BIOLOGICAL OVERVIEW

Serotonin (5-hydroxytryptamine, 5-HT) is a well known monoamine neurotransmitter, mitogen, and hormone that mediates a wide variety of physiological effects, including peripheral and central actions. Because serotonin availability is associated with mood disorders in adult humans, it is somewhat surprising that serotonin and its receptors also play a developmental role in flies and humans. In Drosophila, serotonin synchronizes morphological gastrulation movements (Colas, 1999a); in mammals, serotonin regulates morphogenetic functions of cranial neural crest cells and myocardiac cells (Choi, 1997).

Before examining the role of Drosophila Serotonin receptor 2 in gastrulation, the pathway for serotonin synthesis will be briefly described, since this information is important in evaluating the evidence that Serotonin plays a developmental role in Drosophila. For the biosynthesis of 5-HT in mammals, the rate-limiting step is catalysed by tryptophan hydroxylase (TPH) which hydroxylates tryptophan to generate 5-hydroxytryptophan (5-HTP). Mammalian TPH is a homotetramer that uses as cofactors tetrahydrobiopterin (BH4), iron and molecular oxygen. The physiological concentration of tryptophan is subsaturating for TPH. The active pteridin cofactor is reduced BH4, and it is the enzyme dihydropteridin reductase that catalyses BH4's regeneration by the reduction of the dihydrobiopterin. In Drosophila, as in mammals, GTP cyclohydrolase I (GTP-CH) is the first, rate-limiting enzyme in the biosynthesis of pteridins. In Drosophila, GTP-CH is encoded by a single gene, located at the Punch (Pu) locus (McLean, 1993 and Chen, 1994), so named for the mutant eye colors imparted by pteridin deficiencies. 5-HTP is transformed by the enzyme 5-hydroxytryptophan decarboxylase (identical to Dopa decarboxylase, which also transforms L-DOPA into the catecholamine neurotransmitter Dopamine) into serotonin. The L-alpha-aromatic amino acid decarboxylase AADC is a soluble homodimer enzyme whose activity is determined by the concentration of its substrates, 5-HTP and DOPA. Depletion of 5-HT and dopamine in a Drosophila temperature sensitive mutant lacking DDC, leads to learning abnormalities and to an aberrant pattern of serotonergic neurons (Budnik, 1989). In null alleles there is also embryonic lethality associated with an incomplete sclerotization of the cuticle (Wright, 1987). In early gastrulae, a peak of TPH activity (Colas, 1995) preceded by a peak of TPH mRNA (Neckameyer, 1992) is observed. In addition, a peak of DDC activity with unknown function has been described previously at the same embryonic stage (Konrad, 1987). It is concluded that 5-HT may be synthesised zygotically and that consequently, tryptophan and biopterins should be available (Colas, 1999b and references).

The large variety of 5-HT functions is paralleled by the pharmacological complexity of 5-HT receptors that can be classified into different families depending on their signaling pathways. The family including 5-HT1 and 5-HT5 receptors interacts negatively with adenylyl cyclase; the 5-HT2 receptor family is coupled to the activation of phospholipase C (PLC); the family, including 5-HT4, 5-HT6 and 5-HT7 receptors, activates adenylyl cyclase, whereas the 5-HT3 receptor is a ligand gated ion channel. The 5-HT2B receptors from the mouse (Loric, 1992) and human (Choi, 1994) have been cloned. These receptors are functionally coupled to Gq and to the ras signaling pathway, and can be considered as a ligand dependent oncogene acting on protein kinase C (PKC) and MAP Kinase activation (Launay, 1996). Additionally, the Serotonin receptor 2 in Drosophila (5-HT2), the subject of this essay, is expressed during gastrulation. A study of the pharmacological properties of Drosophila 5-HT2 reveals these properties best correlate with mouse 5-HT2B receptor antagonists and the 5-HT2B agonists and that no significant correlation is seen for the 5-HT2A and 5-HT2C agonists. Although Drosophila 5-HT1 subtypes have already been cloned (Hen, 1992), their sequence and pharmacology appear distinct from 5-HT2 (Colas, 1995 and Colas, 1999a).

Embryos lacking the 5-HT2 locus show abnormal germband extension movements. In embryos bearing a deficiency in the 5-HT2 locus [Df(3R)HTRI], time-lapse video recordings reveal an apparently correct cellularization and stage 6 progression: the cephalic furrow forms and both the mesoderm and the proctodeum start to invaginate. The germband initially extends dorsally in response to the pull from the endoderm primordium on the dorsal side of the embryo. However, in homozygous Df(3R)HTRI embryos (genotyped after recording), the extension movements become rapidly delayed. The rapid phase of germband extension, in 5-HT2 null embryos, occurs but at a clearly reduced speed. Taking the initiation of ventral extension movements as a reference time point, the reduced speed of the extension movements appears clearly in 5-HT2 null embryos at stage 7. After a short phase of contraction that corresponds to mesodermal cell shape changes and invagination (210 min) and to the posterior midgut dorsal shift, the ventral extension movements appear reduced both in the initial forward movements and in all the subsequent backward movements. The pole cells invaginate about 3 min late and are not centered. These observations suggest that for mutant embryos, the dorsal contraction is not relayed properly by intercalation: the movements appear slower, delayed and end prematurely. The resulting effect is desynchronization between germband extension and mesodermal and endodermal invaginations and a premature termination of movements (Colas, 1999a).

Transgenic embryos lacking 5-HT2 binding sites mimic Df(3R)HTRI abnormal germband extension. To assess the specific involvement of 5-HT2 in the Df(3R)HTRI deficiency, the expression of the receptor was abolished using an antisense 5-HT2 cDNA expressed from a heat-shock promoter (Y32 transgenic strain). After establishing the conditions of minimal heat-shock, it was verified that in Y32 embryos, the 5-HT2-specific DOI-binding sites (DOI is a specific ligand for 5-HT2 receptor subtypes) are lost at stage 7. An 8 min heat-shock induction is sufficient to eliminate any 5-HT2-specific binding sites and to trigger specific lethality associated with embryonic defects phenocopying those of embryos lacking the 5-HT2 locus. As in Df(3R)HTRI deficiency, slower germband extension movements, abnormal dorsal pole cells positioning and incomplete ventral closure, were observed. In summary, the similar defects in extension movements strongly suggest that both Df(3R)HTRI and the Y32 transgenic strain have lost the same function: signaling through 5-ht2Dro (Colas, 1999a).

Lack of serotonin synthesis in stage-7 embryos mimics the abnormal germband extension seen in Df(3R)HTRI deficiency embryos. Since the peaks of serotonin and serotonin-specific binding sites precisely coincides with stage 7 when the rapid phase of germband extension begins, documented mutations were sought that could specifically affect this peak of serotonin synthesis in the Drosophila gastrula. This study focused on alleles of the Punch locus, which encodes the GTP-CH enzyme (Reynolds, 1987). This enzyme synthesizes the pteridin cofactor required for aromatic amino acids hydroxylases enzymatic activity, including tryptophan hydroxylase, that catalyses the limiting reaction in serotonin synthesis. One class of allele of the Pu locus, the embryo specific or class V, affects maternal and/or early zygotic GTP-CH activity, suggesting that the resulting lethality is due to a deficit in early pteridin function. In embryos homozygous for the punctual mutation rWE67 in the Punch locus, the pushing force generated by ectoderm convergent extension seems also impaired or lacking. A desynchronization of germband extension from mesoderm and endoderm invaginations is revealed. SEM images similar to those obtained from Df(3R)HTRI embryos were obtained for Punch mutants. These images reveal embryos where pole cells, rather than being centered in the forming proctodeal invagination, are located posteriorly, together with defects in the ventral midline closure and arrested extension (Colas, 1999a).

To further assess the ability of the 5-HT2 receptor to control ectodermal cell movements, the global gain of function effect was examined in transgenic embryos expressing a sense 5-HT2 cDNA ectopically under heat-shock promoter control. Strikingly, even after mild induction (heat shock of 8 min), the ubiquitous expression of the receptor strongly disturbs the ectoderm layer elongation and cuticular organization, both associated with a high level of lethality. Therefore, this effect prevents the performance of any reliable phenotypic rescue experiments. Thus the effect of a more restricted ectopic expression was investigated. In the Kr-UT1 strain, which uses a Kruppel driver to express the Gal4 coupled to a UAS-5-HT2 cDNA, a local overexpression of the 5-HT2 mRNA takes place in the domain of the segmentation gap gene Kruppel. It starts almost synchronously with the 5-HT2 receptor endogenous expression as a large domain (parasegment 5±8) in the region of the weakest endogenous receptor expression (parasegment 8) and includes the mesodermal area. Drivers derived from the pair-rule genes generate expression patterns that appear later (due to the delay in synthesizing the Gal4 protein) and therefore are not useful for these studies. In spite of the low level of lethality displayed by Kr-UT1, time-lapse video observations reveal significant perturbations at the beginning of gastrulation. The first defects appear as an abnormal anterior initiation of the extension movements 7 min before the posterior initiation of movements. The midline ectodermal cells first move forward and only when the movements initiate at the posterior pole of the germband does the backward movement start. The pole cells are positioned ahead of endoderm invagination since dorsal movement seems to initiate 1±2 min before the anterior movements start. The initial delay appears to be compensated by a late increase in the speed of cell movement. Globally, the fact that the movement is not strikingly abnormal in timing or extent may explain the low level of lethality. However, the lethality is enhanced when Kr-UT1 gastrulae are heat-shocked in conditions that do not affect the wild-type control (Colas, 1999a).

The delay between anterior and posterior movements is revealed by scanning electron microscopy in embryos where the mesoderm starts closing anteriorly and pole cells, positioned outside at the front of the endoderm invagination, can also be observed. Later (at stage 8), the ectodermal cells located in the dorsal Kruppel domain do not appear to involute into the normal dorsal transverse folds. Instead this domain seems to form a barrier in such a way as to slow the migrating front of the germband. In conclusion, the local gap-like persistence of 5-HT2 also triggers a global decoordination of germband extension with apparent local inhibition of ectoderm movements (Colas, 1999a).

Changes in ectoderm cohesion have been shown to coincide with the onset of cell intercalation. Also they are accompanied by modifications of cellular apical shape (from round to hexagonal and oblong) due to passive stretching in the direction of the ectodermal extension movements. Ultrastructural observations of wild-type embryos have also revealed that junctions in ectodermal cells become apically concentrated from stage 6 to stage 7. Strikingly, in homozygous Df(3R)HTRI embryos at apparent stage 7 (with respect to cephalic furrow and mesodermal invagination) most ectodermal cells have a round apex presenting only few intercellular connecting structures. This is similar to the ectodermal cells in control stage-6 embryos. Thus, the progression in ectoderm cohesion, which normally occurs between stage 6 and 7, is impaired in 5-HT2 null embryos. At gastrulation stage, E-cadherin constitutes the adhesive part of the adherens junction structures, acting by homophilic and calcium-dependent interactions. Junction clustering in the zonula adherens is dependent upon association of the E-cadherin cytoplasmic tail to the cortical actin cytoskeleton. Given the pivotal role of Armadillo in the regulation of E-cadherin-dependent cell adhesive properties, the fraction of this protein associated with E-cadherin at the membrane was analyzed. Confocal microscopy analysis of heat-fixed Df(3R)HTRI homozygous embryos (at stage 7) reveals that the membrane-associated Armadillo, rather than being located as in normal ectodermal cells at their apex (within a length of less than 2 mm), is distributed along their apical side (over more than 4 mm in length). These results reveal in 5-HT2-null stage-7 embryos a reduction of apical projections and an altered apico-basal localization of junctional structures, presumably due to a delay in their apical concentration (Colas, 1999a).

How can the 5-HT2 receptor affect cell intercalation during germ-band extension? Within the early Drosophila gastrula, maternal and zygotic E-cadherin and catenins are ubiquitously distributed. Modulation of adhesive properties within the time-scale of cell intercalation requires regulatory mechanisms that are faster than can be achieved by transcriptional regulation. In 5-HT2 null embryos, the absence of apical ectodermal projections parallels the lack of an apical concentration of adherens junctions. These observations indicate that 5-HT signaling may control the redistribution of pre-existing junctional elements and imply that it can also generate adhesive constraints. As a consequence, the 5-HT2 striped pattern generates parasegmental adhesive differences necessary for cell intercalation. Modulation of adhesive strength by clustering of spot adherens junctions at the apex in zonula adherens is regulated by phosphorylation of intracellular portions of cadherin and/or beta-catenin. Receptors of the 5-HT2 subfamily signal through the trimeric G protein Gq (Launay, 1996). In tumor cells, adhesion mediated by cadherins can be regulated by receptors coupled to Gq (Williams, 1993). In Drosophila, the link between 5-HT2 and cell movements is, therefore, likely to rely on its control of E-cadherin-cytoskeleton association. Preliminary transmission electron microscopy data confirm the abnormal distribution of adherens junction reported here by confocal microscopy studies (Colas, 1999a and references).

Why does elimination of the segmented 5-HT2 expression lead to a non-segmented phenotype? According to the model proposed by Irvine and Wieschaus (1994), intercalation depends upon the establishment of stripes of pair-rule gene products. When these stripes are widened or eliminated, either by pair-rule mutations, mutations in genes that regulate pair-rule gene expression, or ubiquitous expression of eve, then ectodermal cell intercalation and germband extension is reduced. Although the postulated adhesive molecules remain to be identified, the ability of this model to explain the effects of embryonic patterning mutations on germband extension make it attractive. In its simplest form, this model for cell intercalation requires only two types of adhesive cells, which would be distributed in alternate segments. This model implied that (1) within stripes, relative strengths of adhesion among cells must be equal; (2) cells of different stripes must differ quantitatively in their strength of adhesion, and (3) defects resulting from alterations of the segmented pattern may not necessarily be segmented. Furthermore, it has been reported that within a single tissue, cells expressing an identical cadherin can, on the basis of the expression level of this protein, segregate into distinct populations. The data presented in this study indicate that both the lack and the excess of misexpressed 5-HT2 receptors induce abnormal germband extension accompanied by a change in the subcellular distribution of cellular junctions. By inducing local gap-like ectopic 5-HT2Dro, subtle desynchronized movements restricted to the transgene overexpression domain and loss of ectodermal plasticity are observed in the dorsal region of the embryo. This corresponds to the initial location of the Kruppel domain, a region in which the receptor is normally only weakly expressed. This phenotype is reminiscent of that previously reported for embryos expressing eve ectopically; such embryos also display a reduced germband extension (Irvine, 1994). Combined, these data suggest that the alternate distribution of differentially dosed receptor is required to generate normal germband extension and is consistent with 5-HT signaling as a mechanism generating parasegmental differences in adhesion necessary for cell intercalation. Since the defects do not appear to have a segmented pattern, one has to assume that 5-HT signaling is involved in generating segmental differences in the wild-type Drosophila embryo, although direct evidence for an alternate distribution of cell adhesive structures has not yet been reported. The identification of direct intracellular targets of 5-HT signaling that control cell adhesion should constitute an important contribution to the understanding of how extracellular signals control cell movements (Colas, 1999a).

GENE STRUCTURE

Genomic length - 10 kb

Exons - 6

PROTEIN STRUCTURE

Amino Acids - 868

Structural Domains

5-HT2 displays eight hydrophobic regions, seven of which can be identified as transmembrane domains. The 5-HT2 protein shares homologies with the G protein-coupled receptor family, including (1) potential N-glycosylation sites in its amino terminus; (2) consensus sequences for phosphorylation by different protein kinases in the cytoplamic regions, and (3) an identical location for conserved amino acids within the transmembrane regions. Competative inhibition studies gave the following rank order of potencies for selected drugs: ritanserin > ketansurin > pizotifen > 5-HT > setoperone > spiperone = N-(3-trifluoromethylphenyl)piperazine = cyproheptadine = mesulergine = N-acetyl-5-HT >methiothepine + methysergide. LY 53,857 was noncompetitive, and the binding of chlorpromazine and the other aminses histamine, dopamine, tyramine, and octopamine were not significant (Colas, 1995).

date revised: 24 October 99

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