betaTubulin56D (ß1 tubulin)

Promoter Structure

The ß1 tubulin gene is expressed in the CNS and PNS. Upstream elements for low level expression in the CNS are present between -2.2 kb and the transcription initiation site. Located in the intron between +0.44 kb and +2.5 kb are enhancer elements that drive the expression in the chordotonal organs and the apodemes. Between the start site and +0.44 kb (273 bp) and +2.5 kb and the second exon (315 bp), maternal and CNS enhancers result in full level expression of a lacZ-ß1 reporter gene (Buttgereit, 1991).

During Drosophila embryogenesis ß1 tubulin is expressed during formation of the apodemes, ectodermal attachment sites for the somatic muscles. Expression is first detected at late stage 13 and remains until hatching. By deletion analysis of the intron, a 14-bp element has been found to be present in three copies. This element represents a classical enhancer, as it acts on a heterologous promoter. Separate fragments containing the respective elements yield nearly identical expression patterns, although no cooperativity is observed between the three copies. Thus, the expression of the àß1 tubulin gene in the apodemes is under control of redundant enhancer elements. Double staining for ß1 tubulin gene expression in apodemes and for ß3 tubulin gene expression in muscles allowed the correlation of apodeme and muscle formation. Cells of the apodemes that are in contact with their corresponding muscles show expression of the reporter gene as monitored by antibody staining (Buttgereit, 1993a).

Inductive interactions between cells of distinct fates underlie the basis for morphogenesis and organogenesis across species. In the Drosophila embryo, somatic myotubes form specific interactions with their epidermal muscle attachment (EMA) cells. The establishment of these interactions is a first step toward further differentiation of the EMA cells into elongated tendon cells containing an organized array of microtubules and microfilaments. The molecular signal for terminal differentiation of tendon cells is the secreted Drosophila neuregulin-like growth factor Vein, produced by the myotubes. Although Vein mRNA is produced by all of the myotubes, Vein protein is secreted and accumulates specifically at the muscle-tendon cell junctional site. In loss-of-function vein mutant embryos, muscle-dependent differentiation of epidermal tendon cells, measured by the level of expression of specific markers (Delilah and beta1 tubulin) is blocked. When Vein is expressed in ectopic ectodermal cells, it induces the ectopic expression of these genes. These results favor the possibility that the Drosophila EGF receptor DER/Egfr expressed by the EMA cells functions as a receptor for Vein. Vein/Egfr binding activates the Ras pathway in the EMA cells leading to the transcription of the tendon-specific genes stripe, delilah, and beta1 tubulin. In Egfr1F26 mutant embryos lacking functional Egfr expression, the levels of Delilah and beta1 Tubulin are very low. The ability of ectopic Vein to induce the expression of Delilah and beta1 Tubulin depends on the presence of functional Egfrs. Activation of the Egfr signaling pathway by either ectopically secreted Spitz, or activated Ras, leads to the ectopic expression of Delilah. These results suggest that inductive interactions between myotubes and their epidermal muscle attachment cells are initiated by the binding of Vein, to the Egfr on the surface of EMA cells (Yarnitzky, 1997).

Expression of the ß1 tubulin gene is under complex developmental control. For high levels of transcription in the embryonic central nervous system (CNS) different modules dispersed over 3 kb have to co-operate. Combination of a core promoter, either with far upstream localized enhancer elements or, alternatively, with an enhancer from the intron results in expression limited to only a few neuronal cells. However cooperation of all three modules leads to high level expression in most neuronal cells of the CNS. In the intron, a 6 bp core element has been identified that is essential for transcription in the CNS, as well as an 8 bp element required for maternal expression. Interestingly, both motifs are quite similar, with CAAAAT as the CNS core and CAAAAAT as the maternal enhancer core. Specific binding of proteins from nuclear extracts to the CNS-specific element has been demonstrated. The beta1 tubulin gene represents an ideal marker gene to elucidate connections between pro-neural or neurogenic genes and downstream target genes throughout the CNS (Kohler, 1996).

Stem cell differentiation into mature spermatozoa is a morphogenetic process, highly dependent on microtubular arrays. In early, mitotically active stages of spermatogenesis, only the ß1 tubulin isotype is expressed. This expression is regulated by sequences located between positions -45 and -191 upstream of the transcription initiation site. Furthermore, ß1 tubulin is a major component of cyst cells. Expression in these cells is driven by enhancer elements located in the ß1 tubulin gene intron. In addition, redundant enhancer elements in the intron drive expression in the testis wall. Within a single tissue, the male gonad, expression of the ß1 tubulin gene is under cell-type-specific control mediated by independent cis-acting elements. Therefore in the germ line, control of ß1 tubulin expression is strictly governed by promoter-proximal elements, while for the somatic parts of the testis, enhancer elements confer less stringent expression control (Buttgereit, 1993b).

Transcriptional Regulation

The Egr-type zinc-finger transcription factor encoded by the Drosophila gene stripe (sr) is expressed in a subset of epidermal cells to which muscles attach during late stages of embryogenesis. Loss-of-function and gain-of-function experiments indicate that sr activity provides ectodermal cells with properties required for the establishment of a normal muscle pattern during embryogenesis and for the differentiation of tendon-like epidermal muscle attachment sites (EMA). To interfere with the activity of both longer variants (Stripe a) and shorter variants (Stripe b) of Stripe, a dominant-negative Stripe variant was generated: the unique Stripe DNA-binding domain was fused to the repressor domain of the transcription factor Engrailed. This turns out to be a transcriptional repressor that acts from the Stripe DNA-binding domain. The fusion protein, Striperep, causes mutant stripe phenocopies, strongly disrupting muscle patterns. Striperep expression already specifically perturbs muscle pattern formation during an early stage when endogenous stripe is first expressed in a subset of ectodermal cells. The phenocritical period covers the time window when the myotubes normally undergo their oriented growth along the inner surface of the epidermis. Levels of expression of groovin, delilah and beta1-tubulin are altered in response to Striperep activities. Ectopic stripe induces groovin, delilah and beta1-tubulin only in epidermal cells. Ectopic Stripe b expression in ventral midline cells interfers with the orientation of myotubes, and the effects on the muscle pattern is restricted to muscles of the ventral half of the embryos. Thus, sr encodes a transcriptional activator that acts as an autoregulated developmental switch gene. sr activity controls the expression of EMA-specific target genes in cells of ectodermal but not of mesodermal origin. sr-expressing ectodermal cells generate long-range signals that interfere with the spatial orientation of the elongating myotubes (Vorbruggen, 1997).

Changes in the extracellular matrix (ECM) govern the differentiation of many cell types during embryogenesis. Integrins are cell matrix receptors that play a major role in cell-ECM adhesion and in transmitting signals from the ECM inside the cell to regulate gene expression. In this paper, it is shown that the PS integrins are required at the muscle attachment sites of the Drosophila embryo to regulate tendon cell differentiation. The analysis of the requirements of the individual alpha subunits, alphaPS1 and alphaPS2, demonstrates that both PS1 and PS2 integrins are involved in this process. In the absence of PS integrin function, the expression of tendon cell-specific genes such as stripe and beta1 tubulin is not maintained. In addition, embryos lacking the PS integrins also exhibit reduced levels of activated MAPK. This reduction is probably due to a downregulation of the epidermal growth factor receptor (Egfr) pathway, since an activated form of the Egfr can rescue the phenotype of embryos mutant for the PS integrins. Furthermore, the levels of the Egfr ligand Vein at the muscle attachment sites are reduced in PS mutant embryos. Altogether, these results lead to a model in which integrin-mediated adhesion plays a role in regulating tendon cell differentiation by modulating the activity of the Egfr pathway at the level of its ligand Vein (Martin-Bermudo, 2000).

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