Ferruh Özbudak
email: ozbudak@metu.edu.tr

Zülfükar Saygı
email: zsaygi@etu.edu.tr

Explicit Maximal and Minimal Curves over Finite Fields of Odd Characteristics

Keywords: Algebraic curves, Rational points, Maximal curves, Minimal curves

In this work we present explicit classes of maximal and minimal Artin-Schreier type curves over finite fields having odd characteristics. Our results include the proof of Conjecture 5.9 given in [1] as a very special subcase. We use some techniques developed in [2], which were not used in [1] at all.

Introduction

Algebraic curves over finite fields have various applications in coding theory, cryptography, quasirandom numbers and related areas (see, for example, [START_REF] Niederreiter | Rational Points on Curves over Finite Fields: Theory and Applications[END_REF][START_REF] Niederreiter | Algebraic Geometry in Coding Theory and Cryptography[END_REF][START_REF] Stichtenoth | Algebraic Function Fields and Codes[END_REF][START_REF] Tsfasman | Algebraic Geometric Codes: Basic Notions American Mathematical Society[END_REF]). For these applications it is important to know the number of rational points of the curve. Throughout this paper by a curve we mean a smooth, geometrically irreducible and projective curve over a finite field of odd characteristic.

Let p be an odd prime, e be a positive integer, q = p e and n be a positive integer. Let F q n denote the finite field with q n elements. Let h ≥ 0 and

S(x) = s 0 x + s 1 x q + • • • + s h x q h ∈ F q n [x]
be an F q -linearized polynomial of degree q h in F q n [x]. We consider the Artin-Schreier type curves χ given by χ :

y q -y = xS(x) = h i=0 s i x q i +1 . (1)
These curves are related with the quadratic forms

Q(x) = Tr(xS(x)) (2)
where Tr denote the trace map from F q n to F q . Let N (Q) denote the cardinality

N (Q) = |{x ∈ F q n | Tr (xS(x)) = 0}|
and let N (χ) be the number of F q n rational points of the curve χ. Then using Hilbert's Theorem 90 we have

N (χ) = 1 + qN (Q),
and hence determining N (χ) is the same as determining N (Q). Note that in general, it is difficult to determine N (χ). For the number N (χ), the Hasse-Weil inequality states that

q n + 1 -2g(χ) √ q n ≤ N (χ) ≤ q n + 1 + 2g(χ) √ q n
where g(χ) is the genus of χ. We know that there exist curves attaining the Hasse-Weil bounds. If the upper bound is attained then the curve is called a maximal curve and if the lower bound is attained then the curve is called a minimal curve. Here we note that using [START_REF] Stichtenoth | Algebraic Function Fields and Codes[END_REF]Proposition 3.7.10] the genus of the curve χ in (1) is g(χ) = (q -1)q h 2 .

Using the relations between the curve χ in (1) and the quadratic form Q in (2) some characterizations and classification results on maximal and minimal curves are obtained in [START_REF] Fitzgerald | Highly degenerate quadratic forms over finite fields of characteristic 2[END_REF][START_REF] Fitzgerald | Highly degenerate quadratic forms over over F2[END_REF][START_REF] Özbudak | Quadratic forms of codimension 2 over certain finite fields of even characteristic[END_REF][START_REF] Özbudak | Quadratic forms of codimension 2 over finite fields containing F4 and ArtinSchreier type curves[END_REF][START_REF] Özbudak | On the Number of Quadratic Forms Having Codimension 2 Radicals in Characteristic 2 Giving Maximal/Minimal Curves[END_REF] for the curves over finite fields with even characteristics. Also using similar relations some results are obtained for the curves over finite fields with odd characteristics in [START_REF] Anbar | Quadratic functions and maximal Artin-Schreier curves[END_REF]. Furthermore for all integers n ≡ 0 mod 12 and for all primes p, 5 ≤ p ≤ 29 with gcd(p, n) = 1 the following conjecture is given in [1, Conjecture 5.9].

Conjecture 1.

[1] Let p > 3 be an odd prime and let n be an integer relatively prime to p and divisible by 12. Then the curve χ over F p n defined by

χ : y p -y = x n 12 -1 j=0 x p 4 -x p 3 + x p 2 p 6j
is a minimal curve.

Similar observations and discussions are also given in the last section of [START_REF] Anbar | Quadratic functions and maximal Artin-Schreier curves[END_REF]. In this paper we prove a much more stronger version of the conjecture above. We also give explicit classes of many other maximal and minimal curves. We use some techniques developed in [START_REF] ¸akçak | Some Artin-Schreier type function fields over finite fields with prescribed genus and number of rational places[END_REF], which were not used in [START_REF] Anbar | Quadratic functions and maximal Artin-Schreier curves[END_REF] at all.

Preliminaries

In this section we recall basic definitions and some facts that we use in this paper. A quadratic form on F q n over F q is a map such that -Q(αx) = α 2 Q(x) for all α ∈ F q and x ∈ F q n , and -The related map B(x, y) on F q n × F q n defined by

B(x, y) = Q(x + y) -Q(x) -Q(y) is a bilinear map over F q n . The radical W of Q is an F q -linear subspace of F q n given by W = {x ∈ F q n : B(x, y) = 0 for all y ∈ F q n } .
Let w be the F q -dimension of W . By codimension of the radical we mean the difference n -w.

The following result was proven in [START_REF] Güneri | Artin-Schreier curves and weights of two-dimensional cyclic codes[END_REF] using some tools from algebraic geometry. Here we give a different proof using only elementary tools. We will use the following proposition later. Proposition 1. Let q be a prime power and m ≥ 1 be an integer. Consider the curve χ over F q 2m defined by

χ : y q -y = x a 0 x + a 1 x q + • • • + a m x q m .
Assume that a m = 0 and χ is maximal over F q 2m . Then a 0 = a 1 = • • • = a m-1 = 0 and a m +a m q m = 0. The converse holds as well.

Proof. As a m = 0, the genus of χ is (q-1)q m 2 and hence it has 1 + q 2m+1 many rational points in F q 2m . By Hilbert's Theorem 90 this means that

Tr x a 0 x + a 1 x q + • • • + a m x q m = 0 for all x ∈ F q 2m ,
where Tr is the trace from F q 2m to F q . Let I ⊆ {0, 1, . . . , m -1} be the subset consisting of 0 ≤ i ≤ m -1 with a i = 0. Assume that I = ∅. For each i ∈ I and 0 ≤ ≤ 2m -1 let n i, be the integer 0 ≤ n i, ≤ q 2m -1 with n i, ≡ q (i+1) mod (q 2m -1).

Note that 1, q + 1, . . . , q m-1 + 1, q m + 1 are in distinct q-cyclotomic cosets modulo (q 2m -1). Hence there exists a polynomial H(T ) ∈ F q 2m [T ] defined as

H(T ) = a m + a m q m T q m +1 + i∈I 2m-1 =0 a q i T n i, ,
such that H(x) = 0 for all x ∈ F q 2m . If H(T ) is not the zero polynomial we get a contradiction as H(T ) has degree strictly less that q 2m -1 and has q 2m distinct zeroes. This implies that I = ∅ and a m + a m q m = 0. The converse holds trivially.

Main results

In this section we state and prove our results. Note that in the proof of the following theorem we use the notion of function fields. The theory of algebraic curves is essentially equivalent to the theory of function fields. For a brief survey of the relations between algebraic curves and function fields we refer to [START_REF] Stichtenoth | Algebraic Function Fields and Codes[END_REF]Appendix B].

Theorem 1. Let q be a power of an odd prime and m ≥ 2 be an integer. Let

S(x) = a 1 x q + • • • + a m-1 x q m-1 ∈ F q 2m [x] with a 1 a m-1 = 0.
Assume that the radical of the quadratic form Tr(xS(x)) has dimension 2m -2 over F q . Then the curve

χ : y q -y = xS(x)
is a minimal curve over F q 2m . Proof. Let E 1 = F q 2m (x, y) with y q -y = xS(x) be the function field of χ. As the dimension of the radical is 2m -2 and a m-1 = 0, it is rather well known that E 1 (or equivalently χ) is either maximal or minimal over F q 2m (see, for example, [START_REF] ¸akçak | Some Artin-Schreier type function fields over finite fields with prescribed genus and number of rational places[END_REF]). Using [2, Proposition 5.1] we obtain that an extension field E 2 of E 1 such that

E 2 is maximal (minimal) ⇔ E 1 is maximal (minimal).
Moreover an affine equation for E 2 is also given:

E 2 = F q 2m (z, t) with t q -t = zR(z).
Here [2, Proposition 5.1] proves existence of c ∈ F * q 2m such that

D(x) q = S(x q + cx) and R(x) = cD(x) q + D(x) (3)
in the polynomial ring

F q 2m [x]. Put R(x) = b 0 x + b 1 x q + • • • + b m x q m .
Using (3) we obtain that b 0 = (c q a 1 ) 1/q = 0.

If E 2 is maximal, then b 0 = 0 by Proposition 1. Hence E 2 and E 1 are minimal, which completes the proof.

Using a similar idea we prove the following Theorem 2. Let q be a power of an odd prime and m ≥ 4 be an integer. Let

S(x) = a 2 x q 2 + • • • + a m-2 x q m-2 ∈ F q 2m [x] with a 2 a m-2 = 0.
Assume that the radical of the quadratic form Tr(xS(x)) has dimension 2m -4 over F q . Then the curve

χ : y q -y = xS(x) is a minimal curve over F q 2m .
Generalizing the technique in the above theorems we have the following result.

Theorem 3. Let q be a power of an odd prime and k, m be positive integers with m ≥ 2k. Let

S(x) = a k x q k + a k+1 x q k+1 + • • • + a m-k x q m-k ∈ F q 2m [x] with a k a m-k = 0.
Assume that the radical of the quadratic form Tr(xS(x)) has dimension 2m -2k over F q . Then the curve

χ : y q -y = xS(x) is a minimal curve over F q 2m .
Using the above theorems we obtain the following explicit class of minimal curves. Note that this result includes [1, Conjecture 5.9]. Theorem 4. Let q be a power of an odd prime and let n be an integer divisible by 12. Then the curve χ over F q n defined by

χ : y q -y = x n 12 -1 j=0 x q 4 -x q 3 + x q 2 q 6j
is a minimal curve.

Proof. We have

S(x) = n 12 -1 j=0 x q 4 -x q 3 + x q 2 q 6j .
Then the radical W of the quadratic form Tr(xS(x)) becomes

W =    x ∈ F q n | n 12 -1 j=0 x q 4 -x q 3 + x q 2 q 6j + x q -4 -x q -3 + x q -2 q -6j = 0    =    x ∈ F q n | n 6 -1 j=0 x q 2 -x q + x q 6j = 0    .
Then the corresponding q-associate of n 6 -1 j=0

x q 2 -x q + x

q 6j becomes x 2 -x + 1 1 + x 6 + x 12 + • • • + x n-6 .
As 12|n and x n -1 = (x 6 -1) 1 +

x 6 + x 12 + • • • + x n-6 we have deg gcd x n -1, x 2 -x + 1 1 + x 6 + x 12 + • • • + x n-6 = n -4,
which means that dim Fq W = n -4. Then we complete the proof using Theorem 2.

Using Theorem 2 and similar observation as in the proof of Theorem 4 we obtain the following result.

Theorem 5. Let q be a power of an odd prime and let n be an integer divisible by 12. Then the curve χ over F q n defined by χ : y q -y = x n 12 -1 j=0 x q 4 + x q 3 + x q 2 q 6j is a minimal curve.

In the following theorems we obtain maximal and minimal curves depending on the characteristic of the finite fields. We observe that the results are true for all odd primes of the form p ≡ 1 mod 6 and p = 3. Theorem 6. Let n = 6 and p = 3 or p is a prime satisfying p ≡ 1 mod 6. Then the curve χ over F p n defined by

χ : y p -y = x (-2x p + x)
is a minimal curve if p ≡ 1 mod 4 and a maximal curve if p ≡ 3 mod 4.

Proof. Let S(x) = -2x p + x and c ∈ F p is a root of c 2 -c + 1 = 0. Note that c 2 -c + 1 = 0 has roots c 1 = 1+ √ -3 2 and c 2 = 1- √ -3 2
in F p , since -3 is a quadratic residue as p ≡ 1 mod 6. Then define

D(x) p = S(x p + cx) -cx = -2x p 2 + (1 -2c)x p .
Then we have D

(x) = -2x p + (1 -2c)x. Now define R(x) = cS(x p + cx) + D(x) + cx p = -2cx p 2 -cx.
Then the radical of Tr(xR(x)) becomes

W R = x ∈ F p 6 | (-2c) p 2 x p 4 -2cx p 2 -2cx = 0 = x ∈ F p 6 | x p 4 + x p 2 + x = 0 .
Corresponding p-associate becomes t 4 + t 2 + 1 and as deg gcd t 6 -1, t 4 + t 2 + 1 = 4 we obtain that dim W R = 4. Now we will use the same scenario. Assume that S 1 (x) = 2x p 2 + x. Then define

D 1 (x) p = S 1 (x p + c 1 x) -c 1 x = 2x p 3 + 2c p 2 1 x p 2 + x p .
Then we have D 1 (x) = 2x p 2 + 2c p 1 x p + x. Now define R 1 (x) = c 1 S 1 (x p + c 1 x) + D 1 (x) + c 1 x p = 2c 1 x p 3 + 2 c p 2 +1 1 + 1 x p 2 + 2 (c p 1 + c 1 ) x p + (c 2 1 + 1)x.

Then we know that the curve y p -y = xR 1 (x) is maximal if and only if the following system has a solution in F p 6 (see Proposition 1):

(2c 1 ) + (2c 1 ) p 3 = 0

c p 2 +1
1 + 1 = 0 c p 1 + c 1 = 0 c 2 1 + 1 = 0. Note that this system has a solution in F p 6 if and only if -1 is a quadratic nonresidue modulo p, that is, p ≡ 3 mod 4.

Similarly we obtain the following result.

Theorem 7. Let n = 6 and p = 3 or p is a prime satisfying p ≡ 1 mod 6. Then the curve χ over F p n defined by

is a minimal curve if p ≡ 1 mod 4 and a maximal curve if p ≡ 3 mod 4.

Remark 1. In the proof of Theorem 1 we have used [2, Proposition 5.1], which guarantees the existence of c ∈ F * q 2m such that D(x) q = S(x q + cx) and

Now we want to give explicit c's for our Theorem 4. Assume that q = p ≡ 1 mod 6 and n = 12. Let

Then let us define

Also define

We know that x 2 -x + 1 = 0 has roots in F p , since -3 is a quadratic residue as p ≡ 1 mod 6. If we take c ∈ F p as a root of

since c p = c and c p 3 +1 = c 2 . Then the radical of Tr(xR(x)) becomes

Corresponding p-associate becomes t 10 + t 8 + t 6 + t 4 + t 2 + 1 = t 4 + t 2 + 1 t 6 + 1 , which means that dim W = 10. Note that the same technique is also works for n > 12 for the case q = p ≡ 1 mod 6.


B Schaerlinger

J M Launay

J L Vonesch

L Maroteaux
email: luc.maroteaux@chups.jussieu.fr

A gain of affinity point mutation in the serotonin receptor gene 5-HT 2Dro accelerates germband extension movements during Drosophila gastrulation Running title: Point mutation in 5-HT 2Dro gene

Keywords: Serotonin, receptor, mutation, ectoderm, gastrulation, cuticle, affinity

Serotonin not only works as neurotransmitter in the nervous system, but also as a morphogenetic factor during early embryogenesis. In Drosophila, a previous report showed that embryos that lack the 5-HT 2Dro receptor locus, display abnormal gastrulation movements.

In this work, we screened for point mutations in the 5-HT 2Dro receptor gene. We identified one point mutation that generates a gain of serotonin affinity for the receptor and affects germband extension, 5-HT 2Dro C1644 . Embryos homozygous for this point mutation display a four-fold increase in the maximal speed of ectodermal cell movements during the rapid phase of germband extension. Homozygous 5-HT 2Dro C1644 embryos present a cuticular phenotype including a total lack of denticle belt. Identification of this gain of function mutation shows the participation of serotonin in the regulation of the cell speed movements during the germband extension and suggests a role of serotonin in the regulation of cuticular formation during early embryogenesis.

Introduction

In vertebrates, the biogenic amine serotonin (5-Hydroxytryptamine; 5-HT) affects a wide variety of central and peripheral functions involved in behavioral and physiological activities in addition to embryonic development. Numerous receptor subtypes mediate the many functions of 5-HT. At least 14 different receptors have been molecularly characterized in mammals. All receptors except 5-HT 3 belong to the G protein coupled receptor family and activate different signaling pathways. In insects, biogenic amines are similarly involved as neuromodulators and neurotransmitters, in addition to their role in cross-linking proteins and chitin during the sclerotization of the cuticle [START_REF] Wright | The genetics of biogenic amine metabolism, sclerotization, and melanization in Drosophila melanogaster[END_REF]. In Drosophila, four 5-HT receptors have been described [START_REF] Witz | Cloning and characterization of a Drosophila serotonin receptor that activates adenylate cyclase[END_REF][START_REF] Saudou | A Family of Drosophila Serotonin Receptors with Distinct Intracellular Signalling Properties and Expression Patterns[END_REF][START_REF] Colas | Drosophila 5-HT2 serotonin receptor: coexpression with fushi-tarazu during segmentation[END_REF] in addition to the transporter [START_REF] Park | Cell-type-specific limitation on in vivo serotonin storage following ectopic expression of the Drosophila serotonin transporter, dSERT[END_REF].

We previously reported that the G-protein coupled receptor 5-HT 2Dro is an ortholog of the mammalian 5-HT 2A, B, C receptor subfamily. 5-HT 2Dro receptor mRNA and protein expression starts after cellularization at the blastoderm stage, when major gastrulation movements begin. Further experiments demonstrated that the 5-HT 2Dro receptor is functional during early developmental stages. Concomitant with 5-HT 2Dro receptor expression, there is also a detectable transient peak of 5-HT synthesis [START_REF] Colas | Drosophila 5-HT2 serotonin receptor: coexpression with fushi-tarazu during segmentation[END_REF]. Moreover, the 5-HT 2Dro receptor mRNA is expressed in a pattern similar to the pair-rule gene Fushi tarazu [START_REF] Pankratz | Blastoderm segmentation[END_REF]. In contrast to other pair-rule genes whose expression surrounds the embryo entirely, the seven stripes of 5-HT 2Dro mRNA are restricted to the presumptive ectoderm.

We previously demonstrated that embryos homozygous for the deficiency Df(3R)HTRI, that removes the 5-HT 2Dro receptor locus, display abnormal morphogenetic movements during the germband extension (GBE) and die at the end of embryogenesis or early first instar larval stage (Colas et al., 1999b). However, a contribution of other genes present in the 55 kb of DNA deleted in Df(3R)HTRI could not be completely ruled out. In this work, we looked for a point mutant in the 5-HT 2Dro receptor gene in order to validate the functions of 5-HT in GBE process.

In wildtype gastrulae, peaks of both 5-HT 2Dro receptor expression and 5-HT synthesis coincide precisely with the onset of GBE. We previously reported that the peak of 5-HT synthesis is dependent on the maternal deposition of biopterins, cofactors of tryptophan hydroxylase (the limiting enzyme in 5-HT synthesis) and on the zygotic expression of both tryptophan hydroxylase and DOPA decarboxylase (Colas et al., 1999a). Mutant embryos with an impaired peak of 5-HT synthesis display a particular cuticular organization called "double line" (Colas et al., 1999b), which was similarly observed in Df(3R)HTRI homozygous embryos and in shotgun embryos mutated in the DE-cadherin locus (Colas et al., 1999b). Whether the cuticular phenotype is a direct consequence of a gastrulation defect remained to be determined. GBE results in an approximate doubling in length of the antero-posterior axis of the Drosophila embryo [START_REF] Zallen | Patterned gene expression directs bipolar planar polarity in Drosophila[END_REF]. As in other species, this process of convergent extension relies mostly on cell intercalation [START_REF] Schoenwolf | Roles of neuroepithelial cell rearrangement and division in shaping of the avian neural plate[END_REF].

Irvine and Wieschaus proposed that ectodermal cell intercalation, the main process in GBE, could be driven by adhesion strength, which would be different in adjacent segments [START_REF] Irvine | Cell intercalation during Drosophila germband extension and its regulation by pair-rule segmentation genes[END_REF]. Differences in adhesion strength between cells of alternate segments would control cell intercalation speed. In fact, cells do not delaminate or migrate across the tissue but, instead, rearrange specific contacts after an ordered spatial-temporal pattern of junction remodeling. It has been proposed that axis elongation in Drosophila occurs through stereotyped cell-shape changes across a uniform field [START_REF] Bertet | Myosin-dependent junction remodelling controls planar cell intercalation and axis elongation[END_REF]. However, this model stipulates a single round of intercalation, whereas multiple rounds are required for full elongation. Recently, the asymmetric distribution of cytoskeletal and junctional proteins has been proposed to contribute directly to polarized cell behavior during tissue elongation [START_REF] Bertet | Myosin-dependent junction remodelling controls planar cell intercalation and axis elongation[END_REF][START_REF] Zallen | Patterned gene expression directs bipolar planar polarity in Drosophila[END_REF][START_REF] Blankenship | Multicellular rosette formation links planar cell polarity to tissue morphogenesis[END_REF]. DE-cadherins localize to the apical part of the lateral membranes of ectodermal cells and are involved in cell adhesion [START_REF] Tepass | shotgun encodes Drosophila E-cadherin and is preferentially required during cell rearrangement in the neurectoderm and other morphogenetically active epithelia[END_REF][START_REF] Oda | Dynamic behavior of the cadherin-based cell-cell adhesion system during Drosophila gastrulation[END_REF][START_REF] Greaves | A screen for identifying genes interacting with armadillo, the Drosophila homolog of beta-catenin[END_REF]. DE-cadherin regulation seems to be important for the GBE process. Zallen and Wieschaus have demonstrated that striped expression of the pair-rule genes are both necessary and sufficient to orient planar polarity [START_REF] Zallen | Patterned gene expression directs bipolar planar polarity in Drosophila[END_REF]. The F-actin asymmetry was reported as the primary planar polarity in the GBE, followed by the segregation of Myosin II and Bazooka into complementary surface domains. DE-cadherin and Armadillo/beta-catenin would then be recruited to sites of new cell contacts prior to Bazooka association [START_REF] Blankenship | Multicellular rosette formation links planar cell polarity to tissue morphogenesis[END_REF]. The local destabilization of DE-cadherin-based adhesion indeed may cause the local destabilization of polarized cell behavior during tissue elongation. Regulators of these phenomenon remain to be identified.

In this work, we screened for mutations in the 5-HT 2Dro receptor gene and identified one point mutant that generates an increase in 5-HT affinity for the receptor. Homozygous embryos for this mutation present a defect in cell speed movements during GBE accompanied by cuticular defects.

Results

l(3)82CDb C1644 encodes a mutant 5-HT 2Dro receptor with increased affinity for 5-HT

To understand the embryonic functions of 5-HT, we became interested in mapping mutants in the 5-HT 2Dro receptor. The gene encoding this receptor is localized on the right arm of the third chromosome at 82C5 [START_REF] Colas | Drosophila 5-HT2 serotonin receptor: coexpression with fushi-tarazu during segmentation[END_REF]. Using EMS, Meagraw and Kaufman (FlyBase) screened several homozygous lethal mutants in the 82C4-D5 region. Five mutants, C360 and l(3)82CDc l6 , were mapped in this interval, since they all complement with Df(3R)6-7 (82D5-82F3-6) but not with Df(3R)110 (82C4-82F3). To check whether some of these point mutants affect the 5-HT 2Dro receptor gene, we refined further the mapping using Df(3R)HTRI (82C4-82D5) that deletes 5-HT 2Dro and the Df(3R)HTR6 (82D2-82D5) that leaves the gene intact (figure 1A) (Colas et al., 1999b). L(3)82CDb C1644 , l(3)82CDc C931 , l(3)82CDd C142 and l(3)82CDd C360 were lethal with Df(3R)HTRI but viable with Df(3R)HTR6 indicating that they are in the region of interest. However, l(3)82CDc l6 that is viable with Df(3R)HTRI, does not map at the 5-HT 2Dro locus and were not further analyzed. We found that l(3)82CDd C142 and l(3)82CDd C360 define a distinct complementation group in 82C5, since they fail to complement the lethal insertion j3A4 localized in the vicinity of 5-HT 2Dro (Colas et al., 1999b).

l(3)82CDb C1644 , l(3)82CDc C931 , l(3)82CDd C142 , l(3)82CDd
We then focused on the two remaining mutants l(3)82CDb C1644 and l(3)82CDc C931 . To assess whether they correspond to mutant alleles of the 5-HT 2Dro gene, we extracted genomic DNA from single embryo to check by PCR the presence of the balancer chromosome. We took advantage of a reporter gene (twi-LacZ) of the balancer chromosome to distinguish the homozygous mutant embryos from the others. We sequenced all the coding exons of the 5-HT 2Dro receptor gene of homozygous l(3)82CDb C1644 and l(3)82CDc C931 embryos. The sequence revealed that the l(3)82CDb C1644 chromosome had a mutation in the first coding exon of 5-HT 2Dro gene (Exon2), while the l(3)82CDc C931 issued from the same genetic screen had a wildtype sequence (fig. 1B). The point mutation in l(3)82CDb C1644 changes the proline 52 into a serine in the N-terminus of the receptor (fig. 1B-C).

Up to now, the role of the N-terminus part of the GPCR that recognize biogenic amine is not established. In mammals, the N-terminus domain of the 5-HT receptors is not directly involved in ligand binding. So it was intriguing that a mutation in this domain leads homozygous lethal mutation. We investigated how this point mutation influenced the receptor function. We performed transient expression of the mutated 5-HT 2Dro receptor in COS-1 cells and binding experiments on membrane preparations. This experiment showed that several of the tested ligands, including 5-HT, the endogenous physiological ligand, have an increased affinity for the mutant receptor compared with wildtype receptor. The Ki of 5-HT was 200 nM for the wildtype 5-HT 2Dro receptor and only 11 nM for the mutant receptor, while expression was similar (table 1). These data indicate that the mutation in the N-terminus domain of the 5-HT 2Dro receptor confers to the receptor a gain of 5-HT affinity.

5-HT 2Dro

C1644 homozygous embryos display specific cuticular alterations.

L(3)82CDb C1644 was EMS-induced on a Rucuca chromosome that presents several non-lethal mutations: ru h 1 th st cu sr e s ca. In order to analyze the phenotype induced by the mutation in the 5-HT 2Dro receptor, we first generated a recombinant chromosome that was only mutated in the 5-HT 2Dro gene. After recombination of both arms of the third chromosomes, we verified that the recombinant 5-HT 2Dro C1644 chromosome was still lethal against the original l(3)82CDb C1644 strain and Df(3R)HTRI. This recombined 5-HT 2Dro C1644 chromosome was then used for all subsequent experiments. We analyzed the timing of the lethality in 5-HT 2Dro C1644 mutant embryos.

In order to distinguish the phenotypes, we used a GFP-labeled balancer chromosome to follow the fate of each embryo. With this technique, we observed that about 20% of homozygous 5-HT 2Dro C1644 embryos hatched and died shortly during the first instar larvae (table 2). The majority of the homozygous 5-HT 2Dro C1644 embryos died at the embryonic stages.

We analyzed the cuticular phenotype of the embryos that never hatched in the 5-HT 2Dro C1644 strain (figure 2, table 2). We verified that the homozygous balancer embryos displayed mainly wildtype cuticles (table 2). The 5-HT 2Dro C1644 homozygous embryos presented either "double line" or "ghost" phenotypes (figure 2C-D, 2E-F, table 2). Among the unhatched homozygous

5-HT 2Dro

C1644 embryos 43% present wild-type cuticle, 30% double-line and 22% ghost cuticles.

The "double line" phenotype is characterized by the presence of two rows of denticles in each segment (figure 2E-F) and has been previously observed in homozygous Df(3R)HTRI embryos that lack the 5-HT 2Dro receptor (Colas et al., 1999b). Moreover, we observed that the "ghost" phenotype with no denticle at the surface of the embryo was mainly represented in 5- 2). This phenotype could be distinguished from the non-fertilized embryos as the cuticle had been secreted. Interestingly, the transheterozygous 5-HT 2Dro C1644 /Df(3R)HTRI embryos hatched but lead to small larvae that all died quickly with no obvious cuticular defects, showing a partial cuticular rescue. These data revealed that the 5-HT 2Dro C1644 mutation affects significantly the cuticular development.

HT 2Dro C1644 embryos (figure 2C-D, table

5-HT 2Dro C1644 embryos display altered cell speed movements during GBE.

A previous study showed that Df(3R)HTRI homozygous embryos presented defects in GBE (Colas et al., 1999b). We thus investigated the GBE in 5-HT 2Dro C1644 homozygous and heterozygous mutant embryos. Following the previously described method (Colas et al., 1999b), we video-recorded the rapid phase of the GBE and followed the movements of ectodermal cells around the midline of single embryos. After recording, each embryo was PCR-genotyped. The absence of the balancer chromosome marker was indicative of homozygous mutant chromosome. Using image analysis of the digital video recording, we assessed the speed of cell movements, near the ventral and dorsal midline of the embryo. This was made by using a developed software that allowed the "peeling off" of a single superficial layer of pixels from each recorded video image, then laying them flat after opening from the most rostral point. Accumulating these layers over time allowed the visualization and quantification of the speed and extent of individual cell movement at the surface of the embryo and the evaluation of the synchronization of these movements. Taking the initiation of extension movements as a reference time point, the speed and synchronization of the extension movements can be directly evaluated. The figure 3 displays representative results of these investigations on individual genotyped embryo. As the X-axis represents the position of the cell along the embryo and the Y-axis the time scale, the slope of the curves represents their speed. We followed the migration of ectodermal cells, ventral to the pole cells because they were easy to distinguish in each embryos (fig. 3D). Interestingly, we observed an initial contraction of the ventral ectoderm reflecting the mesoderm invagination in wildtype embryos before ectodermal cells start moving (fig. 3A) that could not be detected in 5-HT 2Dro C1644 homozygous embryos (fig. 3B). For cells close to the pole cells, the highest cell speed (V 2 ) was recorded in 5-HT 2Dro C1644 homozygous embryos and was four times higher than in the wildtype embryos (V 2 = 12.5±1.8, n=6 vs. 3.3±0.5 microm/min, n=6) (fig. 3E). Calculating the speed of similar cells in 5-HT 2Dro C1644 /TM3 heterozygous embryos (V 2 = 5.9±0.3 microm/min, n=5)

showed that it was only two times faster (fig. 3E). The data shows that 5-HT and 5-HT 2Dro receptors are involved in cell speed regulation during the GBE. Moreover, the intermediary phenotype observed for the heterozygous 5-HT 2Dro C1644 /TM3 embryos supports the notion that the mutation leads to a gain of function for the 5-HT 2Dro receptor.

Locally over-expressing 5-HT 2Dro receptor embryos display altered cell speed movements during GBE.

A transgenic strain that overexpresses a wildtype 5-HT 2Dro receptor under the control of the Krüppel promoter, has been previously produced (Kr-Gal4,UAS-5-HT 2Dro ) (Colas et al., 1999b). This transgenic strain displays an overexpression of the wildtype 5-HT 2Dro receptor in the central region of the embryos, where the endogenous receptor expression is low (Colas et al., 1999b). Cell speed calculation in Kr-Gal4,UAS-5-HT 2Dro embryos during GBE showed that the maximal speed was similar to heterozygous 5-HT 2Dro C1644 /TM3 embryos (V 2 = 5.7±0.7, n=3 vs. 5.9±0.3 microm/min, n=5) (figure 3C-E). These data reveals that defects of GBE in heterozygous 5-HT 2Dro C1644 mutant embryos can be phenocopied by 5-HT 2Dro local overexpression.

Discussion

The mutation in the N-terminal domain of 5-HT 2Dro receptor is a gain of 5-HT affinity.

We identified a lethal mutation in a Drosophila strain characterized by a point mutation in the 5-HT 2Dro receptor gene that changes proline 52 to serine in the N-terminal domain of the receptor, increasing 20 fold its affinity for 5-HT. The complete deletion of the N-terminal domain of the 5-HT 2Dro receptor also led to a gain of 5-HT affinity [START_REF] Colas | Evolutionary conservation of the 5-HT2B receptors[END_REF]. These data indicate that the N-terminal domain of the 5-HT 2Dro receptor is involved in binding of 5-HT. Classically, biogenic amines including 5-HT bind to amino-acid side chains within the transmembrane domains of their receptors [START_REF] Manivet | The serotonin binding site of human and murine 5-HT2B receptors: molecular modeling and site-directed mutagenesis[END_REF]. However, in Drosophila, most biogenic amine receptors have a long N-terminal domain [START_REF] Onai | Cloning, sequence analysis and chromosome localization of a Drosophila muscarinic acetylcholine receptor[END_REF][START_REF] Saudou | Cloning and characterisation of a Drosophila tyramine receptor[END_REF][START_REF] Witz | Cloning and characterization of a Drosophila serotonin receptor that activates adenylate cyclase[END_REF][START_REF] Colas | Drosophila 5-HT2 serotonin receptor: coexpression with fushi-tarazu during segmentation[END_REF][START_REF] Gotzes | Functional properties of Drosophila dopamine D1-receptors are not altered by the size of the N-terminus[END_REF]. Few studies have been performed to understand the role of these long domains. Our study demonstrates for the first time a role for the long N-terminal domain of the serotonergic receptor 5-HT 2Dro . Since this mutant receptor is more sensitive for its endogenous ligand 5-HT, 20 fold less 5-HT could be sufficient to reach signaling activity in mutant receptor comparable to that of fully stimulated wildtype receptor.

The mutant 5-HT 2Dro receptor GBE phenotype can be phenocopied by wildtype 5-HT 2Dro receptor ectopic expression.

5-HT 2Dro

C1644 homozygous mutant embryos display an increase in GBE movements compared with wildtype. As previously shown, 5-HT is transiently synthesized in Drosophila embryos, with a peak of synthesis at 3h15 after fertilization (Colas et al., 1999a). Since the mutated 5-HT 2Dro receptor found in 5-HT 2Dro C1644 strain could be activated by 20 fold less 5-HT, it could thus reach a threshold of activity earlier than wildtype receptor, stronger signaling and/or its activity may last longer. 5-HT 2Dro C1644 homozygous embryos seem to respond to an early signal inducing cell movements since GBE starts before detectable mesodermal invagination.

However, the precise measure of the time elapsed between fertilization and the onset of GBE is hardly accessible, thus limiting the possibility to document this putative time shift.

A transgenic fly strain with a restricted ectopic expression of 5-HT 2Dro cDNA under the Krüppel gene promoter (Kr-Gal4, UAS-5-HT 2Dro ) also generates gastrulation defects, supporting a role of 5-HT signaling in GBE (Colas et al., 1999b). This transgenic strain displays (i) an overexpression of the 5-HT 2Dro receptor in the central region of the embryos, where the endogenous receptor expression is low with a local disruption of the receptor segmental expression and (ii) a change in the timing of the 5-HT 2Dro receptor expression as Krüppel is expressed before 5-HT 2Dro . In Kr-Gal4, UAS-5-HT 2Dro embryos, the first defects appear as abnormal anterior initiation of the extension movements before the posterior initiation of movements (Colas et al., 1999b). Cell movements studies during GBE show that cell speed defects of heterozygous 5-HT 2Dro C1644 /TM3, and Kr-Gal4, UAS-5-HT 2Dro embryos are similar. The cuticular phenotype of Kr-Gal4, UAS-5-HT 2Dro embryos is weaker than 5-HT 2Dro C1644 . Indeed, they display either wildtype or mild denticles alterations, like heterozygous 5-HT 2Dro C1644 /TM3 embryos. Moreover, the Kr-Gal4, UAS-5-HT 2Dro strain is viable, indicating that the overexpression of the receptor mimics the phenotype of 5-HT 2Dro C1644 /TM3 embryos. The phenotype of homozygous point mutant is thus stronger than the overexpression of the wildtype 5-HT 2Dro C1644 . The difference between homozygous and heterozygous 5-HT 2Dro C1644 embryos and the similar phenotype of heterozygous 5-HT 2Dro

C1644

and Kr-Gal4, UAS-5-HT 2Dro strongly support the notion that the mutation in 5-HT 2Dro confers a gain of function to the receptor in respect to GBE movements. In mutants depleted of 5-HT (synthesis mutant) (Colas et al., 1999a) or of 5-HT 2Dro receptor {Df(3R)HTRI} (Colas et al., 1999b), although altered, GBE movements still occurs. 5-HT and the 5-HT 2Dro receptor seem thus to be involved in regulating the speed of the cell movements during the GBE process but apparently not the GBE or mesodermal invagination movements themselves.

GBE defects in 5-HT 2Dro

C1644 could be due to cell adhesion defects.

The change in cell speed in the 5-HT 2Dro receptor mutant most probably reflects alterations in ectodermal cell intercalation that was initially proposed to depend upon the difference of adhesion strength between neighboring cells [START_REF] Irvine | Cell intercalation during Drosophila germband extension and its regulation by pair-rule segmentation genes[END_REF]. Cell intercalation of epithelial cells would be driven by simple remodeling of contacts in an ordered directional pattern allowing progressively exchange places with neighboring cells [START_REF] Bertet | Myosin-dependent junction remodelling controls planar cell intercalation and axis elongation[END_REF][START_REF] Zallen | Patterned gene expression directs bipolar planar polarity in Drosophila[END_REF]. In this model, axis elongation in Drosophila occurs through stereotyped cell-shape changes across a uniform field [START_REF] Bertet | Myosin-dependent junction remodelling controls planar cell intercalation and axis elongation[END_REF]. However, multiple rounds are required for full elongation and intercalating cells locally organize to generate multicellular structures (rosette) that form and resolve in a directional fashion. Directionality is disrupted in mutants that lack antero-posterior patterning and fail to elongate. Multicellular structures, and not individual cells or cell interfaces, represent thus the functional units of cell behavior during tissue elongation. The asymmetric distribution of cytoskeletal and junctional proteins has been proposed to contribute directly to polarized cell behavior during tissue elongation [START_REF] Blankenship | Multicellular rosette formation links planar cell polarity to tissue morphogenesis[END_REF]. F-actin enrichment at antero-posterior interfaces is the earliest evidence of planar polarity. Myosin II subsequently accumulates at these interfaces and may coordinate the contraction of linked edges to drive multicellular (rosette) formation. These transient structures resolve in a directional fashion to create contact between cells that were previously separated along the dorso-ventral axis. DE-cadherin association is an early step in new contact formation and coincides with formation of a transient F-actin structure. Bazooka may be recruited later to new interfaces during their eventual stabilization [START_REF] Blankenship | Multicellular rosette formation links planar cell polarity to tissue morphogenesis[END_REF].

We previously reported that the 5-HT 2Dro receptor is expressed with a pair rule-like pattern in ectodermal cells and that the apico-basal localization of Armadillo ( -catenin ortholog), in the ectodermal cells, that links cadherins with the cytoskeleton is altered in homozygous Df(3R)HTRI embryos (Colas et al., 1999b). Moreover, GBE defects are detected both in the 5-HT 2Dro C1644 gain of function strain and in Df(3R)HTRI deficient strain. 5-HT via the 5-HT 2Dro receptor appears thus to be able to influence the polarized adhesion signals controlling the GBE process. DE-cadherin and Armadillo/beta-catenin being recruited to sites of new cell contacts prior to Bazooka association [START_REF] Blankenship | Multicellular rosette formation links planar cell polarity to tissue morphogenesis[END_REF], the 5-HTdependent DE-cadherin-based adhesion may thus regulate the stabilization/destabilization of polarized cell behavior during tissue elongation.

Putative link between GBE defects and 5-HT 2Dro receptor

In embryos injected with Y-27632, a pharmacological inhibitor of Rho-kinase and specific regulator of Myosin-II, intercalation fails to occur [START_REF] Bertet | Myosin-dependent junction remodelling controls planar cell intercalation and axis elongation[END_REF]. Interestingly, the 5-HT 2Dro receptor is orthologous to mammalian 5-HT 2A, B, C receptors, which are involved in smooth muscle cell contraction via a Rho-dependent regulation of myosin light chain kinase [START_REF] Nishikawa | The role of rho and rho-dependent kinase in serotonin-induced contraction observed in bovine middle cerebral artery[END_REF]. The 5-HT 2Dro receptor may thus transduce extracellular signal (5-HT)

to the cytoskeleton and regulate either the timing or intensity of cell intercalation, Rho/Rho kinase being a likely effector of the 5-HT 2Dro receptor although the precise transduction of this pathway remains to be determined in Drosophila.

Many 5-HT 2Dro C1644 homozygous embryos that did not hatch display a "ghost" phenotype that is nearly absent in other studied strains. The previously described "ghost" and "double line" cuticular phenotypes in Df(3R)HTRI (Colas et al., 1999b) have been proposed to result from variations in intensity of GBE defects, the "ghost" phenotype reflecting earlier and/or stronger defects. In both strains, alterations of differential adhesion between cell expressing or not the 5-HT 2Dro receptor may explain the cuticular phenotypes. Interestingly, by in situ hybridization, 5-HT 2Dro probe showed no 5-HT 2Dro mRNA misexpression in 5-HT 2Dro C1644 mutant embryos and by immunohistochemistry, we observed no defects in Fushi tarazu or Engrailed, expression in 5-HT 2Dro C1644 embryos at different developmental stages (data not shown). These observations support an indirect role of 5-HT 2Dro in cuticular formation. It has to be noticed that cell speed appears more variable in 5-HT 2Dro C1644 homozygous embryos (higher SEM, figure 3D), suggesting higher variability in the control of GBE. Together, these observations support the notion that this gain of function mutation in the 5-HT 2Dro receptor increases variability in the embryonic development and that 5-HT is likely involved in setting the time and/or the speed of GBE process by polarizing adhesion signals via

the 5-HT 2Dro receptor transduction.

Experimental procedures

Fly stocks

The following strains have been used in this study. Point mutant lines:

l(3)82CDb C1644 Stocks 0004830, l(3)82CDc C931 Stocks 0004831, l(3)82CDd C142 Stocks 0004832, l(3)82CDd C360
Stocks 0004833, l(3)82CDc l6 Stocks 0004839. Deficiency lines: Df(3R)HTRI, Df(3R)HTR6, Df(3R)110, Df(3R)6-7 (Colas et al., 1999b). Blue balancers: TM3 Sb Ser P[hb-lacZ] and TM3

Ser P [twi-lacZ]; Green balancers: TM3 Sb Ser P[hb-GFP].

To obtain 5-HT 2Dro C1644 strain, we used kar 2 ry 506 , ru,h 1 ,th,st,cu,sr,e s ,Pr,ca (Ruprica), Ki,kar 2 ry 506 , D 1 ,kar 2 ry 506 and w1118 background chromosomes.

We proceeded the crossings as follows to obtain the 5-HT 2Dro C1644 strain:

1) Recombination of the left arm of the third chromosome :

DNA extraction, PCR, sequencing

Unique embryos have been genotyped by PCR. After collection of the embryos (from 3 to 5 hours), DNA was extracted as described (Colas et al., 1999b) and 1/10 of the DNA from a single embryo was amplified using regular PCR amplification conditions for 38 cycles. In order to identify homozygous mutants, embryos were genotyped by PCR using oligonucleotides for Atonal (5'CGTTGGATCCAGCAACATAACACCACCATA3' and 5'ATCGGGATCCGGGAATTCAGCGCAGCAATC3') or (Twist-beta-galactosidase)

(5'GCGTCAGTTGCGTTCCGTAAGTGC3' and 5'CAGTTTGAGGGGACGACGACAGTA3') genes.

In order to identify point mutations in homozygous embryos, each exon of the 5-HT 2Dro receptor had been amplified by PCR using following amplimers: exon 1: 5'GGAATTCCAAAACAAGTATTGTTGATGCTG3' and 5'GGGGTACCTTATCTACTATATTTTACTTGC3'; exon 2, 3 and 4: 5'GGGGTACCAATTTTTTTTGAGATTCACATATA3' and 5'GGGGTACCGCAGTAAGTCACCACCACGTAATT3'; exon 5: 5'GGTGGAAAATTATTCGAAAGT3' and 5'GCGATTCACCACCAAAATAATTTA3'; exon 6: 5'GGGGTACCCTTAACTTTTTGGTACTACTAATA3' and 5'GGGGTACCTGGCACTGGATCCGCGTCTGGGCT3'.

Embryos, whose DNA was not amplified by twist beta-galactosidase amplimers but by atonal (a marker of the third chromosome) primers, were considered as homozygous for a mutation on the third chromosome. Indeed, twist beta-Galactosidase is present on the balancer chromosome. This technique allowed the unambiguous genotyping of all individual embryos.

PCR products of the 5-HT 2Dro receptor were purified with Geneclean kit and directly sequenced.

Binding experiment

[ 125 I] DOI was used as a radioligand to investigate the pharmacological properties of membrane fractions from 5-HT 2Dro R transiently transfected COS-1 cells [START_REF] Colas | Drosophila 5-HT2 serotonin receptor: coexpression with fushi-tarazu during segmentation[END_REF].

Briefly, cell membranes were prepared by four cycles of homogenization (Brinkman P10 disrupter) and centrifugation (48,000 x g for 15 min). The assay was established to achieve steady state conditions and to optimize specific binding [START_REF] Kellermann | Sequential onset of three 5-HT receptors during the 5-hydroxytryptaminergic differentiation of the murine 1C11 cell line[END_REF]. 50 ]g of membrane proteins were incubated with 5 nM [ 125 I]-DOI at 4°C for 60 min. The incubation medium (200 microl) contained 50 microl of radioligand, 50 microl of buffer or of competing drug, and 100 microl of membrane suspension (Protein concentration, 50 microg/ml). The mixture was incubated at 30°C for 30 min. Assays were terminated by vacuum filtration through glass fiber filters (GF/B), which had been pretreated with 0.1% polyethyleneimine, followed by 4 washes of 5 ml of ice-cold buffer. Filters were dried rapidly and their radioactivity determined by liquid scintillation counting. Non specific binding, determined in the presence of 10 microM unlabeled DOI, represented about 30% of total binding.

Competition studies for [ 125 I] DOI binding were performed by adding increasing concentrations of test drug to the reaction. Data were analyzed using The GrapPad software.

Pharmacological profiles of transiently expressed receptors were determined over three independent experiment performed in triplicate.

Cuticle preparation

Embryos were laid on an agar plate, observed at 25°C, 60% humidity. The percent of hatching larvae was evaluated after 24, 48 h and 72 h (25°C) after egg laying and expressed as the percentage of non-hatched embryos in relation to the total number embryos and larvae. After 72 h, the number of non-hatched embryos has been counted. About 25 % of l(3)82CDc C931 embryos (larval lethal on the same chromosome), 25% of 5-HT 2Dro C1644 /Df(3R)HTRI, and 40 % of 5-HT 2Dro C1644 embryos died before hatching. Cuticles from non-hatched embryos were prepared as described in (Colas et al., 1999b). Percentages in table 2 correspond to the proportion of a phenotypic class in relation to the total number of non-hatched embryos.

Time lapse video experiment

Flies laid on apple juice agar for 1 hour, aged for 2 hours, then overlaid with halocarbon 3S oil (Prolabo Voltalef) were staged according to [START_REF] Campos-Ortega | The embryonic development of Drosophila melanogaster[END_REF]. The (speed = slope of the curve). We followed a set of ventral ectodermal cells close to the invaginating endoderm since they can be easily distinguish on each figures. We illustrated the different speed of individual cells (V 1 to V 4 ) as changes in the slope of the curve. The main inflexion point of the curve, the most reliably identifiable point, corresponds to V 2 . Each embryo was individually PCR-genotyped after video recording using balancer chromosome as markers (Colas et al., 1999b). False colors correspond to different grey levels of the recorded images. Statistical significance was assessed by ANOVA and NeumanKeul posthoc test. Affinity determined as competition of 125 I-DOI is expressed as Ki±SEM in nM for each compound for wildtype 5-HT 2Dro or mutant 5-HT 2Dro C1644 receptor cDNA expressed transiently in COS-1 cells. # significant increase compared to wildtype; * significant decrease compared to wildtype; P<0.05, n=9. Following transfection of identical amount of plasmid encoding cDNAs, the expression levels of wildtype and mutant receptors were not significantly different (similar Bmax). 5-HT: 5-Hydroxytryptamine; 5-CT: Carboxytryptamine.

Table 2: Cuticular phenotypic classes of non-hatched embryos in percent.

5-HT 2Dro

l( 3

 3 )82CDb C1644 /TM3 Ser and kar²ry506/kar²ry506 generated l(3)82CDb C1644 /kar²ry506 l(3)82CDb C1644 /kar²ry506 and Ruprica/TM6 Bri,Tb generated recombined l(3)82CDb C1644 */Ruprica recombined l(3)82CDb C1644 */Ruprica and Df(3R)HTRI/TM3 Ser, Sb generated l(3)82CDb C1644 */TM3 Ser, Sb 2) Preliminary crossing to prepare the second recombination : pr, pwn FLP38/CyO ; Ki, kar²ry506 and kar²ry506/kar²ry506 generated +/CyO ; Ki, kar²ry506/kar²ry506 3) Recombination of the right arm of the third chromosome : l(3)82CDb C1644 */TM3 Ser, Sb X and +/CyO ; Ki, kar²ry506/kar²ry506 generated l(3)82CDb C1644 */Ki, kar²ry506 l(3)82CDb C1644 */Ki, kar²ry506 and D, kar²ry506/TM3 Ser, Sb generated recombined l(3)82CDb C1644 **/D, kar²ry506 l(3)82CDb C1644 **/D, kar²ry506 and Df(3R)HTRI/TM3 Ser generated l(3)82CDb C1644 **kar²ry506/TM3 Ser This stock was used in this study as 5-HT 2Dro C1644 .

  development of randomly selected single embryo was video-recorded for 1 hour (one picture every 15 seconds, i.e. 241 pictures/hour) using a Leica MZ12 binocular and digital Sony camera under transmitted light. A surrounding superficial pixel layer of each picture of the video recorded embryos was selected and, after opening at the most rostral point and flattening, accumulated over time. (see supplementary data for detailed procedure). This allows a direct visualization of the movements of individual cells initially located around the midline, the quantification of their speed during gastrulation movements and their relative synchronization

Figure 1 :

 1 Figure 1: l(3)82CDb C1644 is an allele of 5-HT 2Dro that encodes a P52S mutant receptor. (A) The l(3)82CDb C1644 , l(3)82CDc C931 , l(3)82CDd C142 , l(3)82CDd C360 and l(3)82CDc l6 complementation groups are represented. Their location were ordered and refined by deletion mapping with Df(3R)HTRI and Df(3R)HTR6. Only l(3)82CDb C1644 and l(3)82CDc C931 mapped in the vicinity of 5-HT 2Dro . (B) The sequence of the second exon of the 5-HT 2Dro gene (first coding exon) is presented for wildtype (WT) and 5-HT 2Dro C1644 chromosomes. A C to T transition in l(3)82CDb C1644 changes the proline 52 into a serine within the long N-terminus domain of the receptor. (C) Schematic drawing indicating the location of the point mutation in the receptor molecule of the l(3)82CDb C1644 strain. TM: transmembrane domain.

Figure 2 :

 2 Figure 2: Cuticular phenotype of 5-HT 2Dro C1644 mutant embryos. (A, B) Cuticular phenotype of wildtype embryos. (C, D) "Ghost" cuticular phenotype of 5-HT 2Dro C1644 embryos. "Ghost" embryos present no denticle but clear differentiated cuticle. (E, F) "Double line" phenotype of 5-HT 2Dro C1644 embryos. Note that in each segment, there are only 2 rows of denticles. The scale bar in A, C, E corresponds to 80 microm while it corresponds to 20 microm in B, D, F.

Figure 3 :

 3 Figure 3: Quantification of cell movements during GBE in wildtype, 5-HT 2Dro C1644 and Kr-Gal4, UAS-5-HT 2Dro embryos. The gastrulation of embryos has been video recorded and image analysis allowed the direct visualization and quantification of ectodermal cell movements along their relative synchronization The X-axis is the flat projection of a pixel layer at the periphery of the embryos for each video frame, i.e. at a determinate time. This allows to visualize the location of ectodermal cells present around the midline of the embryo before cell movements start (1 pixel layer correspond to 1 picture). As we recorded one picture

  as described in Material and Method and observed with bright field microscope. The fly strains were maintained with the same TM3 balancer chromosome. The l(3)82CDc C931 embryos were used as control of the homozygous balancer phenotype since homozygous l(3)82CDc C931 are late larval lethal. Transheterozygous 5-HT 2Dro C1644 /Df(3R)HTRI and homozygous l(3)82CDc C931 embryos are hatching as weak first instar larvae that are dying with apparent wildtype cuticle. Part (62.5% for 5-HT 2Dro C1644 ) and the near totality (5-HT 2Dro C1644 /Df(3R)HTRI and l(3)82CDc C931 ) of the non-hatched embryos correspond to homozygous TM3 balancer chromosome on the same genetic background. The percent of homozygous 5-HT 2Dro C1644 embryos that hatch and die as first-instar larvae is thus about 20%, and the percent of unhatched homozygous 5-HT 2Dro C1644 embryos presenting wildtype, double-line or ghost cuticular phenotypes is about 43, 30, 22% respectively. Using a GFP -labeled balancer chromosomes, we confirmed that the "ghost" phenotype represented about 1/4 of all 5-HT 2Dro C1644 homozygous embryos. Wildtype, "ghost", and "double line" phenotypes are described in figure 2. Values are presented as percent of embryos with a cuticular phenotype in relation to those that hatch 72 hours after egg laying and (n) corresponds to the total counted embryos.

Table 1 : 5-HT 2Dro pharmacological properties.

 1

	Transfection	5-HT 2Dro cDNA	5-HT 2Dro C1644 cDNA
	Agonists
	Dexfenfluramine	17±1	5.0±0.3 #
	5-HT	200±16	11±1 #
	-methyl 5-HT	420±23	370±18
	5-CT	1700±72	28 000±900 *
	Tryptamin	2040±83	1950±62
	Antagonists
	Ritanserin	8.1±1.1	7.0±0.8
	Ketanserin	40±5	8.1±0.7 #
	Mesulergine	78±6	71±5
	Bufotenine	200±12	1350±45 *
	Yohimbine	310±18	340±16

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Acknowledgements

We are indebted to the Drosophila stocks centers of Bloomington, Szeged, Exelixis, and Umea, Drs. A. Spradling, J. O'Donnell, J. Roote, S. DiNardo, P. Heitzler and G. Reuter for providing fly strains. We wish to acknowledge M. Nullans for fly cares and D. Hentsch for help with video-recordings and image analysis. We thank Drs. V. Setola and P. Heitzler for critical reading of the manuscript, and for helpful discussions. This work has been supported by funds from the Centre National de la Recherche Scientifique, the Institut National de la Santé et de la Recherche Médicale, the Université Louis Pasteur, and by grants from the Fondation pour la Recherche Médicale, the Fondation de France, the French Ministry of Research ACI and Agence Nationale pour le Recherche, the Association pour la Recherche contre le Cancer and by a Region Alsace fellowship to B. S..