Development 122, 783-793 (1996) 783 Printed in Great Britain © The Company of Biologists Limited 1996 DEV2020 Late effects of retinoic acid on neural crest and aspects of rhombomere identity Emily Gale1, Victoria Prince2, Andrew Lumsden3, Jon Clarke4, Nigel Holder1 and Malcolm Maden1 1Developmental Biology Research Centre, The Randall Institute, King’s College London, 26-29 Drury Lane, London WC2B 5RL, UK 2Department of Molecular Biology, Princeton University, Washington Road, Princeton, NJ 08544, USA 3MRC Brain Development Programme, Division of Anatomy and Cell Biology, UMDS, Guy’s Hospital, London SE1 9RT, UK 4Department of Anatomy and Developmental Biology, University College, Windeyer Building, Cleveland St., London W1P 6DB, UK SUMMARY We exposed st.10 chicks to retinoic acid (RA), both ment of the facial ganglion in addition to a mis-direction of globally, and locally to individual rhombomeres, to look at the extensions of its distal axons and a dramatic decrease its role in specification of various aspects of hindbrain in the number of contralateral vestibuloacoustic neurons derived morphology. Previous studies have looked at RA normally seen in r4. Only this r4-specific neuronal type is exposure at earlier stages, during axial specification. Stage affected in r4; the motor neuron projections seem normal 10 is the time of morphological segmentation of the in experimental animals. The specificity of this result, hindbrain and is just prior to neural crest migration. combined with the loss of Hoxb-1 expression in r4 and the Rhombomere 4 localised RA injections result in specific work by Krumlauf and co-workers showing gain of con- alterations of pathways some crest cells that normally tralateral neurons co-localised with ectopic Hoxb-1 migrate to sites of differentiation of neurogenic derivatives. expression, indicates a role for Hoxb-1 and RA in the spec- The r4 crest cells that give rise to mesenchymal derivatives ification of this cell type in normal development. These are unaffected. In addition, r4 gene expression is also results suggest that RA, at st.10, is able to affect some partially altered by RA; within 6 hours of r4 exposure to aspects of segment identity while leaving others unchanged. RA, ectopic expression of Krox-20 is seen in r4 and Hoxb- 1 expression is lost while Hoxa-2 expression continues normally. When we examined these RA-treated animals Key words: chick embryo, retonoic acid, neural crest, rhombomere, later in development, they showed an anterior displace- Hoxb-1 INTRODUCTION Hoxa-2 which in the chick has an anterior expression boundary at the rhombomere 1/2 border (Prince and Lumsden, 1994) and Treatment with retinoic acid at around the time of gastrulation Krox 20, which is expressed in r3 and r5 (Wilkinson et al., and neurulation causes abnormal development of vertebrate 1989). The studies in the mouse show that the RA effect on head structures including the brain. This has been documented the hindbrain is a complex process involving the expression of in mouse (Leonard et al., 1995; Wood et al., 1994), rat regulatory genes and the underlying differentiation of specific (Morriss, 1972), Xenopus (Durston et al., 1989; Lopez and groups of cells characteristic to specific rhombomeres. Corrasco, 1992; Papalopulu et al., 1991; Sive et al., 1990) and During late neurula stages the individual hindbrain zebrafish (Hill et al., 1995; Holder and Hill, 1991) embryos. segments, the rhombomeres (Lumsden and Keynes, 1989; Analysis of phenotypes shows that alterations in the organisa- Simon et al., 1995), contain two morphogenetically distinct tion of the anterior hindbrain are common to all species. It has populations; the neural crest cells and the cells of the neu- been suggested that the spatial organisation of the hindbrain roepithelium. Later in development, these two cell populations region is regulated by the spatial patterning of the Hox genes, differentiate to form a unique combination of elements either as well as other regulatory genes, and since retinoic acid (RA) within or derived from each rhombomere. The first group, the can affect the expression of these genes it may be that RA is neural crest, gives rise to neurogenic and mesenchymal deriv- altering hindbrain organisation by altering regulatory gene atives. Using r4 as an example, the neurogenic derivatives expression. Recent anatomical studies have thus been comple- consist of a few neurons and all the support cells of the acoustic mented by analysis of expression patterns of candidate regula- and vestibular ganglia, including the Schwann cells which tory genes (Conlon and Rossant, 1992; Marshall et al., 1992; support the axonal extensions of the ganglia (Couly and Le Papalopulu et al., 1991) which are normally expressed in the Douarin, 1990; D’Amico-Martel and Noden, 1983; Le Douarin anterior hindbrain region. These genes include Hoxb-1, which et al., 1986). The mesenchymal derivatives of r4 crest include is expressed in rhombomere (r)4 (Wilkinson et al., 1989), entoglossal and branchial cartilages and two of the cartilages 784 E. Gale and others in the middle ear (Lumsden et al., 1991; Noden, 1983). The (Lee et al., 1995) has shown a specific mis-migration of crest. cells of the neuroepithelium will form, among other neuronal In the latter experiment, animals were exposed globally to RA groups, part of the facial motor nucleus (Fraser et al., 1990; and no correlation between altered migration, early gene Lumsden and Keynes, 1989) and the vestibuloacoustic expression, and subsequent alteration in morphology was neurons. The latter is a set of specialised sensory efferent made. In contrast, our study examines how crest is affected by neurons, exclusive to r4, some of whose cell bodies migrate local exposure to RA at the time of the initiation of crest across the midline of the neural tube after axonal outgrowth migration, while also examining alterations in regulatory gene begins (Simon and Lumsden, 1993). expression and later phenotype resulting from the same exper- The capacity of cranial neural crest to form both mesenchy- imental treatment. mal derivatives (dermis, cartilage, bone, connective tissue, and Just prior to crest migration the hindbrain undergoes many some muscle cells) and neurogenic derivatives (neurons, changes. At the 9 somite stage (ss), in chicks, rhombomeres Schwann cells, and support cells of the cranial ganglia) (Couly are forming morphological boundaries and certain homeobox et al., 1992; Noden, 1992) has inspired many decades of inves- genes are developing precise borders of expression which tigation into the location and timing of cell type specification coincide with those boundaries. Krox-20 expression is (Landacre, 1910; Wagner, 1959; for reviews see Anderson, localised to the regions of the two stripes of cells forming r3 1989; de Beer, 1937; Le Douarin et al., 1993). However, there and r5 (Wilkinson et al., 1989), Hoxa-2 is developing its is still no clear picture of when cell fate is determined for anterior border of expression at the r1/r2 border (Prince and neural crest, although there are results supporting each of the Lumsden, 1994) and Hoxb-1 is expressed in the entire posterior various hypotheses. It is very possible that some cells are hindbrain up to the developing r3/r4 border (Kuratani and specified before migration while others retain their pluripoten- Eichele, 1993). In the next few hours, as the rhombomeres tiality until they reach their site of differentiation (Baroffio et develop morphological boundaries and crest migration begins, al., 1988). The hypothesis of prespecification of regions of Krox-20 and Hoxa-2 expression is sharpened to coincide with hindbrain crest by their axial position is supported by three those boundaries, while Hoxb-1 expression is blocked in r3 and experiments with premigratory cranial crest (Kuratani and r5 to leave a precisely defined band of expression in r4. Eichele, 1993; Noden, 1983; Prince and Lumsden, 1994); they Given the variety of developmental events occurring at this demonstrated that heterotopically transplanted crest cells form stage it would be useful to know the effect of RA exposure on cartilage elements and express genes appropriate to their these processes. Despite many detailed studies (Conlon and original position rather than to their host site. The different Rossant, 1992; Cunningham et al., 1994; Holder and Hill, question of prespecification of final tissue type of individual 1991; Kessel, 1993; Marshall et al., 1992; Wood et al., 1994) premigratory crest cells has been addressed by a number of recording RA effects, it is still unclear if RA acts endogenously techniques. Individual cell labelling, culture and transplant to control the normal expression of Hox genes. However, a experiments in chicks have indicated that, in the trunk, pluripo- number of recent lines of evidence suggests that it does; RA is tentiality is not lost until the site of organogenesis is reached present endogenously in Henson’s node (Hogan et al., 1992) (Fraser and Bronner-Fraser, 1991; Le Douarin and Dupin, and the Xenopus dorsal lip during gastrulation (Chen et al., 1992). However, individual cell labelling in cranial neural crest 1994), which is the time of first expression of the Hox genes cells in zebrafish has suggested prespecification of tissue type which later acquire anterior expression boundaries in the before migration (Schilling and Kimmel, 1994) while the trunk hindbrain (e.g. Hoxa-1 and Hoxb-1; Kolm and Sive, 1995; neural crest has shown similar behaviour to that described in Wilkinson et al., 1989). Recent work has also shown that chickens (Riable and Eisen, 1994). In chick cranial neural retinoic acid response elements (RAREs) are present in the reg- crest, results have shown some labelled cells giving rise to only ulatory regions of the Hoxa-1 (Langston and Gudas, 1992) and a few of the many potential crest-derived tissue types but have Hoxb-1 genes (Studer et al., 1994; Marshall et al., 1994). It is given no evidence of specification of an individual final also important to consider the specific stages at which RA phenotype (Bronner-Fraser and Fraser, 1988; Le Douarin et al., exerts its action on the regulation of Hox gene expression in 1993).