A critical period for conversion of ectodermal cells to a neural crest fate

Previously, we found that interactions between neural and nonneural ectoderm can generate neural crest cells, with both the ectodermal and the neuroepithelial cells contributing to induced population (M. A. J. Selleck and M. Bronner-Fraser, 1995, Development 121, 525-538). To further characterize the ability of ectodermal cells to form neural crest, we have challenged their normal fate by transplanting them into the neural tube. To ensure that the ectoderm was from nonneural regions, we utilized extraembryonic ectoderm (the proamnion) and transplanted it into the presumptive midbrain of 1. 5-day-old chick embryos. We observed that the grafted ectoderm has the capacity to adopt a neural crest fate, responding within a few hours of surgery by turning on neural crest markers HNK-1 and Slug. However, the competence of the ectoderm to respond to neural crest-inducing signals is time limited, declining rapidly in donors older than the 10-somite stage. Similarly, the inductive capacity of the host midbrain declines in a time-dependent fashion. Our results show that extraembryonic ectoderm has the capacity to form neural crest cells given proper inducing signals, expressing both morphological and molecular markers characteristic of neural crest cells.     

Hensen's node induces neural tissue in Xenopus ectoderm. Implications for the action of the organizer in neural induction.

The development of the vertebrate nervous system is initiated in amphibia by inductive interactions between ectoderm and a region of the embryo called the organizer. The organizer tissue in the dorsal lip of the blastopore of Xenopus and Hensen's node in chick embryos have similar neural inducing properties when transplanted into ectopic sites in their respective embryos. To begin to determine the nature of the inducing signals of the organizer and whether they are conserved across species we have examined the ability of Hensen's node to induce neural tissue in Xenopus ectoderm. We show that Hensen's node induces large amounts of neural tissue in Xenopus ectoderm. Neural induction proceeds in the absence of mesodermal differentiation and is accompanied by tissue movements which may reflect notoplate induction. The competence of the ectoderm to respond to Hensen's node extends much later in development than that to activin-A or to induction by vegetal cells, and parallels the extended competence to neural induction by axial mesoderm. The actions of activin-A and Hensen's node are further distinguished by their effects on lithium-treated ectoderm. These results suggest that neural induction can occur efficiently in response to inducing signals from organizer tissue arrested at a stage prior to gastrulation, and that such early interactions in the blastula may be an important component of neural induction in vertebrate embryos.     

Cells remain competent to respond to mesoderm-inducing signals present during gastrulation in Xenopus laevis

During gastrulation, the vertebrate embryo is patterned and shaped by complex signaling pathways and morphogenetic movements. One of the first regions defined during gastrulation is the prospective notochord, which exhibits specific cell behaviors that drive the extension of the embryonic axis. To examine the signals involved in notochord formation in Xenopus laevis and the competence of cells to respond to these signals, we performed cell transplantation experiments during gastrulation. Labeled cells from the prospective notochord, somitic mesoderm, ventrolateral mesoderm, neural ectoderm, and epidermis, between stages 9 (pregastrulation) and 12 (late gastrulation), were grafted into the prospective notochord region of the early gastrula. We show that cells from each region are competent to respond to notochord-inducing signals and differentiate into notochordal tissue. Cells from the prospective neural ectoderm are the most responsive to notochord-inducing signals, whereas cells from the ventrolateral and epidermal regions are the least responsive. We show that at the end of gastrulation, while transplanted cells lose their competence to form notochord, they remain competent to form somites. These results demonstrate that at the end of gastrulation cell fates are not restricted within germ layers. To determine whether notochord-inducing signals are present throughout gastrulation, grafts were made into progressively older host embryos. We found that regardless of the age of the host, grafted cells from each region give rise to notochordal tissue. This indicates that notochord-inducing signals are present throughout gastrulation and that these signals overlap with somite-inducing signals at the end of gastrulation. We conclude that it is the change of competence that restricts cells to specific tissues rather than the regulation of the inducing signals.     

Duration of competence and inducing capacity of blastomeres in notochord induction during ascidian embryogenesis

Notochord cells in ascidian embryos are formed by the inducing action of cells of presumptive endoderm, as well as neighboring presumptive notochord, at the 32-cell stage. Studies of the timing of induction using recombinations of isolated blastomeres have suggested that notochord induction must be initiated before the decompaction of blastomeres at the 32-cell stage and is completed by the 64-cell stage. However, it is not yet clear how the duration of notochord induction is strictly limited. In the present paper, the aim was to determine in detail when the presumptive notochord blastomeres lost their competence to respond, and when the presumptive endoderm blastomeres produced inducing signals for the notochord. Presumptive notochord blastomeres and presumptive endoderm blastomeres were isolated from early 32-cell embryos, and were heterochronously recombined at various stages ranging from the early 32-cell stage to the 64-cell stage. Presumptive notochord blastomeres could respond to inductive signals at the early 32-cell stage, and started to lose their responsiveness at the decompaction stage. By contrast, the presumptive endoderm blastomeres persisted in their inducing capacity even at the 64-cell stage. These observations suggest that the loss of competence in presumptive notochord blastomeres limits the duration of notochord induction in intact ascidian embryos.     

Evolution of predetermined germ cells in vertebrate embryos: implications for macroevolution

The germ line is established in animal embryos with the formation of primordial germ cells (PGCs), which give rise to gametes. Therefore, the need to form PGCs can act as a developmental constraint by inhibiting the evolution of embryonic patterning mechanisms that compromise their development. Conversely, events that stabilize the PGCs may liberate these constraints. Two modes of germ cell determination exist in animal embryos: (a) either PGCs are predetermined by the inheritance of germ cell determinants (germ plasm) or (b) PGCs are formed by inducing signals secreted by embryonic tissues (i.e., regulative determination). Surprisingly, among the major extant amphibian lineages, one mechanism is found in urodeles and the other in anurans. In anuran amphibians PGCs are predetermined by germ plasm; in urodele amphibians PGCs are formed by inducing signals. To determine which mechanism is ancestral to the tetrapod lineage and to understand the pattern of inheritance in higher vertebrates, we used a phylogenetic approach to analyze basic morphological processes in both groups and correlated these with mechanisms of germ cell determination. Our results indicate that regulative germ cell determination is a property of embryos retaining ancestral embryological processes, whereas predetermined germ cells are found in embryos with derived morphological traits. These correlations suggest that regulative germ cell formation is an important developmental constraint in vertebrate embryos, acting before the highly conserved pharyngula stage. Moreover, our analysis suggests that germ plasm has evolved independently in several lineages of vertebrate embryos.     

The chicken RaxL gene plays a role in the initiation of photoreceptor differentiation

The paired type homeodomain gene, Rax, was previously identified as a key molecule in early eye formation in mice and humans. We report the expression patterns of two Rax family members from chicken, Rax and RaxL, and on the function of RaxL in photoreceptor development. Both Rax and RaxL are expressed in early retinal progenitor cells, with Rax being expressed at a significantly higher level than RaxL. At the time that photoreceptors begin to form, RaxL appears at a relatively high level in a subset of cells within the zone of proliferating progenitor cells. Subsequently, it is expressed in cells migrating to the photoreceptor layer, where it is highly expressed during the initial, but not late, stages of photoreceptor differentiation. To test the function of RaxL, a putative dominant-negative allele of RaxL comprising a fusion of the engrailed repressor domain and a region of RaxL (EnRaxLDeltaC) was introduced in vivo into the early chick eye using a retroviral vector. EnRaxLDeltaC, but not the dominant negative Rax (EnRaxDeltaC), caused a significant reduction in expression of early markers of photoreceptor cells. Examination of the transactivation activity of RaxL on a reporter construct bearing a canonical photoreceptor-specific enhancer element showed that RaxL exhibited significant activation activity, and that this activity was severely diminished in the presence of EnRaxLDeltaC. The effect on photoreceptor gene expression in vivo was specific in that other cell types were unaffected, as was general proliferation in the retina. The reduction in numbers of cells expressing photoreceptor markers was probably due to decreased survival of developing photoreceptor cells, as there was increased apoptosis among cells of the retina expressing dominant-negative RaxL. We propose that RaxL plays a role in the initiation of differentiation, and also possibly commitment, of photoreceptor cells in the chicken retina.     

Isolation of progenitor cells from GFP-transgenic pigs and transplantation to the retina of allorecipients

Work in rodents has demonstrated that progenitor transplantation can achieve limited photoreceptor replacement in the mammalian retina; however, replication of these findings on a clinically relevant scale requires a large animal model. To evaluate the ability of porcine retinal progenitor cells to survival as allografts and integrate into the host retinal architecture, we isolated donor cells from fetal green fluorescent protein (GFP)-transgenic pigs. Cultures were propagated from the brain, retina, and corneo-scleral limbus. GFP expression rapidly increased with time in culture, although lower in conjunction with photoreceptor markers and glial fibrillary acid protein (GFAP), thus suggesting downregulation of GFP during differentiation. Following transplantation, GFP expression allowed histological visualization of integrated cells and extension of fine processes to adjacent plexiform layers. GFP expression in subretinal grafts was high in cells expressing vimentin and lower in cells expressing photoreceptor markers, again consistent with possible downregulation during differentiation. Cells survived transplantation to the injured retina of allorecipients at all time points examined (up to 10 weeks) in the absence of exogenous immune suppression without indications of rejection. These findings demonstrate the feasibility of allogeneic progenitor transplantation in a large mammal and the utility of the pig in ocular regeneration studies.     

Malformation of Embryonic Neural Retina Elicited by BrdU

Exposure of neural retina tissue from early chick embryos (5 and 6-days) to 5-bromo-2'-deoxyuridine (BrdU) for 24 h irreversibly prevented normal histogenesis and resulted in the formation of chaotically disorganized tissue. The sensitivity of the retina to this effect decreased with embryonic age and declined sharply after the commencement of cell stratification. Examination by electron microscopy revealed the following progressive morphologic changes resulting from BrdU treatment: complete breakdown of the outer limiting membrane due to disappearance of its constituent tight junctions which normally anchor cells at the outer retinal surface; collapse and endocytosis of cilia, resulting in the absence of photoreceptor processes; increasing disorganization of the cells which commenced at the outer surface of the retina and progressed inward, resulting in chaotic distortion of the histologic architecture of the retina. Ultrastructural differences were noted between cells in the malformed retina, indicating the presence of several cell types. Possible mechanisms of this BrdU-elicited malformation are considered in the Discussion.     

Control of photoreceptor development.

Recent studies of cell type determination in the vertebrate retina suggest that rod photoreceptor development involves interactions among cells that are mediated, at least in part, by temporally regulated diffusible signals. In this review the strategies used to generate rods in the vertebrate retina are compared with those described for photoreceptor development in the Drosophila retina.     

Differential CRX and OTX2 expression in human retina and retinoblastoma

The histogenesis of retinoblastoma tumors remains controversial, with the cell-of-origin variably proposed to be an uncommitted retinal progenitor cell, a bipotent committed cell, or a cell committed to a specific lineage. Here, we examine the expression of two members of the orthodenticle family implicated in photoreceptor and bipolar cell differentiation, cone-rod homeobox, CRX, and orthodenticle homeobox 2, OTX2, in normal human retina, retinoblastoma cell lines and retinoblastoma tumors. We show that CRX and OTX2 have distinct expression profiles in the developing human retina, with CRX first expressed in proliferating cells and cells committed to the bipolar lineage, and OTX2 first appearing in the photoreceptor lineage. In the mature retina, CRX levels are highest in photoreceptor cells whereas OTX2 is preferentially found in bipolar cells and in the retinal pigmented epithelium. Both CRX and OTX2 are widely expressed in retinoblastoma cell lines and in retinoblastoma tumors, although CRX is more abundant than OTX2 in the differentiated elements of retinoblastoma tumors such as large rosettes, Flexner-Wintersteiner rosettes and fleurettes. Widespread expression of CRX and OTX2 in retinoblastoma tumors and cell lines suggests a close link between the cell-of-origin of retinoblastoma tumors and cells expressing CRX and OTX2.     

Ultracytochemical study on in vitro differentiation of neural retinal cells of chick embryos

In order to study cell differentiation and morphogenesis of neural retina, ultracytochemical examination for acetylcholine esterase (AChE) was carried out on neural retinal cells from 6-day-old chick embryos cultured in monolayer for 20 days. AChE is a suitable marker for identifying cell specificity and structure of cultured neural retinal cells, because its specific localization in the intact chick neural retina has been established. After about 2 weeks of culturing a number of cell aggregates formed on the monolayer sheet of glial cells, in which cell bodies were generally located on the periphery regions while their cellular processes were in the center, forming neuropil structures. Among such peripherally located cells presumptive ganglion, amacrine, bipolar, and photoreceptor cells could be distinguished. In the neuropil structures, some cellular processes had typical ribbon synapses indicating that these structures correspond to the plexiform layers of the retina. We could also classify the neuropils into two types of both from the AChE activity and from the structure of the nerve terminals. These findings indicate that our cell culture system can be used for the study of cell differentiation and histogenesis of retinal cells.     

Cell disorganization and malformation in neural retina caused by antibodies to R-cognin: ultrastructural study

Retina tissue from 6-day chick embryos was organ-cultured for 3 days in the presence of antibodies to R-cognin, a surface antigen of retina cells. The antibodies which are known to bind to this antigen caused a striking malformation: interruption of the outer limiting membrane and extensive cell disorganization resulting in exteriorization of many cells and forming of chaotic masses on the surface of the tissue. Controls did not show these effects. These results further confirm that R-cognin is involved in the mechanism of histotypic contacts and recognition of retina cells, and that it plays an essential role in cell organization and histogenesis in the retina.     

Sequential genesis and determination of cone and rod photoreceptors in Xenopus

In this study, we addressed the temporal sequence of photoreceptor fate determination in Xenopus laevis by examining a number of key events during early cone and rod development. We compared the relative timing and spatial pattern of cone and rod specification using a number of cell type-specific markers, including probes to a long wavelength-sensitive opsin which is expressed by the major cone subtype. Our results show that cones are initially more numerous, and can arise in less mature regions of the retina than rods, although both types of photoreceptors begin to express their respective opsins at about the same time. We applied these markers to an assay of cellular determination to identify the stages of embryonic development at which the earliest photoreceptor fates are induced in vivo. The relative birth order of the major cone and rod subtypes was revealed by simultaneous labeling with markers of cell proliferation and terminal differentiation. Although there is much temporal overlap between the periods of cone and rod genesis and determination in Xenopus, we could discern that the earliest cones are both born and determined before the first rods. Thus, even in the rapidly developing retina of Xenopus, photoreceptors achieve their identities in a sequential fashion, suggesting that the inductive cues which determine specific photoreceptor fates may also arise sequentially during development. © 1998 John Wiley & Sons, Inc. J Neurobiol 35: 227–244, 1998     

Role of N-cadherin cell adhesion molecules in the histogenesis of neural retina

We investigated the role of N-cadherin cell adhesion molecules in the histogenesis of the chicken neural retina. In the undifferentiated retina of early embryos, N-cadherin is almost evenly distributed. With differentiation, N-cadherin was gradually localized in particular cell layers. In the 8.5 to 10.5 day embryos, N-cadherin was most abundant in the optic nerve fiber layer, the plexiform layers and the outer limiting membrane. Thereafter, this molecule gradually diminished from most parts of the retina, except in the outer limiting membrane. When incubated with Fab fragments of a polyclonal antibody to N-cadherin, retinas of early embryos tended to dissociate and could not be maintained as a tissue mass. Retinas from older embryos were not dissociated by the Fab, but their morphogenesis was severely affected. We conclude that N-cadherin is essential for maintaining the overall structure of the undifferentiated retina, but during development, its role becomes restricted to maintaining more specific regions of the tissue. We also suggest that there might be additional, unidentified cadherin-like molecules in the retina.     

Two cellular inductions involved in photoreceptor determination in the Xenopus retina.

Cellular determination in the Xenopus retina is not a strict consequence of cell lineage or cell birthdate. This suggests that a retinal cell gets its fate by either local cellular interactions, diffusible factors, or an indeterminate stochastic mechanism. We have performed an in vitro experiment in which cellular contact is controlled to test the first possibility directly. We use these experiments to demonstrate that two cellular inductions are involved in photoreceptor determination in vitro and that these inductions also occur during development in the retina in vivo. The first interaction is responsible for biasing cells toward either a generic photoreceptor or a cone fate, while the second directs cells toward a rod cell fate.