Vertical regulation of En-2 expression and eye development by FGFs and BMPs.

The formation of the midbrain region depends mainly on the activity of a signalling center located in the isthmus region, on the border between the prospective mesencephalon and metencephalon. FGF-8 has been proposed as a signalling molecule responsible for this specification because of its expression pattern and its ability to elicit duplication of the midbrain region when expressed ectopically in the neuroepithelium. Here we present evidence that members of the FGF family of growth factors when released in the cephalic mesenchyme are able to extend the expression of the mesencephalic marker En-2 to both the anterior and the posterior regions of its original landmark. This alteration in the expression pattern of En-2 is not accompanied by a significant alteration in the later development of the midbrain-cerebellar anlage, although the eye development is severely altered. Members of the bone morphogenetic protein family ectopically released from the mesenchyme down-regulate the expression of En-2 and also have an effect on the development of the eye. These results demonstrate that growth factor molecules produced in the mesenchyme (vertical signalling) participate in the correct establishment of the antero-posterior patterning of the cephalic nervous system during development.

Pax2, Otx2, Gbx2 and Fgf8 expression in early otic vesicle development.

The inner ear is a suitable system to study the mechanisms involved in the specification of different functional domains during morphogenesis. Using single and double in situ hybridization (ISH) we show that three transcription factors (Otx2, Gbx2and Pax2) and a member of the fibroblast growth factor family (Fgf8) could participate in the compartmentalization of the otic vesicle and in the formation of the acoustic-vestibular ganglion.

Monoclonal antibody GL1 and its possible involvement in the morphogenesis of the otic vesicle.

In a previous study, a monoclonal antibody (MAB) named GL1 was identified that is expressed in a precise pattern during gastrulation and early neurulation stages in chick embryos. In this article we have further investigated the expression pattern of this MAB in the chick embryo. GL1 antigen is present in several organs that seem not to be related developmentally. Among them, GL1 is present during the early steps of the otic placode formation, in the pharyngeal endoderm, in some neural crest cells, in the somites, and in the ventricular surface of the nervous system. The distribution in the nervous system is well patterned with two broad lines of expression in the ventricular side of the metencephalic region, a unique and centered expression in the border between the metencephalon and the myelencephalon and again in two lines running along the myelencephalon and the rostral spinal cord. Additionally, GL1 can be induced by members of the FGF family, and we have used this system to elucidate its role in otic placode formation. The results obtained reveal that GL1 can be a useful marker for the study of developmental processes in the endoderm, the otic anlage, and the apical surface of the nervous system. Biochemical analysis of the antigen recognized by this MAB must be carried out to elucidate the molecular nature of the antigen.

Morphological and quantitative studies in the otic region of the neural tube in chick embryos suggest a neuroectodermal origin for the otic placode.

Careful histological observation of the development of the anlage of the inner ear in chicken embryos led us to question the traditional view of otic placode (OP) formation. First, morphological studies in the cephalic region carried out on stages preceding the appearance of the placodal epithelium revealed that the medial placodal cells are continuous temporally and spatially with cells belonging to the neural fold (NF). Second, both the formation of the basal lamina between the dorsal region of the neural tube (NT) and ectoderm and the pattern of formation of the neural crest present distinctive characteristics between otic levels and regions located anteriorly and posteriorly. Third, numerical comparisons of parameters for the NT and the OP between different levels of the rhombencephalon allowed us to assign a differential behaviour in the growth pattern of the otic region. These results indicated that the medial part of the OP is not derived from already independent ectoderm that increases in thickness under the influence of the NT (as previously accepted) but that it develops directly from the NFs. Although we do not exclude other possibilities, we propose that at least a proportion of the OP cells originate directly from cells committed to be neural crest. After this incorporation, basal laminal formation would delimit the NT from the OP without transition of the otic cells to ectoderm. This hypothesis would imply that part of the otic cells originate directly from neuroepithelial cells having a neuroectodermal (rather than the previously established ectodermal) origin.

Neural induction in whole chick embryo cultures by FGF.

FGFs are well known as mesodermal inducers and they have been reported to have neural inducing and/or caudalizing activity in Xenopus. To evaluate the role of FGFs in neural induction and patterning of the nervous system in chick embryos, we have targeted the ectopic expression of these factors by applying FGF-soaked beads to extended primitive streak chick embryos developing in culture. The whole embryo culture system allows to directly assessing the neural inducing activity on nonneural ectodermal cells. Our results show that FGFs induce the production of ectopic neural structures in the epiblast cell layer which are independent of the endogenous neural tube. The induced neural plates express several neural positional markers (Otx-2, Krox-20, EphA4, EphA7, and cHox-B9), although they lack anterior neural markers such as BF-1. These effects are very likely to be exerted by the direct action of FGF on epiblast cells because we have found no evidence of the induction of an organizer-like activity or of the presence of new axial mesoderm induced by the growth factor. We conclude that FGFs can act as direct neural inducers and that this action is exerted independently from the axial mesoderm.

Targeted over-expression of FGF in chick embryos induces formation of ectopic neural cells.

Fibroblast growth factors (FGFs) are known to be involved mainly in mesoderm formation in Xenopus embryos but their participation in other inductive mechanisms such as neural induction has not been clearly established and is now under study. Here, we provide evidence that targeted over-expression of members of this family of growth factors in the periphery of full-length primitive streak chick embryos produces the formation of ectopic neural cells that are able to differentiate into neurons. The supernumerary neural plate obtained derives from the epiblast layer of the blastoderm and show signs of neural differentiation 24 h after the application of FGF. We have used cell labeling and have examined the expression of mesodermal markers to ascertain how this expansion of the neural forming region of the epiblast takes place. We conclude that the new neural cells formed are originated in the region of the epiblast fated to be epithelia and that the induction of the ectopic neural tissue is not mediated by an increase, migration or new formation of axial mesoderm. This strongly suggests that FGF is acting directly on epiblast cells, changing their fate from epidermal ectoderm to neural ectoderm. Therefore, our results show that FGF can induce neural ectoderm when acting on still uncommitted cells and, therefore, it is a putative candidate for acting in normal neural induction during development.

Evidence that Shh cooperates with a retinoic acid inducible co-factor to establish ZPA-like activity.

Patterning across the anteroposterior axis of the vertebrate limb bud involves a signal from the polarizing region, a small group of cells at the posterior margin of the bud. Retinoic acid (RA; Tickle, C., Alberts, B., Wolpert, L. and Lee, J. (1982) Nature 296, 554-566) and Sonic hedgehog (Shh; Riddle, R. D. Johnson, R. L., Laufer, E. and Tabin, C. J. (1993) Cell 25, 1401-1416; Chang, D. T., Lopez, A., von Kessler, D. P., Chiang, C., Simandl, B. K., Zhao, R., Seldin, M. F., Fallon, J. F. and Beachy, P. A. (1994 Development 120, 3339-3353) have been independently postulated as such signals because they can mimic the mirror image digit duplication obtained after grafting polarizing cells to the anterior of limb buds. Here we show that a embryonal carcinoma cell line, P19, transfected with a Shh expression vector shows low polarizing activity, but when cultured with retinoic acid, duplications like those induced by the polarizing region (ZPA) arise. Complete duplications are also obtained by cotransfecting P19 Shh cells with a constitutively active human retinoic acid receptor (VP16-hRARalpha). These data suggest that Shh and RA cooperate in generating ZPA activity and that Shh, while essential, may not act alone in this process.

Locations of the ectodermal and nonectodermal subdivisions of the epiblast at stages 3 and 4 of avian gastrulation and neurulation.

A prospective fate map of the avian epiblast at late gastrula and early neurula stages has been generated through the construction of quail/chick transplantation chimeras. This map shows the subdivisions of the prospective ectoderm, mesoderm, and endoderm, both within the epiblast prior to their ingression and within the primitive streak. The map demarcates the locations and extents of the prospective surface ectoderm, otic placodes, neural crest, and neural plate--including its postnodal levels--in prospective ectoderm of the epiblast; prospective foregut, within the prospective endoderm of the epiblast and primitive streak; and prospective notochord, somites, intermediate mesoderm, lateral plate mesoderm, and extraembryonic mesoderm in the prospective mesoderm of the epiblast and/or primitive streak. Prospective cardiogenic cells are apparently absent from the primitive streak at these stages, and contributions of the epiblast to the heart are relatively scant and inconsistent with the expected timing and directions of migrations of prospective cardiogenic cells. Mapping of the primitive streak at earlier stages in another study (García-Martinez and Schoenwolf: Developmental Biology, in press) reveals that the ingression of cardiogenic cells through the primitive streak occurs prior to late gastrula stages, suggesting that contributions of epiblast to the heart at later stages are artifactual. Tests of prospective potency, based on the projected locations of origin of various cell groups provided by the new prospective fate map, are underway.

Monoclonal antibodies identifying subsets of ectodermal, mesodermal, and endodermal cells in gastrulating and neurulating avian embryos.

The goal of our laboratory research is to elucidate the mechanisms underlying gastrulation and neurulation, using the avian embryo as a model system. In previous studies, we used two approaches to map the morphogenetic movements involved in these processes: (1) we constructed quail/chick transplantation chimeras in which grafted quail cells could be identified within chick host embryos by the presence of nucleolar-associated heterochromatin, and (2) we microinjected exogenous cell markers. However, it would be advantageous to be able to detect endogenous markers to demarcate various subsets of cells within the unmanipulated embryo. To elucidate such a series of natural markers, we have used monoclonal antibodies to identify epitopes found on subsets of ectodermal, mesodermal, and endodermal cells. Antibodies were made by immunizing mice against either homogenized ectoderm (i.e., prospective neural plate and surface ectoderm) or primitive streak, which had been microdissected from stage 3 chick embryos. Additionally, we screened a panel of antibodies made against soluble protein obtained from isolates of cell nuclei from late embryonic chick brain. Here, we describe the labeling patterns of three monoclonal antibodies, called MAb-GL1, GL2, and GL3 (GL, germ layer), during avian gastrulation and neurulation. Our results show that labeling early avian embryos with monoclonal antibodies can reveal previously undetected distributions of cells bearing shared epitopes, providing new labels for subsets of cells in each of the three primary germ layers.

Expansion of surface epithelium provides the major extrinsic force for bending of the neural plate.

Neurulation, formation of the neural tube, requires both intrinsic forces (i.e., those generated within the neural plate) and extrinsic forces (i.e., those generated outside the neural plate in adjacent tissues), but the precise origin of these forces is unclear. In this study, we addressed the question of which tissue produces the major extrinsic force driving bending of the neural plate. We have previously shown that 1) extrinsic forces are required for bending and 2) such forces are generated lateral to the neural plate. Three tissues flank the neural plate prior to its bending: surface epithelium, mesoderm, and endoderm. In the present study, we removed two of these layers, namely, the endoderm and mesoderm, underlying and lateral to the neural plate; bending still occurred, often with complete formation of a neural tube, although the latter usually rotated toward the side of tissue depletion. These results suggest that the surface epithelium, the only tissue remaining after microsurgery, provides the major extrinsic force for bending of the neural plate and that the mesoderm (and perhaps endoderm) stabilizes the neuraxis, maintaining its proper orientation and position on the midline.

Specification of neurepithelium and surface epithelium in avian transplantation chimeras.

Previous studies of the avian blastoderm have revealed that extensive displacements occur within the epiblast during gastrulation and neurulation. The present study had two main purposes: (1) to map the origin and movement of prospective surface epithelial cells, and (2) to ask whether neurepithelial and surface epithelial cell fates are determined prior to cell movement, or whether they arise later as a result of the ultimate position attained by cells through their movement. Our results show that the rostral and lateral intraembryonic and extraembryonic surface epithelium originates as far laterally as at the area pellucida-area opaca interface of the early epiblast. Intraembryonic surface epithelial cells rearrange relative to one another, extending medially to contribute to the formation of the neural folds, whereas extraembryonic surface epithelial cells maintain their lateral positions, spreading uniformly as the epiblast expands. Our results further show that surface epithelial and neurepithelial cell fates are labile at the onset of neurulation, suggesting that cell fate is specified following cell movement.

Patterns of neurepithelial cell rearrangement during avian neurulation are determined prior to notochordal inductive interactions.

In the epiblast of elongating primitive-streak-stage avian embryos, MHP cells--short wedge-shaped neurepithelial cells contained within the median hinge point of the bending neural plate--arise from the midline prenodal and nodal area, whereas L cells--tall spindle-shaped neurepithelial cells constituting the lateral neural plate--arise from paired areas flanking the cranial primitive streak. These characteristic differences in neurepithelial cell shape are acquired as a result of inductive interactions with the notochord. Both MHP and L cells undergo extensive rearrangement (intercalation) during shaping and bending of the neural plate, but their pattern of rearrangement differs. MHP cells intercalate with other MHP cells and the population always spans the midline, whereas L cells intercalate with other L cells, remaining in bulk lateral to the midline. The following experiment was performed to establish whether these distinctive rearrangement patterns are determined prior to notochordal inductive interactions. Quail prospective MHP and L cells were transplanted isochronically and heterotopically to chick host blastoderms at stages prior to formation of the notochord (to wit, prospective MHP cells were transplanted into prospective L cell territory and vice versa) and the distribution, fate, and morphological characteristics of grafted cells were determined in chimeras collected 24 hr later. Our results demonstrate that heterotopic MHP and L cells do not adopt the rearrangement pattern characteristic of their new site; rather, they change their position so that grafted MHP cells intermix with MHP cells of the host and grafted L cells intermix with L cells of the host. Thus, patterns of neurepithelial cell rearrangement are determined prior to notochordal inductive interactions. When and how this determination occurs are topics for further studies.

Shaping, invagination, and closure of the chick embryo otic vesicle: scanning electron microscopic and quantitative study.

Scanning electron microscopy, light microscopy, and morphometric analysis were used to study the morphological changes of the otic placode and vesicle before and during invagination and closure processes. Our results reveal that the otic placode undergoes shaping between stages HH9 and HH12; during this period the rostrocaudal axis is shortened, while the lateromedial axis of the placode lengthens. The presence of long cytokinesis bridges during this period suggests that cellular displacements after mitosis may participate in the shaping of the otic placode. The shaping process appears to facilitate the approach of the otic placode to the neural tube. From stage HH12 on, the otic anlage gradually becomes a U-shaped structure with its medial portion in intimate apposition to the rhombencephalic neural tube. The coincidence in time between the beginning of intimate otic anlage-rhombencephalon contact and active invagination suggests that these two processes are related. Changes occurring at the edges of the otic vesicle until their disappearance in stage HH17 suggest that, in addition to a process of invagination, the edges of the otic anlage become bent. During closure, cells at the edges of the otic vesicle differ in apical morphology according to their topographical location: The cells between the rostral and lateral edges have elongated apices, in contrast with the polygonal shape of the cell apices in other places of the edges. In the opposite side (between the caudal and medial edges) cell death is observed. Closure of the otic vesicle conceptualized as a zipper-like model is discussed. We propose that early development of the otic anlage takes place in four stages: 1) shaping (stages HH9-11); 2) triggering of the invagination (stage HH12); 3) early invagination and lateral bending (stages HH13-15); and 4) late invagination and closure (stages HH16-17).

Cell proliferation during early development of the chick embryo otic anlage: quantitative comparison of migratory and nonmigratory regions of the otic epithelium.

During development of the otic anlage, a certain proportion of epithelial cells migrate toward the mesenchymal compartment to form part of the acoustic-vestibular ganglion. The migrating cells are observed only in the zone of the otic anlage that will make contact with the acoustic-vestibular ganglion (so-called ganglion zone). In Hamburger and Hamilton's stages 13 to 16, the number of epithelial cells that migrate is relatively low, but it becomes steadily higher from stage 17 on. In the otic anlage of chick embryos, between developmental stages 9 and 21 (48 to 94 hours of incubation), mitotic index, apical or basal localization within the epithelium of dividing cells, and orientation of the mitotic spindles were analyzed. These features in the ganglion zone were compared with observations in the rest of the otic epithelium, where migratory processes do not take place. In stages 13 to 15, when few epithelial cells are migrating, the mitotic index (MI) in the ganglion zone of the otic anlage is similar to that in nonmigratory regions. In more advanced stages, however, when cell migration becomes accelerated, the MI in the migratory zone of the otic wall is significantly higher than that in the rest of the otic epithelium. This suggests an intimate relationship between the migration of otic epithelial cells and a high rate of cell proliferation, the possible nature of which is discussed. Although the majority of mitoses in the otic anlage are located at the apical surface of the epithelium, from stage 13 onward, a few dividing cells are seen in the basal third of the epithelium. Furthermore, these basal mitoses appear exclusively in the migratory zone of the otic anlage, thus suggesting a possible relationship between epithelial cell migration and basal mitosis. During the developmental period prior to stage 18, no significant differences in mitotic spindle orientation are noted between migratory and nonmigratory zones of the otic anlage. In contrast, in stages of maximal otic epithelial cell migration (stages 19 to 21), the frequency of mitoses with the spindle axis oriented radially is significantly higher in the migratory zone. These findings point toward a close correlation between increased frequency of radial mitotic spindle orientation and intense cell migration, although the exact nature of this relationship is as yet unknown.

Roles of neuroepithelial cell rearrangement and division in shaping of the avian neural plate.

Shaping of the neural plate, one of the most striking events of neurulation, involves rapid craniocaudal extension. In this study, we evaluated the roles of two processes in neural plate extension: neuroepithelial cell rearrangement and cell division. Quail epiblast plugs of constant size were grafted either just rostral to Hensen's node or paranodally and the resulting chimeras were examined at selected times postgrafting. By comparing the size of the original plug, the number of cells it contained and the distribution of cells within it to those same features of the grafts in chimeras, we were able to ascertain that, during transformation of the flat neural plate into the closed neural tube (a period requiring 24 h), the graft undergoes a maximum of 3 rounds of craniocaudal extension (each round of craniocaudal extension was defined as a doubling of graft length, so 3 rounds equaled an 8-fold increase in length). Such extension is accompanied by 2 rounds of cell rearrangement and 2-3 rounds of cell division (cell rearrangement occurred mediolaterally, so each round was defined as a halving of the number of cells in the width of the graft and a doubling of the number of cells in its length; each round of cell division was defined as a doubling of graft cell number). Modeling studies demonstrate that these amounts of cell rearrangement and division are sufficient to approximate the shaping of the neural plate that normally ensues during neurulation, provided that some of the cell division occurs within the longitudinal plane of the neural plate and some within its transverse plane: longitudinal cell division results in craniocaudal extension of the neural plate, whereas transverse cell division results in lateral expansion of the neural plate such as that occurring at its cranial end; cell rearrangement results in craniocaudal extension of the neural plate as well as in its narrowing. In conclusion, our results provide evidence that shaping of the neural plate involves mediolateral cell rearrangement and cell division, with the latter occurring within both the longitudinal and transverse planes of the neural plate.

Quantitative studies of mitotic cells in the chick embryo optic stalk during the early period of invasion by optic fibres.

In addition to mitoses of neuroepithelial cells at the ventricular surface of the chick embryo optic stalk, mitoses in nonventricular stalk zones begin to be observed from stage 19 on. These latter represent the division phase of glioblasts detached from the ventricular surface. Thus, the topographical location of mitotic cells could be considered a morphological marker of neuroepithelial and glioblast populations in the optic stalk. Quantitative analysis of ventricular (VMCs) and extraventricular (EMCs) mitotic cells revealed that the total number of VMCs decreases through the developmental stages studied, while the number of EMCs simultaneously increases exponentially. These results suggest that the glioblast population arises from both division of the early glioblasts and progressive transformation of neuroepithelial cells. The first EMCs in the ventral region of the stalk wall are observed in stage 19, previous to the stages in which the first EMCs appear in the dorsal region. Moreover, EMCs are much more numerous in the ventral than in the dorsal stalk wall in all stages analysed. Keeping in mind that the invasion of the stalk by optic fibre fascicles occurs essentially in the ventral region, these results suggest that EMCs are strongly related to axon fascicle outgrowth in the stalk. Cell division features are different in neuroepithelial cell and glioblast populations, as the proportions of the mitotic phases differ in VMCs and EMCs. In addition, the patterns of mitotic spindle orientation in VMCs and EMCs are also different. In the former, orientations are predominantly longitudinal parallel and transverse parallel, with a smaller proportion of radial mitoses, which are slightly more frequent through stages 23 to 28 than in earlier development.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell death in suboptic necrotic centers of chick embryo diencephalon and their topographic relationship with the earliest optic fiber fascicles.

The structural features of suboptic necrotic centers (SONCs) in the floor of the chick embryo diencephalon were studied. These necrotic areas were observed lateral to the prospective zone of the optic chiasm through developmental stages 14 to 24. The relationship between SONCs and the earliest optic fiber fascicles also was studied in an attempt to determine the possible significance of these cell death areas in the mechanism of optic pathway development. In SONCs, healthy neuroepithelial cells contain primary lysosomes and phagocytose fragments of dead cells. Discrete regions within the cytoplasm of some cells show electron-transparent vacuoles in contact with dense contents of ruptured lytic bodies. The cytoplasm of dying cells and dead cell fragments are notably electron dense, with numerous secondary lysosomes and electron-transparent vacuoles. These observations are interpreted on the assumption that after autophagic processes, condensation and fragmentation take place in dying cells of the SONCs. In the ventricular lumen adjacent to the SONCs, numerous more or less spherical bodies are observed that appear to be shed from the tip of the cells constituting the SONCs. Three different types of intraventricular bodies can be distinguished: loose, moderately dense, and highly dense. The first type appears to originate from apical portions of cells that undergo autolytic processes. Moderately dense fragments are interpreted as originating from dying cells in which the cytoplasm is undergoing condensation. Finally, highly dense intraventricular bodies appear to be fragments of dead cells that are shed into the ventricular lumen. SONCs separate the prospective area of the optic chiasm from lateral regions of the diencephalic floor. Extracellular spaces are poorly developed within the wall of the SONCs, whereas the neuroepithelium of the presumptive optic chiasm and regions located rostral and caudal to SONCs show abundant and extensive extracellular spaces. These are bounded by long marginal processes of neuroepithelial cells. Sagittal sections of embryonic heads at stages 22-24 reveal optic fiber fascicles penetrating the SONCs asymmetrically, as they are found only in its caudal half. These observations suggest that the SONCs function as doorways made of compact neuroepithelium, to be traversed by the earliest optic fibers before they reach the middle zone of the floor of the diencephalon through which they travel to the contralateral optic tract within large extracellular spaces.

Cell death in the ventral region of the neural retina during the early development of the chick embryo eye.

The present study deals with morphologic and quantitative changes that take place in the area of cell death in the ventral part of the presumptive retinal wall of the chick embryo. These changes were followed from the optic vesicle stage until the first optic fiber fascicles leave the neural retina. Our results show that both the volume occupied by the area of cell death and the density of its pyknotic fragments undergo considerable variation during the period between Hamburger and Hamilton's (1951) stages 12 to 20. In the optic vesicle stages, cell death in the ventral wall of the vesicle was observed in 50 to 75% of the embryos studied. During stages 14 and 15, this zone was seen in more than 90%. By the time invagination of the optic cup was complete, the ventral retinal zone of cell death had disappeared entirely in a large proportion of embryos; in all others, it shrank significantly both in volume and density of pyknotic fragments. In stage 19, when the first optic fiber fascicles begin to emerge from the retina, a dramatic increase occurs in the number of pyknotic fragments in the posterior pole of the retina. The appearance of dying cells, in a region shortly to be traversed by developing ganglion cell axons, supports the hypothesis that cell death processes are apparently somehow related to the creation of a suitable environment for the emergence of fibers toward the optic stalk. Densities of mitotic and interphasic cells as well as the mitotic index were determined in both the retinal zone of cell death and in areas devoid of dead cells. In all developmental stages analyzed, the mitotic index was notably lower in the former than in non-necrotic zones, suggesting that cell proliferation is partially inhibited in retinal areas of cell death.