BIOLOGICAL OVERVIEW

Two transcription factors, Tailless and Huckebein, constitute the regulatory proteins that are the final products of the terminal system. "Terminal" refers to the anterior and posterior ends of the embryo. A terminal gene is one that works and affects development at the terminal ends of the embryo. tll and hkb are transcribed at these terminals, or ends of the embryo, and therefore are considered terminal genes. Early and transient expression at the posterior pole is required to establish a domain from which arise the eighth abdominal segment, telson and posterior gut.

tailless is also considered one of the gap genes, so called because gap gene mutants have gaps in their structures. In tailless mutants, these gaps are found in the head and the posterior. Therefore, tailless controls terminal genes that result in normal development of head and posterior.

Just a few nuclear cycles after activation of tailless in the tail, a brain-specific domain is initiated at the anterior; expression in this domain is maintained with complex modulations throughout embryogenesis. Expression of tailless in this domain is required to establish the most anterior region of the brain. To understand the function and regulation of these different domains of expression, a detailed description of tailless expression in brain neuroblasts is provided (Rudolph, 1997).

Transcription of tll is initiated early in the syncytial blastoderm stage (stage 4) in two symmetrical caps: these two caps depend entirely on activity of the maternally encoded terminal system. Expression in the posterior cap is required for posterior patterning of the blastoderm stage embryo. Altough expression in both caps is transient, that in the posterior cap reaches a higher level and persists longer, i.e., through most of the blastoderm stage. Expression in the posterior cap largely disappears during gastrulation and is undetectable by the end of germband extension. By the beginning of the cellular blastoderm stage, the anterior cap of expression is replaced by a horseshoe-shaped stripe that straddles the dorsal midline between 76 and 89% egg length. This stripe covers the entire brain anlage, i.e., the procephalic neuroectoderm expression in this stripe continues in domains that will become the brain. Later, TLL mRNA is present in all proneural domains of the protocerebrum; the level of transcript in a domain is highest shortly before and during the stage at which neuroblasts delaminate from that domain. During gastrulation, the horseshoe-shaped stripe of tll expression becomes tilted backwards and undergoes a process of internal differentiation into regions with different levels of expression. A group of cells in the dorsal midline gradually ceases expressing tll, resulting in a split of the horseshoe into two dorsolateral domains. Within each of these domains, a roughly triangle-shaped, anterodorsal region shows the highest level of expression and is designated HL: posteroventral to HL is a domain with a lower level of expression, designated LL. The HL domain during stages 7 and 8 covers the dorsocentral part of the protocerebral neurectoderm. From within this region, the first groups of protocerebral neuroblasts delaminate during late stage 8. Subsequently other tll expressing neuroblasts delaminate in a well defined pattern. During stage 12, tll is expressed at a high level in a new region: the primordium of the optic lobe. This structure arises by invagination of the posterior procephalic ectoderm. tll expression remains high in the optic lobe throughout late embryonic development and can also be seen in the optic lobe in the late third instar larva (Rudolph, 1997).

tailless is activated by the torso pathway. Torso, a receptor for the putative ligand Trunk, causes a phosphorylation cascade that ultimately results in the activation of transcription of tailless. Two additional factors are involved: activation and repression by Bicoid in the anterior domain and repression by Dorsal in the central domain (Liaw, 1993).

Which genes are the targets of tailless? In the anterior domain, tailless represses fushi tarazu, hunchback and deformed, as well as hedgehog, and helps to establish the borders of expression of head gap genes, like orthodenticle.

In the posterior, Tailless acts on gap genes knirps, Krüppel and giant, setting up the posterior borders of expression (Kuelskamp, 1991). POU domain genes pdm1 and pdm2 are also regulated by tailless. tailless appears to activate caudal, forkhead and hunchback in the posterior, and is implicated in the activation of the seventh stripes of even-skipped, hairy, paired and fushi tarazu . Tailless also appears to activate T-related gene, a Drosophila Brachyury homolog.

The transcription factors encoding genes tailless (tll), atonal (ato), sine oculis (so), eyeless (ey) and eyes absent (eya), and Efgr signaling play a role in establishing the Drosophila embryonic visual system. The embryonic visual system consists of the optic lobe primordium, which, during later larval life, develops into the prominent optic lobe neuropiles, and the larval photoreceptor (Bolwig's organ). Both structures derive from a neurectodermal placode in the embryonic head. Expression of tll is normally confined to the optic lobe primordium, whereas ato appears in a subset of Bolwig’s organ cells that are called Bolwig’s organ founders. Phenotypic analysis of tll loss- and gain-of-function mutant embryos using specific markers for Bolwig’s organ and the optic lobe, reveals that tll functions to drive cells to an optic lobe fate, as opposed to a Bolwig’s organ fate. Similar experiments indicate that ato has the opposite effect, namely driving cells to a Bolwig’s organ fate. Since tll and ato do not regulate one another, a model is proposed wherein tll expression restricts the ability of cells to respond to signaling arising from ato-expressing Bolwig’s organ pioneers. The data further suggest that the Bolwig’s organ founder cells produce Spitz (the Drosophila TGFalpha homolog) signal, which is passed to the neighboring secondary Bolwig’s organ cells where it activates the Epidermal growth factor receptor signaling cascade and maintains the fate of these secondary cells. The regulators of tll expression in the embryonic visual system remain elusive: no evidence for regulation by the 'early eye genes' so, eya and ey, or by Egfr signaling is found (Daniel, 1999).

The Drosophila visual system comprises the adult compound eye, the larval eye (Bolwig’s organ) and the optic lobe (a part of the brain). All of these components are recognizable as separate primordia during late stages of embryonic development. These components originate from a small, contiguous region in the dorsal head ectoderm. During the extended germband stage, the individual components of the visual system can be distinguished morphologically as well as by spatially localized expression of the homeobox gene so and the adhesion molecule Fas II. Initially centered as an unpaired, oval domain straddling the dorsal midline, the anlage of the visual system subsequently elongates in the transverse axis and narrows in the anteroposterior axis. By late gastrulation (stage 8), the anlage occupies two bilaterally symmetric stripes that are anterior and adjacent to the cephalic furrow. The domain of so expression at this stage contains two regions with a high expression level [ol_ex (the external fold of the optic lobe) and ol_in]. Only these two regions will ultimately give rise to the optic lobe and Bolwig’s organ; the so-positive cells dorsal and posterior to these domains will either form part of the dorsal posterior head epidermis (dph) or undergo apoptotic cell death. During the extended germband stage, the anlage of the visual system expands further ventrally until, around stage 10, it reaches the equator (50% in the dorsoventral axis) of the embryo. Shortly thereafter, ol_in, the portion of the anlage that will give rise to most of the optic lobe and Bolwig’s organ, reorganizes into a placode of high cylindrical epithelial cells that differ in shape from the surrounding more squamous cells of the head ectoderm. During stage 12, this placode starts to invaginate, forming a V-shaped structure with an anterior lip (ol_al) and a posterior lip (ol_pl). Bolwig’s organ, which consists of a small cluster of sensory neurons, derives from the basal part of the posterior lip and can be recognized during stage 12 as a distinct, dome-shaped protrusion. During stage 13, invagination of the optic lobe separates it from the head ectoderm; only the cells of Bolwig’s organ remain in the ectoderm. The ectodermal region ol_ex is also internalized and forms an external 'cover' of the optic lobe; many cells of this population undergo apoptosis (Daniel, 1999).

The tll gene is expressed in a dynamic pattern in the protocerebral neurectoderm. In the posterior, this region overlaps part of the anlage of the visual system, in particular that part that will give rise to the anterior lip of the optic lobe. The anterior lip of the optic lobe upregulates expression of tll during stage 12. In addition, the posterior lip of the optic lobe, does not expressed tll at an earlier stage, now switches on this gene. Expression of tll in the posterior lip is patchy, with some cells expressing the gene at a higher level than others. The Bolwig’s organ primordium does not express tll. During later embryonic stages and during larval development, tll expression remains strong in the optic lobe, but is never detected in the Bolwig’s organ. Also the primordium of the eye disc, which expresses tll during larval stages, is devoid of this expression during embryonic development (Daniel, 1999).

tll controls a switch between optic lobe and Bolwig’s organ cell fate. Loss of zygotic tll activity results in an absence of most of the protocerebrum of the brain (Younossi-Hartenstein, 1997). In addition, the visual system of the late tll embryo shows a dramatic phenotype, namely the transformation of optic lobe into Bolwig’s organ. In wild-type embryos, the neuronal marker 22C10 (see Futsch) labels 12 neurons and their axons that project towards the optic lobe. In tll mutant embryos, the number of cells in the Bolwig’s organ is dramatically increased (by a factor of 2-3), while the optic lobe, marked by anti-Crumbs or a PlacZ insertion in so, is absent. Use of antibodies to FasII and Crb, which label the apical surface of the optic lobe, and not that of the Bolwig’s organ, allowed an analysis of how the phenotype unfolds in the tll mutant embryo. Abnormalities first become apparent during stage 11, when FasII expression increases strongly in the domain of the anterior lip of the optic lobe placode, a region that normally ceases to express FasII. In spite of this abnormal expression, the optic lobe placode appears to invaginate normally. As a results, in stage 13 tll mutant embryos, a Crb-positive vesicle can be seen subjacent to the head epidermis. During stage 14, all cells of this aberrant vesicle activate expression of the neuronal marker 22C10 and lose Crb expression, revealing that these cells are Bolwig’s organ cells. Overexpression of tll under the control of a heat-shock promoter has an effect opposite that seen in the absence of tll activity. An additional consequence of tll overexpression is that the optic lobes become located more dorsally and fused in the dorsal midline. This 'cyclops' phenotype most likely arises because the dorsomedial cells, which normally die or form part of the head epidermis, now express optic lobe markers and become an integral part of the optic lobe. The results of both loss and gain of tll expression are consistent with the interpretation that tll is required to drive cells of the anlage, which would otherwise become photoreceptor neurons of Bolwig’s organ, to develop as optic lobe cells (Daniel, 1999).

atonal is expressed in and required for the development of Bolwig’s organ. ato is expressed in the head in several small cell clusters, one of which is a group of three to four cells that is part of the Bolwig’s organ primordium. Expression of ato in this domain begins during stage 11 and continues until stage 12. Initially, a group of 6-8 cells faintly expresses ato. By stage 12, their number has decreased to 3 cells. During this period, ato-expressing cells can be seen as a small group of cells within the dome-shaped Bolwig’s organ primordium. Loss of ato function results in the absence of Bolwig’s organ. Thus, similar to what has been demonstrated for the compound eye, even though only a small subset of photoreceptors actually expresses ato, lack of ato function results in absence of all photoreceptors. Since Bolwig’s organ is enlarged in a tll mutant background, it was asked whether tll inhibits ato expression. The number and pattern of ato-positive cells in tll mutants is found to be normal. These results suggest that tll functions in parallel with, or downstream of ato in the development of the Bolwig’s organ/optic lobe primordium (Daniel, 1999).

Epidermal growth factor receptor is activated in midline regions of the head neurectoderm, in particular in the anlage of the visual system. Moreover, increased and/or ectopic activation of Egfr results in a 'cyclops' phenotype very similar to what has been described for ectopic tll expression. Egfr signaling has been shown to be required in both chordotonal organs and compound eye for the inductive signaling triggered by ato expression. Two questions raised by these observations have been investigated: (1) is Egfr signaling required for tll expression in the optic lobe and (2) is Egfr signaling involved in the recruitment of the secondary (non-atonal-expressing) Bolwig’s organ cells? The answer to both these questions is no. tll expression is unaltered when levels of Egfr signaling are manipulated, suggesting that Egfr signaling is not required for tll expression. To investigate the second question, a test was performed for the presence of Egfr-relevant mRNAs or proteins: Rhomboid mRNA, which would be expected to be present only in the signaling cells, and phosphorylated MAPK, Pointed and Argos mRNAs, which would be expected to be expressed in all cells receiving an Egfr-mediated signal. In stage 12 embryos, rho is expressed only in the small group of Bolwig’s organ founder cells (the same cells expressing ato). In contrast, activated (phosphorylated) MAPK is present in a larger cluster of cells including the entire Bolwig’s organ and part of the adjacent optic lobe. Consistent with this, pnt and aos, both known to be switched on in cells receiving the Spi signal, are expressed at the same stage throughout the entire Bolwig’s organ primordium. These gene expression and MAPK activation patterns are consistent with the idea that the Spi signal is activated by rho in the Bolwig’s organ founders and passed to the neighboring secondary Bolwig’s organ cells where it activates the Egfr signaling cascade. Supporting this view, only 3-4 photoreceptor neurons are found in the Bolwig’s organ of embryos lacking rho or spi; furthermore, the size of the posterior lip of the optic lobe is reduced in such embryos. The fact that absence of secondary Bolwig’s organ cells in rho or spi mutant embryos can be rescued by blocking cell death in the background of a deficiency that takes out the reaper complex of genes indicates that the Spi signal is not necessary for the specification (recruitment) of secondary Bolwig’s organ cells, but rather, for their maintenance (Daniel, 1999).

While the maternal patterning systems that regulate tll during its blastoderm expression have been determined, the genes required to turn on tll at a later stage in the visual system are not known. Candidates are the 'early eye genes', so, eya and ey, which encode transcription factors expressed in the embryonic visual system and in the larval eye disc in front of the morphogenetic furrow. The expression of these genes was analyzed in the visual system anlage, and tll expression was examined in embryos mutant for each of these genes. tll expression in the optic lobe does not depend on any of these three genes. It was also concluded that ey and so, which have been shown to interact with each other during eye disc determination, must act independently in embryonic visual system development, since they are expressed in those primordia in non-overlapping patterns (Daniel, 1999).

Although so is expressed initially in the entire visual system anlage, during later stages its expression becomes increasingly restricted to subsets of visual system progenitors. Thus, during stage 11, when a morphologically distinct optic lobe placode first becomes visible, the domain of so expression retreats to the posterior lip of this placode; slightly later its expression is limited to only the Bolwig’s organ, where it is maintained until stage 13. eya expression is initiated during the late blastoderm stage in a trapezoidal field in the dorsomedial head region that includes the visual system anlage, as well as progenitors of the medial brain. Beginning during gastrulation (stage 6/7), the eya domain becomes divided into an anterior stripe and a narrow posterior stripe immediately anterior to the cephalic furrow that widens laterally; this posterior domain will become part of the posterior lip of the optic lobe, including Bolwig’s organ. eya expression continues in the optic lobe until stage 12 and in Bolwig’s organ until stage 13. Embryos that lack either so or eya exhibit defects in the portions of the visual system where these genes are expressed. In both mutants, development proceeds normally until stage 11, when the posterior lip of the optic lobe (ol_pl) would normally start to invaginate. In eya and so embryos, invagination of the optic lobe placode does not take place and differentiation markers characteristic of the lobe are not expressed. In conclusion, so and eya, although expressed coincidental with tll, are not required for its activation. ey plays no role in the embryonic visual system (Daniel, 1999).

Since tll is a nuclear receptor transcription factor, it must function to block the effect of signaling from the founder cells (which is mediated at least in part by Egfr) at the transcriptional level. In the posterior of the blastoderm stage embryo, tll has been shown to function directly as both a repressor (of Kruppel, knirps and Ubx) and as an activator (of hunchback). Additional activator effects of tll (not yet demonstrated to be direct) have been shown for brachyenteron at the posterior of the blastoderm embryo, and for the proneural gene lethal of scute in the brain. Thus, in the optic lobe, tll could repress genes that would in its absence be activated by Egfr signaling, and/or activate genes that would block receipt, or execution, of the signal (Daniel, 1999 and references).

GENE STRUCTURE

Bases in 5' UTR - 189

Bases in 3' UTR - 442

PROTEIN STRUCTURE

Structural Domains

The Tailless repressor has an N-terminal DNA binding domain and a C-terminal hormone binding domain. It is identified as a steroid receptor superfamily protein (Pignoni, 1990). Tailless is an orphan nuclear receptor, meaning that although it possesses a hormone binding domain, no hormone has been identified that can bind to this domain. Although the ligand binding domain is conserved, the effects of mutation are felt most strongly in the DNA binding domain. The ligand binding domain of Tailless and mammalian homolog contains a conserved putative dimerization domain (consisting of seven heptad repeats). There is some evidence that the protein binds DNA as a dimer (Diaz, 1996).

date revised: 15 August 98

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