CANAL DE APROXIMACIÓN

The retinoid-deficient quail em bryo, in addition to num erous neural crest defects (trunk and cranial) an d a dram atic reduction in som ite size, show s evidence of a respecification of hindbrain tissue such that rhom bom eres 4-7

are lost w hile rhom bom eres 1-3 expand to take their place (M aden et a l, 1996;

Gale et a l, 1999). There are only three rhom bom ere boundaries and two

bulges visible rather than the usual six and five respectively. The r4 band of

Hoxbl expression fails to appear, as do the r5 Krox-20 stripe, expression of

FGF-3 (norm ally present in r4-6), and MafB (the chick hom ologue of m ouse

kreisler, norm ally expressed in rh o m bom eres 5 an d 6). The h in d b rain

expression of Hoxb4, w hich ordinarily reaches to the r 6 /7 border, is also

absent. Evidence that w hat rem ains of the hindbrain are expanded versions of

rhom bom eres 1-3 comes from the expression of Hoxa2 w hich retains its

norm al pattern relative to them (M aden et a l, 1996) and the r3 band of Krox-

20 expression w hich occupies the posterior third of the altered hindbrain

(Gale et a l, 1999).

Expression of m id b rain / hindbrain b oundary m arkers, FgfS and Pax2,

are largely unaffected in the retinoid-deficient anim als. Interestingly, the phenotype can be rescued by a single injection of all-frans-retinol into the album in layer of the egg as late as the 5 somite stage (though m ore optim ally at the 2-4 som ite stage), pointing to this as the critical w indow for retinoid involvem ent in the régionalisation of the hindbrain. This indication of the im portance of the 5 somite stage in hindbrain developm ent is also supported by the discovery that rhom bom eric fate is respecified in the avian embryo

w hen rhom bom eres are transplanted caudally at this time (Grapin-Botton et

al, 1995; G rapin-Botton et a l, 1997; G rapin-B otton et a l, 1998; Itasaki et a l,

1996).

The endogenous supply of retinoic acid has been disrupted in Xenopus

by overexpression of XCYP26 (Hollem ann et a l, 1998) and in the m ouse by

targeted d isruption of Raldh2 (N iederreither et a l, 1999). Both approaches

mimic certain effects of R A -deprivation in anteriorising the developing hindbrain, b u t w ith some im portant differences. XCYP26 overexpression was found to rescue R A -induced dev elo p m en tal defects an d to selectively

anteriorise aspects of the h indbrain (Hollemann et ah, 1998). D epending on the dose, both the r3 and r5 stripes of Krox-20 expression w ere shifted

posteriorally by one or two rhom bom eric units. Pax6, norm ally expressed in

r3 and r5, also shifted posteriorally by one rhom bom ere at the lower dose, but at the higher dose only one stripe (presum ed to be r3) rem ained - in the norm al position of r5. Hoxb3, w hich also m arks r5, w as similarly affected but both the anterior and posterior boundaries of the hindbrain appeared normal. Intriguingly, the trigem inal (V‘^) ganglion, rath er th an sim ply relocating posteriorally, is duplicated - indicating that not all aspects of A-P identity are affected in the same w ay as Krox-20 expression.

R a ld h l null m u tan ts do n o t survive bey o n d 10.5 dpc an d show

m ultiple abnorm alities consistent w ith RA deprivation (N iederreither et al,

1999). The hin d b rain s of RaldhI m u tan t mice ap p ear to be unsegm ented

caudally and poorly segm ented rostrally (Niederreither et a l, 2000). Krox-20 is

expressed in a single broad dom ain throughout the caudal hindbrain, which

lacks a sharp posterior boundary. There is a low level of Fgf-3 expression

th roughout this dom ain and patchy expression of Hoxbl is also found, but

kreisler expression is entirely absent. Similarly, Hoxa3 and Hoxb3 (normally expressed u p to the r4 /5 boundary) are dram atically dow nregulated while

Hoxa4, b4 and d4 (norm ally expressed up to rhom bom ere 7) are no longer expressed in the hindbrain. Thus it appears that at least some portion of the caudal hindbrain is m issing. Rhom bom eres 2 and 3 appear to be enlarged,

b a se d on th e e x p re ssio n of M eisl, CRABPI a n d H oxal, b u t the

m id b rain / h indbrain b o u n d ary is not affected. These defects are partially rescued by the addition of sub-teratogenic doses of RA. The effects of the

Raldh-2 m utation are largely analogous to those seen in the retinoid-deficient quail em bryo, w ith the im p o rtan t exception of the persistence of post-r3

m arkers such as H o x b l. This m ay indicate differences of regulation of this

gene in the m ouse or the continued synthesis of RA by an alternative enzyme

in the Raldh-2 m u ta n t anim al. In d eed , an o th er enzym e w ith retinal

dehydrogenase activity - class I aldehyde dehydrogenase (ALDH-I) - has

been show n to be expressed in m any of the same tissues as Raldh-2 from 7.5

1.3.3.21 Retinoic acid receptors

RA acts as a ligand to the retinoic acid receptors (RAR/RXRs). Ligand binding m akes them transcriptionally active - providing direct regulation of target genes. One experim ental approach to this system has been the use of

dom inant-negative form s of these receptors in Xenopus, another m ethod of

reducing retinoid signalling levels. Several such studies have in common the fact that embryos overexpressing the dom inant-negative (DN) constructs are

protected from the teratogenic effects of exogenous RA (Blumberg et al, 1997;

v an d er Wees et al, 1998; K olm et a l, 1997). This dem onstrates th at these

receptors really are the agents th at m ediate the effects of retinoic acid and th at they efficiently block th is signalling. Significant differences arise depending on which m em ber of the receptor family is used - reflecting the com plexity of th eir o v e rla p p in g expression p a tte rn s a n d functions. O v erex p ressio n of a D N v e rsio n of RARa2.2 com pletely elim inates

expression of H oxD l, w hose dom ain of expression it shares (Kolm et a l,

1997). It also leads to a dow nregulation of Krox-20 expression in r5, w hile

leaving m ore anterior m ark ers such as En-2 and Otx2 unaffected. This

contrasts slightly w ith another stu d y using a DN version of another RARa

isotype - R A R al (Blum berg et a l, 1997). In this case, although Krox-20 is

sim ilarly affected, there is an expansion of the Otx2 and a posterior shift of

the En-2 expression dom ains. Electroporation of this construct into early

chick em bryos (7-10 som ite stage) results in a failure to p ro p erly induce

Hoxb4 expression in the hindbrain and spinal cord (Gould et al, 1998).

More severe effects on the hindbrain are produced by the use of a DN

RARP construct (van d er W ees et a l, 1998). This leads to a reduction in

h in d b ra in length an d , in m ore extrem e exam ples, the loss of visible rhom bom ere boundaries. Supernum erary M authner neurons are produced

(see section 1.3.3.lii), appearing in rhom bomeres 4, 5 or 6, suggesting partial

anteriorisation of these rhom bom eres. Krox-20 expression is considerably

d isru p ted , appearing in an extrem ely patchy ectopic dom ain throughout

rhom bom eres 4, 6, 7, an d even 8. Expression of Hoxbl in r4, by contrast,

rem ain s unaffected, even th o u g h it lies rig h t in the m id d le of these perturbations.

It is clear that functional red u n d an cy exists betw een the receptor classes. This is m ost evident in targeted inactivations of the RAR genes in the m ouse, w here various single null RAR m u tan ts are largely norm al b u t com b in atio n of m ultiple n u ll m u ta n ts leads to serious effects w hich

recapitulate aspects of vitam in A deficiency (Lohnes et ah, 1994). D isruptions

in the post-otic hin d b rain are observed in R A R a/R A R P double-m utants

(Dupé et ah, 1999). D ouble-m utant anim als exhibit disorganisation of the

post-otic cranial nerves and fusion of the and 4^ branchial arches along

w ith associated arteries and nerves. Rhom bom ere 5 is expanded, and the

morphological boundary betw een r6 and r7 is absent. Correlated w ith this are

the enlargem ent of the r5 Krox-20 stripe and its expansion into r6 territory, as

w ell as p osterior expansion of kreisler expression an d the induction of

supernum erary otic vesicles. This suggests a norm al role for retinoic acid in restricting the caudal expression lim it of these genes. The expression of

sev eral H ox genes is also affected. H o xb l gains an ectopic, posterior

expression dom ain w ith an anterior lim it at the prospective r6 /7 boundary,

suggesting that this is norm ally repressed by retinoic acid. HoxbS, norm ally

specifically upregulated in r5, now shows high expression throughout r5 and

r6 and Hoxd4 expression, norm ally extending to the r 6 /7 b o undary, now

recedes to the caudal-most region of the neural tube. These characteristics are strikingly similar to those of rat embryos carried by vitam in A deficient dams

m aintained on a m inimal dose of all-trans-RA required to su p p o rt pregnancy

(White et ah, 1998). These anim als also p resen t w ith su p ern u m erary otic

vesicles, as well as loss of post-otic pharyngeal arches and cranial nerves IX -

X l l .