A Role for Midline Closure in the Reestablishment of Dorsoventral Pattern Following Dorsal Hindbrain...

DEVELOPMENTAL BIOLOGY 183, 150–165 (1997)ARTICLE NO. DB968460

A Role for Midline Closure in the Reestablishmentof Dorsoventral Pattern FollowingDorsal Hindbrain Ablation

Paul Buxton, Paul Hunt, Patrizia Ferretti, and Peter Thorogood1

Developmental Biology Unit, Institute of Child Health (University College London), 30,Guilford Street, London WC1N 1EH, Britain

The cellular and molecular study of dorsal neural tube ablation reported here demonstrates a critical role for midlineclosure in hindbrain repatterning. This was revealed by detailed analysis of the transcriptional response of two genes, Pax-3 and slug, during repair of the neural tube following ablation. The reexpression of Pax-3 appears to rely on a single surfaceectoderm/neuroepithelial contact, while this is insufficient for reexpression of slug. In fact, slug up-regulation only occurredupon midline closure and, strikingly, corresponded to down-regulation of Pax-3. We examined whether a candidate dorsaliz-ing molecule, Bmp-4, was responsible for this reciprocal regulation of Pax-3 and slug at midline closure. However, Bmp-4 was not reexpressed following ablation, indicating not only that it is not responsible for the observed repatterning butthat it lies in regulatory pathways distinct from Pax-3 and slug. We additionally examined the expression of Pax-6, which,together with assessment of the pattern of cranial ganglia, roof plate morphology, and positioning of branchiomotor exitpoints, demonstrates that neural crest regeneration is accompanied by reestablishment of a normal dorsoventral patternwithin the neural tube. Thus, both local and longer range patterning appears to be restored following ablation, which isreliant dorsally on midline closure of the neural tube. q 1997 Academic Press

INTRODUCTION activity of the surface ectoderm can be substituted by BMP4and BMP7 signaling molecules which are expressed in thesurface ectoderm, or dorsal midline, of the trunk (Liem etThe neural tube of the vertebrate embryo develops intoal., 1995). These results extend the earlier findings of Mourythe central nervous system (CNS) of the adult and, as such,and Jacobson (1989, 1990) in which axolotl neural crest cellsit possesses many neuronal cell types, specifically posi-were formed from juxtaposition of epidermis and neuraltioned within both the dorsoventral and anterior-posteriorplate, crest cells arising from both tissues, and similar con-axes. The initial inductive event that leads to formation ofclusions have been reached in the chick (Selleck and Bron-the neuroepithelial structure occurs early in developmentner-Fraser, 1995). While extensive studies have been per-in response to a combination of vertical (from underlyingformed on the dorsal specification of the spinal cord, culmi-mesoderm) and planar (ectodermal) signals (reviewed bynating in the identification of candidate causal moleculesRuiz i Altaba, 1993; Kelly and Melton, 1995). At the mor-(Liem et al., 1995), relatively little work has been done onphological level, induced epiblast (neuroepithelium) can bethe dorsoventral patterning of the hindbrain (Simon et al.,distinguished from uninduced (surface ectoderm) as neuro-1995; Sechrist et al., 1995). The normal structures of theepithelial cells become organized into a columnar array,hindbrain and trunk display quite different organizationsfollowing regression of Hensen’s node. An additional rolewhich may derive either from the action of different pat-for the surface ectoderm in neural tube patterning is in theterning molecules or from spatio-temporal differences ininduction of several dorsal neural tube markers; slug, Paxthe deployment of identical molecules. That the patterning3, msx-1, dsl-1, wnt-1, and wnt 3a (Dickinson et al., 1995;influences on the hindbrain may differ from those of theLiem et al., 1995). It has also been demonstrated that thistrunk is illustrated by the findings of Graham et al. (1994)using BMP4 protein. The protein elicits apoptosis in rhomb-encephalic crest cells (rhombomeres 3 and 5) rather than1 To whom correspondence and reprint requests should be ad-

dressed. Fax: 0171-831-4366. E-mail: [email protected]. dorsalizing the hindbrain neural tube. While the key mole-

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151Neural Crest Regeneration and DV Patterning

England) to give stage 9 embryos, all staging was carried outcules may differ in the two systems, the surface ectodermaccording to Hamburger and Hamilton (1951). From each egg, 2appears to play the same dorsalizing role (Sechrist et al.,ml of albumin was withdrawn, the egg windowed and a small1995).amount of India Ink (Pelikan Fount, Hanover, Germany), dilutedThe phenomenon of neural crest regeneration can help1:9 with sterile phosphate-buffered saline (PBS), injected underus to understand hindbrain dorsoventral patterning in thatthe embryo to aid visualization. The vitelline membrane was

it demonstrates the formation of a dorsal cell type in the then reflected from the cranial part of the embryo and a bilateralventro-lateral region of the hindbrain as a response to crest dorsal neural tube ablation was carried out using flame-sharp-ablation (Scherson et al., 1993; Hunt et al., 1995). The regen- ened tungsten needles. This involved making transverse inci-erating hindbrain therefore provides a means to study dorso- sions at the anterior end of R1 (rhombomere 1), and at a levelventral specification of this region since surgical ablation adjacent to the posterior aspect of the first somite (R6/7) followed

by longitudinal incisions along the dorsolateral part of the neuralwould be expected to remove the putative dorsal signalingtube and adjacent surface ectoderm in order to remove the dorsalstructure. Reestablishment at a more ventral site in thethird of the neural tube (Fig. 1). Two or three drops of Tyrodesneural tube of a new surface ectoderm/neuroepithelial (SE/solution with antibiotic/antimycotic agents (50 units/ml peni-NE) interface then occurs (Sechrist et al., 1995). The factcillin, 50 mg/ml streptomycin, 250 mg/ml fungizone; GIBCO)that one dorsal cell type (the neural crest) can be regeneratedwere then added to the embryo, the window sealed with Sellotapeleads to the possibility that other dorsal cell types may alsoand reincubated at high humidity and 387C for the desired period.

be respecified by the same events. Analysis of embryos in Viability was assessed by an increase in somite number, closurewhich the dorsal neural tube has been ablated may thus of the neural tube at the hindbrain level, and presence of a beatingreveal the mechanisms underlying dorsoventral patterning heart (when applicable). For barrier insertions, a unilateral R1–in the hindbrain. R6/R7 level ablation was carried out followed by the midline

Recently, Sechrist et al. (1995) have conducted an abla- insertion of a length of aluminium foil (WBS, UK).tion study on rostral hindbrain and midbrain regions of the Embryos were fixed in 4% paraformaldehyde (PFA) overnight

and stored in 100% methanol at 0207C. Expression patterns ofhead which revealed the necessity for an interaction be-stage-matched normal embryos were used for comparison. For eachtween surface ectoderm and neuroepithelium in neuralgroup, as defined by the gene analyzed, developmental stage, andcrest cell regeneration. In order to address whether furtheroperative procedure, between four and eight embryos were as-cell types are respecified by this interaction, and its precisesessed. A total of 120 operated embryos were used in the analysis.nature, we have carried out bilateral dorsal neural tube abla-

Whole mount in situ hybridization. Riboprobes for wholetions at hindbrain levels (Hunt et al., 1995) in conjunctionmount in situ hybridization were prepared with digoxygenin-la-

with gene expression analysis to monitor the molecular/ beled UTP as described in Wilkinson (1992), including treatmentcellular events taking place within the neural tube during with DNase I. The slug probe used was as described by Nieto etregeneration. In order to do so we selected the following as al. (1994). The Pax-3 probe was derived from the partial cDNAmarkers for dorsoventral position within the neural primor- cloned by Goulding et al. (1993) and spanned from the internaldium: Pax-3 and Pax-6, previously used in trunk neural tube EcoRV site to the HindIII site of the 5* distal polylinker. The Pax-

6 probe used was as described by Goulding et al. (1993). The dor-patterning studies (Goulding et al., 1993); dorsalin-1, Bmp4,salin-1 probe was produced by subcloning the cDNA of Basler etBmp7 (TGF-b superfamily; Basler et al., 1993; Houston etal. (1993) to remove the poly(T) stretch at the 3* end (a PstI/KpnIal., 1994; Francis et al., 1994), and slug (a zinc-finger tran-deletion). The Bmp4 probe was synthesized from the cDNA ofscription factor; Nieto et al., 1994). Additionally, the mono-Francis et al. (1994) with T3 polymerase after linearizing with XbaI.clonal antibody 3A10, recognizing a neurofilament-associ-

The whole mount procedure used was essentially as describedated protein (Furley et al., 1990) was used to determine theby Wilkinson (1992), with the following alterations: 60 –657C hy-

effects of dorsal neural tube ablation on both cranial gangliabridization temperature (51 SSC/50% formamide), no RNase-A

pattern and the dorsoventral position of the associated treatment was performed and posthybridization washes were 51branchiomotor exit points. SSC/50% formamide followed by 21 SSC/50% formamide (both at

We provide evidence that the patterning properties of the 607C); 1% sodium dodecyl sulfate was included in both washes tosurface ectoderm alone are insufficient for crest regenera- prevent embryos adhering to the tubes. The alkaline phosphatasetion in vivo and that midline closure is the prerequisite for reaction was stopped by rinsing in PBS containing 1% Tween.

Embryos were embedded in a gelatine:albumin mix (1:2) and 50-the respecification of a normal dorsoventral pattern withinmm sections were cut with a Pelco 101 Vibratome (St. Louis, MO).the hindbrain neural tube following dorsal neural tube abla-Embryos and sections were viewed with Nomarski differential in-tion. Additionally we show that slug and Pax 3 expression,terference contrast optics using Zeiss Axiophot and Olympus BH2while inducible in vitro by a common signal, BMP4/7 (Liemphotomicroscopes. Alternatively, embryos were dehydrated in anet al., 1995), reflect the presence of distinct cell populationsethanol series, wax embedded, microtome sectioned at 20 mm, andin both the hindbrain and trunk, and display opposite re-viewed under bright field conditions. Images were either recorded

sponses to midline closure following ablation. photographically or captured electronically using a KontronProgRes 3012 digital camera, version 2.0 of the associated software,and stored as Adobe Photoshop v3.0 files.MATERIALS AND METHODS

Immunochemistry. The whole mount procedure used was es-sentially as described by Furley et al. (1990), using the 3A10 anti-In ovo microsurgery. Fertilized White Leghorn eggs were in-

cubated at 387C in a forced draft incubator (Curfew Ltd., Essex, body which recognizes a phosphorylated neurofilament-associated

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152 Buxton et al.

FIG. 1. Stage 20 control and 48-hr postablation embryos with neurofilament-associated protein visualized by 3A10 antibody (A, C, wholemounts; B, D, transverse sections/TS). (A) control embryo demonstrating the normal organization of hindbrain ganglia; (B) shows a sectionthrough the IX ganglion region (R6) of this embryo with the exit points of the branchiomotor axons indicated by the arrowheads. (C)Postoperative embryo; note the relatively normal patterning of the ganglia. (D) Section through level of ganglion IX revealing that thebranchiomotor exit points (arrowheads) are, proportionately, in the correct lateral locations following ablation of the dorsal neural tube.Note that the roof plate (asterisk in B and D) forms normally in D. Bars, 200 mm.

protein, and obtained as a supernatant from the Developmental lowed us to assess both the pattern of the cranial ganglia andBiology Hybridoma Bank (St. Louis, MO). The peroxidase reaction the positioning of the branchiomotor exit points followingwas halted via refixation in 4% PFA and clearing in benzyl alcohol/ ablation.benzyl benzoate. Selected embryos were ethanol dehydrated, wax Since the proximal parts of the cranial ganglia are derivedembedded, sectioned at 20 mm, and examined using bright field from neural crest cells (Moody and Heaton, 1983; reviewedoptics. Noden, 1993), the ganglia pattern might be expected to be

aberrant in the absence of crest. However, comparison be-tween control, intact embryos at stage 20, and 48 hr postop-

RESULTS erative embryos revealed that the normal metameric rela-tionship between the rhombomeres and ganglia is main-tained in operated embryos (Figs. 1A and 1C). In additionNeurofilament Immunostaining Reveals Thatto normal registration of ganglia pattern in the anterior–Dorsoventral Polarity Is Reestablishedposterior axis, sectioning of the embryos enabled us to as-

We chose to use the 3A10 antibody since it detects a form sess the dorsoventral position of branchiomotor exit pointsof neurofilament-associated protein expressed by a wide and somatic motor nuclei. Branchiomotor neurones arise inrange of neuronal cell types (Furley et al., 1990). At stage the ventral part of the hindbrain and their axons travel ei-20 (40–43 somites), it recognizes neurones of the cranial ther rostrally or caudally (depending upon the rhombomere)ganglia, branchiomotor neurones, somatic motor nuclei, to a specific exit point from which they (Fig. 1B) extend

to the appropriate cranial ganglion (Lumsden and Keynes,and the dorsal root ganglia (Fig. 1A). This antibody has al-


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of this continuity and the closure of the neural tube (albeitwith a reduced diameter) constitutes repair of the neuraltube. Histological analysis of embryos harvested 0, 4, 8, 24,and 48 hr postoperatively revealed that the tissue move-ments necessary to achieve repair were completed at someaxial levels by 8 hr after the operation and were completeat all levels by 24 hr. Serial vibratome sections at 8-hr timepoint revealed the details of the repair process and enabledus to identify three events (Fig. 2) that comprised the repairof the neural tube: (1) Closure of the remaining NE, withoutmidline apposition of SE; (2) lateral annealing of the NE andSE to form a new interface, while the hindbrain remainsopen; (3) midline apposition of the contralateral edges ofthe SE in addition to neural tube closure (midline closure).

We found that (1) and (2) occurred independently andcould be seen at different axial levels in an individual em-bryo, while (3) was an essential condition for crest cell re-generation. In addition, limited repatterning was found tobe initiated by (2), but not by (1). Of the 14 embryos fixedand sectioned at 8 hr after surgery, only one showed themorphology shown in (1) (Fig. 2), suggesting that this event,while possible, is rare.

Molecular Events of Early Hindbrain Patterning

We examined the expression patterns of genes implicatedin early neural tube patterning and/or crest genesis; of these,slug and Pax-3 proved to be the most informative (see later).In addition, we assessed the expression of dorsalin-1, Bmp4,and Pax-6. Using a whole mount procedure we could detectFIG. 2. Diagram illustrating ablation strategy, as seen in trans-dorsalin-1 mRNA only in the trunk regions of embryos atverse section. (A) intact hindbrain at stage 9. (B) Dorsal third re-both stages 10 and 14 (Figs. 3A and 3B; stage 14, 22 somites,moved. (C) SE/NE annealing prior to neural tube closure (left) ordata not shown), and in a pattern correlating well with pre-closure prior to midline apposition of SE/NE (right). (D) Repaired

neural tube with regenerated crest migrating—occurs by approxi- vious reports (Basler et al., 1993); however, absence of ex-mately 8 hr postablation. (E) Illustrates barrier insertion into the pression in the hindbrain has not previously been noted (Fig.lumen of the neural tube together with unilateral ablation (see 3A). In contrast, we confirm that Pax-6 expression follows atext), this experiment was carried out to assess the necessity for complex pattern in the hindbrain (Li et al., 1994; Simon etmidline closure in hindbrain repatterning. (SE, surface ectoderm, al., 1995). Thus at stage 14, rhombomeres 2, 4, and 6 (anddark grey; NE, neuroepithelium, light grey; notochord, black).

more caudally) show expression in the lateral and ventralneural tube, whereas expression in rhombomeres 3 and 5extends more dorsally (Fig. 3C). This pattern is recapitulated

1989). Despite removal of the dorsal third of the hindbrain in the 24 hr postoperative embryo (Fig. 3D), demonstratingneural tube, somatic motor nuclei (data not shown) and that dorsoventral pattern (i.e., dorsal exclusion from rhom-exit points are still maintained in the correct dorsoventral bomeres 2, 4, and 6) has been reestablished (n Å 8).location (Fig. 1D). It is also evident (Figs. 1B and 1D) that As dorsalin-1 is not expressed in the hindbrain it was not48 hr after the operation a normal roof plate is restored. considered to be an appropriate genetic marker for these

The distribution of the neurones and ganglia revealed the experiments and Pax-6 expression was assessed only at theregeneration of a variety of cell types in their correct posi- 24 hr postoperative time point (see above). The expressiontions and led us to conclude that a proportionately normal patterns of Pax-3 and slug were examined at 0, 8, and 24 hrdorsoventral pattern is reestablished within the regulated time points, which were informative with regard to crestneural tube. regeneration and dorsal neural tube patterning. Bmp4 ex-

pression was analyzed at the 8 hr postoperative time only.Hindbrain Patterning Relies on Specific Tissue Pax-3 and slug expression in the embryonic hindbrain.Interactions Expression of the Pax-3 gene in the hindbrain of the chick

embryo is here described for the first time; slug expressionRemoval of the dorsal third of the hindbrain neural tuberesults in discontinuity between the surface ectoderm, SE, data are included for comparative purposes since it is also

documented elsewhere (Nieto et al., 1994; Liem et al.,and the neuroepithelium, NE (Fig. 2). The reestablishment


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FIG. 3. Dorsalin-1 and Pax-6 expression; rostral is to the left in all whole mounts (A and B, -dorsalin-1; C and D, -Pax-6). (A) Transversesection (TS) at hindbrain level to show lack of the dorsalin-1 domain of 10 somite stage normal embryo (staining on luminal surface isartefactual trapping). (B) TS at trunk level showing characteristic dorsalin-1 domain in roof of trunk level neural tube from the sameembryo. (C) and (D), respectively, show Pax-6 expression in control (stage 14) and postoperative embryo—24 hr after bilateral dorsal neuraltube ablation of rhombomeres 1–7; reexpression within the ablated hindbrain includes the more dorsal expression seen in R3 and R5 ofthe normal (arrows in C, and arrowheads in D, respectively). Bars, 100 mm.

1995). There is significant expression of Pax-3 in the open 4G). Not all the presumptive hindbrain neural tube is posi-tive and there is notably reduced expression in the myelen-neural tube of the trunk at early stage 8 (3-somites), but no

expression in the cranial neural folds (Fig. 4A). This pattern cephalon, adjacent to the presumptive otocyst. By stage 9,dorsal midline expression of slug is intense (Fig. 4H), andpersists through stage 8 (4-somite stage) (Figs. 4B and 4C),

in marked contrast to the expression of slug at this stage at midbrain levels slug-positive crest cells which have leftthe neural tube are visible (Fig. 4I). At the 10 somite stage(Figs. 4D and 4E), which is expressed in the head and not

the trunk neural tube. expression of Pax-3 has extended ventrally in the wall ofthe hindbrain (Fig. 4J) and the absence of midline expressionThe earliest detectable expression of Pax-3 in the head

occurs in the lateral wall of the hindbrain and surface ecto- is still obvious (Fig. 4K). There is some evidence of faintPax-3 expression adjacent to the neural tube (data notderm of the midbrain at stage 9 (6 somites) (Figs. 4F and

FIG. 4. Comparative expression of slug and Pax-3 in the hindbrain. (A, B, C, F, G, J, K, Pax-3; D, E, H, I, L, M, slug). (A) Dorsal view of2 somite embryo showing Pax-3 expression in the somites and open neural plate, but absent from the hindbrain. (B) Dorsal view of 4somite embryo, again showing Pax-3 expression in the trunk but not the hindbrain. (C) Section through the rostral hindbrain —no Pax-3 expression. (D) Section through rostral hindbrain of slug embryo shown in E; (E) slug expression in a 4 somite embryo —confined to thehindbrain and more rostral regions (dorsal view). (F) Dorsal view of Pax-3 expression in the hindbrain of a 7 somite embryo. (G) Transversesection through hindbrain of a similar embryo showing lateral wall expression but no dorsal midline expression. (H) Slug expression at a


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similar level to (G) of the embryo shown in I. (I) Dorsal view of 7 somite embryo showing exclusively dorsal midline expression of slug.(J) Pax-3 expression in the hindbrain of a 10 somite stage normal embryo showing diffuse surface ectoderm expression adjacent torhombomeres 1 and 2, but no obvious migratory crest cell expression. (K) Section through hindbrain level of a similar 10 somite embryodemonstrating ventral shift in the Pax-3 expression domain toward the notochord, but note that there is no dorsal shift in expressiontoward the midline, which remains Pax-3 negative. (L) A section through a 10 somite embryo showing slug expression in the hindbrainat the same level as K. (M) Dorsal view of hindbrain showing slug expression at the 10 somite stage and evidence of migrated crest cellsin the rostral hindbrain and midbrain. Bars, 100 mm.


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156 Buxton et al.

shown). The pattern of slug expression at stage 10 (10 so- ham et al., 1994). At stage 9 (seven somites) it shows someoverlap with slug expression in the hindbrain (Fig. 8A).mites) is similar to that at stage 9, except that the wave of

crest emigration is now more caudal and the domain at However, it will be noted from sections through the sameembryo (Figs. 8B and 8C) that Bmp4 expression is extin-the midline appears to be broader (Fig. 4L), particularly at

rhombomeres 2 and 4 (Fig. 4M). This analysis reveals a mu- guished by the crest cells at the point of emigration fromthe neural tube. In more caudal regions (Fig. 8D) transcriptstual exclusivity of Pax-3 and slug expression domains at all

stages studied in intact, control embryos. are present in the dorsal midline, becoming most abundantin R3 and R5 (see also Graham et al., 1994). This pattern isAssessment of the extent of ablation. Zero hour proce-

dural control embryos (unilateral ablation) demonstrate in no way recapitulated in the eight hours postoperativeembryo (Fig. 8E), either prior to (Fig. 8F), or following mid-that both slug and Pax-3-positive domains of the neural

tube, the neighboring surface ectoderm, and immediately line closure (Figs. 8F and 8G). Therefore, Bmp4 is not in-volved in the early stages of repatterning that occur afteradjacent mesenchyme were all removed by the bilateral ab-

lation (Figs. 5A–5C). ablation (n Å 4) and is not responsive to the same signalsas Pax-3 and slug during regeneration.Initial regeneration is apparent after 8 hr. The 8 hr

postoperative data revealed that Pax-3 is reexpressed in the In the twenty four hours post-operative embryo normalpatterns of gene expression are evident. In control em-NE before closure of the operated neural tube (Figs. 6A and

6D) and that SE/NE interfaces are evident at all sites of Pax- bryos at stage 14 (22 somites), all Pax-3 expression in thehindbrain is confined to the NE, with the exception of a3 reexpression (nÅ 8). This results in the expression of Pax-

3 at more ventral regions of the neural tube that had not patch of superficially located positive cells at the site of thepresumptive rostral lobe of the trigeminal ganglion (Fig. 9A).been expressing Pax-3 at the time of the ablation (compare

Fig. 5C with Figs. 6D–6F). However, as unilateral SE/NE We confirmed that these Pax-3-positive cells were part ofthe opthalmic lobe of the trigeminal ganglion by carryinginterfaces were typically observed, Pax3 expression could

not be unambiguously correlated with SE/NE apposition. out 3A10 immunostaining on embryos which had alreadybeen processed for Pax-3 expression by in situ hybridizationIn contrast, the role of midline closure in the down-regula-

tion of Pax-3 expression was not in doubt, since it only (data not shown). Their position is consistent with the ear-lier expression of Pax-3 adjacent to R1 and R2 (Fig. 6E),occurred when midline closure took place. This finding was

true irrespective of the axial level in the hindbrain; compare which demonstrates that Pax-3 is marking a neurogeniclineage outside of the CNS. At the same stage, slug expres-Fig. 6B, -rostral, and 6C, -caudal. In some embryos, the neu-

ral tube had not fully repaired (Figs. 6D–6F) and this allowed sion is widespread within mesenchymal tissues of the head,including the branchial arches, but is almost entirely absentthe duration of SE/NE interfaces to be excluded as a possible

factor in the down-regulation of Pax-3. Transverse sections from the NE (and other epithelia) at this level (Fig. 9B).Mesenchymal expression is not homogeneous and appearsthrough the open neural tube (Fig. 6F) demonstrate not only

the ventral expansion of Pax-3 expression but also that, in to be stronger in core cells of the branchial arches. In con-trast, the roof of the neural tube in the hindbrain remainsthe absence of midline closure, it extends to the dorsal tips

of the neural tube. Pax-3-negative and there is no evidence of migrating, orpostmigratory, neural crest cells expressing Pax-3 in theIn contrast, slug reexpression follows a different course,

with reexpression of slug occurring along only some of the branchial arches at stage 14.Twenty-four hours after the operation Pax-3 expressionlength of the deletion (Fig. 6G). Transverse sections revealed

that when SE/NE junctions form, without closure of the in the operated embryo is virtually indistinguishable fromthat in the normal counterpart at stage 14 (compare Figs.neural tube, then slug is not reexpressed (Fig. 6H). Thus,

the same critical event that leads to Pax-3 down-regulation, 9A and 9C). In ablated embryos (n Å 4), the relative ventralextent of positive cells in the hindbrain neural tube is com--midline closure, also leads to slug up-regulation (n Å 8). It

is only at regions which have undergone midline closure parable to that in the normal, although the absolute diame-ter of the tube has been reduced. The trigeminal patch ofthat neural crest cell regeneration is evident, as determined

by slug expression. These results were consistent among all Pax-3 expression is still present although displaced slightlyand again was found to be positive for 3A10 anti-neurofil-12 embryos assessed for slug expression at this stage (6 were

serially sectioned). It will be noted that a low level of expres- ament associated antibody (data not shown). Most strik-ingly there is no evidence of Pax-3 expression in thesion was also evident in the head mesoderm adjacent to the

ventral part of the hindbrain; this appears to be independent branchial arches, despite the fact that the most dorsal aspectof the open hindbrain, the source of the regenerated neuralof slug expression derived from crest cells. Significantly,

circularization of the neuroepithelium in the absence of crest cells (Fig. 6D), is expressing Pax-3 8 hr after ablationand before midline closure (Fig. 6E).surface ectoderm fusion at the midline does not lead to slug

reexpression (Figs. 7A–7D). Slug expression in operated embryos is distinguishablefrom the normal (Fig. 9B) only by a slight diminution of theAbsence of Bmp4 expression during hindbrain regenera-

tion. The normal expression of Bmp4 has been previously signal in the branchial arches (Fig. 9D). Areas of mesen-chyme adjacent to the ablated region are positive for slugdescribed and is restricted to the dorsal midline of the neural

tube in both trunk (Liem et al., 1995) and hindbrain (Gra- and it is only at the most distal regions of the branchial


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of this expression may also derive from the head mesodermthat weakly expresses slug.

Barrier Insertion Prevents Crest Regeneration

To investigate the requirement for midline apposition ofSE/NE in the induction of slug reexpression, we adoptedanother experimental strategy (Fig. 2E). Instead of a bilateralablation, a unilateral ablation was carried out, followed byinsertion into the midline of an inert barrier (aluminiumfoil). This prevented closure of the neural tube but did notprevent SE/NE annealing on the ablated side, allowing usto assess the inductive influence of each interaction inde-pendently.

Embryos were collected at 8 and 11 hr postoperative timepoints. The later time point was included to ensure that itwas not merely the amount of time that a single SE/NEjunction had been present which resulted in slug reexpres-sion in regions that had circularized (previous bilateral abla-tions; Fig. 6I). Results were consistent with those of 8 hrbilateral ablations (nÅ 4). Reexpression of slug did not occurin the absence of dorsal midline apposition of SE/NE inter-faces at either time interval (Fig. 7E). The contralateral un-ablated neural fold of the embryo demonstrated that mid-line apposition of SE/NE was not required for maintenanceof slug expression once already established. This also dem-onstrates that the foil did not affect slug expression nonspe-cifically. Pax-3 expression was not assessed in this waysince midline apposition was known not to be a prerequisitefor Pax-3 reexpression (see Fig. 6F), indeed it causes down-regulation.

Is a Patterning Role for Midline Closure a Featureof Normal Development?

A role for midline closure is also supported by the normalpattern of expression of Pax-3 and slug in the trunk neuraltube, where slug is expressed in the midline at the time ofcrest specification and Pax-3 is expressed more laterally(Figs. 10A and 10B). However, prior to this timepoint Pax-3 is expressed in the dorsal tips of the trunk neural plate

FIG. 5. Extent of ablation as assessed by slug and Pax-3 expression (Goulding et al., 1993; Liem et al., 1995; data not shown).at zero hours. (A) and (B) are whole mount in situs of unilateral Pax-3 and slug transcripts appear to label discrete cell popu-procedural controls demonstrating that ablation entirely removes

lations in both cephalic and trunk regions of the embryo.the slug (A) and Pax-3 (B and C) domains, respectively. AblatedHowever, far from having independent domains of expres-level covers R1-R6/R7 on left side of neural primordium, indicatedsion there is a marked complementarity. In the hindbrainby arrows (A) and arrowheads (B). (C) Vibratome TS of the embryothis is manifest in the lateral expression of Pax-3 at stagein B, showing complete unilateral removal of the Pax-3 domain by

ablation. Bars, 100 mm. 9, compared to dorsal midline expression for slug (compareFigs. 4G and 4H with 3K and 3L). The different nature ofthe SE/NE interactions necessary for the reexpression ofeach gene confirm this theme (Figs. 6F, 6H, and 6I). Thefact that midline closure causes up-regulation of slug andarches that slug expressing cells are absent (n Å 4). This is

consistent with a delay in crest migration due to the abla- down-regulation of Pax-3 is also indicative of the reciprocalway these two genes are regulated.tion, predicted in our earlier work (Hunt et al., 1995). Some


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A Unique Patterning Role for Midline ClosureDISCUSSIONIn the case of slug, a single SE/NE interface was notDorsoventral Organization within the

sufficient for reexpression; it had to be accompanied byRegenerating Hindbrainapposition of the SE at the dorsal midline (i.e., midlineOur combined analysis of slug and Pax-3 expression re-closure). The role of midline closure in dorsal neural tubeveals the existence of distinct cell populations in the devel-patterning has not been demonstrated previously al-oping hindbrain by stage 9. Removal of these cell popula-though critical developmental events have been ascribedtions by surgical ablation results in a neuroepitheliumto it, e.g., crest migration (Le Douarin, 1982). However,containing only ventral and lateral cell types. Our investiga-due to the physical fragility of the initial midline apposi-tions have centered upon the expression of these and other,tion, the exact state of closure in embryos which havemarkers following ablation and how it arises. We found thatbeen subjected to either embedding or processing forslug, Pax-3, and Pax-6 were all expressed, and the cranialwhole mount in situ hybridization is always in doubtganglia (as assessed by 3A10 antibody) present, in patternsand cannot be accurately correlated with gene expres-normal and consistent with the developmental stage of thesion. This is not the case within the confines of the hind-operated embryos; Bmp4 was found not to be reexpressedbrain DNT ablation as the tube is sealed both rostrallyafter ablation. Twenty-four hours after ablation of the dorsaland caudally and hence is much less likely to unzip. Asthird of the hindbrain neural tube, regulation had occurredwe are confident of the exact state of closure, we cansuch that a normal dorsoventral polarity and pattern wasalso be confident of causal changes in gene regulationreestablished, albeit within a neural tube of reduced diame-coincident with its presence, e.g., up-regulation of slugter. We have demonstrated that repatterning was not solelyand down-regulation of Pax-3. In addition to providingconfined to neural crest cells (Scherson et al., 1993; Huntthe evidence of an unforeseen patterning role for midlineet al., 1995; Sechrist et al., 1995) as additionally Pax-3 andclosure, these data are, as far as we know, the first dem-Pax-6 expression domains, roof plate morphology and theonstration of a reciprocal activation/repression responseposition of the branchiomotor exit points were all indistin-within the chick hindbrain. This obligate complemen-guishable from normal. It may be significant that this oc-tarity between Pax-3 and slug was later manifest at thecurred in the absence of Bmp4 reexpression (see below).24-hr postoperative timepoint when Pax-3 was only ex-The normal relative position of the exit points is strikingpressed in the neural tube and trigeminal placode, andas it shows that their dorsoventral location is not deter-not in the mesenchyme of the arches, which was slug-mined by a single signal but most likely by coordinationexpressing.between dorsal and ventrally located signals (see Sechrist

That slug reexpression, and Pax-3 down-regulation, re-et al., 1995). It has been shown that the effect of dorsalizingquires midline closure reveals a qualitative difference be-influences on neuroepithelial cells can be modulated bytween cells at the dorsal midline and those which areventral signals either by using tissue recombination in vitromerely dorsal. This could have occured either by a greateror by ectopic grafting in vivo (Dickinson et al., 1995; Liemlevel of signal being present at the apposition of two SE/et al., 1995). However, this previous work has been entirelyNE interfaces (the dorsal midline), or as the result of aconcerned with trunk neural plate, whereas here we focusnovel signal being produced on midline closure. This re-on the hindbrain using the regenerating neural tube as asult apparently contrasts with the data of Dickinson etmodel.al. (1995), Liem et al. (1995), and Sechrist et al. (1995) inOur detailed comparative analysis of the normal expres-which a single SE/NE interface suffices for slug expres-sion domains of slug and Pax-3 in the hindbrain (and em-sion. However, whereas these investigations have utilizedbryo-wide) has revealed that they mark quite distinct celleither trunk neural plate (Liem et al., 1995) or midbrainpopulations, a fact not recognized in previous studiesand rostral hindbrain (Sechrist et al., 1995) as their model(Goulding et al., 1993; Liem et al., 1995; Sechrist et al.,system, our study was carried out in the caudal hindbrain1995). In summary, slug expression characterizes those cellsand at a later developmental stage. Also it should be notedwhich will migrate from the dorsal neural tube as head crestfrom the normal expression of Pax-3 reported here that(Nieto et al., 1994) and Pax-3 marks a more ventrolateralin the area (and stage) ablated by Sechrist et al., 1995,population that remains in the neural tube. This differencethere is no Pax-3 expression; hence, in the light of ouris manifest in the different criteria that must be met forresults, there would be no need for midline closure fortheir reexpression following ablation. Pax-3 expression ap-slug reexpression.pears to be up-regulated in ventrolateral neuroepithelial

cells by a unilateral SE/NE interface, not requiring neuraltube closure. This interpretation concurs with previous Cellular Response of the Neuroepithelium tostudies on the induction of other dorsal markers (Dickinson Dorsalizing Signalset al., 1995; Liem et al., 1995; Sechrist et al., 1995), but thefact that Pax-3 expression shifts ventrally over the same 8- The absolute size of the ablated neural tube remains

smaller than that of normal control embryos (at least untilhr time course in the normal hindbrain indicates it may beindependent of the surface ectoderm. the 48 hr postoperative time point, Fig. 1), indicating that


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FIG. 6. The effect of midline closure on the reexpression of Pax-3 and slug; all embryos are 8 hr postbilateral ablation (A–F, Pax-3; G–I, slug). Level of section through the whole mounts is indicated by the symbols. (A) Pax-3 is reexpressed throughout the ablated region,but is down-regulated at the dorsal midline, see sections from the same embryo. (B) Rostral and (C) caudal hindbrain. (D) Another embryoshowing alternative morphology; here the hindbrain is open for the majority of the ablated region—Pax-3 expression is evident in thelateral walls. Sections of the embryo reveal (E) that at the R1/2 level down-regulation has occurred (note also normal ectoderm expression),and, in contrast, expression extends to the dorsal tips of the open neural tube in more caudal regions (F). (G) Slug expression shows theopposite pattern, with reexpression is only evident at points of midline closure. (H) Section through the rostral part of the ablation showingno neuroepithelial expression of slug, although the slightly oblique section shows some crest-derived expression from a more rostral,intact, region. (I) More caudal levels where midline closure has occurred slug expression is intense in the neuroepithelium, but there isweaker expression in the ventral head mesoderm. SE, surface ectoderm. Bars, 100 mm.


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lateral neural tube cells are changing fate rather than extra case that the underlying mechanism is also a recapitulationof normal. Indeed, it may not occur by the same signalingcells being produced in significant numbers from existing

tissue (see below). As patterning in the operated embryos is mechanism as in the normal hindbrain, but may insteadresemble that of the trunk. It does not, however, confineessentially normal, dorsal cell phenotype must therefore be

induced in cell populations previously destined to be of a regenerated head crest to trunk fates (Hunt et al., 1995) oraffect the characteristic hindbrain pattern of ganglia.lateral phenotype, confirming earlier claims of plasticity of

dorsoventral patterning and cell fate specification (Hunt etal., 1995; Simon et al., 1995). Given the timetable of reap-pearance of dorsally located expression domains for slug and Potential Signals in Hindbrain RegenerationPax-3, only changes in cell fate could accomodate the speed

The flexibility of the hindbrain in terms of order of dorsalwith which these cells are respecified. Indeed, the time inter-gene expression, and its ability to follow a ‘‘trunk’’ se-val, a maximum of 8 hr, is sufficient only for a single neuro-quence, led us to explore the action of proposed signalingepithelial cell division (Guthrie et al., 1991). The reason cellcandidates in regeneration. Liem et al. (1995) have demon-fate can be considered, rather than simply gene expression,strated, in vitro, the ability of BMPs 4 and 7 to substitutestems from the fact that slug and Pax-3 mark different cellfor surface ectoderm in the dorsalization of trunk neuralpopulations in the hindbrain (and other parts of the embryo;plate, including the induction of slug and Pax-3 expression.P.B., personal observation). This supports and extends theOur discovery that Bmp4 was not reexpressed followingdata of Sechrist et al. (1995) which reports similarly rapidablation of the dorsal neural tube demonstrates that its rolechanges in dorsal cell fate following ablation.in dorsalizing the neuroepithelium (Liem et al., 1995) iswholly replaceable and indicates that the control of BMP4expression is not in the same signaling pathways as slug,Repatterning Does Not Iterate NormalPax3, and Pax-6. We also examined Bmp7 expression butDevelopmentsince its predominant hindbrain expression was in the oticplacodes (data not shown) we did not examine further itsIt follows from our study of normal slug and Pax-3 expres-

sion that the sequence of gene expression is opposite in the role in hindbrain regeneration. Roles have been proposedfor BMP4 in diverse embryonic patterning events, includinghead versus the trunk in the early embryo (3–6 somites).

Slug was expressed in the head folds and absent from the apoptosis in the chick hindbrain (Graham et al., 1994).Whatever the role of BMP4 in the hindbrain, the fact thattrunk and Pax-3 expression showed the converse (i.e., ex-

pression in trunk not head). This reveals an influence of it is not reexpressed, when Pax-3 and slug are, demonstratesits transcriptional regulation is clearly different from theseaxial level on DV patterning and also provides a paradigm

for the phenomenon of regeneration that occurs after abla- genes and is unaffected by the cues which lead to theirreexpression. However, the functional promiscuity of fac-tion of the dorsal neural tube in the hindbrain. That is, in

the trunk Pax-3 is expressed first (chronologically) in the tors belonging to the TGF-b superfamily (Basler et al., 1993;Liem et al., 1995) suggests that another member may welldorsal tips of the neural tube (Goulding et al., 1993; Liem

et al., 1995) and is subsequently supplanted by slug expres- be responsible for the dorsalizing action of the surface ecto-derm in the hindbrain.sion at midline closure (approximately, see caveat above).

This is the same succession forced upon the neural tube via Here we have shown that Dorsalin-1, previously consid-ered a likely signal for trunk crest induction, is excludedthe ablation of the dorsal third of the hindbrain (the early

slug expression domain) and its replacement by a Pax-3 from a role in cranial crest development since it is not ex-pressed in the hindbrain or overlying ectoderm. Bmp4 isdomain; this is in turn overridden by slug gene expression

at midline closure (see Figure 11). The pattern that thus likewise an unlikely candidate as it is not reexpressed fol-lowing ablation. Additionally the complementary expres-emerges appears to be normal, but it is not necessarily the

FIG. 7. Relationship between slug expression and stage of repair following ablation; Transverse sections of 8-hr postoperative embryosthroughout (A, B, C, and D, slug). (A) No slug expression is seen prior to midline closure, caudal hindbrain level. (B, C, and D) Vibratome serialsections taken through a whole mount embryo similar to that shown in Fig. 6G. (B) Rostral hindbrain level showing slug reexpression wheremidline closure has taken place (arrowhead). (C) 100 mm more caudally, where only closure of neural tube has occurred and surface ectodermedges remain unapposed (arrows) and, on one side, unannealed with the NE, no slug expression is detectable. (D) 100 mm further caudally, ata point where there is full midline closure (including the surface ectoderm), dorsal midline slug expression is up-regulated. Bars, 100 mm.FIG. 8. Absence of Bmp-4 expression in regeneration. (A) Dorsal view of stage 9 normal embryo showing Bmp-4 expression in the dorsalmidline of the hindbrain. (B, C) Section through rostral hindbrain of the same embryo showing down-regulation of Bmp-4 expression ascrest cells emigrate; (C) close up of (B). (D) More caudal level showing expression in the midline neuroepithelium prior to crest emigration.(E) Dorsal view of bilaterally ablated embryo assessed for Bmp-4 expression 8 hr after operation—there is no reexpression. (F) Sectionthrough open part of the hindbrain confirming no expression. (G) More caudal section showing no neuroepithelial expression despiteclosure; some expression is evident in the surface ectoderm of the otocyst. Bars, 100 mm, except in (C) where the bar is 25 mm.


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162 Buxton et al.

FIG. 9. Stage 14 control and 24 hr postoperative embryos (A and C, Pax-3; B, D, and E; slug). (A) and (B) illustrate expression of Pax-3and slug, respectively, in control embryos, with Pax-3 positive cells contributing to the trigeminal ganglion indicated by an arrow in A(1, 2, and 3, first, second, and third branchial arches; ot, otocyst). (C) and (D) 24-hr postoperative counterparts, displaying reexpression ofPax-3 and slug, respectively, in normal patterns. (E) The result of barrier insertion on slug expression in an 8-hr postoperative embryoafter a unilateral left side ablation; the slug-negative area, indicated by arrow, correlated with the location of the barrier. Before fixation,this was present immediately medial within the neural tube lumen preventing midline closure (see Fig. 2E for schematic of procedure).Bars, 100 mm.

sion pattern of slug and Pax-3 in the hindbrain argues reformed following ablation due to midline closure and, asboth genes encode transcription factors, it is possible thatagainst a single signal being responsible in this region, un-

less differing threshold concentrations can produce the dis- they interact directly. If so slug would be the predominantpartner since its expression at the midline coincides withcrete domains. This complementary expression pattern is


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FIG. 10. Complementary expression of Pax-3 and slug at the trunk level; Pax-3 (A) and slug (B), in transverse section. Note the absenceof Pax-3 expression at the dorsal midline (A), contrasting with midline expression of slug (B). Bar Å 100 mm.

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FIG. 11. Schematic diagram demonstrating the sequence of slug and Pax-3 expression in the cranial, regenerating, and trunk neuraltubes. While initially the order of gene expression is reversed in the head compared with the trunk the final pattern is similar. Note thatin hindbrain regeneration, the sequence follows that of the trunk but still results in normal hindbrain repatterning and derivatives. Slugis in black, Pax-3 is in gray.

kindly provided by the following people: A. Graham and A. Lums-down-regulation of Pax-3 in the same cells. In any event,den, Pax-3 and Pax-6; M. Sargent, slug; B. Houston, bmp-7; P. Brick-it is likely that preexisting A-P specification, such as thatell, bmp-4, dorsalin-1 with permission of T. Jessell.seen in the branchial Hox genes (Hunt et al., 1991), modu-

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A Role for Midline Closure in the Reestablishment of Dorsoventral Pattern Following Dorsal Hindbrain...

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