The lens: a classical model of embryonic induction providing new insights into cell determination in early development

The lens was the first tissue in which the concept of embryonic induction was demonstrated. For many years lens induction was thought to occur at the time the optic vesicle and lens placode came in contact. Since then, studies have revealed that lens placodal progenitor cells are specified already at gastrula stages, much earlier than previously believed, and independent of optic vesicle interactions. In this review, I will focus on how individual signalling molecules, in particular BMP, FGF, Wnt and Shh, regulate the initial specification of lens placodal cells and the progressive development of lens cells. I will discuss recent work that has shed light on the combination of signalling molecules and the molecular interactions that affect lens specification and proper lens formation. I will also discuss proposed tissue interactions important for lens development. A greater knowledge of the molecular interactions during lens induction is likely to have practical benefits in understanding the causes and consequences of lens diseases. Moreover, knowledge regarding lens induction is providing fundamental important insights into inductive processes in development in general.     

Changes in neural and lens competence in Xenopus ectoderm: evidence for an autonomous developmental timer.

The ability of a tissue to respond to induction, termed its competence, is often critical in determining both the timing of inductive interactions and the extent of induced tissue. We have examined the lens-forming competence of Xenopus embryonic ectoderm by transplanting it into the presumptive lens region of open neural plate stage embryos. We find that early gastrula ectoderm has little lens-forming competence, but instead forms neural tissue, despite its location outside the neural plate; we believe that the transplants are being neuralized by a signal originating in the host neural plate. This neural competence is not localized to a particular region within the ectoderm since both dorsal and ventral portions of early gastrula ectoderm show the same response. As ectoderm is taken from gastrulae of increasing age, its neural competence is gradually lost, while lens competence appears and then rapidly disappears during later gastrula stages. To determine whether these developmental changes in competence result from tissue interactions during gastrulation, or are due to autonomous changes within the ectoderm itself, ectoderm was removed from early gastrulae and cultured for various periods of time before transplantation. The loss of neural competence, and the gain and loss of lens competence, all occur in ectoderm cultured in vitro with approximately the same time course as seen in ectoderm in vitro. Thus, at least from the beginning of gastrulation onwards, changes in competence occur autonomously within ectoderm. We propose that there is a developmental timing mechanism in embryonic ectoderm that specifies a sequence of competences solely on the basis of the age of the ectoderm.     

Early tissue interactions leading to embryonic lens formation in Xenopus laevis

Our previous research has demonstrated that lens induction in Xenopus laevis requires inductive interactions prior to contact with the optic vesicle, which classically had been thought to be the major lens inductor. The importance of these early interactions has been verified by demonstrating that lens ectoderm is specified by the time it comes into contact with the optic vesicle. It has been argued that the tissues which underlie the presumptive lens ectoderm during gastrulation and neurulation, dorsolateral endoderm and mesoderm, are the primary early inductors. We show here, however, that these tissues alone cannot elicit lens formation in Xenopus ectoderm. Evidence is presented that presumptive anterior neural plate tissue (which includes the early eye rudiment) is an essential early lens inductor in Xenopus. The presence of dorsolateral mesoderm appears to enhance this response. These findings support a model in which an essential inductive signal passes through the plane of ectoderm during gastrula and early neurula stages from presumptive anterior neural tissue to the presumptive lens ectoderm. Since there is evidence for such interactions within a tissue layer in mesodermal and neural induction as well, this may be a general feature of the initial stages of determination of many tissues.     

Inductive interactions in the spatial and temporal restriction of lens-forming potential in embryonic ectoderm of Xenopus laevis

The process of lens cell determination in amphibians is currently viewed as one involving a series of inductive interactions. On the basis of previous investigations, these interactions are thought to begin during gastrulation when the presumptive foregut endoderm and then the heart mesoderm come into contact with the presumptive lens ectoderm. This earlier period of induction is followed by the later interaction of the optic vesicle with the lens-forming ectoderm. Transplantation experiments were performed to determine the relative significance of the early and later periods of induction in the process of lens cell determination in the anuran Xenopus laevis. Various ectodermal tissues were transplanted either into the lens-forming region of open neural plate stage host embryos or over the newly formed optic vesicle of later neurula stage embryos. All transplanted tissues were labeled with the intracellular marker horseradish peroxidase to assess the exact origins of any induced lens structures. The results indicate that all nonneural ectodermal tissues have some lens-forming potential early during gastrulation; however, this potential is restricted to the lens-forming region, and perhaps nearby regions, later in development during the time of neurulation. Furthermore, the results show that the optic vesicle is not a substantial inductor of the lens in tissues that have not been previously exposed to the earlier series of inductive interactions that take place during gastrulation and neurulation. Since the optic vesicle does not appear to be a sufficient inductor of the lens, these earlier inductive interactions are, therefore, essential in the process of lens cell determination in Xenopus. These earlier inductive interactions lead to a steady increase in what may be called a lens-forming bias in the presumptive lens ectoderm during this period of development. The eventual loss in the ability of nonlens ventral ectoderm to respond to these lens inductors is presumably the result of other determinative processes that occur in this tissue.     

The chemical nature of the factor responsible for embryonic induction

The discovery by Hans Spemann of the “organizer” tissue and its ability to induce the formation of the amphibian embryo’s neural tube inspired leading embryologists to attempt to elucidate embryonic induction’s underlying mechanisms. Since then several studies have described several developmental model system to better understand the role of specific signaling molecules, the interplay of different signals and tissue interactions in regulating tissue induction and patterning events. Different groups of workers set out to subject embryonic amphibian tissues and inductive adult organs to various extraction methods in the hope that the active agents could be isolated and chemically identified. In addition, a large number of well characterized chemical compounds were tested.     

The restriction of the heart morphogenetic field in Xenopus laevis

We have examined the spatial restriction of heart-forming potency in Xenopus laevis embryos, using an assay system in which explants or explant recombinates are cultured in hanging drops and scored for the formation of a beating heart. At the end of neurulation at stage 20, the heart morphogenetic field, i.e., the area that is capable of heart formation when cultured in isolation, includes anterior ventral and ventrolateral mesoderm. This area of developmental potency does not extend into more posterior regions. Between postneurula stage 23 and the onset of heart morphogenesis at stage 28, the heart morphogenetic field becomes spatially restricted to the anterior ventral region. The restriction of the heart morphogenetic field during postneurula stages results from a loss of developmental potency in the lateral mesoderm, rather than from ventrally directed morphogenetic movements of the lateral mesoderm. This loss of potency is not due to the inhibition of heart formation by migrating neural crest cells. During postneurula stages, tissue interactions between the lateral mesoderm and the underlying anterior endoderm support the heart-forming potency in the lateral mesoderm. The lateral mesoderm loses the ability to respond to this tissue interaction by stages 27-28. We speculate that either formation of the third pharyngeal pouch during stages 23-27 or lateral inhibition by ventral mesoderm may contribute to the spatial restriction of the heart morphogenetic field.     

The restriction of the heart morphogenetic field in Xenopus laevis

We have examined the spatial restriction of heart-forming potency in Xenopus laevis embryos, using an assay system in which explants or explant recombinates are cultured in hanging drops and scored for the formation of a beating heart. At the end of neurulation at stage 20, the heart morphogenetic field, i.e., the area that is capable of heart formation when cultured in isolation, includes anterior ventral and ventrolateral mesoderm. This area of developmental potency does not extend into more posterior regions. Between postneurula stage 23 and the onset of heart morphogenesis at stage 28, the heart morphogenetic field becomes spatially restricted to the anterior ventral region. The restriction of the heart morphogenetic field during postneurula stages results from a loss of developmental potency in the lateral mesoderm, rather than from ventrally directed morphogenetic movements of the lateral mesoderm. This loss of potency is not due to the inhibition of heart formation by migrating neural crest cells. During postneurula stages, tissue interactions between the lateral mesoderm and the underlying anterior endoderm support the heart-forming potency in the lateral mesoderm. The lateral mesoderm loses the ability to respond to this tissue interaction by stages 27–28. We speculate that either formation of the third pharyngeal pouch during stages 23–27 or lateral inhibition by ventral mesoderm may contribute to the spatial restriction of the heart morphogenetic field.     

The role of the dorsal lip in the induction of heart mesoderm in Xenopus laevis

We have examined the tissue interactions responsible for the expression of heart-forming potency during gastrulation. By comparing the specification of different regions of the marginal zone, we show that heart-forming potency is expressed only in explants containing both the dorsal lip of the blastopore and deep mesoderm between 30 degrees and 45 degrees lateral to the dorsal midline. Embryos from which both of these 30 degrees-45 degrees dorsolateral regions have been removed undergo heart formation in two thirds of cases, as long as the dorsal lip is left intact. If the dorsal lip is removed along with the 30 degrees-45 degrees regions, heart formation does not occur. These results indicate that the dorsolateral deep mesoderm must interact with the dorsal lip in order to express heart-forming potency. Transplantation of the dorsal lip into the ventral marginal zone of host embryos results in the formation of a secondary axis; in over half of cases, this secondary axis includes a heart derived from the host mesoderm. These findings suggest that the establishment of heart mesoderm is initiated by a dorsalizing signal from the dorsal lip of the blastopore.     

The chemical nature of the factor responsible for embryonic induction: An historical overview

The discovery by Hans Spemann of the “organizer” tissue and its ability to induce the formation of the amphibian embryo’s neural tube inspired leading embryologists to attempt to elucidate embryonic induction’s underlying mechanisms. Since then several studies have described several developmental model system to better understand the role of specific signaling molecules, the interplay of different signals and tissue interactions in regulating tissue induction and patterning events. Different groups of workers set out to subject embryonic amphibian tissues and inductive adult organs to various extraction methods in the hope that the active agents could be isolated and chemically identified. In addition, a large number of well characterized chemical compounds were tested.     

A re-examination of lens induction in chicken embryos: in vitro studies of early tissue interactions

Early studies on lens induction suggested that the optic vesicle, the precursor of the retina, was the primary inducer of the lens; however, more recent experiments with amphibians establish an important role for earlier inductive interactions between anterior neural plate and adjacent presumptive lens ectoderm in lens formation. We report here experiments assessing key inductive interactions in chicken embryos to see if features of amphibian systems are conserved in birds. We first examined the issue of specification of head ectoderm for a lens fate. A large region of head ectoderm, in addition to the presumptive lens ectoderm, is specified for a lens fate before the time of neural tube closure, well before the optic vesicle first contacts the presumptive lens ectoderm. This positive lens response was observed in cultures grown in a wide range of culture media. We also tested whether the optic vesicle can induce lenses in recombinant cultures with ectoderm and find that, at least with the ectodermal tissues we examined, it generally cannot induce a lens response. Finally, we addressed how lens potential is suppressed in non-lens head ectoderm and show an inhibitory role for head mesenchyme. This mesenchyme is infiltrated by neural crest cells in most regions of the head. Taken together, these results suggest that, as in amphibians, the optic vesicle cannot be solely responsible for lens induction in chicken embryos; other tissue interactions must send early signals required for lens specification, while inhibitory interactions from mesenchyme suppress lens-forming ability outside of the lens area.     

Reinvestigation of the role of the optic vesicle in embryonic lens induction

The induction of the lens by the optic vesicle in amphibians is often cited as support for the view that a single inductive event can lead to determination in a multipotent tissue. This conclusion is based on transplantation experiments whose results indicate that many regions of embryonic ectoderm which would normally form epidermis can form a lens if brought into contact with the optic vesicle. Although additional evidence argues that during normal development other tissues, acting before the optic vesicle, also contribute to lens induction, it is still widely held, on the basis of these transplantation experiments, that the optic vesicle alone can elicit lens formation in ectoderm. While testing this conclusion by transplanting optic vesicles beneath ventral ectoderm in Xenopus laevis embryos, it became apparent that contamination of optic vesicles by presumptive lens ectoderm cells can generate lenses in these experiments, illustrating the need for adequate host and donor marking procedures. Since previous studies rarely used host and donor marking, it was not clear whether they actually demonstrated that the optic vesicle can induce lenses. Using careful host and donor marking procedures with horseradish peroxidase as a lineage tracer, we show that the optic vesicle cannot stimulate lens formation in neurula- or gastrula-stage ectoderm of Xenopus laevis. Since the general conclusion that the optic vesicle is sufficient for lens induction rests on studies in many organisms, we felt it was important to begin to test this conclusion in other amphibians as well. Similar experiments were therefore performed with Rana Palustris embryos, since it was in this organism that optic vesicle transplant studies had originally argued that this tissue alone can cause lens induction. Under conditions similar to those used in the original report, but with careful controls to assess the origin of lenses in transplants, we found that the optic vesicle alone cannot elicit lens formation. Our data lead us to propose that the optic vesicle in amphibians is not generally sufficient for lens induction. Instead, we argue that lens induction occurs by a multistep process in which an essential phase in lens determination occurs as a result of inductive interactions preceding contact of ectoderm with the optic vesicle.     

Cell lineages in the embryonic kidney: their inductive interactions and signalling molecules.

The first signalling genes acting in the inductive interactions in the kidney have now been identified. Differentiation of the permanent kidney or the metanephros is critically dependent on inductive signalling between the nephrogenic mesenchyme and ureteric bud epithelium. Further inductive interactions occur between developing nephrons, interstitial stroma, endothelial cells and neurones. Glial-cell-line-derived neurotrophic factor is a signal for the ureteric bud initiation and branching, and Wnt4 is an autocrine epithelializing signal at the pretubular stage of nephron formation. The signals for renal angiogenesis and innervation are less well defined, but seem to include vascular endothelial growth factor and neurotrophins, at least. The ureteric-bud-derived signal for induction of the nephrogenic mesenchyme (to bring the cells to the condensate stage) is not yet known, but fibroblast growth factor 2 is a good candidate. None of the signalling genes identified from the embryonic kidney is specific to the organ, which raises some general questions. How do the organs develop from similar rudiments to various patterns with different cell types and functions? Does the information for organ-specific differentiation pathways retain in the epithelial or mesenchymal compartment? The present, rather fragmentary molecular data would favour the view that similar molecules acting in different combinations and developmental sequences, rather than few organ-specific master genes, could be responsible for the divergence of patterning.     

The specification of heart mesoderm occurs during gastrulation in Xenopus laevis

The establishment of heart mesoderm during Xenopus development has been examined using an assay for heart differentiation in explants and explant combinations in culture. Previous studies using urodele embryos have shown that the heart mesoderm is induced by the prospective pharyngeal endoderm during neurula and postneurula stages. In this study, we find that the specification of heart mesoderm must begin well before the end of gastrulation in Xenopus embryos. Explants of prospective heart mesoderm isolated from mid- or late neurula stages were capable of heart formation in nearly 100% of cases, indicating that the specification of heart mesoderm is complete by midneurula stages. Moreover, inclusion of pharyngeal endoderm had no statistically significant effect upon either the frequency of heart formation or the timing of the initiation of heartbeat in explants of prospective heart mesoderm isolated after the end of gastrulation. When the superficial pharyngeal endoderm was removed at the beginning of gastrulation, experimental embryos formed hearts, as did explants of prospective heart mesoderm from such embryos. These results indicate that the inductive interactions responsible for the establishment of heart mesoderm occur prior to the end of gastrulation and do not require the participation of the superficial pharyngeal endoderm.     

Simulation study on effects of signaling network structure on the developmental increase in complexity

The developmental increase in structural complexity in multicellular lifeforms depends on local, often non-periodic differences in gene expression. These, in turn, depend on a network of gene-gene interactions coded within the organismal genome. To see what architectural features of a network (size, connectivity, etc.) affect the likelihood of patterns with multiple cell types (i.e. patterns where cells express > or = 3 different combinations of genes), developmental pattern formation was simulated in virtual blastoderm embryos with small artificial genomes. Several basic properties of these genomic signaling networks, such as the number of genes, the distributions of positive (inductive) and negative (repressive) interactions, and the strengths of gene-gene interactions were tested. The results show that the frequencies of complex and/or stable patterns depended not only on the existence of negative interactions, but also on the distribution of regulatory interactions: for example, coregulation of signals and their intracellular effectors increased the likelihood of pattern formation compared to differential regulation of signaling pathway components. Interestingly, neither quantitative differences in strengths of signaling interactions nor multiple response thresholds to different levels of signal concentration (as in morphogen gradients) were essential for formation of multiple, spatially unique "cell types". However, those combinations of architectural features that greatly increased the likelihood for pattern complexity tended to decrease the likelihoods for pattern stability and developmental robustness. Nevertheless, elements of complex patterns (e.g. genes, cell type order within the pattern) could differ in their developmental robustness, which may be important for the evolution of complexity. The results show that depending on the network structure, the same set of genes can produce patterns of different complexity, robustness and stability. Because of this, the evolution of metazoan complexity with a combinatorial code of gene regulation may have depended at least as much on selection for favorable distribution of connections between existing developmental regulatory genes as on the simple increase in numbers of regulatory genes.     

Induction of the Eye Lens

In general terms embryonic induction involves the association of embryonic tissues and leads to tissue differentiation. It is one of the known essential processes leading to the normal development of embryos. However, despite its importance, very little is known about the mechanisms of inductive interactions. For example, what is the nature of communication between tissues, how does this communication effect the synthetic activity of the cells, and once a new pattern of synthesis has been established how is the sequence of events leading to tissue differentiation co-ordinated? The answers to these questions will come only from the intensive study of inductive interactions and tissue differentiation at all levels from the morphological to the molecular.
One of the best known examples of induction, at least superficially, is the differentiation of lens from head ectoderm after its interaction with optic vesicle. The popularity of this tissue with embryologists may be attributed to its accessibility of manipulation because of its position on the outside of the embryo. In addition, its distinct morphology and specific biochemical composition make it relatively easy to determine whether the lens differentiates after experimental treatment. About the turn of this century lens differentiation was thought to depend on the specific interaction of just two embryonic tissues, head ectoderm and neuro-ectoderm (optic vesicle). However, experimental analysis since then has revealed that this oversimplified view of lens induction is incorrect. In fact there is evidence that a large number of other tissues besides embryonic head ectoderm can differentiate into lens and that other conditions besides the presence of optic vesicle can induce lens differentiation. The purpose of this work is to review the evidence on lens induction and based on this, to determine what we know about the mechanism(s) controlling this process.     

Repeated evolution of bacteriocytes in lygaeoid stinkbugs

Host–microbe symbioses often evolved highly complex developmental processes and colonization mechanisms for establishment of stable associations. It has long been recognized that many insects harbour beneficial bacteria inside specific symbiotic cells (bacteriocytes) or organs (bacteriomes). However, the evolutionary origin and mechanisms underlying bacterial colonization in bacteriocyte/bacteriome formation have been poorly understood. In order to uncover the origin of such evolutionary novelties, we studied the development of symbiotic organs in five stinkbug species representing the superfamily Lygaeoidea in which diverse bacteriocyte/bacteriome systems have evolved. We tracked the symbiont movement within the eggs during the embryonic development and determined crucial stages at which symbiont infection and bacteriocyte formation occur, using whole-mount fluorescence in situ hybridization. In summary, three distinct developmental patterns were observed: two different modes of symbiont transfer from initial symbiont cluster (symbiont ball) to presumptive bacteriocytes in the embryonic abdomen, and direct incorporation of the symbiont ball without translocation of bacterial cells. Across the host taxa, only closely related species seemed to have evolved relatively conserved types of bacteriome development, suggesting repeated evolution of host symbiotic cells and organs from multiple independent origins.     

Epidermal patterning and induction of different hair types during mouse embryonic development

An intriguing question in developmental biology is how epidermal pattern formation processes are established and what are the molecular mechanisms involved in these events. The establishment of the pattern is concomitant with the formation of ectodermal appendages, which involves complex interactions between the epithelium and the underlying mesenchyme. Among ectodermal appendages, hair follicles are the “mini organs” that produce hair shafts. Several developmental and structural features are common to all hair follicles and to the hair shaft they produce. However, many different hair types are produced in a single organism. Also, different characteristics can be observed depending on the part of the body where the hair follicle is formed. Here, we review the mechanisms involved in the patterning of different hair types during mouse embryonic development as well as the influence of the body axes on hair patterning. Birth Defects Research (Part C) 87:263–272, 2009. © 2009 Wiley‐Liss, Inc.