seminars in THE NEUROSCIENCES, Vo14, 1992 : pp 307-315

Patterning the rostrocaudal axis of the hindbrain

Anthony Graham

During earb development the hindbrain is segmentally organised along the rostrocaudal axis into units termed rhombomeres. This segmental organisation is underlain by patterns of cellular differentiation. A relationship between the organisation of the branchiomotor and somatic motor nerues and the segmented ground plan has been found. It has also been shown that neural crest cells have a segmental origin. The expression patterns of a number of putative regulatory

genes relate to the rhombomere pattern. Foremost amongst these are the Hox gene farnib. This family of genes are particularly interesting as they are thought to be involved in rostrocaudal patterning. A number of recent experiments, both embryological and genetic, support such a role for these genes in the rhombencephalon.

that each of the three principal branchiomotor nerves originates within the confines of alternate rhombomeres and that their early nuclei lie opposite their target branchial arch (Figure 1). The somatic motor nerves

Key words: rhombomeres / Hox genes I segmentation -

THE CELLULAR and molecular mechanisms involved in specifying pattern along the rostrocaudal axis of the nervous system are the subject of current interest. Because of its relatively simple, segmented organisation and because it is the site of expression of a number of developmentally interesting genes, the hindbrain is a system that offers considerable promise. This review will therefore consider the structure of the hindbrain region and the Hox genes that are expressed there. Details of patterning in other regions of the brain are few and these will therefore be considered only briefly.

The hindbrain is a segmented structure

During the early development of the hindbrain the neural epithelium becomes transiently organised into a rostrocaudal series of eight segments, or rhombomeres. Anatomical analysis of the hindbrain has revealed that the rhombomere pattern is related to underlying cellular organisation. 1 It was observed

From the MRC Brain Development Program, Division of Anatomy and Cell Biology, UMDS, Guy’s Hospital, London SE1 9RT, UK

01992 Academic Press Ltd 1044.5765/92/050307 + 09$8.00/O

Figure 1. Schematic representation of the major an- atomical features of the chick hindbrain. Note that the rhombomeres are numbered 1-8 on the left hand side. The branchiomotor nerves are depicted inside the hindbrain on the right hand side and their exit points marked (mV, trigeminal; mVI1, facial; mIX, glosso- pharyngeal; mX/XI, vagal/accessory). The somatic motor nerves and their exit points are on the left (mIV, trochlear; mV1, abducens). The cranial ganglia are beside the brain on the left. Note the position of the otic vesicle (OV) at r6 and r5. The arrows to the right of the brain represent the streams of neural crest from the hindbrain. Note that rhombomeres 3 and 5 produce no neural crest.

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are also generated segmentally (Figure 1) . Anothercharacteristic of rhombomeres is that they are lineagerestricted compartments, as shown by single cellmarking experiments . 2 These demonstrated thatwhile neural epithelial cells disperse freely andwidely within the territory of a single rhombomere,they do not mix with cells of the neighbouringrhombomeres . Thus rhombomeres can be consideredas polyclonal lineage restriction units somewhat akinto the compartments of insects . Early segregation ofcell populations along the rostrocaudal axis suggeststhat cell lineage plays some role in the generationof specific cell patterns in the rhombencephalon .

Positional cues are carried by the neuro-ectoderm in the rhombencephalic

region

Embryological experiments have suggested that inthe rhombencephalic/brachial arch region positionalcues are not carried by the mesoderm but insteadby the neural epithelium . That this information istransmitted to the surrounding tissues by the neuralcrest was demonstrated by transplanting presumptivefirst arch crest in the place of presumptive secondor third arch crest . 3 The result of these manip-ulations was the formation of first arch structures,by both transplanted neural crest and host mesodermcells, in inappropriate sites . It can therefore beconcluded that the first arch neural crest cellsare different from those that will colonise the secondor third arches . This difference does not concernthe range of differentiated cell types that areproduced, rather it concerns the morphogeneticcapacity of the cells and their ability to patternthe mesodermal cells of these separate axial levels .These results also suggested that the neural crestcells acquire their spatial programming prior toemigration from the neural epithelium .

Neural crest has a segmented origin inthe hindbrain region

The origin and migration of rhombencephalic neuralcrest cells have been mapped by labelling thepremigratory crest in chick embryos with a vitaldye .4 This analysis revealed a segmental origin forneural crest in the hindbrain, the crest originatingat three discontinuous positions and migrating inthree corresponding streams (Figure 1) . The first was

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from rl and r2, the second from r4 and the thirdfrom r6. Thus the three streams of neural crest wereinterrupted by two regions that did not produce crest,r3 and r5 . The dorsal midline of r3 and r5 was foundto be associated with enhanced levels of cell death,suggesting the possibility that neural crest cellproduction is uninterrupted along the rostrocaudalaxis but that the cells are eliminated in r3 andr5 before emigration . The consequence of thisarrangement is the specific filling of the appropriatebranchial arch and the organisation of the cranialganglia and the entry/exit points for cranial nerves,both of which are associated with the even numberedrhombomeres .

Krox 20 is segmentally expressed

The first report of a gene expression pattern thatmatches the segmented organisation of the hindbrainwas for the zinc finger gene Krox 20 . This gene isexpressed initially in a single stripe in the 8 .0 dpcmouse embryo hindbrain, corresponding to thepresumptive r3 . By 8 .5 dpc a second stripe appears inthe presumptive r5 and expression is maintainedin these stripes until after rhombomeres 3 and 5become distinct morphologically (Figure 3) . 5 Thegene is also expressed in neural crest cells and laterin the neural crest-derived boundary cap cells ofcranial and dorsal root ganglia . 5 Importantly, thedomains of Krox 20 precede the morphologicalappearance of rhombomere boundaries and it istherefore possible that Krox 20 is involved in definingsegmental territories or possibly a two segment repeatwithin the hindbrain . Yet the fact that Krox 20 isexpressed in neural crest cells along the entire axis,in addition to being expessed throughout r3 and r5which are crest free, could suggest that this gene isinvolved in the discontinuous production of neuralcrest cells . These two possible roles for Krox 20 neednot be mutually exclusive .

Whatever the role of Krox 20 it would be unlikelyfrom its expression pattern that it could play a directrole in the specification of rostrocaudal identity in thehindbrain . Possible and more attractive candidatesfor such a role are members of the Hox gene family .

Hox genes and rostrocaudal patterning

The homeobox, an 180 by sequence that can encodea 60 amino acid DNA binding domain, was first

Rostrocaudal axis of the hindbrain

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isolated from genes in the Drosophila homeotic genecomplex, HOM-C, which have been shown tobe involved in the specification of rostrocaudalidentity . 6 The vertebrate genes are clustered, asthey are in the fly, but there are four clusters invertebrates with each cluster mapping to a separatechromosome (Figure 2) . All the clusters share acorrelation between the order of genes on thechromosome and their order of expression along therostrocaudal axis . On the whole, each successivelymore 3' gene is expressed more rostrally . Structuralcomparisons have suggested that the HOM-C andeach of the four Hox clusters share a commonancestral gene complex .',

These studies have been important in supportingassertions that the vertebrate Hox gene are involvedin rostrocaudal patterning . They also imply thatthis family of genes is an ancient and conservedelement of rostrocaudal patterning in a largenumber of metazoan organisms . Indeed the recentreport of a similar complex in Caenorhabditis elegansstrengthens this suggestion . 8 It is also importantto note that each of the four vertebrate Hox clustersare related through duplication and divergence

1 .1

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Figure 2 . Genomic organisation of the mammalian Antp-class Hox genes (based on mouseand human) . The genes are assigned to cognate groups, based on sequence identity, termedparalogues . These groups are numbered in roman numerals . 5' is to the left, 3' to the right .Modified from refs within McGinnis and Krumlauf . 6

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and therefore these clusters contain between themparalogous genes which are highly related .

The products of the Hox genes have been shownto be transcription factors, probably asserting theireffects through transcriptional regulation of down-stream genes- 9 Hox genes also have the potentialfor auto- and cross-regulation, capabilities that couldallow the four Hox clusters to interact and to respondto each other in a regulatory network .

Hox gene expression in the rhombencephalon

respects segment boundaries

The rostral limits of expression of genes at the 3' endof the Hox clusters map to the hindbrain andcoincide with rhombomere boundaries (Figure 3) .Interestingly, paralogous genes of each of the clustersdisplay the same spatial and temporal dynamics ofexpression . 10 Those of the Hox 2.6 family (X) areexpressed up to the r7/6 boundary, the Hox 2. 7family (XI) to r5/4, the Hox 2.8 family (XII) tor3/2 and 2 members of the Hox 2.9 family (XIII)to the r4/3 boundary (Figure 3) . The fact that these

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XlIxnIxI x Figure 3. A schematic represtentation of the expression patterns of the Krox 20 gene and those members of the mammalian Hox gene family that are expressed in the hindbrain. The columns within the hindbrain represent the expression patterns within the neuroepithelium while those to the left relate to the expression of these genes outside the rhombencephalon. The roman numerals at the base refer to the Hex gene paralogous groups (see Figure 3): XIII- Hox 1.6, 2.9, 4.9; XII- Hox 1.11, 2.8; XI- Hox 1.5, 2.7, 4.1; X- Hox 1.4, 2.6, 4.2.

rostral limits do not emerge until after the rhombo- meres become defined morphologically would suggest that these genes are not involved in producing the segmental arrangement. It would be possible, however, that they could confer rostrocaudal identity on the developing rhombomeres, in an analogous fashion to their Drosophila counterparts.

A Hox code

The specification of segmental identity could be achieved through the combination of functionally

active Hox genes, in a ‘Hox code’. ‘t Thus, r3 \vould be defined by the expression of Hox 2. H and Hex I 1 I while r5 would be specified by these together with Hox 2.7, Hox 1.5 and Hox 4.1. Rostrocaudal identity as specified by the Hox genes would probably not relate exclusively to the development of individual aspects of hindbrain structure, rather to the whole phenotype of each segmental level.

Paralogous genes from different clusters could act independently of the others, both spatially and temporally, to increase the complexity of any putative Hox code. It should be noted that they exhibit different levels of expression in different rhombomeres. This is seen, for example, in the Hox 2.7 family, where Hox 2.7 and Hox 1.5 exhibit a high level of expression in r5 with lower expression posteriorly while Hox 4. I displays a complementary pattern. lo The coincident rostra1 expression limits of family members may reflect a degree of redundancy in the system. Alternatively, there may be differences in expression of paralogous genes down the dorso- ventral axis. I2 It is conceivable that Krox 20 could act to upregulate specific Hox genes in rhombomeres 3 and 5 and therefore to produce areas of higher level within their domain of expression.

There are also notable differences between paralogues in the Hox 2.9 family. First, while Hox 2.9 and Hox 1.6 are expressed up to the r4l3 boundary at 8.0 dpc in the mouse, Hox 4.9 is posteriorly restricted.‘O Second, the early expression of both Hox 1.6 and Hox 2.9 in a continuous domain up to the r4l3 boundary is not maintained past 8.5 dpc. Hox 1.6 expression retreats from this rostra1 limit while Hox 2.9 continues to be expressed in r4, at migh higher levels, while being down regulated posterior to the r5/4 boundary. Thus there would appear to be two phases in the expression of Hox 2.9-one early phase which is coincident with that of its paralogue Hox 1.6 and a later phase where it is only expressed within r4. This suggests that Hox 2.9 has two roles in the developing rhombencephalon. lYi

Offset expression of Hox genes in the hindbrain versus the adjacent tissue

Some of the 3’ members of the Hox gene clusters that are expressed in the rhombencephalon are also expressed in similar but offset domains in the adjacent branchial tissues (Figure 3). t4 Hox 2.8 is expressed in the facial-acoustic ganglion and in the second branchial arch mesenchyme, while Hox 2.7 is

Rostrocaudal axis of the hindbrain

expressed in the glossopharyngeal/vagal complexand in the third branchial arch .

These genes are expressed in more rostral domainsin the rhombencephalon compared with the surround-ing tissues, an offset that may result from the lackof neural crest cells from rhombomeres 3 and 5,although this absence has not been shown for themouse embryo . However, Hox 2.6 is also posteriorlyoffset in the surrounding tissues with respect tothe hindbrain yet there would not appear to beany corresponding crest free area . This could beaccounted for if the expression patterns of these geneswere established separately in the migrating neuralcrest and not carried from the neural tube .

While these descriptive studies are consistent withthe Hox genes having a role in rostrocaudalpatterning they do not prove such a role nor do theyreveal any precise function . Recently, however,both embryological and genetic experiments havebegun to address the function of the Hox genes inhindbrain patterning .

Embryological analysis of Hox 2.9 expression

The expected link between Hox 2.9 gene expressionand phenotypic commitment in the segmentingrhombencephalon has recently been demonstratedby embryological analysis . 13 If the rostrocaudalidentity of a region of the hindbrain is dependentupon the expression of Hox genes, their expressionshould be autonomous from the time that develop-ment becomes committed in that region . In thisexperiment the presumptive r4 region of the earlyrhombencephalon (HH st 9-), was transplanted,with or without the surrounding mesenchyme, tothe more rostral position of r2 and after furtherdevelopment was phenotypically assayed by retrogradetracing of efferent nerves and in situ hybridisationwith a Hox 2.9 probe .

The results of this study demonstrated that evenfrom this early time (six somites) both segmentidentity and the later high level of Hox 2.9 expressionare autonomous and independent both of axialposition within the neural epithelium and of anyeffect from the surrounding mesoderm . The up-regulation of Hox 2. 9 suggests that the later highlevel of expression of this gene is specific to r4 andseparate from the early low level phase . Thisinterpretation is backed up by the finding that thereexists a regulatory region that can drive expressionof a lac Z reporter gene specifically in r4 in transgenic

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mice and that this control region is distinct fromthat responsible for the early expression pattern . 13

Hox gene knockouts

Gene targeting by homologous recombination inmouse embryo-derived stem cells has recently beenused to generate mice with disrupted Hox genes,specifically Hox 1 .5 and Hox 1 .6, which both havetheir rostral limits of expression in the developingrhombencephalon (Figure 3) . 15-17 No heterozygousphenotype was observed for either null mutationwhile the homozygotes died at or shortly after birthand displayed defects in the branchial/hindbrainregion .The Hox 1.5 gene disruption was generated by the

insertion of exogenous DNA in the middle of thehomeobox which would have produced a non-functional protein . 15 The Hox 1 .5-/Hox 1.5- animalsdeveloped multiple defects in the thymus, para-thyroid, thyroid and submaxillary tissue and had awide range of throat abnormalities . Other defectivestructures included the heart and arteries as well asthe craniofacial region . The underlying theme ofthese defects is that the affected tissues derivefrom the four branchial arches .

Defects associated with Hox 1.6 ablation werecentred around the level of rhombomeres 4 to 7, therostral domain of normal expression . Lufkin et all 6reported delayed neural tube closure at the level of thehindbrain and absence of certain cranial nerve roots,the VIIth motor nucleus and the VIIIth and thesuperior Xth cranial sensory ganglia, together with theloss of connection between the IXth and Xth nervesand the hindbrain . Other defects reported by theseauthors were malformations of the inner ear andbones of the skull . The Hox 1.6-/Hox 1.6- mutantgenerated by Chisaka et all? displayed similar but alsodifferent defects : both the middle and the external ears(in addition to the inner) were affected, the cranialganglia VII through X were displaced towards thetrigeminal, the otocyst was displaced rostrally, andthe superior olivary complex was absent .

The differences between the two mutant pheno-types could result from the strategies employedto disrupt the gene . Lufkin et al replaced theendogenous 5' end of Hox 1 .6 with exogenous DNAwhile Chisaka et al disrupted the Hox 1.6 gene in thesame way as they disrupted Hox 1 .5, by insertingexogenous DNA in the middle of the homeobox . TheHox 1.6 gene produces two transcripts-one that

3 1 2

could direct the synthesis of a full length protein thatincludes the homeodomain and a second that wouldproduce a truncated protein that shares the sameN-terminus but lacks the homeodomain . The defectgenerated in the Hox 1. 6 gene by Lufkin et al wouldhave abolished both transcripts while that producedby Chisaka et al would only have disrupted thetranscript that contained the homeobox .

While these results are of great importance, thepublished reports are preliminary analyses ofthe mutant phenotypes; more detailed probing of theanatomical defects of these mice will undoubtedlyyield a clearer picture of their disorders .

Gene targeting supports a role for Hoxgenes in rhombencephalic patterning

The defects described in these mutants do certainlyprovide evidence that Hox 1 .5 and Hox 1 .6 play arole in patterning the hindbrain and the surroundingbranchial region . The mutations in each of thesegenes result in distinct and non-overlapping pheno-typic alterations suggesting that each gene isplaying a separate role and that the genes are notinteracting with each other . Each set of defects waslimited to a specific rostrocaudal region, with theHox 1 .6 defects having a more anterior site of originthan those of Hox 1 .5, consistent with their respectiveexpression domains (Figure 3) .

In the light of Noden's3 experiments, it issignificant that the Hox 1 .5 mutant defect wasfocussed on neural crest derivatives or tissues thatrequire neural crest interactions . By contrast, theHox 1.6 mutants exhibited rhombencephalic defectsin addition to those in the periphery . Hox 1 .5 ablationaffected mesenchymal derivatives of neural crestbut not neurogenic derivatives while the Hox 1 .6defects included both non-neurogenic and neurogeniccrest derivatives, with a bias towards neurogenic crestdefects .

Individual neural crest cells are multipotent,capable of producing both neural and mesectodermaldescendants in vitro ; it would therefore seem unlikelythat there exists a pre-specified mesectodermalpopulation of pre-migratory neural crest . 18 It istherefore also unlikely that Hox 1 .5 acts on thepre-migratory neural crest ; rather it would actindependently, after neural crest emigration . Thiswould be consistent with the offset Hox 2 expressionin the branchial tissues with respect to the neuralepithelium .

A . Graham

One peculiarity of the Hox 1 .6 mutants is that theaffected structures never themselves express thisgene . Hox 1 .6 is expressed up to the r4/3 boundarybut it almost immediately recedes from this position(by 8.5 dpc) and is thus not expressed in the facialnerve or the superior olivary complex nor is itexpressed in the neural crest cells of this region . Itwould seem, therefore, that Hox 1 .6 is required veryearly for the correct patterning of the hindbrain andthat its effects are transmitted to the neural crest-asomewhat different situation than that observedfor Hox 1 .5 .The Hox 1 .6 results are also interesting given the

early and late expression patterns of the paralogousHox 2.9 and suggest that the early pattern of Hox 2.9expression is required, as Hox 1 .6, for the basicpattern of r4 while the later expression may relatemore specifically to details of r4 phenotype .

Gene targeting provides support for posterior

prevalence and for a Hox code

It was noted that defects in the Hox 1.5 and Hox 1 .6mutants were not observed in all the sites ofexpression-for example in the lung, stomach, spleenand kidneys for Hox 1 .5 or in the gut for Hox 1 .6 . 19,20This would suggest that there must be someredundancy in the Hox gene complexes . Given thatHox genes have the capabilities for auto- andcross-regulatory activity the removal of one gene mayresult in the abnormal regulation of another gene,possibly that gene's paralogue(s), and therefore inthe rescue of that gene's functions (Figure 2) . Specificdefects were found for each gene, however, at therostral limit of expression . This suggests that eachHox gene only exerts its function within its rostraldomain of expression, a situation that is observedfor the Drosophila abdominal homeotic genes andthat has been termed posterior prevalence . 21 Thisconcept asserts that the posterior genes impose theirdevelopmental programme in a dominant fashion onoverlapping anterior gene expression . Therefore inthe case of rhombomere 4 which expresses Hox 1 .6and Hox 2.9 at their rostral limits and the genes ofthe Hox 2.8 family which extend more anteriorly itis those genes which have the more posteriorexpression pattern, i .e . Hox 1.6 and Hox 2.9 whichimpose their developmental function .

These results also have implications for theexistence of a 'Hox code' . Given that the defectswere only observed at the rostral limit of expression

Rostrocaudal axis of the hindbrain

then the Hox code clearly is not relevant throughoutthe whole domain of gene expression but only at therostral limit .

Morphological and molecular aspects offore- and midbrain development

Segmental organisation has been proposed for thefore- and midbrain regions, based on the appearanceand disappearance of successive bulges in theanterior neural tube and also on the appearance ofacetylcholinesterase in the middle of diencephalicneuromeres . 22 However, the status of segmentationin these regions remains uncertain and must awaitcell marking studies .

A number of genes with interesting expressionpatterns at the rostral end of the neural tubehave been described . These genes also containhomeoboxes and may therefore encode transcriptionfactors . The homeoboxes in these genes are distinctfrom the Antp-like homeobox of the Hox genes .The engrailed homologue En 2 first expressed during

early stages of embryogenesis (HH stlO- chick) inthe rostral rhombencephalon and caudal mes-encephalon, slightly later in the forming tectumand in the adult cerebellum . 23 Interestingly, theEn 2 protein exhibits a graded distribution with highlevels in rl and declining levels towards and throughthe mesencephalon . This gene has been knocked outand again the phenotype did not seem to correlatewith the expression pattern . 24 The only defect foundwas abnormal foliation in the adult cerebellum . Thefact that no more extreme phenoype was producedis thought to be the result of the rescue of En 2function by the closely related En 1 gene .

An antibody has been produced which recognisesthe En 2 protein and this has proven to be veryuseful in embryological studies . Itasaki et a1 23 havegrafted pieces of the rnesencephalic alar plate,which expresses En 2, into the diencephalon . Theresults from this study were somewhat surprising ;the level of expression of En 2 in the graft dependedon the proximity of the graft site to the mesencephalic/diencephalic border, such that the nearer thegraft was to the boundary the lower the levelof expression. This would suggest that there is arepressive influence on En 2 expression aroundthis boundary and it may thereby control the gradedlevel of normal expression .

Spatially-restricted patterns of expression of twogenes related to the Drosophila NK family of

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homeobox genes, TTF 1 (Nkx 2.1) and Nkx 2.2,and one related to the Drosophila D11 gene, Dlxhave recently been reported for the mammalianforebrain . 25 The three genes were found to haveexpression domains that partially overlap and abut .While the TTF 1 and Nkx 2.2 genes were expressedacross all layers of the neuroepithelium it was foundthat Dlx was restricted to more peripheral areas andthat the expression of this gene was excluded fromthe ventricular region . These expression patternswould be consistent with these genes playing a rolein the regionalisation of the forebrain ; however, Dlxis expressed one day later and therefore it could beresponding to effects created by Nkx 2.2 and TTF 1 .

What organises the rostrocaudal pattern?

Specification of the different brain territories probablyoccurs during neural induction from a seriesof processes, well characterised in amphibianembryos, that involve both vertical and horizontalsignals . 26 The first step involves the specification ofanteroposterior identity in the mesoderm duringgastrulation. The ectoderm is then thought to becomeregionalised in response to the signals that areemitted from the underlying mesoderm . A gradedsignal also passes through the plane of the neural platefrom the organiser region, the dorsal blastopore lip . Itwould seem that during these steps the posterior cuesare dominant and therefore that regionalisationproceeds in a posterior to anterior direction .

Expression of the En 2 marker is induced morefrequently by anterior notochord than by posteriornotochord and neither presumptive somitic nor headmesoderm were strong inducers of this marker . 27This suggests that the mesodermal population respons-ible for patterning the rostral neural plate may be theanterior notochord . The situation regarding therhombencephalon would seem to be inconsistent sinceit has been demonstrated that in this area positionalcues are intrinsic to the neuroepithelium . This maypossibly reflect a situation wherein the hindbrain regionis specified as a whole by extrinsic influences but thatit subsequently develops as a separate embryonic fieldand organises itself .

Posterior dominance and posteriorprevalence

The posterior dominance inferred from Hox genestudies has interesting parallels with the posterior

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prevalence that has been deduced from embryo-logical experiments in amphibia . It was observed thatanterior archenteron roof would induce the productionof anterior neural tissue while the posterior roofwould induce posterior neural tissue . 26 Yet whenboth anterior and posterior tissue was used asthe inducer the induced tissue has a posteriorcharacter. This, and a number of other findings,would suggest that posterior is prevalent. Thisis obviously intriguing given the idea of posteriordominance and how it relates to the Hox genes .These two phenomena may be two aspects ofthe same process and may relate to the mannerof establishment of the overlapping Hox geneexpression .

The nature of the rostrocaudal information

A number of factors suggested that the Hox genesrespond to pre-existing cues . First, these genes en-code nuclear proteins which could not responddirectly to the sort of cues which organise thevertebrate embryo since this is achieved in a cellularenvironment . Second, by the time that Hox genesare expressed in the hindbrain some degree ofpatterning must have occurred since there arealready two stripes of Krox 20 expression in r3 and r5 .

Experiments in Xenopus have identified moleculesthat are implicated in rostrocaudal pattern formationsuch as peptide growth factors and retinoids . Thetreatment of explanted animal caps with basicfibroblast growth factor (bFGF) tends to result inthe formation of posterior structures while thetreatment of animal caps with activin results inanterior structures . 26 Treating dissociated animalcap cells with differing concentrations of activin 28has demonstrated that cells can distinguish sharplybetween different concentrations of activin and thatthe activin effect can be stably recorded . High dosesof activin were found to induce dorsal-anterior tissuewhile low doses produced tissue of a ventral-posteriorstate. These are the properties one would expect ofa morphogen; activin could signal rostrocaudalpositional information, probably in concert withother molecules, such as bFGF .

The treatment of embryos with retinoic acid hasquite profound effects on the rostrocaudal patternof the developing CNS . It was reported that retinoicacid treatment resulted in the truncation of embryosat their anterior ends . At high doses the forebrainand midbrain are lost while the treatment of embryos

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with low doses of retinoic acid resulted in a compressionand disruption of anterior hindbrain structures . 29,3 n

A number of authors have suggested that bothgrowth factors and retinoic acid have effects on theexpression of Hox genes . The anteriorly expressedgene XlHbox 1 is selectively activated by activin whilebFGF selectively activates the posteriorly expressedgene XlHbox 6 . 31

The effects of retinoic acid on Hox gene expressionis even more dramatic . It has been demonstrated inmurine EC cells that there is a direct correlationbetween the gene order and the relative doseresponsiveness of each gene to retinoic acid alongthe Hox 2 cluster . 32,33 Thus genes at the 5' end ofthe cluster are less sensitive to retinoic acid than thoseat the 3' end . This correlation between the positionof a Hox gene in the cluster and its responsivenessto retinoic acid has also been observed in vivo bytreating whole Xenopus embryos with retinoic aeid . 33

It should be noted that different results have beenobtained comparing XlHbox 6 with XlHbox 1 andcomparing XlHbox 6 with XlHbox 3 . 31,34 In theseinstances it was found that the more posteriorlyexpressed XlHbox 6 increased upon retinoic acidtreatment while both XlHbox 1 and XlHbox 3 remainedunaffected . Either things are not as simple as theyappear or these results reflect the fact that each ofthese genes is from a separate cluster and theytherefore do not function in a coordinated way ;XlHbox 6 is the equivalent of Hox 2.5, XlHbox 1 theequivalent of Hox 3.3 and XlHbox 3 of Hox 1 .1 . 3 '

Both of these results suggest ways in which theoverlapping Hox gene expression patterns could beorganised . In the first case the growth factors couldsignal rostrocaudal positional information and theHox genes respond to those cues . The differentialresponsiveness of Hox genes to retinoic acid alsosuggests a manner in which the overlapping rostro-caudal domains of these genes could be organised . Yetwhether these systems are actually used, either aloneor in conjunction, still remains to be determined .

Future prospects

Questions regarding Hox genes and how they act areby no means answered, yet the most importantoutstanding problem is the fundamental basis ofrostrocaudal pattern and how this involves the Hoxgenes . Future research will focus on the early periodsof neural patterning both at a morphological andmolecular levels . In terms of morphology it wouldbe particularly interesting to analyse the role that

Rostrocaudal axis of the hindbrain

Hensen's node and the notochord play in patterningthe hindbrain and other regions of the CNS . It will beimportant to assess what we can learn from theexperiments in Xenopus and whether retinoids and/orgrowth factors play a role in organising the highervertebrate CNS . Experiments with growth factors onthe avian epiblast already suggest that they domediate similar steps .

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