betaTubulin60D (beta3 tubulin) part 2/2


Promoter Structure

The ß3-tubulin gene is expressed in somatic and pharyngeal musculature, and the dorsal vessel. This expression is mediated by sequences lying far upstream. The first intron of the ß3-tubulin gene bears a tissue-specific enhancer element that is required for expression in the visceral muscles. It also functions efficiently when cloned downstream of an indicator gene. The separability of elements driving ß3-tubulin expression in the somatic and visceral mesoderm facilitates the investigation of the different programs involved in regulating the early differentiation of this germ layer (Gasch, 1989)

The results of a more detailed analysis of the regulatory capabilities of the intron reveal not only a certain degree of redundancy in the cis-acting elements (which act at different developmental stages in the same mesodermal derivatives) but also demonstrates in the visceral mesoderm (which forms a continuous epithelium along the body axis of the embryo) an early action of regulators guiding gene expression along the anterior-posterior axis of the embryo. An enhancer element in the intron leads to expression in a subdomain restricted along the anterior-posterior axis. This pattern is altered in mutants for the homeotic gene Ultrabithorax (Ubx), whereas ectopic Ubx expression leads to activity of the enhancer in the entire visceral mesoderm (Hinz, 1992).

The beta 3 tubulin gene is a structural gene expressed during mesoderm development from the extended germ band stage onward. Expression within the individual mesodermal derivatives is guided by different control elements. The upstream regions allow expression in the dorsal vessel and the somatic mesoderm, while enhancers localized in the first intron guide expression in the visceral mesoderm. In addition to this tissue-specific mode of transcriptional control, beta 3 tubulin is regulated in a distinct stage-specific manner. From stage 10 to stage 12, expression in the visceral mesoderm is regulated along the anterior-posterior axis by a first intron localized enhancer; this regulation is mediated by homeotic selectors genes like Ubx. After stage 12, an additional farther 3'-located enhancer mediates expression in the visceral and somatic mesoderm. In the epidermis, transcription of the beta 3 tubulin gene is repressed by Engrailed (Damm, 1998).

Deletion analysis carried out in transgenic flies reveals independent regulatory elements for the dorsal vessel and the somatic mesoderm. These elements are located upstream of the transcriptional start site. For expression in the somatic mesoderm, a 279-bp region is absolutely essential. This region contains a binding site for the Drosophila myocyte-specific enhancer binding factor 2 (D-MEF2). Deletion or mutation of this D-MEF2 binding site strongly reduces transcription. This pattern is consistent with the strongly reduced expression of beta 3 tubulin in D-mef2 mutant embryos. This analysis furthermore reveals that the D-MEF2 binding site acts in concert with nearby cis regulatory elements; D-MEF2 cannot, on its own, direct beta 3 tubulin expression. These data show that the upstream control region of the beta 3 tubulin gene is an early target of the D-MEF2 transcriptional activator (Damm, 1998).

The beta 3-tubulin gene is a direct target of Engrailed. The cytological location of beta 3-tubulin, 60C, is a strong site for Engrailed binding on polytene chromosomes. Immunostaining analysis of a transgenic line containing a P[beta 3-tubulin-lacZ] construct shows an additional site for Engrailed binding at the location of the transgene. Molecular analysis allows identification of several Engrailed binding sites, both in vitro and in vivo, within the first intron of the beta 3-tubulin locus. Sequence analysis of beta 3-tubulin fragments that bind En reveal the presence of ten sites related to the En consensus binding sequence TTAATTGCAT. Engrailed binding sites identified in vitro are active in larvae. Expression of beta 3-tubulin has been shown to be derepressed in the ectoderm of engrailed mutant embryos (Serrano, 1997).

During embryogenesis, expression in the visceral and late somatic musculatures is regulated by sequences contained within the first intron of beta 3-tubulin, while upstream sequences are necessary for early expression in somatic musculature, suggesting that beta 3-tubulin regulation is achieved by an early transient program until stage 12 and a more stable late program in later stages. Two different sets of Engrailed binding sites are shown to be involved in the early and late regulation of beta 3-tubulin by Engrailed during embryogenesis. The intronic beta 3-tubulin region can be subdivided into 5 subregions (A-E), each containing one to four En binding sites. Evidence is provided that fragments A to D are involved in late en expression, while sequences in the E fragment are implicated in the early regilation of beta 3-tubulin expression by En. Sequences in the E fragment are able to respond in vivo to differences in En protein concentration and might be part of the regulation of beta 3-tubulin expression in one half of the larval hindgut. It is suggested that En might be responsible for the observed repression of beta 3-tubulin, at least in the posterior compartment, through its binding to the D region. Repression of beta 3-tubulin by Engrailed is obtained when Engrailed is ectopically expressed in embryonic mesoderm. A 500 base pair F fragment (which does not contain En binding sites and is located between region D and E), is shown to be sufficient to confer a pattern similar to the endogenous beta 3-tubulin expression in the visceral mesoderm and in the late somatic mesoderm. The addition of the D fragment to this 500 base pair F fragment shows a particularly high level of expression in the chordotonal organs, a place for the normal expression of beta 3-tubulin. The absence of En binding sites in the F fragment points to a complicated network of regulation of beta 3-tubulin in different types of cells in early development (Serrano, 1997).

Ecdysone (See Ecdysone receptor) regulation of promoter constructs of the ß3 tubulin gene takes place 0.91 kb upstream from the transcription start site and 360 bp from the first large intron: repression is found in the absence of ecdysone and derepression-activation in the presence of the hormone. This 360 bp fragment contains several enhancer and silencer sequences. The regulation of the expression of the ß3 tubulin gene results from the combined activity of all the positive and negative regulatory sequences of the first intron, and a dialogue with the promoter sequences. The nucleotide sequence of this intronic regulatory-fragment has been established and several EcRE (ecdysone responsive element) consensus sequences have been identified (Tourmente, 1993).

In Drosophila Kc cells at the transcriptional level, the expression of the ß3 tubulin gene is regulated by the steroid hormone ecdysone. Essential for conferring ecdysone inducibility are 360 bp extending from the first intron of the ß3 tubulin gene and associated with the 5' flanking sequences. The 5' flanking region contains ecdysone-independent cis-positive elements in close proximity to the promoter. Deletion analysis of the 360 bp intronic region reveals that a fragment of 57 bp is crucial for the ecdysone response of the ß3 tubulin gene. This fragment contains 5'-TGA(A/C)C-3' motifs homologous to ecdysone responsive elements (EcRE) half sites. Band shift assays show that this 57-bp fragment is bound by three specific complexes. One of them appears to be involved in the level of the ecdysone response (Bruhat, 1993).

During Drosophila embryogenesis, the beta3 tubulin gene is expressed in the visceral and somatic mesoderm as well as in the dorsal vessel. Transcription of the gene is limited to four pairs of cardioblasts per segment. Its expression in the dorsal vessel (dv) is mediated by a 333-bp enhancer located upstream of the gene (between -21705 and -21385 bp). The homeodomain protein Tinman is expressed in these cardioblasts, implying that Tinman might be a key regulator of the beta3 tubulin gene. Gel retardation and footprint assays indeed has revealed two Tinman binding sites within the dv-specific enhancer. The relevance of the Tinman binding sites was analyzed in a transgenic fly assay and distinct functions for both sites were observed. The BS(Tin-1460) site is absolutely required for expression in cardioblasts, while BS(Tin-1425) is needed for high-level expression. Thus, these two Tinman binding sites act in concert to drive beta3 tubulin gene expression during heart development. Tinman initially functions in the specification of visceral mesoderm and heart progenitors, but remains expressed in cardioblasts until dorsal closure. Overall, these data demonstrate a late function for Tinman in the regulation of beta3 tubulin gene expression in the forming heart of Drosophila (Kremser, 1999a).

The beta3 tubulin gene of Drosophila is expressed in the major mesodermal derivatives during their differentiation. The gene is subject to complex stage- and tissue-specific transcriptional control by upstream as well as downstream regions. Analysis of the vm1 enhancer, which is responsible for tissue-specific expression in the visceral mesoderm and is localized in an intron, reveals a complex modular arrangement of regulatory elements. In vitro and in vivo experiments uncovered two binding sites [termed UBX1 and UBX2, for the product of the homeotic gene Ultrabithorax(Ubx)] that are required for high-level expression in pPS6 and PS7. Further analysis of the vm1 enhancer has revealed that deletion of a specific element, termed element 7 (e7), abolishes transcription of the lacZ reporter gene in all parasegments except pPS6/PS7. Gel-retardation and footprint analysis has identified a binding site for the homeodomain protein Tinman, which is essential for the specification of the dorsal mesoderm, within e7. Simultaneous deletion of two further sequence blocks in the vml enhancer, named elements 3 (e3), and 6 (e6), results in a reduction analogous to that caused by removal of e7. The e6 sequence contains conserved motifs also found in the visceral enhancer of the Ubx gene. It is therefore concluded that these elements act in concert with the Tinman binding site to achieve high expression levels. Thus the vm1 enhancer of the beta3 tubulin gene contains a complex array of elements that are involved in transactivation by a combination of tissue- and position-specific factors, including Tinman and UBX (Kremser, 1999b).

The Drosophila hindgut develops three morphologically distinct regions along its anteroposterior axis: small intestine, large intestine and rectum. Single-cell rings of 'boundary cells' delimit the large intestine from the small intestine at the anterior, and the rectum at the posterior. The large intestine also forms distinct dorsal and ventral regions; these are separated by two single-cell rows of boundary cells. Boundary cells are distinguished by their elongated morphology, high level of both apical and cytoplasmic Crb protein, and gene expression program. During embryogenesis, the boundary cell rows arise at the juxtaposition of a domain of Engrailed- plus Invected-expressing cells with a domain of Delta (Dl)-expressing cells. Analysis of loss-of-function and ectopic expression phenotypes shows that the domain of Dl-expressing cells is defined by En/Inv repression. Further, Notch pathway signaling, specifically the juxtaposition of Dl-expressing and Dl-non-expressing cells, is required to specify the rows of boundary cells. This Notch-induced cell specification is distinguished by the fact that it does not appear to utilize the ligand Serrate and the modulator Fringe (Iwaki, 2002).

At its anterior, the hindgut joins the posterior midgut; at its posterior, it forms the anus. Along this AP axis, the hindgut of the mature embryo consists of three morphologically distinct domains: the wide, looping small intestine, the long and narrow large intestine, and the tapered rectum. Beginning at stage 13, these domains are demarcated at their junctions by rings of unusually high accumulation of the apical surface protein Crumbs (Crb). The ring at the small intestine/large intestine junction is designated the anterior boundary cell ring, and the ring at the large intestine/rectum junction is designated the posterior boundary cell ring (Iwaki, 2002).

Patterning of the hindgut in the DV axis is detected at stage 10 (germ band extension) when the hindgut develops an interiorly directed (dorsal) convexity. The side of the hindgut closest to the interior of the embryo is dorsal and expresses both En and Inv; that closest to the exterior is ventral and expresses dpp. By the completion of germ band retraction, the convexity at the anterior of the hindgut has shifted toward the left side of the embryo. Thus at the anterior of the hindgut, the initially dorsal, En- and Inv-expressing side comes to lie on the outer (left-facing) curve, while the initially ventral, Dpp-expressing side of the hindgut comes to lie on the inner (right-facing) curve; the DV relationship is retained at the posterior connection to the rectum. These initially DV patterned domains of the large intestine persist to the end of embryogenesis and into the larval stages; they are referred to as large intestine dorsal (li-d) and large intestine ventral (li-v). At each of the two boundaries between li-d and li-v, there is a single row of cells with high levels of Crb expression running the length of the large intestine, from the anterior boundary cell ring to the posterior boundary cell ring. These are designated the 'boundary cell rows'. In addition to their high level of Crb expression, the boundary cell rows and rings express the nuclear protein Dead ringer (Dri). Double antibody staining reveals that boundary cell rows at the border of the En/Inv-expressing li-d domain and the Dpp-expressing li-v domain express Dri in their nuclei and have strong Crb expression at their apical surfaces (Iwaki, 2002).

In addition to expressing Dpp, the li-v domain expresses the Notch ligand Delta (Dl); Dl is also expressed in the anterior of both the rectum and the small intestine. Fringe (Fng), a modulator of Notch signaling, is expressed opposite Dl in the Drosophila wing and eye; in the hindgut, Fng is expressed in li-d and the boundary cell rows, opposite the domain of Dl expression in li-d (Iwaki, 2002).

Interestingly, the Dri- and Crb-expressing boundary cells delimit both AP and DV boundaries in the hindgut. The rings form borders at the anterior and posterior ends of the large intestine, while the rows form borders between the dorsal (li-d) and ventral (li-v) regions of the large intestine. This study focusses primarily on the establishment and characteristics of the boundary cell rows (Iwaki, 2002).

Staining with both anti-Crb and anti-ßHEAVY Spectrin shows that the boundary cell rows are significantly more elongated along the AP axis than other hindgut epithelial cells. Staining of bynapro/+ embryos (containing a P-element insert in byn) with anti-ß-Gal antibody reveals that the nuclei of the cells of the boundary rows (identified by strong staining with anti-Crb) are also elongated in the AP axis (Iwaki, 2002).

The dramatically higher level of Crb expression in the boundary cells (both rings and rows) suggests that their apical surface may differ from that of other hindgut epithelial cells, and/or that, in the boundary cells, Crb may be present in cellular compartments in addition to the apical surface. Both of these expectations are borne out by a higher magnification examination of the boundary cells. In cross-sections of the large intestine viewed by electron microscopy, short microvilli on the apical surfaces of two cells on opposite sides of the hindgut lumen were observed; these cells most likely correspond to the boundary cell rows. The microvilli of the presumed boundary cell rows appear more organized and parallel than the irregular protrusions on the surfaces of the other cells of the hindgut epithelium. Because of their apical microvilli, the presumed boundary cell rows have a larger apical membrane surface and are expected to be labeled more strongly with anti-Crb. Consistent with this, cross-sections of anti-Crb-stained embryos viewed by light microscopy reveal two cells on opposite sides of the large intestine lumen with a higher level of Crb on their apical surfaces. In addition to their stronger apical labeling with anti-Crb, these presumed boundary cell rows also display an accumulation of Crb in their cytoplasm; this is strongest apical to the nucleus. The cytoplasmic accumulation of Crb suggests that Crb is produced at a higher level, or is more stable, in the boundary cells (Iwaki, 2002).

In conclusion, differences in gene expression demonstrate that the boundary cells are a separately patterned (fated) group of cells in the large intestine. The unique fate of the boundary cells is manifested both molecularly, in their expression of Dri and high cytoplasmic accumulation of Crb, and morphologically, in their marked AP elongation and development of apical microvilli (Iwaki, 2002).

The boundary cell rows form at the junction of the li-d and li-v domains, which express different genes. To investigate whether the spatially restricted gene expression observed in these domains is essential for establishment of boundary cell rows, embryos homozygous for loss-of-function alleles of en, inv, dpp, dri, Dl, Ser, Notch, or fng were examined. The presence or absence of boundary cells was assessed by anti-Crb staining, since this delineates their characteristic morphology, and also detects one of their unique differentiated features (i.e. the cytoplasmic accumulation of Crb) (Iwaki, 2002).

In embryos homozygous for a strongly hypomorphic dri allele (dri null mutants lack a discernable hindgut), the hindgut is of roughly normal diameter but only about one-third its normal length. Even in these severely reduced dri hindguts, however, boundary cells can still be observed; this phenotype is similar to that described for embryos lacking both maternal and zygotic dri function. Since reduced hindgut size is observed in embryos that lack zygotic, but retain maternal dri function, it is concluded that zygotic expression of dri (most likely the uniform expression at the blastoderm stage) is required to establish or to maintain the normal-size hindgut primordium. Neither blastoderm expression of dri, nor its later expression in the boundary cells, however, appears to be required to establish the boundary cells (Iwaki, 2002).

In dpp embryos, the large intestine is shorter; this is believed to be due to a requirement for dpp in DNA endoreplication in the large intestine. Although the hindgut is variable and severely deformed in dpp mutant embryos (only rudimentary hindguts are detected in the strongest dpp alleles), boundary cell rows were detectable in the hindguts of embryos carrying several different strongly hypomorphic dpp alleles. Thus even though it is required for normal hindgut development, dpp activity does not appear to be required to establish the boundary cell rows (Iwaki, 2002).

In embryos lacking only en, the boundary cell rows and rings form normally. Similarly, many embryos lacking only inv form boundary cell rows and rings. In a significant number of inv embryos, however, gaps were observed in the posterior of the boundary cell rows. This is the only embryonic phenotype known for inv. When both en and inv are removed [in Df(enE) embryos], the phenotype is much more dramatic: boundary cell rows and rings are completely absent. Consistent with previous studies demonstrating a functional redundancy of en and inv, it is concluded that en and inv are required largely redundantly to establish the boundary cells. However, while inv can substitute completely for en, there is a requirement for inv that cannot be completely substituted by en. This is likely not due to a difference in protein structure, but rather to the fact that, in the hindgut, inv is expressed earlier and at a higher level than en. As their functions are so closely intertwined, the activities of en and inv, and the highly related proteins that they encode, are referred to as single entities: en/inv and En/Inv (Iwaki, 2002).

Embryos lacking Dl function are extremely deformed and do not always have a recognizable hindgut, indicating that function of Dl early in embryogenesis is required to establish and/or maintain the hindgut. Since Dl encodes a ligand for Notch, embryos lacking the zygotic contribution of Notch were examined. Strikingly, Notch mutant hindguts completely lack both boundary cell rows and rings, revealing that Notch signaling is required to establish the boundary cells. The data demonstrate that formation of the boundary cell rows at the border of Dl expression requires the Notch receptor; however, Fng does not appear to be required for this process (Iwaki, 2002).

To further investigate the required role of Dl in establishing the boundary cells, a dominant-negative form of Dl was expressed throughout the hindgut. bynGal4:UAS-Dl.DN embryos show a complete absence of boundary cell rows and rings; this phenotype closely resembles that seen in Notch loss-of-function embryos. Expression of a dominant negative Notch receptor throughout the hindgut results in a similar absence of boundary cell rows and rings. Furthermore, bynGal4 driven expression of UAS-Hairless, which acts to suppress activity of Su(H) also results in an absence of boundary cells. This last result indicates that the Notch signaling required to establish the boundary cells must act through Su(H). In summary, the above results demonstrate required roles in boundary cell specification of the following Notch pathway components: the ligand Dl, the receptor Notch, and the downstream transcription factor Su(H). It is therefore concluded that the Notch signaling pathway is required for boundary cell induction (Iwaki, 2002).

An intriguing observation, given the demonstrated role of the LIN-12/Notch signaling pathway in generation of left¯right asymmetry in the Caenorhabditis elegans intestine is that a large portion of 455.2Gal4:UAS¯Su(H)VP16 hindguts display a reversal of left¯right looping (Iwaki, 2002).

Ectopic expression experiments, taken together with the loss-of-function experiments, demonstrate that establishment of the boundary cell rows requires the juxtaposition of Dl-expressing and Dl-non-expressing cells and signaling via Notch and Su(H). In addition to Notch and spatially restricted Dl, establishment of the anterior ring requires localized activity of Dpp; the posterior ring requires En/Inv activity (which does not need to be localized) and the localized activity of Dl (Iwaki, 2002).

Since the experiments described in the preceding sections show that both spatially localized En/Inv and a boundary of Dl expression are required to establish the boundary cells, it was asked whether En/Inv might control the boundary of Dl expression. In Df(enE) embryos, Dl is not restricted to li-v, but rather is uniform in the hindgut circumference, indicating that en/inv is required to repress Dl. In the large intestine, uniform expression of En/Inv results in an absence of Dl expression. Expression of En/Inv in li-d is thus both necessary and sufficient to restrict Dl expression to li-d. While it represses Dl throughout the large intestine, ectopic En/Inv does not affect Dl expression in the rectum. Embryos with ectopic En/Inv not only express Dl at the anterior of the rectum, they also form the posterior boundary cell ring. Thus a boundary of Dl-expressing with Dl-non-expressing cells is required not only to establish the boundary cell rows but also likely to establish the posterior ring; the posterior ring also requires En/Inv activity, but this activity does not need to be localized (Iwaki, 2002).

Consistent with observations that En and Inv are repressors with the same targets, the data presented in this study demonstrate that Dl expression in the large intestine is restricted to the li-v domain by the repressive activity of En/Inv in li-d (Iwaki, 2002).

The data presented here support the following model. En/Inv is expressed in li-d and represses Dl in that domain; Dl expression is thereby restricted to the li-v domain. At the li-v/li-d transition, the Dl-expressing cells induce, by Notch signaling, a row of Dl-non-expressing cells to become a boundary cell row. Since En/Inv is not detected in differentiated boundary cells, Notch activation likely represses En/Inv expression. Notch activation also leads to Dri expression and an upregulation of Crb expression. While all of these transcriptional changes could be mediated by Su(H), they could also be further downstream (Iwaki, 2002).

In summary, three steps in the establishment of the Drosophila hindgut boundary cell rows are similar to steps characterized in other Notch dependent boundary-forming systems. (1) A homeodomain transcription factor (En/Inv in the case of the boundary cells) is expressed on one side of the forming boundary; (2) this transcription factor defines two domains, one which expresses Dl and one which does not; (3) Notch activation in the Dl-non-expressing cells that confront Dl-expressing cells leads to a unique cell fate (Iwaki, 2002).

Given the essential role of spatially restricted En/Inv expression in establishing the boundary cells, it is of interest to consider how En/Inv expression is restricted to the li-d domain. The activation of en expression in the large intestine at stage 10 requires the T-domain transcription factor brachyenteron (byn), which is expressed uniformly in the hindgut. Since dissection of the en regulatory region has identified fragments that drive reporter expression in all hindgut cells, en expression is likely restricted to li-d by a repressor that remains to be identified (Iwaki, 2002).

Boundary cells could be imagined to provide adhesive differences important for cell rearrangement; alternatively, their AP elongation might provide a mechanical force to drive hindgut elongation. In spite of these tempting scenarios, however, the normal appearance (overall size, diameter, and length) of Notch and Df(enE) hindguts, which completely lack both boundary cell rows and rings, demonstrates conclusively that the boundary cell rows and rings are not required to establish normal hindgut morphology (Iwaki, 2002).

Rather than playing a required role in hindgut morphogenesis, the boundary cells most likely contribute to the ion and water absorption function of the larval hindgut. In the adult insect, this function is carried out by cells in the rectum that are distinguished by their extensive, mitochondria-rich apical membrane leaflets. In the Drosophila larval hindgut, this characteristic ultrastructure is found not in the rectum, but rather in the cells of li-d, leading to the conclusion that water and ion absorption in the larva occurs in the large intestine. Associated with the absorptive cells of the Dipteran rectum is a distinct cell type referred to as 'junctional cells'; these form a collar surrounding the absorptive cells, have extensive intercellular junctional complexes, and are thought to play an isolating and supportive role. The Drosophila boundary cell rings and rows similarly constitute a collar surrounding the absorptive li-d cells of the larval hindgut and, based on their intensive Crb staining, have unusual membrane characteristics. It is therefore proposed that, like the junctional cells in the adult insect rectum, the boundary cells serve to isolate and support a domain of ion and water absorbing cells in the Drosophila larval hindgut (Iwaki, 2002).

The NK homeobox gene bagpipe and the FoxF fork head domain gene biniou have been identified as essential regulators of visceral mesoderm development in Drosophila. Additional genetic and molecular information is presented on the functions of these two genes during visceral mesoderm morphogenesis and differentiation. Both genes are required for the activation of ß3Tub60D in the visceral mesoderm. A 254 bp derivative of a previously defined visceral mesoderm-specific enhancer element, vm1, from ß3Tub60D contains one specific in vitro binding site for Bagpipe and two such sites for Biniou. While the wild-type version of the 254 bp enhancer is able to drive significant levels of reporter gene expression within the entire trunk visceral mesoderm, mutation of either the Bagpipe or the Biniou binding sites within this element results in a severe decrease of enhancer activity. Moreover, mutation of all three binding sites for Bagpipe and Biniou, respectively, results in the complete loss of enhancer activity. Together, these observations suggest that Bagpipe and Biniou serve as direct, partially redundant, and tissue-specific activators of the terminal differentiation gene ß3Tub60D in the visceral mesoderm (Zaffran, 2002).

To test whether the expression of ßTub60D in the trunk visceral mesoderm depends on the activity of two known visceral mesoderm regulators, bap and bin, ßTub60D protein expression was examined in embryos that were mutant for the respective gene. In addition, the embryos carried a bap-lacZ transgene as an independent marker for the early visceral mesoderm which; in a wild-type background bap-lacZ is co-expressed with ß3 tubulin. In embryos lacking bap, ß3 tubulin expression is severely reduced in the visceral mesoderm and at early stage 12 only trace amounts remain detectable in this cell layer. Likewise, loss of bin activity also results in an almost complete loss of ß3 tubulin expression in the visceral mesoderm layer. These data show that the activities of both bap and bin are required for normal ß3-tubulin expression in the trunk visceral mesoderm (Zaffran, 2002).

A visceral mesoderm-specific enhancer element from the ßTub60D gene, vm1, has been described that is contained in the reporter construct pWHß3-14 and consists of 515 bp of enhancer sequences from the first intron of this gene (+3154 to +3669). While two Ubx binding sites within this enhancer are involved in increasing enhancer activity within parasegments (PS) 6 and 7, bap and/or bin may act as direct regulator(s) of the broad basal activity of this enhancer in the entire trunk visceral mesoderm. To test this possibility a derivative of pWHß3-14, in which the Ubx sites were deleted (pWHß3-14/DeltaUbx1+2), was crossed into bap and bin mutant backgrounds. While in the wild-type background this enhancer derivative is driving significant (though anteroposteriorly graded) levels of ßgal expression in a continuous row of visceral mesoderm cells, in a bap null mutant background enhancer activity is completely lost in this tissue. Likewise, a strong reduction of enhancer activity driven by pWHß3-14/DeltaUbx1+2 is also observed in a bin null mutant background, although in this case some residual visceral mesoderm cells are still expressing low levels of the reporter gene (Zaffran, 2002).

In order to clarify whether these genetic interactions reflect any direct interactions of the bap or bin products with vm1 enhancer sequences in vitro DNA-binding experiments were performed with the two proteins. DNaseI protection assays with bacterially expressed Bin fusion proteins revealed two strongly protected sequences, termed BIN-I and BIN-II, within vm1. Closer inspection of these sequences showed that BIN-I contains overlapping tandem copies and BIN-II a single copy of a canonical binding motif for fork head domain proteins. The specificities of these in vitro binding activities are further corroborated by the results from gel mobility shift experiments. In particular, these data show that both BIN-I and BIN-II oligonucleotides can compete for binding of Bin to vm1, whereas analogous oligonucleotides in which the canonical fork head domain binding sequence was mutated fail to compete (Zaffran, 2002).

Bap fusion proteins also produce a strongly protected region in DNaseI footprinting experiments. The protected sequence contains an overlapping tandem repeat of a canonical NK-homeodomain binding motif, which has been shown to bind Tinman. Indeed, the footprints produced with Bap and Tin on this sequence are almost indistinguishable (Zaffran, 2002).

In preparation for functional tests of the Bin and Bap binding sites in vivo, a shorter version of vm1, termed ß3-17, was generated that lacks 5' and 3' sequences that have been shown to be dispensable for driving basal levels of visceral mesoderm expression (+3252 to +3506). As predicted, ß3-17-driven ßgal expression occurs in a uniform pattern and at intermediate levels within the visceral mesoderm. Next the effects of mutant Bin and Bap binding sites on the in vivo activity of the ß3-17 enhancer element were tested. Mutation of either BIN-I (ß3-17 bin-Imt) or BIN-II ß3-17 (bin-IImt) results in a strong decrease of ß3-17 enhancer activity. Although it was expected that the activities of BIN-I and BIN-II may be partially redundant, simultaneous mutation of both binding sites did not result in a significant reduction of enhancer activity beyond the levels seen with mutations in either binding site alone, particularly BIN-II, (ß3-17 bin-I+IImt) (Zaffran, 2002).

Mutation of the Bap binding site also results in a strong reduction but not a complete loss of enhancer activity within the visceral mesoderm. To determine whether the residual enhancer activity of the mutated elements is due to functional redundancy between the Bin or Bap binding sites the effects of mutations in all three binding sites were tested. Simultaneous disruption of all Bin and Bap binding sites within the ß3-17 enhancer element results in the complete loss of enhancer activity, thus confirming that Bin and Bap have partially redundant roles in activating the vm1 enhancer of ßTub60D (Zaffran, 2002).

The residual enhancer activity upon mutation of Bin or Bap binding sites is largely observed in the middle portion of the visceral mesoderm, suggesting the influence of spatially restricted regulator(s). Indeed, the close spatial correlation between residual enhancer activity and Dpp-signaling activity as well as the presence of putative Smad binding sites within vm1(+3265: GGGCCG; +3289: CAGAC; +3431: CAGACGGCAGAC) suggests a role for direct inputs from Dpp in the regulation of vm1 enhancer activity. Thus, Smad complexes and Bap bound to vm1 sequences may act in a synergistic fashion, a situation that may be analogous to the synergistic activity of Smad and Tin during the induction of the Dpp-responsive enhancer of the tin gene. However, the fact that this effect is only observed with a weakened version of the enhancer indicates that the Dpp-input plays a minor role during the normal activation of the ßTub60D gene in the visceral mesoderm. Additional inputs, which may also be insignificant for ßTub60D regulation in the normal situation, could come from Wg and/or Hh and result in low levels of metameric expression with weakened enhancer constructs (Zaffran, 2002).

Activation of the vm1 enhancer during stage 11 is restricted to the ventral row of visceral mesodermal cells, but is missing in the remaining cells of this tissue that also express Bap and Bin. Hence, the combination of Bap and Bin is required, but not sufficient for activating ßTub60D expression through vm1. Previous observations have shown that the region defined by deletion 3 (e3, +3439 to +3471), which neither contains Bap nor Bin binding sites, is also required for normal enhancer activity. Therefore, this sequence may be a target of an as yet unknown activity within the ventral row of visceral mesodermal cells that is required in combination with Bap and Bin to trigger vm1 activation. Recent reports have shown that these ventral cells are the equivalent of founder cells in the visceral mesoderm; these cells subsequently fuse with adjacent dorsal cells into binucleate syncytia. Similar to the expression of dpp in PS 7 of the visceral mesoderm, vm1-lacZ expression spreads throughout the visceral mesoderm only upon fusion of founders with fusion-competent cells (Zaffran, 2002).

Combined with previous data, the current results define a continuous regulatory cascade of gene activation that initiates with the regulation of genes which pattern the early mesoderm; this process concludes with the activation of a terminal differentiation gene in the visceral mesoderm. Specifically, this pathway involves the activation of tin by twist, followed by the induction of dorsal mesodermal tin by dpp, then activation of bap by tin and dpp, activation of bin by bap and dpp, and finally activation of ßTub60D by the combined action of bap and bin. A second gene that is activated at the end of this cascade in the visceral mesoderm with a similar temporal, albeit more restricted spatial pattern as compared to ßTub60D, is dpp. In the case of dpp, a visceral mesoderm-specific enhancer requires only Bin, but not Bap, as a direct activator. Hence, genes controlling morphogenesis or differentiation of the visceral mesoderm differ in their requirement for either one or both of the ubiquitously distributed visceral mesoderm activators, Bap and Bin, as direct regulators. These differences may depend on the particular involvement of additional regulators, which in the case of dpp includes spatially-restricted activities such as Ubx, that may obviate a requirement for Bap in addition to Bin as a direct activator (Zaffran, 2002).

Transcriptional Regulation

MEF2 is a positive regulator of Drosophila Tromomyosin I in the body-wall muscles of embryos, larvae, and adults. Ectopic expression of MEF2 in the epidermis and ventral midline cell in embryos activates the expression of TmI and other muscle genes in these tissues; this activation is stage-dependent, suggesting a requirement for additional factors. Among the genes activated by MEF2 are ß3-Tubulin, a cytoskeletal protein found in myoblasts, muscle cells and some nonmuscle cells and DMLP, a Drosophila homolog of the vertebrate muscle LIM protein, which is a positive regulator of myogenic differentiation. Mesodermal nautilus expression is diminished, but there is no indication of ectopic MEF2 activating nautilus in the ectoderm or ventral midline. Ectopic expression of DMLP1 is observed only in the ventral midline cells but not in the epidermis of embryos that ectopically express MEF2 in the epidermis and ventral midline. Furthermore, ectopic expression of MEF2 in the epidermis results in a decrease in the expression of signaling molecules in the epidermis and a failure of the embryo to properly form body-wall muscles. Muscle VA1 of the ventral group body-wall muscles is missing and muscle VA2 is much shorter than that found in wild type embryos. Muscles VO 4 to 6 are also much shorter than those found in wild-type embryos and often fuse to one another. LT1 to LT4 of the lateral group body-wall muscles are disorganized. These results indicate that MEF2 can function out of context in the epidermis to induce the expression of muscle genes and interfere with a requirement for the epidermis in muscle development. The level of MEF2 in the mesoderm and/or muscles in embryos is critical to body-wall muscle formation; however, no effect is observed on the development of the visceral muscle or dorsal vessel (Lin, 1997)

Cultured Kc cells of Drosophila are sensitive to the insect molting hormone ecdysone. The morphological changes of Kc-treated cells observed by electron microscopic analysis of pseudopodia show a large increase in the number of microtubules, all arranged in the same orientation. The 60 C beta tubulin gene that is expressed only in ecdysone treated cells encodes a 2.6-kb mRNA, essentially cytoplasmic and polyadenylated. The corresponding premessenger is 7 kb long and is absent in untreated cells. Two peaks of expression of the 60 C beta tubulin gene are observed during Drosophila development: at midembryogenesis (stage 8-13 h) and at the late third instar larvae-early pupae stage. By use of the Ecdysone 1 mutant, 60 C beta tubulin gene expression has been demonstrated to be regulated in part by ecdysone during Drosophila development (Sobrier, 1989).

The prothoracicotropic hormone (PTTH, see Bombyx and Manduca prothoracicotropic hormone) stimulates the specific synthesis of three proteins in the prothoracic glands of the tobacco hornworm Manduca sexta (See Bombyx prothoracicotropic hormone section in the Ecdysone receptor site). One of these proteins (p50) is identified as a beta tubulin. The ability of PTTH to stimulate beta tubulin synthesis increases dramatically late on Day 3 of the 10-day fifth larval instar. At this time and still later, beta tubulin synthesis in response to PTTH in vitro can be detected in some prothoracic glands 5-10 min after the onset of stimulation.  Newly synthesized beta tubulin then enters the microtubule pool within 12 min. Levels of beta tubulin in the glands of fifth instar larvae change in a tissue-specific manner that parallels or presages circulating ecdysteroid levels. The accumulation of beta tubulin in PTTH-stimulated prothoracic glands results from increased transcription and translation and not from a decreased protein turnover rate. Pulse-chase experiments indicate that the newly synthesized beta tubulin has a very short half-life in vitro (approximately 0.5 hr). Studies with cycloheximide and actinomycin D indicate that beta tubulin synthesis and ecdysteroid synthesis are coregulated and that beta tubulin synthesis is regulated in a unique manner relative to most other prothoracic gland proteins. Beta tubulin levels may play an important role in ecdysteroidogenesis, perhaps by influencing the dynamics of microtubule-dependent secretion or interorganelle movement of ecdysteroid precursors (Rybczynski, 1995).

Protein Interactions

For information about Kinesins, Dyneins and MAPs, see beta 1 Tubulin.


While the beta 1 tubulin gene is constitutively expressed during development, ß3 mRNA is restricted to two distinct phases: mid embryogenesis and metamorphosis. The transcription initiation sites of ß3-tubulin are identical in both these stages; comparison of presumptive promoter regions reveals no extensive homologies between the genes beta 1 and beta3. In situ localization shows beta 1 tubulin mRNA to be maternally expressed in the nurse cells of the egg chambers and evenly distributed during early embryogenesis. In contrast, during later stages of embryogenesis, beta 1 tubulin transcripts are predominantly expressed in neural derivatives. The ß3 tubulin gene expression is also spatially regulated: ß3 mRNA is restricted to the mesoderm. ß3-tubulin is also expressed during the 4 days of pupal development (Gasch, 1988 and Kimble 1989).

ß3 tubulin protein is first detectable in the cephalic mesoderm at maximal germband extension. Shortly afterwards, ß3 tubulin is expressed in single cells at identical positions of the thoracic and abdominal segments. These cells could represent Drosophila muscle pioneer cells. During later embryonic development the somatic musclature, visceral musculature, dorsal vessel and macrophages contain ß3 tubulin. In dorsalizing mutants (dorsal, snail and twist), which do not form a ventral furrow during gastrulation, ß3 expression is greatly reduced but not completely abolished (Leiss, 1988).


Early in pupal development ß3 is expressed in the imaginal discs, while at later times ß3 is expressed in the epidermal cells of the wing blade, the optic lobe, the ovaries, and the testes. The expression of ß3 tubulin ceases by the end of pupal development in all of these tissues except the ovaries and testes where expression persists into the adult. In both developing muscles and wings these results indicate that ß3-tubulin is utilized in populations of specialized but transient cytoskeletal microtubules that are involved in establishing the final form of the tissue (Kimble, 1989).

Drosophila beta3-tubulin is an essential isoform expressed during differentiation of many cell types in embryos and pupae. During pupal development transient b3 expression demarcates a unique subset of neurons in the developing adult visual system. beta3 is coassembled into microtubules with beta1, the sole beta-tubulin isoform in the permanent microtubule cytoskeleton of the adult eye and brain. Examination of beta3 mutant phenotypes show that beta3 is required for axonal patterning and connectivity and for spatial positioning within the optic lobe. Comparison of the phenotypes of beta3 mutations with those that result from disruption of the Hedgehog signaling pathway shows that beta3 functions early in the establishment of the adult visual system. These data support the hypothesis that beta3 confers specialized properties on the microtubules into which it is incorporated. Thus a transient specialization of the microtubule cytoskeleton during differentiation of a specific subset of the neurons has permanent consequences for later cell function (Hoyle, 2000).

Only two beta-tubulin isoforms are expressed in the developing brain, beta3 and beta1 (the predominant Drosophila beta-tubulin). In considering possible functional roles played by the beta3 isoform, it is important to note that beta3 represents only a minor part of the total beta-tubulin, primarily beta1, present in the developing eye and brain. This is illustrated by comparing beta3 accumulation with total beta-tubulin in brains of the same stages. Onset of beta3 expression coincides with differentiation of the compound eye. Photoreceptor differentiation in the eye is marked by the morphogenetic furrow, a wave of cell shape change that moves posterior to anterior across the eye disc. beta3 tubulin is first expressed at the posterior margin of the furrow in the cell bodies of photoreceptor 8 (R8), the first type of photoreceptor to differentiate. Subsequently all photoreceptors express beta3 as they differentiate. In the second row posterior to the furrow and all older rows beta3 is expressed in the photoreceptor cell bodies and their axons projecting down the optic stalk. R1-R6 axons terminate at the presumptive optic lamina, while the R7, 8 axons pass through the nascent lamina and terminate at the presumptive outer optic medulla. beta3 is still present in the photoreceptor cell bodies and axons as the axons begin to fasciculate. The lamina precursor cells themselves never express beta3. In the optic lobe, beta3 expression begins in the inner optic medulla. Maximal beta3 accumulation occurs 2-2.5 days after pupariation. In the retina, beta3 is present in all photoreceptor cells and their axons, plus the eye bristles and bristle axons. beta3 staining in the termini of R1-R6 reveals the complex architecture of the optic lamina. No beta3-expressing lamina neurons have been identified. beta3 expression is limited to only a subset of cells in the inner medulla. beta3 is also present in the inner optic chiasma, the last structure in the developing visual system to express the beta3 isoform. beta3, which is present only during differentiation of the visual system, is no longer present in the eye or brain by 3.5 days postpupariation. This is in agreement with the expression pattern of beta3 mRNA in the brain. The disappearance of beta3 protein from the visual system generally follows the same order as its appearance. beta3 is lost first from the photoreceptor cell bodies and axons and last from the inner medulla and internal chiasma, the last structures in which it was expressed. The shortest photoreceptor axons, R1-R6, lose beta3 staining before the longer R7, 8 axons, even though R8 was the first to express beta3. In R7, 8 axons, beta3 staining is first lost from the axon termini, the location of the 'plus ends' of axonal microtubules. Retrograde loss of beta3 indicates disassembly of beta3-containing microtubules. Since all beta3 staining disappears, beta3 must also be lost from the soluble tubulin pool, raising the possibility that there may be specific degradation of beta3 (Hoyle, 2000).

The transient expression of beta3 defines a unique grouping of cell types, as illustrated by comparison of beta3 localization with an epitope present in all sensory neurons, recognized by the 22C10 antiserum (see Futsch). The early 22C10 staining pattern in the photoreceptors overlaps the beta3 staining pattern, but unlike beta3, the 22C10 epitope is also present in many other cell types, including the larval light-sensing organ, Bolwig’'s organ, and Bolwig’'s nerve. Bolwig’'s organ differentiates during embryogenesis and at that time Bolwig’'s organ does express beta3 tubulin, but beta3 does not persist in Bolwig’'s organ during larval development. The 22C10 epitope is also present in the sensory neurons that project to the brain via the segmental nerves, which do not express beta3 at any stage (Hoyle, 2000).

What is the nature of the cellular function provided by beta3? The data suggest that beta3 does not function primarily to augment the cellular tubulin pool. It is proposed that beta3 confers unique functional properties to the microtubules into which it is incorporated. This hypothesis is consistent with other situations in which the identity of tubulin subunits in the pool determines cellular microtubule function. For example, the protofilament structure of microtubules in the axoneme may be controlled by a component beta-tubulin. Similarly, both meiotic and early mitotic divisions in the Drosophila embryo require that the predominant alpha84B-tubulin isoform and the maternally expressed divergent alpha67C-tubulin isoform are both present and in the correct ratio (Hoyle, 2000).

What might be the specialized properties of beta3- containing microtubules? The timing of beta3 expression and the similarity of the patterning phenotypes in the developing visual system that result from both beta3 mutations and the hedgehog mutation clearly place beta3 function in early differentiative events. Incorporation of beta3 into the microtubule cytoskeleton may confer general properties on microtubules that allow optimal axonal motility required for successful axonal pathfinding and establishment of correct contacts with target cells. For example, microtubule assembly may regulate actin assembly in nerve growth cones. Alternatively, beta3 may play a more direct role, for example if the beta3 subunit provides unique binding sites for specific microtubule motors or other microtubule-associated proteins, perhaps for specific aspects of axonal transport. A precedent for this model is provided by the finding that the maternal alpha67C-tubulin is essential for the function of the kinesin-related Ncd motor protein (Hoyle, 2000).

Observations on embryonic beta3 function reveal differential properties of beta3-containing microtubules compared to microtubules supported primarily by beta1. In the embryo, beta3 is transiently coexpressed with beta1 in the cap cells of chordotonal organs, while other cells in the chordotonal organs contain only beta1. There are many more cross bridges between microtubules in the cells in which beta1 is the sole beta-tubulin than in the beta3-containing cap cells. However, in beta3 mutant animals, the cap cell microtubules are more extensively cross-linked, similar to non-beta3-expressing cells. This observation definitively shows that beta3 can modulate microtubule architecture. Incorporation of beta3 into the chordotonal support cell microtubules might serve to inhibit microtubule associations with specific proteins involved in forming cross bridges. Another possibility is that beta3-containing microtubules are more dynamic than microtubules with only beta1, thus precluding stabilization by cross-bridging. The latter possibility is consistent with the general features of beta3 expression. That is, in both embryos and pupae, beta3 is transiently expressed and is not utilized in construction of long-term microtubule structures. A more labile microtubule cytoskeleton might more readily allow for the rapid changes in cell shape typical of beta3-expressing cells, including the beta3-expressing neurons in the eye and optic lobe. This idea is supported by the finding that experimentally increasing the stability of axonal microtubules inhibits growth cone motility and pathfinding in cultured vertebrate neurons (Hoyle, 2000).

An essential role for the microtubule cytoskeleton has been demonstrated in many different signaling pathways. In the developing visual system, delivery of the Hedgehog protein by the incoming photoreceptor axons is necessary for proliferation of the lamina precursor cells. Although the molecular mode of action of the Hedgehog protein in stimulating optic lobe development has not been elucidated, it has been shown that in the embryo, receipt of the Hedgehog signal by the target cells is a microtubule-mediated process. The data reveal essential microtubule function mediated by the beta3 isoform in the photoreceptor axons that deliver the Hedgehog signal (Hoyle, 2000 and references therein).

Another example of a requirement for microtubule-mediated processes in patterning in the eye and optic lobe is provided by the p150Glued protein, a key member of the dynactin complex required for regulation of cytoplasmic dynein activity. The phenotypes of Glued mutations reveal that the p150Glued protein is required for several different microtubule-mediated events in Drosophila eye development. Moreover, in addition to the 'glued' retinal phenotype, Glued mutant animals exhibit spatial displacement of the medulla, a phenotype that is remarkably similar to that exhibited by both beta3 and hedgehog mutants. Utilization of a transient beta3-containing microtubule cytoskeleton in several different cell types during embryogenesis and metamorphosis suggests that beta3 is involved in an array of determinative events, perhaps involving multiple molecular pathways. Supporting this idea, the data show that beta3 is expressed during several different events in the development of the optic lobe that are not mechanistically linked, including Hedgehog-mediated stimulation of the lamina precursor cells and the Hedgehog-independent formation of the medulla in the absence of retinal innervation of the presumptive optic lobe. Expression of the unique beta3-tubulin isoform thus provides a link between regulatory pathways that specify cell fate and the final executors of permanent differentiated cell structure (Hoyle, 2000).

Effects of mutation or deletion

The small deficiency Df(2R)Px2, which deletes the 60C5,6-60D9,10 region of chromosome 2, removes all of the ß3-tubulin coding sequences. The distal breakpoint of the deficiency is approximately 2 kb upstream from the start of transcription of the ß3 gene. Analysis of the homozygous and transheterozygous phenotypes of five ß3 mutations (alleles designated B3t1-B3t5) demonstrates that beta 3-tubulin is essential for viability and fertility (Kimble, 1990).

The testes-specific isoform, beta 2 Tubulin, is conserved relative to major metazoan beta tubulins, while the developmentally regulated isoform, beta 3, is considerably divergent in sequence. beta 3-tubulin is normally expressed in discrete subsets of cells at specific times during development, but is not expressed in the male germ line. beta 2-Tubulin is normally expressed only in the postmitotic germ cells of the testis, and is required for all microtubule-based functions in these cells. The normal functions of beta 2-tubulin include assembly of meiotic spindles, axonemes, and at least two classes of cytoplasmic microtubules, including those associated with the differentiating mitochondrial derivatives. A hybrid gene was constructed in which 5' sequences from the beta 2 gene were joined to protein coding and 3' sequences of the beta 3 gene. Drosophila transformed with the hybrid gene express beta 3-tubulin in the postmitotic male germ cells. When expressed in the absence of the normal testis isoform, beta 3-tubulin supports assembly of one class of functional cytoplasmic microtubules. In such males the microtubules associated with the membranes of the mitochondrial derivatives are assembled and normal mitochondrial derivative elongation occurs, but axoneme assembly and other microtubule-mediated processes, including meiosis and nuclear shaping, do not occur. These data show that beta 3 tubulin can support only a subset of the multiple functions normally performed by beta 2, and also suggest that the microtubules associated with the mitochondrial derivatives mediate their elongation. When beta 3 is coexpressed in the male germ line with beta 2, at any level, spindles and all classes of cytoplasmic microtubules are assembled and function normally. However, when beta 3-tubulin exceeds 20% of the total testis beta tubulin pool, it acts in a dominant way to disrupt normal axoneme assembly. In the axonemes assembled in such males, the doublet tubules acquire some of the morphological characteristics of the singlet microtubules of the central pair and accessory tubules. These data therefore unambiguously demonstrate that the Drosophila beta tubulin isoforms beta 2 and beta 3 are not equivalent in intrinsic functional capacity, and furthermore show that assembly of the doublet tubules of the axoneme imposes different constraints on beta tubulin function than does assembly of singlet microtubules (Hoyle, 1990).

The developmental and cellular roles played by differential expression of distinct ß-tubulins have been examined. Drosophila ß3-tubulin (ß3) is a structurally divergent isoform transiently expressed during midembryogenesis. Severe ß3 mutations cause larval lethality resulting from failed gut function and consequent starvation. However, mutant larvae also display behavioral abnormalities consistent with defective sensory perception. Embryonic ß3 expression has been detected in several previously undefined sites, including different types of sensory organs. It is concluded that abnormalities in foraging behavior and photoresponsiveness exhibited by prelethal mutant larvae reflect defective ß3 function in the embryo during development of chordotonal and other mechanosensory organs and of Bolwig's organ and nerve. Microtubule organization in the cap cells of chordotonal organs is altered in mutant larvae. Thus transient zygotic ß3 expression has permanent consequences for the architecture of the cap cell microtubule cytoskeleton in the larval sensilla, even when ß3 is no longer present. These data provide a link between the microtubule cytoskeleton in embryogenesis and the behavioral phenotype manifested as defective proprioreception at the larval stage (Dettman, 2001).

Nonmuscle ß3 expression in stage 15-16 embryos demonstrates a potential role for ß3 in differentiation of sensory organs. Further underscoring this conclusion is the finding that sequences within the large first intron of the ß3 gene are capable of driving expression of a LacZ reporter in cap cells of chordotonal organs in late stage 16 embryos. ß3 staining was examined in late stage embryos and it was found that ß3 accumulates in the cap cells of chordotonal and other mechanosensory organs during stage 17, the final stage of embryogenesis. The Drosophila embryo forms several types of mechanosensory organs, including external campaniform sensilla and the internal chordotonal organs. These organs serve as proprioreceptors through which the larva can sense strain or deformation of the cuticle or internal body structures and movements or stretching of the viscera and muscles. Chordotonal sensilla consist of a bipolar neuron surrounded by three nonneural support cells, the scolopale, ligament, and cap or attachment cells. Different classes of chordotonal organs contain one or more four-celled sensilla. ß3 accumulation is observed in cap cells of many types of mechanosensory organs. ß3 accumulates in cap cells of the lateral abdominal pentascolopidial chordotonal organs, lateral abdominal monoscolopidial chordotonal organs, dorsal triscolopidial chordotonal organs, and ventral campaniform sensilla. Morphogenesis of the mechanosensory organs of the larva begins prior to stage 13, after which morphologically mature organs first appear. Elongation of cap cells begins during stage 14 and continues throughout larval development. No ß3 staining was detected in cap cells until stage 17. Thus the ß3 isoform is present only during final stages of differentiation of the sensilla (Dettman, 2001).

In wild-type embryos, the transient presence of ß3 in the tubulin pool during late stages in differentiation of the sensory organs decreases the capacity of microtubules to form crosslinks with other microtubules. Transient ß3 expression thus confers permanent features of microtubule organization that persist in the fully differentiated cell, even after ß3 is no longer present. The sensilla in which ß3 is expressed function as stretch receptors and mechanosensors that allow the larva to sense the state of the cuticle and the viscera. The continuous foraging behavior of ß3 mutant larvae indicates that they are incapable of sensing when they are in the presence of food, or when the gut is filled. These data support the hypothesis that the misorganization of microtubules in the cap cells of the chordotonal organs causes a functional defect in these sensilla, contributing to the sensory deficits exhibited by ß3 mutant larvae (Dettman, 2001).

In pupae, transient expression of the ß3 isoform in the photoreceptor neurons of the compound eye and neurons within the optic lobe is required for neuronal patterning and connectivity in the developing adult visual system. The observation of zygotic ß3 expression in Bolwig's organ and nerve suggests that ß3 might also be essential for development of the larval photoreceptors. The morphologies of Bolwig's organ and Bolwig's nerve were examined at the light microscope level in newly hatched wild-type and ß3 mutant larvae utilizing transgenic lines in which green fluorescent protein (GFP) was expressed under control of Kruppel gene regulatory elements. Kruppel is expressed throughout development of Bolwig's organ and nerve; the transgenic animals accumulate GFP in Bolwig's organ and nerve, allowing detection by fluorescence confocal microscopy. Consistent with the late timing of the onset of ß3 expression in the developing larval photoreceptor system, the overall morphology of Bolwig's organ and targeting of Bolwig's nerve to the brain were normal in larvae that were homozygous or heterozygous for the class I ß3 mutant alleles ß3t2 and ß3t10 and the class II allele, ß3tSK. Thus, similar to the situation with the chordotonal organ cap cells, it is concluded that ß3 is not required for morphogenesis of the larval photosensory organs (Dettman, 2001).

Whether ß3 is required for function of the larval photosensory system was examined. From the second half of the first instar until late in the third instar, foraging wild-type Drosophila larvae exhibit a strong tendency toward photonegativity, such that ~75% avoid light. However, glass mutant larvae, which lack larval photoreceptors, were photoneutral. The larval photoresponse is thus dependent on the larval photoreceptors. Tests of the photobehavior of ß3 mutant larvae revealed that a feature of the abnormal foraging behavior of ß3 mutant larvae is that they do not avoid light as do wild-type larvae. Foraging wild-type first instar larvae display variable photobehavior when newly hatched, but become photonegative by ~12 hr after hatching. Homozygous ß3 mutant larvae are also photonegative, but hemizygous ß3 mutant larvae are photoneutral. The loss of normal larval photonegativity in the ß3 mutant hemizygotes is not attributable either to the inability of the mutant larvae to grow and molt or to hemizygosity for any of the other genes deleted by Df(2R)Px2. It is concluded that the defective photoresponse displayed by hemizygous mutant animals results from defective function of the larval photoreceptors caused by insufficient ß3 function during differentiation of Bolwig's organ and Bolwig's nerve (Dettman, 2001).

The ß3 dose-dependent response resembles the ß3 dose-dependent microtubule phenotype in chordotonal organs. Similarly, decreasing the ß3 dose affects some but not all ß3-dependent functions in the developing adult visual system. Together, these observations suggest that different cell types may have differing requirements either for the absolute amount of ß3 or for a given ratio of ß3 to ß1, the predominant Drosophila ß-tubulin isoform present with ß3. The three light-sensing systems in the fly -- Bolwig's organ and Bolwig's nerve in larvae and the compound eyes and ocelli of the adult -- are based on different developmental mechanisms, but all utilize ß3 during differentiation. An intriguing question is whether a ß3-containing microtubule cytoskeleton provides a common function for differentiation of distinct photosensory cells (Dettman, 2001).


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date revised:  30 August 2002 

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