Mechanical roles of apical constriction,cell elongation,and cell migration during neural tube formation in Xenopus

Neural tube closure is an important and necessary process during the development of the central nervous system. The formation of the neural tube structure from a flat sheet of neural epithelium requires several cell morphogenetic events and tissue dynamics to account for the mechanics of tissue deformation. Cell elongation changes cuboidal cells into columnar cells, and apical constriction then causes them to adopt apically narrow, wedge-like shapes. In addition, the neural plate in Xenopus is stratified, and the non-neural cells in the deep layer (deep cells) pull the overlying superficial cells, eventually bringing the two layers of cells to the midline. Thus, neural tube closure appears to be a complex event in which these three physical events are considered to play key mechanical roles. To test whether these three physical events are mechanically sufficient to drive neural tube formation, we employed a three-dimensional vertex model and used it to simulate the process of neural tube closure. The results suggest that apical constriction cued the bending of the neural plate by pursing the circumference of the apical surface of the neural cells. Neural cell elongation in concert with apical constriction further narrowed the apical surface of the cells and drove the rapid folding of the neural plate, but was insufficient for complete neural tube closure. Migration of the deep cells provided the additional tissue deformation necessary for closure. To validate the model, apical constriction and cell elongation were inhibited in Xenopus laevis embryos. The resulting cell and tissue shapes resembled the corresponding simulation results.

     

Distinct modes of floor plate induction in the chick embryo

To begin to reconcile models of floor plate formation in the vertebrate neural tube, we have performed experiments aimed at understanding the development of the early floor plate in the chick embryo. Using real-time analyses of cell behaviour, we provide evidence that the principal contributor to the early neural midline, the future anterior floor plate, exists as a separate population of floor plate precursor cells in the epiblast of the gastrula stage embryo, and does not share a lineage with axial mesoderm. Analysis of the tissue interactions associated with differentiation of these cells to a floor plate fate reveals a role for the nascent prechordal mesoderm, indicating that more than one inductive event is associated with floor plate formation along the length of the neuraxis. We show that Nr1, a chick nodal homologue, is expressed in the nascent prechordal mesoderm and we provide evidence that Nodal signalling can cooperate with Shh to induce the epiblast precursors to a floor-plate fate. These results indicate that a shared lineage with axial mesoderm cells is not a pre-requisite for floor plate differentiation and suggest parallels between the development of the floor plate in amniote and anamniote embryos.     

Early neurogenesis in Amniote vertebrates

Labelling of Hensen's node in a 6-somite stage chick embryo by the quail/chick chimera method has revealed that, while moving caudalwards as the embryo elongates, the node leaves in its wake not only the notochord but also the floor plate and a longitudinal strand of dorsal endoderm. The node itself contains cells endowed with the capacity to yield midline cells (i.e. notochord and floor plate) along the whole length of the neural axis. Caudal node cells function as stem cells. They are responsible for the apical growth of the cord of cells that are at the origin of the midline structures since, if removed, neither the notochord nor the floor plate, are formed caudally to the ablation. The embryo extends however in the absence of midline cells and a neural tube develops posterior to the excision. Only dorsal molecular markers are detectable on this neural tube (e.g. Pax3 and Slug). The posterior region of the embryo in which the structures secreting Shh are missing undergo cell death within the 24 to 48 hours following its formation. Unpublished results indicate that rescue of the posterior region of the embryo can be obtained by implantation of Shh secreting cells. One of the critical roles of floor plate and notochord is therefore to inhibit the cell death programme in the axial and paraxial structures of the embryo at gastrulation and neurulation stages.     

Studies on the mechanisms of neurulation in the chick: morphometric analysis of force distribution within the neuroepithelium during neural tube formation

Changes in the shape of neuroepithelial cells, particularly apical constriction, are generally thought to play a major role in generating the driving forces for neural tube formation. Our previous study [Nagele and Lee (1987) J. Exp. Zool., 241:197-205] has shown that, in the developing midbrain region of stage 8+ chick embryos, neuroepithelial cells showing the greatest degree of apical constriction are concentrated at sites of enhanced bending of the neuroepithelium (i.e., the floor and midlateral walls of neural tube), suggesting that driving forces resulting from apical constriction are concentrated at these sites during closure of the neural tube. In the present study, we have used morphometric methods to 1) measure regional variations in the degree of apical constriction and apical surface folding at selected regions along the anteroposterior axis of stage 8+ chick embryos, which closely resemble the various ontogenetic phases of neural tube formation, and 2) investigate how forces resulting from apical constriction are distributed within the neuroepithelium during transformation of the neural plate into a neural tube. Results show that, during neural tube formation, driving forces resulting from apical constriction are not distributed uniformly throughout the neuroepithelium but rather are concentrated sequentially at three distinct locations: 1) the floor (during transformation of the neural plate to a V-shaped neuroepithelium), 2) the midlateral walls (during transformation of the V-shaped neuroepithelium into a C-shaped neuroepithelium), and 3) the upper walls (during the transformation of the C-shaped neuroepithelium into a closed neural tube).     

Cell surface beta 1,4-galactosyltransferase functions during neural crest cell migration and neurulation in vivo

Mesenchymal cell migration and neurite outgrowth are mediated in part by binding of cell surface beta 1,4-galactosyltransferase (GalTase) to N-linked oligosaccharides within the E8 domain of laminin. In this study, we determined whether cell surface GalTase functions during neural crest cell migration and neural development in vivo using antibodies raised against affinity-purified chicken serum GalTase. The antibodies specifically recognized two embryonic proteins of 77 and 67 kD, both of which express GalTase activity. The antibodies also immunoprecipitated and inhibited chick embryo GalTase activity, and inhibited neural crest cell migration on laminin matrices in vitro. Anti-GalTase antibodies were microinjected into the head mesenchyme of stage 7-9 chick embryos or cranial to Henson's node of stage 6 embryos. Anti-avian GalTase IgG decreased cranial neural crest cell migration on the injected side but did not cross the embryonic midline and did not affect neural crest cell migration on the uninjected side. Anti-avian GalTase Fab crossed the embryonic midline and perturbed cranial neural crest cell migration throughout the head. Neural fold elevation and neural tube closure were also disrupted by Fab fragments. Cell surface GalTase was localized to migrating neural crest cells and to the basal surfaces of neural epithelia by indirect immunofluorescence, whereas GalTase was undetectable on neural crest cells prior to migration. These results suggest that, during early embryogenesis, cell surface GalTase participates during neural crest cell migration, perhaps by interacting with laminin, a major component of the basal lamina. Cell surface GalTase also appears to play a role in neural tube formation, possibly by mediating neural epithelial adhesion to the underlying basal lamina.     

Myocardial cell relationships during morphogenesis in normal and cardiac lethal mutant axolotls, Ambystoma mexicanum

Sarcomere formation has been shown to be deficient in the myocardium of axolotl embryos homozygous for the recessive cardiac lethal gene c. We examined the developing hearts of normal and cardiac mutant embryos from tailbud stage 33 to posthatching stage 43 by scanning electron microscopy in order to determine whether that deficiency has any effect on heart morphogenesis. Specifically, we investigated the relationships of myocardial cells during the formation of the heart tube (stage 33), the initiation of dextral looping (stages 34-36), and the subsequent flexure of the elongating heart (stages 38-43). In addition, we compared the morphogenetic events in the axolotl to the published accounts of comparable stages in the chick embryo. In the axolotl (stage 33), changes in cell shape and orientation accompany the closure of the myocardial trough to form the tubular heart. The ventral mesocardium persists longer in the axolotl embryo than in the chick and appears to contribute to the asymmetry of dextral looping (stages 34-36) in two ways. First, as a persisting structure it places constraints on the simple elongation of the heart tube and the ability of the heart to bend. Second, after it is resorbed, the ventral myocardial cells that contributed to it are identifiable by their orientation, which is orthogonal to adjacent cells: a potential source of shearing effects. Cardiac lethal mutant embryos behave identically during these events, indicating that functional sarcomeres are not necessary to these processes. The absence of dynamic apical myocardial membrane changes, characteristic of the chick embryo (Hamburger and Hamilton stages 9-11), suggests that sudden hydration of the cardiac jelly is less likely to be a major factor in axolotl cardiac morphogenesis. Subsequent flexure (stages 38-43) of the axolotl heart is the same in normal and cardiac lethal mutant embryos as the myocardial tube lengthens within the confines of a pericardial cavity of fixed length. However, the cardiac mutant begins to exhibit abnormalities at this time. The lack of trabeculation (normally beginning at stage 37) in the mutant ventricle is evident at the same time as an increase in myocardial surface area, manifest in extra bends of the heart tube at stage 39. Nonbeating mutant hearts (stage 41) have an abnormally large diameter in the atrioventricular region, possibly the result of the accumulation of ascites fluid. In addition, mutant myocardial cells have a larger apical surface area compared to normals.     

Embryonic brain enlargement requires cerebrospinal fluid pressure

The brain of the chick embryo begins to enlarge abruptly on the second day of incubation. Shortly thereafter, major flexures and torsions of the brain occur, and many bulges and furrows appear. The onset of enlargement coincides with closure of the spinal canal which makes the neural tube a closed compartment filled with cerebrospinal fluid. We propose that cerebrospinal fluid pressure is a necessary driving force for normal brain enlargement. We have experimentally tested this hypothesis by intubating brains of chick embryos and comparing brain cavity and tissue volumes in normal and intubated embryos. The increase in cavity volume is greatly reduced, whereas brain tissue continues to grow at a reduced rate and folds into the ventricles.     

Experimental Analyses of the Shaping of the Neural Plate and Tube

Understanding the changing morphology of an embryo presentsspecial challenges. Analyses of neurulation in vertebrate embryosdescribed here required observation from sectioned materialand from time-lapse movies, modeling, computer simulation, andexperiments. All these approaches were essential, and each approachhelped guide the use of the others. Experiments have the specialrole of letting the embryo decide between our alternative hypotheses. In the newt embryo, induction and patterning events establishin the ectoderm boundaries between epidermis and neural plate,and between neural plate and the notoplate at its midline. Thedifferentdomains of cells thus established—epidermis, neural plateand notoplate—develop different adhesive properties suchthat cell motility behavior along the notoplate boundary andalong the spinal cord/epidermis boundary produces forceful intercalationof cells which lengthens the boundaries and distorts (lengthens)the neuroepithelium. Neural plate cells also attempt to crawlbeneath the epidermis along their common boundary, raising neuralfolds and producing a rolling moment directed mediad that islargely responsible for neural tube formation. Both cell motilitythat leads to columnarization of neural plate cells and contractionof organized subapical microfilament bundles reduce the apicalsurface area of the neural plate cells and produce an apicaltension that aids neural tube formation. Cell relocation reducesthe width of the neural plate and increases its length, andthe Poisson buckling forces resulting from this elongation ofthe plate also aid neural tube formation. The newt embryo accomplishes neurulation without growth, butbird and mammal embryos grow during neurulation. Understandingthe organization of the products of growth in the amniote neuralplate is critical in determining whether growth helps or hindersneurulation.     

Elongation of axolotl tailbud embryos requires GPI-linked proteins and organizer-induced, active, ventral trunk endoderm cell rearrangements

Application of phosphatidylinositol-specific phospholipase C to early tailbud stage axolotl embryos reveals that a specific subset of morphogenetic movements requires glycosylphosphatidylinositol (GPI)-linked cell-surface proteins. These include pronephric duct extension, "gill bulge" formation, and embryonic elongation along the anteroposterior axis. The work of Kitchin (1949, J. Exp. Zool. 112, 393-416) led to the conclusion that extension of the notochord provided the motive force driving anteroposterior stretching in axolotl embryos, elongation of other tissues being a passive response. We therefore conjectured that axial mesoderm cells might display the GPI-linked proteins required for elongation of the embryo. However, we show here that removal of most of the neural plate and axial and paraxial mesoderm prior to neural tube closure does not prevent elongation of ventrolateral tissues. Tissue-extirpation and tissue-marking experiments indicate that elongation of the ventral trunk occurs via active, directed tissue rearrangements within the endoderm, directed by signals emanating from the blastopore region. Extension of both dorsal and ventral tissues requires GPI-linked proteins. We conclude that elongation of axolotl embryos requires active cell rearrangements within ventral as well as axial tissues. The fact that both types of elongation are prevented by removal of GPI-linked proteins implies that they share a common molecular mechanism.     

Developmentally regulated expression and functional role of alpha 7 integrin in the chick embryo

Integrin alpha 7 beta 1 is a specific cellular receptor for laminin. In the present work, we studied the distribution pattern of the alpha 7 subunit by immunofluorescence and immunoprecipitation and the role of the integrin by blocking antibodies in early chick embryos. alpha 7 immunoreactivity was first detectable in the neural plate during neural furrow formation (stage HH5, early neurula, Hamburger & Hamilton 1951) and its expression was upregulated in the neural folds during primary neurulation. The alpha 7 expression domain spanned the entire neural tube by stage HH8 (4 somites), and was then downregulated and confined to the neuroepithelial cells in the germinal region near the lumen and the ventrolateral margins of the neural tube in embryos by the onset of stage HH17 (29 somites). Expression of alpha 7 in the neural tube was transient suggesting that alpha 7 functions during neural tube closure and axon guidance and may not be required for neuronal differentiation or for the maintenance of the differentiated cell types. alpha 7 immunoreactivity was strong in the newly formed epithelial somites, although this expression was restricted only to the myotome in the mature somites. The most intense alpha 7 immunoreactivity was detectable in the paired heart primordia and the endoderm apposing the heart primordia in embryos at stage HH8. In the developing heart, alpha 7 immunoreactivity was: (i) intense in the myocardium; (ii) milder in the endocardial cushions of the ventricle; (iii) intense in the sinus venosus; (iv) distinct in the associated blood vessels; and (v) undetectable in the dorsal mesocardium of embryos at stage HH17. Inhibition of function of alpha 7 by blocking antibodies showed that alpha 7 integrin-laminin signaling may play a critical role in tissue organization of the neural plate and neural tube closure, in tissue morphogenesis of the heart tube but not in the directional migration of pre-cardiac cells, and in somite epithelialization but not in segment formation in presomitic mesoderm. In embryos treated with alpha 7 antibody, the formation of median somites in place of a notochord was intriguing and suggested that alpha 7 integrin-laminin signaling may have played a role in segment re-specification in the mesoderm.     

Direct action of the nodal-related signal cyclops in induction of sonic hedgehog in the ventral midline of the CNS

The secreted molecule Sonic hedgehog (Shh) is crucial for floor plate and ventral brain development in amniote embryos. In zebrafish, mutations in cyclops (cyc), a gene that encodes a distinct signal related to the TGF(beta) family member Nodal, result in neural tube defects similar to those of shh null mice. cyc mutant embryos display cyclopia and lack floor plate and ventral brain regions, suggesting a role for Cyc in specification of these structures. cyc mutants express shh in the notochord but lack expression of shh in the ventral brain. Here we show that Cyc signalling can act directly on shh expression in neural tissue. Modulation of the Cyc signalling pathway by constitutive activation or inhibition of Smad2 leads to altered shh expression in zebrafish embryos. Ectopic activation of the shh promoter occurs in response to expression of Cyc signal transducers in the chick neural tube. Furthermore an enhancer of the shh gene, which controls ventral neural tube expression, is responsive to Cyc signal transducers. Our data imply that the Nodal related signal Cyc induces shh expression in the ventral neural tube. Based on the differential responsiveness of shh and other neural tube specific genes to Hedgehog and Cyc signalling, a two-step model for the establishment of the ventral midline of the CNS is proposed.     

Anti-apoptotic role of Sonic hedgehog protein at the early stages of nervous system organogenesis

In vertebrates the neural tube, like most of the embryonic organs, shows discreet areas of programmed cell death at several stages during development. In the chick embryo, cell death is dramatically increased in the developing nervous system and other tissues when the midline cells, notochord and floor plate, are prevented from forming by excision of the axial-paraxial hinge (APH), i.e. caudal Hensen's node and rostral primitive streak, at the 6-somite stage ( Charrier, J. B., Teillet, M.-A., Lapointe, F. and Le Douarin, N. M. (1999). Development 126, 4771-4783). In this paper we demonstrate that one day after APH excision, when dramatic apoptosis is already present in the neural tube, the latter can be rescued from death by grafting a notochord or a floor plate fragment in its vicinity. The neural tube can also be recovered by transplanting it into a stage-matched chick embryo having one of these structures. In addition, cells engineered to produce Sonic hedgehog protein (SHH) can mimic the effect of the notochord and floor plate cells in in situ grafts and transplantation experiments. SHH can thus counteract a built-in cell death program and thereby contribute to organ morphogenesis, in particular in the central nervous system.     

Distribution of dorsal-forming activity in precleavage embryos of the Japanese newt, Cynops pyrrhogaster: effects of deletion of vegetal cytoplasm, UV irradiation, and lithium treatment

Two types of axis-deficient embryos developed after deletion of the vegetal cytoplasm: wasp-shaped embryos and permanent-blastula-type embryos. In situ hybridization revealed that neither type of axis-deficient embryo expressed goosecoid or pax-6. brachyury was expressed in the constricted waist region of the wasp-shaped embryos but was not expressed in the permanent-blastula-type embryos. Further, we examined the effect of UV irradiation on Japanese newt embryos. Surprisingly, UV-irradiated Japanese newt eggs formed hyperdorsalized embryos. These embryos gastrulated in an irregular circular fashion with goosecoid expression in the circular equatorial region. At tailbud stage, these embryos formed a proboscis which is very reminiscent of that formed in hyperdorsalized Xenopus embryos. Transplantation of the marginal region of the UV-irradiated embryos revealed that the entire marginal zone had organizer activity. Thus we conclude that UV hyperdorsalizes Japanese newt embryos. Finally, lithium treatment of normal embryos at the 32-cell stage also resulted in hyperdorsalization. Lithium treatment of vegetally deleted embryos had two distinct results. Lithium treatment of permanent-blastula-type embryos did not result in the formation of dorsal axial structures, while the same treatment reinduced gastrulation and dorsal axis formation in the wasp-shaped embryos. Based on these results, we propose a model for early axis specification in Japanese newt embryos. The model presented here is fundamentally identical to the Xenopus model, with some important modifications. The vegetally located determinants required for dorsal development (dorsal determinants, DDs) are distributed over a wider region at fertilization in Japanese newt embryos than in Xenopus embryos. The marginal region of the Japanese newt embryo at the beginning of development overlaps with the field of the DDs. Gastrulation is very likely to be a dorsal marginal-specific property, while self-constriction is most probably a ventral marginal-specific property in Japanese newt embryos.     

Loss of Occludin and Functional Tight Junctions, but Not ZO-1, during Neural Tube Closure—Remodeling of the Neuroepithelium Prior to Neurogenesis

Neuroepithelial cells can generate nonepithelial cells, the neurons. Here we have investigated, for chick and mouse embryos, the epithelial character of neuroepithelial cells in the context of neurogenesis by examining the presence of molecular components of tight junctions during the transition from the neural plate to the neural tube. Immunoreactivity for occludin, a transmembrane protein specific to tight junctions, was detected at the apical end of the lateral membrane of neuroepithelial cells throughout the chick neural plate. During neural tube closure, occludin disappeared from all neuroepithelial cells. Correspondingly, the addition of horseradish peroxidase to the apical side of the neuroepithelium by injection into the amniotic cavity of mouse embryos revealed the presence of functional tight junctions in the neural plate (Embryonic Day 8), but not the neural tube (Embryonic Day 9). In contrast to occludin, expression of ZO-1, a peripheral membrane protein of tight junctions, increased from the neural plate to the neural tube stage, also being confined to the apical end of the lateral neuroepithelial cell membrane. This localization coincided with that of N-cadherin, whose expression increased concomitantly with the disappearance of occludin. We propose that the loss of tight junctions from neuroepithelial cells reflects an overall decrease in their epithelial nature, which precedes the generation of neurons.     

Chick CFC controls Lefty1 expression in the embryonic midline and nodal expression in the lateral plate

Members of the EGF-CFC family of proteins have recently been implicated as essential cofactors for Nodal signaling. Here we report the isolation of chick CFC and describe its expression pattern, which appears to be similar to Cfc1 in mouse. During early gastrulation, chick CFC was asymmetrically expressed on the left side of Hensen's node as well as in the emerging notochord, prechordal plate, and lateral plate mesoderm. Subsequently, its expression became confined to the heart fields, notochord, and posterior mesoderm. Implantation experiments suggest that chick CFC expression in the lateral plate mesoderm is dependent on BMP signaling, while in the midline its expression depends on an Activin-like signal. The asymmetric expression domain within Hensen's node was not affected by application of FGF8, Noggin, or Shh antibody. Implantation of cells expressing human or mouse CFC2, or chick CFC on the right side of Hensen's node randomized heart looping without affecting expression of genes involved in left-right axis formation, including SnR, Nodal, Car, or Pitx2. Application of antisense oligodeoxynucleotides to the midline of Hamburger-Hamilton stage 4-5 embryos also randomized heart looping, but in contrast to the overexpression experiments, antisense oligodeoxynucleotide treatment resulted in bilateral expression of Nodal, Car, Pitx2, and NKX3.2, whereas Lefty1 expression in the midline was transiently lost. Application of the antisense oligodeoxynucleotides to the lateral plate mesoderm abolished Nodal expression. Thus, chick CFC seems to have a dual function in left-right axis formation by maintaining Nodal expression in the lateral plate mesoderm and controlling expression of Lefty1 expression in the midline territory.     

Bone morphogenetic proteins regulate neural tube closure by interacting with the apicobasal polarity pathway

During neural tube closure, specialized regions called hinge points (HPs) display dynamic and polarized cell behaviors necessary for converting the neural plate into a neural tube. The molecular bases of such cell behaviors (e.g. apical constriction, basal nuclear migration) are poorly understood. We have identified a two-dimensional canonical BMP activity gradient in the chick neural plate that results in low and temporally pulsed BMP activity at the ventral midline/median hinge point (MHP). Using in vivo manipulations, high-resolution imaging and biochemical analyses, we show that BMP attenuation is necessary and sufficient for MHP formation. Conversely, BMP overexpression abolishes MHP formation and prevents neural tube closure. We provide evidence that BMP modulation directs neural tube closure via the regulation of apicobasal polarity. First, BMP blockade produces partially polarized neural cells, which retain contact with the apical and basal surfaces but where basolateral proteins (LGL) become apically localized and apical junctional proteins (PAR3, ZO1) become targeted to endosomes. Second, direct LGL misexpression induces ectopic HPs identical to those produced by noggin or dominant-negative BMPR1A. Third, BMP-dependent biochemical interactions occur between the PAR3-PAR6-aPKC polarity complex and phosphorylated SMAD5 at apical junctions. Finally, partially polarized cells normally occur at the MHP, their frequencies inversely correlated with the BMP activity gradient in the neural plate. We propose that spatiotemporal modulation of the two-dimensional BMP gradient transiently alters cell polarity in targeted neuronal cells. This ensures that the neural plate is flexible enough to be focally bent and shaped into a neural tube, while retaining overall epithelial integrity.     

Convergent extension, planar-cell-polarity signalling and initiation of mouse neural tube closure

Planar-cell-polarity (PCP) signalling is necessary for initiation of neural tube closure in higher vertebrates. In mice with PCP gene mutations, a broad embryonic midline prevents the onset of neurulation through wide spacing of the neural folds. In order to evaluate the role of convergent extension in this defect, we vitally labelled the midline of loop-tail (Lp) embryos mutant for the PCP gene Vangl2. Injection of DiI into the node, and electroporation of a GFP expression vector into the midline neural plate, revealed defective convergent extension in both axial mesoderm and neuroepithelium, before the onset of neurulation. Chimeras containing both wild-type and Lp-mutant cells exhibited mainly wild-type cells in the midline neural plate and notochordal plate, consistent with a cell-autonomous disturbance of convergent extension. Inhibitor studies in whole-embryo culture demonstrated a requirement for signalling via RhoA-Rho kinase, but not jun N-terminal kinase, in convergent extension and the onset of neural tube closure. These findings identify a cell-autonomous defect of convergent extension, requiring PCP signalling via RhoA-Rho kinase, during the development of severe neural tube defects in the mouse.     

Neural tube occlusion precedes rapid brain enlargement

Histological examination of early vertebrate embryos during rapid brain enlargement (an event partially driven by fluid pressure) reveals that the spinal cord lumen is occluded. Occlusion (if it is not merely a fixation artifact) may confine neural tube fluid to brain regions and seal off the ventricles before posterior neuropore closure. We injected neural tubes of living chick embryos with dyes, asking (1) is occlusion real; and (2) does occlusion precede brain enlargement? Both questions were answered affirmatively. Experimental analyses of occlusion and brain enlargement are in progress.     

The cellular environment controls the expression of engrailed-like protein in the cranial neuroepithelium of quail-chick chimeric embryos.

We have previously shown that one of two chicken engrailed-like genes, chick En-2, is expressed in a restricted region of the early chick embryo brain: the mes/metencephalon (Gardner et al. 1988). In this study, we examine the role of the cellular environment in regulation of engrailed-like (En) protein expression in quail-chick chimeric embryos. Two types of transplant surgery were performed at the 9-15 somite stage to produce chimeric embryos. In the first, the mid-mesencephalic vesicle or caudal mesencephalic vesicle alar plate (which is En protein-positive) was transplanted from a quail embryo into an En protein-negative region of chick neuroepithelium, the prosencephalon (mMP and cMP grafts, respectively). In the second reciprocal surgery, prosencephalic alar plate which is En protein-negative, was transplanted into the En protein-positive mesencephalic vesicle (PM grafts). A polyclonal antiserum, alpha Enhb-1, which recognizes chick En proteins (Davis et al. 1991) was used to identify En-positive cells 48 h after surgery. In mMP embryos, 71% of integrated grafts had lost En expression (n = 17). In contrast, in cMP grafts, 93% of integrated grafts continued to stain with the antiserum (n = 14). In addition, in 86% of these embryos, the graft induced adjacent chick host diencephalic cells to become En protein-positive as well. All PM grafts contained aEnhb-1-positive cells; such cells never expressed this protein in their normal environment. These early changes in En protein expression correlate well with the morphological changes observed in similar graft surgeries assayed later in development. Thus, our results are consistent with the hypothesis that En genes play a role in the regionalization of the early cranial neuroepithelium.