The patterning function of purines in the brine shrimp Artemia

0 Academic

des sciences

Development

biology

/ Elsevier, Paris

I Biologic

du dkveloppement

The patterning function shrimp Artemia

of purines

in the brine

Le rdle despurines dims Ia morphoge&se d’kemia Arantxa Museum

Hernandorena” national

d’histoire

naturelle,

plateau de I’Atalaye, 64200

(Received 6 July 1998, accepted after revision 2 1 December

Biarritz,

France

1998)

Abstract - In two Artemia wild-type sibling species originating from the Old and New Worlds, the processes underlying the construction of the naupliar body during embryonic development and the construction of the adult body during postembryonic development are disrupted by specific nutritional deficiencies and/or the administration of metabolic inhibitors. The species-specific phenotypic outcomes of these experimental disruptions on the construction of segments and the establishment of their identity, permit us to outline a model in which Hox genes would act as intermediary cogwheels fastened to a mechanism put in gear upstream by purine-mediated processes which would trigger downstream folic acid-mediated processes. The prevalent view that Hox genes can select for different developmental programmes, is challenged by this model whose relevancy is analysed in the context of our present knowledge on the master functions ascribed to Hox genes in developmental and evolutionary processes. (0 Academic des sciences / Elsevier, Paris.) Arfemia

/ pattern

formation

/ epigenetic

regulation

/ Hox

genes

/ purines

R&urn& - Chez deux espkces juntelles et sauvages d’Artemia originaires de I’Ancien et du Nouveau Monde, les processus qui rkgissent la construction du corps du nauplius pendant le dkveloppement embryonnaire et la construction du corps de I’adulte pendant le developpement postembryonnaire sont perturb& par des dtfkiences alimentaires spkifiques et/au I’administration d’inhibiteurs mkaboliques. Les conskquences phknotypiques propres a chaque esptce de ces perturbations expkrimentales sur la construction des segments et l’ttablissement de leur identite permettent d’esquisser un modkle dans lequel les genes HOX interviendraient en tant que rouages intermediaires assujettis B un mkcanisme enclench en amont par des processus rkgis par les putines et qui dtclencherait en aval des processus rigis par I’acide folique. La pertinence de ce modPIe qui remettrait en cause le r61e h+$monique attribuk aux genes Hox est analyske dans le contexte de nos connaissances actuelles sur le dkeloppement et I’kvolution. (0 AcadCmie des sciences I Elsevier, Paris.) Artemia

I morphogenk

I r&hion

Cpigkdtique

I g&e How I purines

Version abrCgt?e Le genre Artemia comporte nog&Ctiques. La spkiation

Note

communicated

by Pierre

des espltces bisexuees et parthtdu genre s’est produite sans

8user

* Correspondence and reprints: Elissaltenia, Les R&oilets, 64600 C. R. Acad. Sci. Paris, Sciences de la vie / Life Sciences 1999.322,289-301

changement morphologique et les diffkrentes espkes sont considkrkes comme &ant jumelies. Les experiences d&rites ici montrent qu’en rkalitk, les espkces originaires de 1’Ancien et du Nouveau Monde, ont subi une divergence kolutive au

Ciboure

289

A. Hernandorena niveau dun mecanisme de regulation des proliferations cellulaires qui regit de la morphogenese. La strategic mise en oeuvre par Artemia pour construire le corps de I’adulte inclut deux programmes. Le programme embryonnaire aboutit a la construction du corps du nauplius segment6 dans la region cephalique et le programme postembryonnaire qui se deroule en partie chez des larves qui s’alimentent, aboutit a la construction du corps de l’adulte segment6 dans les regions cephalique, thoracique, genitale et abdominale. Uutilisation d’une alimentation de synthese dans des conditions d’elevage axeniques permet de montrer que les larves appartenant B l’espece bisexuee americaine Utah et a I’esptce parthenogenetique espagnole La Mata necessitent une alimentation riche en purine pour se developper en adultes normaux. La deficience alimentaire en purine peut dtclencher l’expression de potentialitts morphogenetiques latentes propres a chaque region du corps et propres a chaque espece dont la construction d’yeux surnumtraires dans la region cephalique et la construction d’appendices surnumeraires dans la region abdominale normalement apode. Ces transformations fournissent des informations utiles pour comprendre I’organisation segmentaire de la partie anterieure de la tite et la diversification des segments au tours de l’tvolution des arthropodes mais leur dtterminisme genetique chez Artemia reste a dechiffrer. En revanche, les transformations localisees dans la region genitale, domaine d’expression du gene AM B, peuvent &tre attribuees a l’incapacite de ce gene de jouer le role qui lui est assign&, role qu’il n’est capable d’assurer correctement qu’avec une alimentation riche en

purine. Ces transformations correspondent a la multiplication anarchique des cellules epidermiques de l’ovisac chez l’espece parthenogenetique et a la multiplication contr6ke de ces cellules chez I’espece bisexuee aboutissant B la construction de soies sur les appendices gtnitaux qui en sont normalement dtpourvus. Les soies &ant des structures typiques des appendices thoraciques, leur construction est assimilable B une transformation homeotique. Un modele capable d’expliquer le role morphogenetique des purines peut Ctre esquisse a partir de l’observation des consequences phenotypiques de carences alimentaires en acide folique et/au en thymidine et de I’administration de differents inhibiteurs qui bloquent la dihydrofolate reductase, la thymidylate synthetase, I’IMP deshydrogtnase et la xanthine dtshydrogenase. Dans ce modele, les genes Hox interviendraient en tant que rouages intermediaires assujettis au fonctionnement d’un mecanisme qui, enclenche en amont par des processus regis par les purines, dtclencherait en aval des processus rtgis par l’acide folique. MCme si la drosophile fournit des arguments genetiques, biochimiques et nutritionnels solides en faveur de ce modele, le fonctionnement du mecanisme purines-genes Hex-acide folique qui remettrait en cause le role hegemonique attribue aux genes Hox, reste a demontrer par une analyse moleculaire. Ce mecanisme s’accorderait avec I’attribution de la diversite morphologique des especes au redeploiement dans des contextes differents dun systeme genetique ancestral. II expliquerait que des corps differents aient pu etre construits au tours de l’kolution avec des genes Hox identiques.

1. Introduction

Artemia affords a peculiar nutritional approach to the problem of pattern formation due to a developmental strategy which includes two programmes. The embryonic one whose genetic determinism remains unknown, results in the construction of the naupliar body segmented in the cephalic region. The postembryonic one, which takes place in feeding larvae, results in the construction of the adult body further segmented in the cephalic, thoracic, genital and abdominal regions. Artemia larvae reared axenically using an artificial nutritive medium with hypoxanthine as the dietary source of purines and treated with mycophenolic acid (MA) from 24 to 96 h posthatch, develop into adults presenting anteriorward transformations of genital segments into thoracic-like segments, of abdominal segments into genital-like segments and an abnormal abdominal segmentation. Dietary guanosine restores normal pattern in adults developing from larvae MA-treated during early stages (61. MA interferes with guanylate production and reduces Artemia larval nucleotide pools which are significantly depleted 96 h posthatch [7] long before the final differentiation of genital and abdominal segments. Apart from the general recognition that guanylate is the precursor of the pteridine pathway, the intricacy between purines and pteridines arises from the fact that xanthine dehydrogenase (XDH) which is induced by hypoxanthine, one of its purine substrates, acts on both purines and pte-

The Artemia genus, which belongs to the primitive order of Anostraca, includes bisexual and parthenogenetic species. The apparition of parthenogenetic species from the European bisexual one, occurred about six million years ago and is a more recent event in the evolutionary history of this crustacea than the separation of Old and New World species. The speciation occurred without any morphological changes and the different Artemia species are considered as siblings [l]. Their identical morphology implies the execution of genetically determined similar developmental programmes. Artemia is presently the only crustacea in which several Hex genes are identified and their domain of expression in trunk segments [2] and thoracopods [3] established. The phenotypic outcomes of their mutational inactivation remain unknown but their expression limited to welldefined regions, is consistent with a developmental role which in spite of sustained interest and countless efforts initiated by the pioneer work of Lewis [4] is not yet exactly understood. In order to progress in our understanding of pattern formation, we may need a knife that cuts deeper than the mere observation of transcription factor expression patterns [5], sharp enough to reach products of housekeeping genes as shown by present and previous nutritional studies performed with Artemia.

290

C. R. Acad. Sci. Paris, Sciences

de la vie / Life Sciences 1999.322,289-301

Epigenetics ridines [8]. A new the Spanish La

set of experiments was performed Mata strain, which belongs

species and with the American belongs to the A. franciscana species an inhibitor of XDH. Results show

strain which allopurinol,

Artemia

sibling species have undergone

Worlds

originating from an evolutionary

development

with the

to

A. parthenogenetica

of Artemia

Utah using that

the Old and New divergence at the

level of a mechanism which regulates the localized capacity of cells to proliferate in domains of Hox gene expression and which governs pattern formation. A tentative model is outlined to explain the operation of the mechanism our present to Hex esses.

and its relevancy analysed knowledge on the master

genes

in

2. Material The

techniques

larvae medium reared

developmental

in the functions

and

evolutionary

to

produce,

per 100 mL by a chemically

pyrimidine reared from the quantity

proc-

and methods handle

and

under axenic culture conditions and C were detailed previously [gl.Here, at 25 + 0.5 “C and 7 %O salt concentration

sea water replaced

context of ascribed

of media) defined

Artemia

rear

to prepare larvae were (20 mL

with Torula yeast mixture of purine

RNA and

compounds. Utah and La Mata larvae were hatching to adulthood to study the effects of and quality of dietary purines on postembry-

onic patterning with inosine or guanosine as the dietary source of purines in cytidine-rich (100 mg CR per 100 mL), allopurinol-free media. Utah larvae were reared from urinol

hatching to adulthood on postembryonic

cient GR, were

or guanosine-rich, cytidine-rich media (40 or 80 mg 100 mg CR per 100 mL). Utah and La Mata larvae reared from hatching until the release of the first

brood to patterning

to study patterning

study the effects in inosine-rich,

the effects of allopin guanosine-defi-

of allopurinol cytidine-rich,

on embryonic thymidine-con-

taining media (120 mg IR, 100 mg CR, 40 mg TdR). Their growth performances were assessed by the percentage of larvae capable of reaching adulthood and their reproductive performances by the number of nauplii counted in the first brood released by the first fecund culture tube. The number of individuals

female into each per experimental

condition is indicated in the tables. checked by a microscopical examination ogy of nauplii and adults. Some naupliar

Development was of the morpholand adult anom-

alies were easier to detect in unfixed photographed alive. Fixed animals DAPI as described previously [lo].

animals were

which stained

were with

During

Artemia

adult media

body

postembryonic

are added progressively diately in front of the

C. R. Acad. Sci. Paris, Sciences 1999. 322,289-301

instar

larvae

stained

segmentation

and

growth

Feeding

begins

this

stage.

during

nauplii reared grow beyond When moults

in purine-free the stage reached

cytidine-rich by unfed

showing

internal

media do not larvae (figure 1).

start feeding after expense of vitelline

having performed two reserves and become

amenable to nutritional treatments, segments are internally delineated vation explains why the disruptive ments with mycophenolic acid

thoracic obsereffects of larval treat[6] and with allopurinol

(see

below),

posterior

adult

body.

The

larvae at the

with DAPI (arrow).

zone

are

dietary

restricted

purine

to the

most

requirement normal capable

of their

(figure 7). This

segments

is essential development of reaching

for (table adulthood

of the

optimal I).

The and

the percentage of adults with a normal phenotype decrease when the dietary purine supply is reduced. La Mata larvae are more sensitive to a purine deficiency than

patterning

development,

from a growth telson (figure I).

1. Second

larval growth and percentage of larvae

3. Results 3.1. Postembryonic in allopurinol-free

Figure thoracic

zone Utah

segments lying immeand La Mata

de la vie / life Sciences

Utah ones. Larvae adversely affected purine requirement to induce XDH adults produced

belonging to both species are more by a purine deficiency when their is met by guanosine whose capacity is inferior to that in purine-deficient

of inosine. Abnormal media, manifest seg-

291

A. Hernandorena Table

1. Growth

Artemia

and

developmental

performances

of Utah

and

La Mata

strain

dietary

Number Percentage Percentage

in relation

to the quantity

and

quality

of the

inosine

mg per 100 mL of larvae reared of larvae reaching adulthood of abnormal

adults

guanosine

purine

inosine

0

20

40

80

20

40

80

0

245 63

390 81

200 96

235 33

635 73

100 97

-

52

18

59

35

140

dietary

supply.

La Mata

Utah

purine

Concentration

larvae

0

0

0 100

20 200

guanosine 40

80 200 76

0

0

260 54

-

-

51

20 200

40

0

0

80

200 6

-

38

95 72 7

c Figure

2. Phenotypic

A: Normal pliar eye,

292

outcome

naupliar and ce: compound

of inosine

deficiency

compound eye (La Mata eye, se: supernumerary

in the

cephalic

region

P); B-D: supernumerary eye, de: duplicated eye.

eyes

(La Mata

0); C: Duplication

C. R. Acad.

Sci. Paris,

of compound

Sciences

eye

de

(Utah

d);

ne: nau-

la vie / Life Sciences 1999.322,289-30

1

Epigenetics merit-specific and species-specific developmental anomalies. They are detailed below for adults produced in inosine-deficient media containing 40 mg IR per 100 mL. The rarest and most striking anomaly is the construction of adventitious eyes (figure 2A) in addition to normal compound eyes (figure 26, D). These supernumerary eyes are constructed in 4.2 % of the abnormal La Mata females in a more anterior domain than that of normal compound eyes and have a reduced but complete array of ommatidia in contrast to the duplicate eye observed only once in an adult male from Utah produced under standard conditions (figure 2C). The construction of supernumerary eyes was never observed in La Mata females produced from larvae reared in purine-rich media nor in Utah adults. The second species-specific anomaly affects the genital appendages, i.e. the two latero-ventral penes in Utah males and the medio-ventral ovisac in Utah and La Mata females. In 19 % of Utah abnormal adults, genital appendages are more or less completely transformed into thoracic-like appendages. The transformation can be limited to the construction of setae on the ovisac (figure 3A) or the penes (figure 3B) never observed in normal genital appendages constructed in purine-rich media (figure 3.Q. In 47 % of La Mata abnormal females, the ovisac manifests an overgrowth (figure 3C, D) owing to the disorganized and increased proliferation of epidermal cells evident in the histological section of the ovisac (figure 3F). This anomaly is the only lethal one. The third developmental anomaly common to both species is the most frequent one. in 68 % of Utah and 53 % of La Mata abnormal adults, a supernumerary appendage is constructed on the first abdominal segment. Their position medio-ventral in females and latero-ventral in males corresponds to that of genital appendages. The fourth developmental anomaly is also common to both species. In 19 % of Utah and 5 % of La Mata abnormal adults, the number of abdominal segments is reduced. The third and fourth anomalies were previously illustrated [61. 3.2. Postembryonic supplemented with

adult body patterning 40 mg allopurinol per

in media 100 ml

Utah larvae manifest a greater sensitivity to allopurinol when reared in guanosine-deficient media than in guanosine-rich media (table II). The inhibitor changes the phenotypic outcomes of the dietary guanosine supply. Larvae reared in guanosine-deficient, allopurinol-supplemented media develop into adults in which the entire set of genital and abdominal segments can be deleted. The telson is normal but has no furca (figure 4). In less extreme cases, abdominal segmentation is severely disrupted with a reduced number of abnormal segments. A great percentage of larvae reared in guanosine-rich, allopurinolsupplemented media develop into abnormal adults presenting in addition to anteriorward transformations of genital and abdominal identities, a posteriorward transformation of the last thoracic appendage into a pene-like C. R. Acad. Sci. Paris. Sciences de la vie / Life Sciences 1999.322.289-301

of Artemia

development

appendage (figure 5A). In extreme cases, the transformed eleventh thoracopods adopt a ventral position which corresponds to that of normal genital appendages (figure 58). In Artemia, patterning along the antero-posterior axis of the body and patterning along the proximo-distal axis of appendages are simultaneous processes 161. Thoracopod defects not scored in table II, were observed in adults produced from allopurinol-treated larvae manifested by the construction of an extra endite-like outgrowth (figure 6) in addition to the four endites which correspond to distalless expression domains [31. 3.3. Embryonic in allopurinol-free

naupliar body media

patterning

When Utah larvae are reared in inosine-rich media containing 120 mg inosine per 100 mL, normal adults are obtained in 8-10 d. From day 10 onwards, males grasp females showing oocyte vitellogenesis in their two ovaries observable through the transparent cuticule with a handlens. By days 12-l 4, mature oocytes migrate from the lateral oviducts to the medio-ventral ovisac where fertilization occurs. Females release their first brood from days 16-19 with a mean number of 43.9 nauplii per brood (tab/e 110. The body of newly hatched nauplii (figure 7a), first (figure 7e) and second (figure 717 instar larvae, comprises the segmented cephalic region with antennules, biramous antenna and mandibules and the externally unsegmented trunk. 3.4. Embryonic supplemented

naupliar body patterning in media with 40 mg allopurinol per 100 ml

When Utah larvae are reared in inosine-rich media containing 120 mg inosine per 100 mL, allopurinol has no detrimental effect on larval survival rates to adulthood, no disruptive effect on postembryonic adult body patterning and does not reduce fertility. Allopurinol greatly reduces the mean number of nauplii per brood (table 111).There is a great heterogeneity with respect to the number of nauplii (O-52), the percentage of abnormal nauplii (O-100 %) and types of naupliar defects in each brood. Broods including at least one hatched nauplius attesting fecundation were scored. Females releasing broods exclusively composed of unhatched potentially lethal embryos were not included in table 111. In addition to unhatched embryos and normal nauplii, scored broods include hatched and live abnormal nauplii presenting defects described in a presumed decreasing order of severity. a) Stunt nauplii: - with abnormal establishment of polarity axes and unrecognizable cephalic segmentation (figure 7b); - with abnormal terminal parts, abnormal dorso-ventral polarity, abnormal fused (figure 7c) or unfused cephalic appendages (figure 7d). b) Short larvae: -with missing post-mandibular region (figure 7fl; - with missing pre-antenna1 region and short posterior region (figure 7g).

293

A. Hemandorena

Figure

3. Phenotypic

outcomes

of inosine

deficiency

in the genital

region.

A. Extra setae (arrow) (La Mata 9); E: normal

on the ovisac genital and

(Utah 9); 6: extra setae (arrow) on one out of two devaginated abdominal phenotype (Utah 0); F: histological section through

0~:

Al,

first,

ovisac,

294

P: penes,

A2, A3:

second

and

third

abdominal

pene (Utah 6); C-D: overgrowth of the ovisac the ovisac (La Mata 0); Tll: 1 lth thoracopod,

segments. C. R. Acad.

Sci. Paris,

Sciences

de

/ Life Sciences ?999.322,289-301

la vie

Epigenetics Table

11. Growth

lopurinol

per

Mg guanosine Allopurinol Number

developmentalperformances

and 100 per

mL (+) in relation 100

to the

of Utah

dietary

mL

of larvae

Percentage

of larvae

Percentage

of abnormal

reaching

larvae

guanosine

adulthood

adults

reared

in allopurinol-free

media

of Arfemia

development

supplemented

(-) or in media

with 40 mg al-

supply.

20 -

40

235 33

635 73

59

35

60 -

80 -

20

40

+

100 93

400

100

+ 620

97

9

0

0

Figure

17

-

5. Phenotypic

100

outcomes

of allopurinol

60 +

80 +

100 38

340 66

88

78

using

guanosine-rich

media. Transformation (Utah 8).

of

A: The transformed Figure

4. Phenotypic

outcome

cient media. Deletion of genital mal telson with no furca (Utah

Table Ill. Reproductive per

100

mL (+) reared

of allopurinol and 9).

abdominal

performances of Utah at 25” (-) or submitted

using

guanosine-defi-

segments.

Note

nor-

formed thoracopods left-transformed showing racopods,

females produced to 32” (+) during

thoracopods

thoracopod assume

Number

appendages

with

40 mg allopurinol

+ +

Vitellogenesis Total Mean

genital-like

B: the transposition. The bears a few setae (arrow) Tl 0, Tl 1: 10th and 11 th tho-

supplemented

+ -

shift

into

keeps a lateral position; a latero-ventral pene-like

pene-like thoracopod, transformation;

in allopurinol-free media (-) or in media 48 h before (-) or during (+) vitellogenesis.

-

Allopurinol Temperature

incomplete P: penes.

1 lth

+ t t

of females

number number

Percentage

C. R. Acad. 1999.322,289-30

of naupli of naupli of abnormal

16 702 per

brood

naupli

Sci. Paris, Sciences 1

de la vie / Life Sciences

62 789

43.9

12.7

0

10.3

11

16

176 16

89

4.5

5.6 38

295

A. Hernandorena

Figure Extra

6. Phenotypic endite-like

outcome outgrowth

of allopurinol (asterisk)

in thoracopods.

in thoracopod

stained

with

DAPI

(Utah

adult).

A?

El

El ?a

._

__-

__. I

___---

------

-.

-

;

__-

-

__-

., 1

A?

,...

Figure

7. Morphology

of normal

naupliar

(a) first (e) and

second

(i) instar

larval

phenotypes

compared

to allopurinol-induced

abnormal

pheno-

types. b, c, d: Stunt

296

nauplii,

f, g: short

larvae,

h: bifid

larva,

j, k, I: abnormal

cephalic

appendages.

Al : Antennule,

C. R. Acad.

A2:

Sci. Paris,

biramous

Sciences

antenna,

de

M: mandibule.

la vie / Life Sciences 1999.322,289-30

1

Epigenetics c) Bifid larvae: - with bifidated described previously d) Larvae

with

gers telson (figure [l I]. various

appendages: - with appendicular

7h);

types fusions

this

of

phenotype

abnormal

showing

was cephalic

failure

of proper

cephalic segmentation: left-side fusion of antenna with mandibule and right-side fusion of antenna with antennule (figure 7j) or with fused antennules (figure 7k); - with stunt uniramous antenna (figure 71). As seen in table 111, the percentage of abnormal released by allopurinol-treated females reduced

nauplii when

adults are reared at 25 “C, can be increased when adults are incubated at 32 “C from days 10 to 12 and returned at 25 “C. The 48 h period of heat treatment coincides with that of the onset of vitellogenesis. When the 48 h heat treatment at 32 “C is imposed day 8 to day 10, reproductive (table

on immature performances

females from are improved

111).

When experiments ditions with La Mata sively composed data ascribable fecundation

are performed larvae, females

under release

identical broods

conexclu-

of unhatched potentially lethal embryos, to a greater sensitivity to allopurinol since cannot

be incriminated

(results

not

shown).

are concerned it work 1121,

genes is not restricted phenotypic outcomes

with building but the role

to making of specific

the body rather of housekeeping

it work as shown by the nutritional deficiencies

and of treatments with metabolic inhibitors observed during present and previous experiments in two wild-type Artemia sibling species. Allopurinol disrupts both embryonic and postembryonic patterning suggesting common underlying biochemical mechanisms. The discussion will focus on postembryonic patterning because embryonic not be analysed in terms of functions zygotic genes which are not yet identified allopurinol-sensitive struction of the

naupliar

mechanism body

involved is at work

of

patterning maternal in Artemia.

canand The

in the conat the onset of

vitellogenesis as shown by temperature shift experiments and is disrupted before the end of gastrulation because Arfemia embryos are permeable until the first gastrula stage and impermeable once the second reached ]13]. Allopurino] interferes with both naupliar body axes and with cephalic

gastrula stage is polarization of segmentation.

Some abnormal naupliar phenotypes are reminiscent of those of Drosophila maternal effect mutant embryos with missing anterior and posterior terminal parts; their temperature-dependent production and erratic frequency even inside reminiscent

a brood released by the same oi Drosophila maternal effect

mother are also mutations 114,

151. In order adequate

correspond the cephalic

to develop normally, Artemia larvae require purine supply. A dietary purine deficiency

C. R. Acad. Sci. Paris, Sciences 1999.322,289-301

de la vie / Life Sciences

an trig-

and

struction region mations

localized

results in phenotypic

capacity

development of cells

segment-specific outcomes.

to the construction region of La Mata

and These

of supernumerary females and

to pro-

several outcomes

eyes in to the con-

of supernumerary appendages in the abdominal of both La Mata and Utah adults. These transforprovide phylogenetic informative data to under-

stand the segmental organization of the anterior the head so far intractable to both morphological, ological and molecular analyses ]I61 the diversification of body segments evolution, but their genetic determinism, Drosophila the genital deficiency B which requires

portion of embry-

and to understand during arthropod deciphered

in

(see below), remains unknown in Artemia. In region, the phenotypic outcomes of a purine can be analysed in terms of the function of Abd defines purine-rich

its downstream ital appendages

Artemia media

genital in order

target genes in La Mata

identity [2]. to correctly

Abd B regulate

and to construct normal genand Utah adults. A dietary

purine deficiency permits us to disclose an evolutionary divergence between sibling species at the level of a mechanism which regulates cell proliferation in the domain of Abd B expression. The following to explain this mechanism. Previous shown that

4. Discussion Hex genes than making

an increased

liferate which species-specific

of Artemia

results obtained the establishment

discussion

will

with the Utah strain of genital fate is an

postembryonic event. Pulse MA treatments imposed on larvae 24-76 h posthatch, result formation appendages. they and

of

interfere with the

interval between

attempt

genital appendages When identical treatments

lasting in the

into are

have early 24 h trans-

thoracic-like postponed,

with the formation of abdominal segments establishment of their identity [6]. The 24 h

between larval

MA moults.

pulses, These

paralleles the 24 h interval data suggest that the same

MA-sensitive mechanism is sequentially activated as new segments are added from the growth zone and is used to pattern the posterior region of the adult body. The activation of this postembryonic comes. Previous

mechanism stages,

results

obtained

at consecutive steps results in segment-specific

with

the

Utah

strain

less efficient have shown

but more refined that the construction

appendages to synthesize

is insured by the folic acid-mediated thymidylate. Normal appendiculated

racic and are reared or in folic

medium than of thoracic

of early out-

using

a

medium C and genital capacity tho-

genital segments are constructed when in thymidine-free, folic acid-containing acid-free, thymidine-containing media.

larvae media When

larvae are reared in folic acid-free, thymidine-free media, they develop into adults with genital segments and posterior thoracic segments transformed into apodous segments ] 171, the construction of their anterior appendiculated

thoracic

maternal folic acid 1181. typic outcomes of larval which blocks dihydrofolate

segments

being

insured

by

The observation of the phenotreatments with aminopterin reductase (DHFR) and trans-

297

A. Hernandorena struction

of gap-like

and

multiple

homeotic-like

pheno-

types. These data suggest that the same XDH-sensitive mechanism is activated at different thresholds to construct segments

and

to

establish

their

identity.

In Drosophila,

allopurinol This result a nutritional

blocks the production of isoxanthopterin I21 1. was obtained with yeast-fed Drosophila larvae, condition which in Artemia precludes the

disclosure

of any

lsoxanthopterin

is a by-

product of tetrahydrobiopterin (BH4) synthesis, end-product devoid of biological activity. deep-orange (dor) mutant females characterized abnormally high isoxanthopterin content are

but not an Drosophila with an sterile [221.

The death in the egg,

disruptive

effect.

of dor embryos, can be prevented

from unfertilized normal embryos, isoxanthopterin [24] and forms a complex import of cytoplasmic the pteridine between

ascribed by the

to a pteridine defect injection of cytoplasm

eggs [23]. In can bind cytoplasmic which facilitates

Oncopeltus proteins the nuclear

proteins due to the intercalation the base pairs of the DNA

of helix

[251. The

regulation

of the subcellular

localization

of Exd,

protein encoded by extradenticle (exdi, a homologue the human proto-oncogene pbxl [26], is an important nal-dependent mechanism for controlling Hox target expression abdominal repressing

]27]. In appendages Distal-less

absent from the The construction

Drosophila, is blocked (D/l [28]

the of siggene

the construction by BX-C expression and Exd is apparently

nuclei of most cells that express of ectopic eyes is blocked by

of by

D/l 1271. homofh-

orax Ihth) expression by directing the nuclear localization of Exd [29]. In the eye disc, eye development is determined by the cytoplasmic localization of Exd [29] and requires

wild-type

function

of eyeless,

the

eye

sefector

gene 1301. The eye size of eyeless mutant adults is highest when larvae are reared in RNA-free or RNA-deficient media and lowest when they are reared in RNA-rich or in Figure 8. Phenotypic outcome of a double deletion using purine rich media.

folic

acid plus thymidine

Transformation of normally appendiculated segments into apodous segments. Note normal formation of thoracic, genital and abdominal segments (Utah young S).

forms appendiculated segments and with 5-fluoro-deoxyuridine synthetase and results regulation of thymidylate level of DHFR activity. of segments transformed ble folic acid dietary purine

into which

apodous blocks

ones [I 81 thymidylate

in teratogenesis [I 91, shows that the biosynthesis is performed at the Now most importantly, the number into apodous segments by a dou-

plus thymidine deletion, increases when supply is increased 1201 (figure 8). This

suggests that the folic acid-mediated size thymidylate required to decreases in purine-rich media.

the data

capacity to syntheconstruct appendages,

adenylic differently

290

strain have patterning in the con-

acid-rich acid and

media. Larvae to guanylic acid

respond [31]. In

the antenna1 disc, hth is the antenna selector gene Exd is nuclear 1321. The antenna to leg transformation Nasobemia mutant adults is highest when larvae reared These

in adenosine-deficient, results permit us to link

with media

phenotypic equate

outcomes nuclear Exd

cytidine-rich genetic and

and show with repression

media nutritional that

remarkable ble that the

evolutionary conservation diverse morphogenetic

observed in multiple tissues homeotic selector gene activity other animals 1351 including foregoing

discussion

permits

C. R. Acad.

Sci. Paris. Sciences

[33]. data

of the

of Exd, it is possifunctions of this gene

of Drosophila [26, 341 may Artemia.

consistent with the experimental the scheme proposed by Duboule strong linkage between patterning

and of are

purine-rich of the expres-

sion of morphogenetic potentialities and that purine-deficient media equate cytoplasmic Exd with de-repression the expression of morphogenetic potentialities. Given

The Present results obtained with the Utah shown that allopurinol disrupts postembryonic in a purine-dependent manner and results

acid, cytidylic to adenylic

that require be present in

us to outline

a model

data and founded upon [36, 371 to explain the and growth control in de la vie / Life Sciences 1999.322.289-301

Epigenetics vertebrate

model

control through

systems

of proliferation a feedback

where

positive

and

negative

insured by epigenetic factors, act loop on the Hox complexes. In the

Arfemia

In this

simplifying

model,

the regulation

of the

capacity to synthesize thymidylate that is the regulation of cell proliferation would be governed in the localized domains of Hox gene expression by the purine-mediated XDH induction which controls the isoxanthopterindependent

subcellular

localization

Artemia contribution patterning

is presently of purines has been

the only organism in which to embryonic and postembryonic disclosed. Two peculiarities

of Exd. the of

Artemia

can explain this uniqueness. The first one is the essentiality of its dietary purine requirement which translates an incapacity of larvae [381 and adults 1391 to syn-

thesize

the purine

ring

de novo

and

permits

the

control

of

the entry point of purines in the purine metabolic pathway. Drosophila can synthesize the purine ring 1401. Purine biosynthesis requires the participation of cofactors derived from folic acid [8]. acid but no dietary source

Drosophila of

purine

adults require [41]. When

folic the

hypothesized

mechanism is put in gear by folic acid as in Drosophila and not by purines as in Artemia, the system is self-maintained by an auto-activation circuit and the cofactor functions of folic acid in purine biosynthesis are difficult to divorce from the cofactor min in thymidylate biosynthesis. The Artemia is its developmental strategy. tems,

segmentation

process

which Nutricarried

nutrition this decision

is an embryonic

vitaof sys-

takes place in a nutritionally closed environment. tional studies should be, but usually are not, beyond one cycle of larval growth. In Myzus maternal wings,

body

function of this second peculiarity In insect model

influences the being mediated

decision to the

persicae,

to construct larvae by their

mother 1421. DHFR activity is higher in presumptive alates than in larvae destined to develop as apteres and the apterizing effect of aminopterin is reversed by thymidine [43]. The execution of the early decision to construct wings is thus insured by the folic acid-mediated capacity to synthesize favours switch

thymidylate.

A dietary

phenylalanine

the production of alates and mechanism of wing dimorphism

excess

is able to keep the set at the alate

course of development [&I]. In Drosophila, dietary phenylalanine excess increases the BH4 demand and decreases isoxanthopterin production 1451. A decreased isoxanthopterin-dependent favour DHFR transcription

nuclear import of Exd and wing construction.

would

The purine-Hox gene-folic-acid mechanism would be consistent with the ascription of morphological (limb) diversity to the redeployment in different contexts of an ancestral genetic regulatory system used to construct a body

wall

outgrowth

Hox gene-folic

acid

[46]. The deployment system in different

C. R. Acad. Sci. Paris, Sciences 1999.322,289-30 1

of the purineXDH contexts

de la vie / Life Sciences

bodies.

lowered to the

leads

The

illustrated morphological to explain

model, Hox genes would act on DHFR transcription as cogwheels fastened to the activation of a mechanism put in gear upstream by purine-mediated processes which would trigger downstream folic acid-mediated processes.

experimentally allopurinol,

of Arfemia

by purine construction

experimental

development

deficiencies of different

production

of the

or

by

Artemia phenotypes

figures 4 and

in

8 shows the feasibility of the changes surmized by Averof and Akam [21 arthropod evolution and indicates the possible

mechanisms by which phology were achieved.

these evolutionary The entire thorax

changes in morof Artemia (I

1

segments) would be homologous with the pre-genital (three thoracic and eight abdominal segments) region of insects, a model making many assumptions which remain to be tested [471. terior segments

The

model

assumes

the

deletion

out of eleven appendiculated thoracic segments dous segments (figure 8). The possibility that context of Drosophila is different from that of suggested by the fact that the allopurinol equal to 95 mg per 100 mL whose effect limited to the modification of wild-type adult

flies

Mollusc port the embryos ence of

of posof eight

(figure 4) and the transformation

Artemia

1211

kills

and

chelicerate

larvae embryos

into apothe XDH

Artemia

is

concentration in

(result provide

Drosophila eye

colour

not

shown).

data

Artemia

model. The polar lobe which represent an early example localized cytoplasmic determinants,

is in

to sup-

of mollusc of the existis highly

enriched in nucleotides 1481. The authors have manifested their reticence in ascribing a status of morphogenetic determinants to “such common metabolites”. In horseshoe crab embryos, inhibitors the number of body segments sistent with results in Artemia

of DNA synthesis increase 1491. These data are conwhere metabolic conditions

which ment

result

favour number.

DNA

synthesis

in a reduction

in seg-

Results obtained with Artemia should direct attention on XDH, “an enzyme that appears to have assumed design so effective as to merit maintenance throughout the evolutionary xanthopterin which may homeoproteins

is

hierarchy” released

explain the reviewed

[SOI. In lower in supernatants specificity by Akam

a

eukaryotes, iso151 I, a data

of the

distribution

of

1471. In Drosophila

embryos, the XDH function as mesoderm was discovered fortuitously 1521, may nuclear localization of Exd in visceral

enhancer contribute mesoderm

which to the 1271.

Variations in the XDH protein revealed by electrophoretic analysis are documented between different Drosophila species and between different strains of the same species 153-551.

These

variations

significance 1561 would be ascribable

of real

genetic

which affect enzyme to post-translational

and

evolutionary

activity modifications

1571, of

the XDH protein by the gene products of several distinct and independently acting loci 1561. The characterization at the molecular level of XDH variants in rosy mutants has proved to be a difficult task [58, 591. In Drosophila, it is not known if the observed have any causal relationship in various morphological According lutionary

to the divergence

Artemia between

differences in XDH activity, to the observed differences and fitness characters 1571. model, the

they do, and the evosibling Artemia species

299

A. Hernandorena originating from the Old and New Worlds which occurred during the last six million years, would be ascribable to XDH. Information on the actual role of Hox genes in developmental and evolutionary processes can be obtained not

Acknowledgements: pathologiques

(Biarritz)

only with arthropods exhibiting a wide range of body plans 1601, but paradoxically with Artemia sibling species in which a subtle epigenetic evolutionary divergence interferes with the regulation of cell proliferation and with pattern formation.

I would like to thank Dr Darasse from the Laboratoire d/analyses for the histological preparation of La Mata females. Part of this work

de biologic was financially

medicale supported

d’anatomie by the

et de CNES.

cytologic

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