment form is duplicated posteriorly, while the inner ear is hypoplastic and shows neither a vestibular apparatus nor a coiled cochlear duct. We propose that Tbx1 acts in the manner of a selector gene to control neural and sensory organ fate specification in the otocyst.     

pitx3 defines an equivalence domain for lens and anterior pituitary placode

Hedgehog signaling is required for formation and patterning of the anterior pituitary gland. However, the role of Hedgehog in pituitary precursor cell specification and subsequent placode formation is not well understood. We analyzed pituitary precursor cell lineages and find that pitx3 and distal-less3b (dlx3b) expression domains define lens and pituitary precursor positions. We show that pitx3 is required for pituitary pre-placode formation and cell specification, whereas dlx3b and dlx4b are required to restrict pituitary placode size. In smoothened mutant embryos that cannot transduce Hedgehog signals, median pituitary precursors are mis-specified and form an ectopic lens. Moreover, overexpression of sonic hedgehog (shh) blocks lens formation, and derivatives of lens precursors express genes characteristic of pituitary cells. However, overexpression of shh does not increase median pituitary placode size nor does it upregulate patched (ptc) expression in pituitary precursors during early somitogenesis. Our study suggests that by the end of gastrulation, pitx3-expressing cells constitute an equivalence domain of cells that can form either pituitary or lens, and that a non-Hedgehog signal initially specifies this placodal field. During mid-somitogenesis, Hedgehog then acts on the established median placode as a necessary and sufficient signal to specify pituitary cell types.