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Understanding the process of regeneration has been one of the longstanding scientific aims, from a fundamental biological perspective, as well as within the applied context of regenerative medicine. Because regeneration competence varies greatly between organisms, it is essential to investigate different experimental animals. The free-living marine flatworm Macrostomum lignano is a rising model organism for this type of research, and its power stems from a unique set of biological properties combined with amenability to experimental manipulation. The biological properties of interest include production of single-cell fertilized eggs, a transparent body, small size, short generation time, ease of culture, the presence of a pluripotent stem cell population, and a large regeneration competence. These features sparked the development of molecular tools and resources for this animal, including high-quality genome and transcriptome assemblies, gene knockdown, in situ hybridization, and transgenesis. Importantly, M. lignano is currently the only flatworm species for which transgenesis methods are established. This review summarizes biological features of M. lignano and recent technological advances towards experimentation with this animal. In addition, we discuss the experimental potential of this model organism for different research questions related to regeneration and stem cell biology.

The idea of regenerating injured body parts has captivated human imagination for centuries, and the topic still remains an area of extensive scientific research. This review focuses on the process of lens regeneration: its history, our current knowledge, and the questions that remain unanswered. By highlighting some of the milestones that have shaped our understanding of this phenomenon and the contributions of scientists who have dedicated their lives to investigating these questions, we explore how regeneration enquiry evolved into the science it is today, and how technological advances accelerated our understanding of these remarkable processes.

Humans (at least a select few) have long known about the cephalochordate amphioxus, first as something to eat and later as a subject for scientific study. The rate of publication on these animals has waxed and waned several times. The first big surge, in the late nineteenth century, was stimulated by Darwin's evolutionary ideas and by Kowalevsky's embryologic findings suggesting that an amphioxus-like creature might have bridged the gap between the invertebrates and the vertebrates. Interest declined sharply in the early twentieth century and remained low for the next 50 years. An important contributing factor (in addition to inhibition by two world wars and the Great Depression) was the indifference of the new evolutionary synthesis toward broad phylogenetic problems like the origin of the vertebrates. Then, during the 1960s and 1970s, interest in amphioxus resurged, driven especially by increased government support for basic science as well as opportunities presented by electron microscopy. After faltering briefly in the 1980s (electron microscopists were running out of amphioxus tissues to study), a third and still-continuing period of intensive amphioxus research began in the early 1990s, stimulated by the advent of evolutionary developmental biology (evo-devo) and genomics. The volume of studies peaked in 2008 with the publication of the genome of the Florida amphioxus. Since then, although the number of papers per year has dropped somewhat, sequencing of additional genomes and transcriptomes of several species of amphioxus (both in the genus Branchiostoma and in a second genus, Asymmetron) is providing the raw material for addressing the major unanswered question of the relationship between genotype and phenotype.

The amphioxus (lancelet) was first described by Pallas in 1774 and incorrectly assigned to mollusks. Since then, amphioxus attracted generations of zoologists. It took however almost one hundred years until Alexander Kowalevsky recognized that the larval stages of amphioxus had much in common with vertebrate embryos. Widely studied around 1900 as the 'elementary vertebrate', amphioxus as a model went out of fashion with the decline of comparative anatomy. Due to the scarcity of taxa at the invertebrate-to-vertebrate transition, amphioxus nevertheless remained the species with a privileged position in animal phylogeny. Its resurrection as the popular model of evolutionary developmental biology came with the advent of modern molecular biology and genomics. In the 1990s amphioxus developmental control genes were identified and characterized at a fast pace with the hope that such studies could provide novel insight into an important evolutionary transition: the origin of vertebrates. Indeed, amphioxus was found to be vertebrate-like but much simpler. Its body resembles that of the vertebrate, but it lacks most of the complexities associated with typical vertebrate organs. Its genome is only 1/6 of the human genome and it has not undergone the whole genome duplications that occurred in the vertebrate lineage. For all of these reasons, amphioxus became widely regarded as a useful proxy for the primitive ancestor of all vertebrates. A persistent problem interpreting amphioxus in the phylogenetic context is the difficulty to distinguish ancestral features, and those that are secondarily derived. There is no doubt that an integrative approach combining information from various disciplines is needed in order to help resolve such issues. Anatomy and comparative morphology has always been strong since the dawn of amphioxus research. Recent developments such as the availability of genomic sequences for three Branchiostoma species, established laboratory cultures of amphioxus that can be spawned at the investigator's will, or techniques allowing transgenesis and gene knockouts represent a major leap for studies on how the genotype generates a phenotype. These advances also enable the smooth transition of amphioxus from the model system of a distinguished past into the one with a very bright future.

The cloning and embryonic expression analysis of an amphioxus Hox gene in 1992 marked the start of molecular analysis of cephalochordate development. Other papers quickly followed, including a description of the amphioxus Hox gene cluster in 1994, fuelling a resurgence of interest in a long-forgotten animal. I describe the academic background, laboratory experiments and field work leading to the earliest publications in amphioxus molecular developmental biology and explain their scientific impact. The story of amphioxus biology in the 1990s involved collaboration, team work, opportunistic meetings, serendipity, incredulous journal editors, stingrays, airport departure lounges and popcorn.

The rabbit has long been used as a laboratory animal model for developing reproductive and stem cell-related technologies, as well as for studying human disease. The Oct4 transcription factor plays a crucial role in the maintenance and regulation of pluripotency in embryos and stem cells. We constructed a reporter plasmid containing the gene encoding the enhanced green fluorescent protein (EGFP) under the control of the rabbit Oct4 promoter (prOG) and transfected it into E14 mouse stem cells and rabbit ESCs. In addition, prOG transgenic fibroblasts were derived and prOG transgenic rabbits were produced by somatic cell nuclear transfer (SCNT). The pattern of expression of ectopic EGFP was similar in E14 mouse stem cells whether under the control of the rabbit (prOG) or mouse Oct4 promoter (pmOG). EGFP expression was observed in rabbit ESCs following prOG transfection. Both prOG transgenic SCNT embryos and F1 prOG transgenic embryos derived from adult transgenic rabbits expressed green fluorescence at the morula and blastocyst stages. EGFP was clearly detected in gonads isolated from fetuses at 27 dpc. The prOG transgenic rabbit represents a new model for studying the derivation and maintenance of rabbit pluripotent cells, and for investigating rabbit embryo development.

A predominant theme in much of evolutionary biology is that organisms are the product of relentless and precise natural selection among them, and that life is about the competition of all-against-all for success. However, developmental genetics has rapidly been revealing a very different picture of the nature of life. The organizing principles by which organisms are made are thoroughly based on complex hierarchies of molecular interactions that require multiple factors to be relentlessly cooperating with each other. Reconciling these two points of view involves changing the scale of observation, and a different understanding of evolution, in which cooperation and tolerance are more important than competition and intolerance.

Developmental Biology has established itself as a solid field of teaching and research in Portugal. Its history is recent, generally considered to have started with the pioneering work of Augusto Celestino da Costa at the beginning of the 20th century. However, research groups were very few and, until the early 1990s, teaching beyond morphological and comparative embryology was uncommon. In 1994, the first university course dedicated to Developmental Biology as a separate field from Embryology was created at the Faculty of Sciences of the University of Lisbon and a course on Plant Differentiation and Morphogenesis was also initiated. A Masters programme in Developmental Biology followed at the Lusofona University in 1996. Subsequently, modules of Developmental Biology were included in many Embryology courses and eventually more Developmental Biology courses were created. From 1999 onwards, the number of research groups working in Developmental Biology started to increase, many of which were initiated by researchers who had had the opportunity to pursue their PhD and/or post-doc studies abroad. The Instituto Gulbenkian de Cincia (Gulbenkian Institute of Science) became the first home of most of these groups, but several later spread to other institutions. This increased activity in turn has stimulated teaching of Developmental Biology and more students have been getting interested in the field. This positive feedback loop makes it a nice time to be teaching and working in Developmental Biology in Portugal.

This paper presents a novel perspective of Robotic Stem Cells (RSCs), defined as the basic non-biological elements with stem cell like properties that can self-reorganize to repair damage to their swarming organization. Self here means that the elements can autonomously decide and execute their actions without requiring any preset triggers, commands, or help from external sources. We develop this concept for two purposes. One is to develop a new theory for self-organization and self-assembly of multi-robots systems that can detect and recover from unforeseen errors or attacks. This self-healing and self-regeneration is used to minimize the compromise of overall function for the robot team. The other is to decipher the basic algorithms of regenerative behaviors in multi-cellular animal models, so that we can understand the fundamental principles used in the regeneration of biological systems. RSCs are envisioned to be basic building elements for future systems that are capable of self-organization, self-assembly, self-healing and self-regeneration. We first discuss the essential features of biological stem cells for such a purpose, and then propose the functional requirements of robotic stem cells with properties equivalent to gene controller, program selector and executor. We show that RSCs are a novel robotic model for scalable self-organization and self-healing in computer simulations and physical implementation. As our understanding of stem cells advances, we expect that future robots will be more versatile, resilient and complex, and such new robotic systems may also demand and inspire new knowledge from stem cell biology and related fields, such as artificial intelligence and tissue engineering.

It is more than fifty years since Alan Turing first presented the reaction-diffusion (RD) model, to account for the mechanism of biological pattern formation. In the paper entitled "The chemical basis of morphogenesis", Turing concluded that spatial patterns autonomously made in the embryo are generated as the stationary wave of the chemical (cellular) reactions. Although this novel idea was paid little attention by experimental biologists, recent experimental data are suggesting that the RD mechanism really functions in some of the course of animal development. Among the phenomena in which involvement of the RD mechanism is suspected, the striped pigment pattern of zebrafish has been highlighted as an ideal model system for the following reasons: the stationary wave made by the RD mechanism stays alive and can be observed only in the fish skin; and in zebrafish, we can utilize genomic information and molecular genetic techniques to clarify the molecular basis of pattern formation. In this review, we summarize recent progresses in the study of zebrafish pigment pattern formation that is uncovering how the RD wave is made and maintained in the skin.

This article considers the role played by a core set of "dynamical patterning modules" (DPMs) in the origination, development and evolution of complex organisms. These consist of the products of a subset of the genes of what has come to be known as the "developmental-genetic toolkit" in association with physical processes they mobilize. The physical processes are those characteristic of chemically and mechanically excitable mesoscopic systems like cell aggregates: cohesion, viscoelasticity, diffusion, spatiotemporal heterogeneity based on activator-inhibitor interaction, and multistable and oscillatory dynamics. We focus on the emergence of the Metazoa, and show how toolkit gene products and pathways that pre-existed the metazoans acquired novel morphogenetic functions simply by virtue of the change in scale and context inherent to multicellularity. We propose that DPMs, acting singly and in combination with each other, constitute a "pattern language" capable of generating all metazoan body plans and organ forms. This concept implies that the multicellular organisms of the late Precambrian-early Cambrian were phenotypically plastic, fluently exploring morphospace in a fashion decoupled from both function-based selection and genotypic change. The relatively stable developmental trajectories and morphological phenotypes of modern organisms, then, are considered to be products of stabilizing selection. This perspective solves the apparent "molecular homology-analogy paradox," whereby widely divergent modern animal types utilize the same molecular toolkit during development, but it does so by inverting the neo-Darwinian principle that phenotypic disparity was generated over long periods of time in concert with, and in proportion to genotypic change.

The development of multicellular organisms involves the partitioning of the organism into territories of cells of specific structure and function. The information for spatial patterning processes is directly encoded in the genome. The genome determines its own usage depending on stage and position, by means of interactions that constitute gene regulatory networks (GRNs). The GRN driving endomesoderm development in sea urchin embryos illustrates different regulatory strategies by which developmental programs are initiated, orchestrated, stabilized or excluded to define the pattern of specified territories in the developing embryo.

Differentiation of the Drosophila compound eye from the eye imaginal disc is a progressive process: columns of cells successively differentiate in a posterior to anterior sequence, clusters of cells form at regularly spaced intervals within each column, and individual photoreceptors differentiate in a defined order within each cluster. The progression of differentiation across the eye disc is driven by a positive autoregulatory loop of expression of the secreted molecule Hedgehog, which is temporally delayed by the intercalation of a second signal, Spitz. Hedgehog refines the spatial position at which each column initiates its differentiation by inducing secondary signals that act over different ranges to control the expression of positive and negative regulators. The position of clusters within each column is controlled by secreted inhibitory signals from clusters in the preceding column, and a single founder neuron, R8, is singled out within each cluster by Notch-mediated lateral inhibition. R8 then sequentially recruits surrounding cells to differentiate by producing a short-range signal, Spitz, which induces a secondary short-range signal, Delta. Intrinsic transcription factors act in combination with these two signals to produce cell-type diversity within the ommatidium. The Hedgehog and Spitz signals are transported along the photoreceptor axons and reused within the brain as long-range and local cues to trigger the differentiation and assembly of target neurons.

In systems undergoing chemical reaction and diffusion, a remarkable variety of spatially structured patterns, stationary or moving, local or global, can arise, many of them reminiscent of forms and phenomena seen in living systems. Chemical systems offer the advantage that one can often control the parameters that determine the patterns formed and can thereby probe fundamental issues about pattern formation, with possible insights into biologically relevant phenomena. We present experimental examples and discuss several mechanisms by which such spatiotemporal structure may arise, classifying the mechanisms according to the type of instability that results in pattern formation. In some systems, the pattern that emerges depends not only on the chemical and physical parameters but also on the initial state of the system. Interactions between instabilities can result in particularly complex patterns.

Hox genes are known to specify spatial identities along the anterior-posterior axis during embryogenesis. In vertebrates and most other deuterostomes, they are arranged in sets of uninterrupted clusters on chromosomes, and are in most cases expressed in a "colinear" fashion, in which genes closer to the 3-end of the Hox clusters are expressed earlier and more anteriorly and genes close to the 5-end of the clusters later and more posteriorly. In this review, we summarize the current understanding of how Hox gene clusters have been modified from basal lineages of deuterostomes to diverse taxa of vertebrates. Our parsimony reconstruction of Hox cluster architecture at various stages of vertebrate evolution highlights that the variation in Hox cluster structures among jawed vertebrates is mostly due to secondary lineage-specific gene losses and an additional genome duplication that occurred in the actinopterygian stem lineage, the teleost-specific genome duplication (TSGD).

The developing limb is a major model for pattern formation in vertebrate embryos. Many of the seminal discoveries of the mechanisms involved in patterning have been made using chick embryos because of the ease of manipulating their developing limbs. More recently, the molecular basis of limb pattern formation has been increasingly uncovered and now, with the availability of genomic resources, the genetic approaches available are even more powerful. Nevertheless, since the limb is ultimately built of cells, gene action must ultimately be translated into cell behaviour and a major challenge will be to integrate genetics with molecular and cellular biology. In this review, we will first outline the stages in limb development, the major interacting signalling pathways that pattern the limb and the molecules involved. We will describe fate maps of the developing limb, and discuss what is known about cellular activities including proliferation, death, adhesiveness, communication and migration during the patterning process. Finally we will explore how these cell activities produce form.

Basic research in pattern formation is concerned with the generation of phenotypes and tissues. It can therefore lead to new tools for medical research. These include phenotypic screening assays, applications in tissue engineering, as well as general advances in biomedical knowledge. Our aim here is to discuss this emerging field with special reference to tools based on zebrafish developmental biology. We describe phenotypic screening assays being developed in our own and other labs. Our assays involve: (i) systemic or local administration of a test compound or drug to zebrafish in vivo; (ii) the subsequent detection or "readout" of a defined phenotypic change. A positive readout may result from binding of the test compound to a molecular target involved in a developmental pathway. We present preliminary data on assays for compounds that modulate skeletal patterning, bone turnover, immune responses, inflammation and early-life stress. The assays use live zebrafish embryos and larvae as well as adult fish undergoing caudal fin regeneration. We describe proof-of-concept studies on the localised targeting of compounds into regeneration blastemas using microcarriers. Zebrafish are cheaper to maintain than rodents, produce large numbers of transparent eggs, and some zebrafish assays could be scaled-up into medium and high throughput screens. However, advances in automation and imaging are required. Zebrafish cannot replace mammalian models in the drug development pipeline. Nevertheless, they can provide a cost-effective bridge between cell-based assays and mammalian whole-organism models.

Patterns are orders embedded in randomness; they may appear in spatial arrangements or in temporal sequences, and each element may appear identical or with variations. Patterns exist in the physical world as well as in living systems. In the biological world, patterns can range from simple to complex, forming the basic building blocks of life. When we see patterns in peacock feathers, leopard spots or zebra stripes, we are fascinated by the order, the variations and the beauty. The process that generates this ordering in the biological world has been termed Pattern Formation. Since Lewis Wolpert promoted this concept four decades ago, scientists from molecular biology, developmental biology, stem cell biology, tissue engineering, theoretical modeling and other disciplines have made remarkable progress towards understanding its underlying mechanisms. We have learned that both molecular processes and physico-chemical principles are important for biological Pattern Formation and as Guest Editors, felt that it is time to review and re-integrate our understanding of this fundamental and fantastic process.

Developmental Biology in Portugal and Spain has been running along mostly parallel tracks - until recently. It is the youngest generations who, driven by an increasing interest in the science and culture of each other's country, are bringing the situation to its natural state: one of strong and fruitful collaboration between Iberian labs.

In this article we will discuss the integration of developmental patterning mechanisms with waves of competency that control the ability of a homogeneous field of cells to react to pattern forming cues and generate spatially heterogeneous patterns. We base our discussion around two well known patterning events that take place in the early embryo: somitogenesis and feather bud formation. We outline mathematical models to describe each patterning mechanism, present the results of numerical simulations and discuss the validity of each model in relation to our example patterning processes.

Pallid anchovy fillet, friendly filtering, peacefully laying and little lancelet are some of the nicknames and adjectives the cephalochordate amphioxus has received throughout the last two centuries. Traditionally regarded as the living representative of the last ancestor of vertebrates, amphioxus has recently been promoted to the privileged position of being the most ancient chordate. The preliminary analysis of its prototypical genome is nearly completed, and its hidden secrets towards the understanding of the primitive chordate and deuterostome genomes will soon see the light. Amphioxus embryonic development and body plan have remained in evolutionary stasis since the cephalochordate lineage split from the chordate ancestor about 500 million years ago. In contrast, amphioxus research is far from being at a standstill; in Europe, thanks to the international cooperation and the Banyuls Oceanographic Station, amphioxus embryos are obtained on demand during the spawning season. We summarise here our progress towards the dream of the experimental manipulation of the amphioxus embryo, to enter the era of Experimental Evo-Devo.

Developmental biology and/or embryology prospectuses in Spanish universities are reviewed. From old masterly classes to virtual teaching, a variety of methods are being used nowadays to teach these subjects. In a country like Spain, adapting to the European Higher Education Space, old and new teaching methods are educational alternatives in a university model which respects both tradition and modernity. In this report, consensus guidelines, concluded from this general survey, are suggested for teachers to ensure future progress.

The control of hair growth in the adult mammalian coat is a fascinating topic which has just begun to be explored with molecular genetic tools. Complex hair cycle domains and regenerative hair waves are present in normal adult (> 2 month) mice, but more apparent in mutants with cyclic alopecia phenotypes. Each hair cycle domain consists of initiation site(s), a propagating wave and boundaries. By analyzing the dynamics of hair growth, time required for regeneration after plucking, in situ hybridization and reporter activity, we showed that there is oscillation of intra-follicular Wnt signaling which is synchronous with hair cycling, and there is oscillation of dermal bone morphogenetic protein (BMP) signaling which is asynchronous with hair cycling. The interactions of these two rhythms lead to the recognition of refractory and competent phases in the telogen, and autonomous and propagating phases in the anagen. Boundaries form when propagating anagen waves reach follicles which are in refractory telogen. Experiments showed that Krt14-Nog mice have shortened refractory telogen and simplified wave dynamics. Krt14-Nog skin grafts exhibit non-autonomous interactions with surrounding host skin. Implantation of BMP coated beads into competent telogen skin prevents hair wave propagation around the bead. Thus, we have developed a new molecular understanding of the classic early concepts of inhibitory "chalone", suggesting that stem cells within the hair follicle micro-environment, or other organs, are subject to a higher level of macro-environmental regulation. Such a novel understanding has important implications in the field of regenerative medicine. The unexpected links with Bmp2 expression in subcutaneous adipocytes has implications for systems biology and Evo-Devo.

The origins of modern Plant Developmental Biology in Spain can be traced back to a handful of scientists settled in Madrid, Barcelona, Valencia and Sevilla, who devoted themselves to plant biochemistry, molecular biology and genetics, and also to Drosophila developmental biology, which influenced, often unintentionally, the pioneers of this field. To reach the present day situation, the experience acquired in centres abroad has also been important, especially in plant research institutes in the USA, Germany (Max-Planck Institute für Züchtungsforschung) and United Kingdom (John Innes Centre). The contributions of Spanish scientists to the advancement of Plant Developmental Biology appears to be imbalanced towards reproductive biology, although relevant publications have also been reported on embryogenesis and seed development, shoot branching, tuberization, vascular morphogenesis, leaf development, regulation of development by light, signal transduction and hormone action and the connection between growth and development. Plant Developmental Biology in Spain is going through a flourishing time, with its future being highly dependent on i) appropriate funding conditions to its young scientists, ii) the opening of new areas of research, iii) the incorporation of technological breakthroughs into laboratories and iv) the carrying out of cooperative research by means of networking. Currently, besides many Departments of the Spanish universities, several centres in which competitive research in plant Developmental Biology can be accomplished, exist: the CNB and CBGP in Madrid, the LGMV CSIC-IRTA in Barcelona, the IBMCP CSIC-UPV, in Valencia and the IBVF CSIC-USE in Sevilla. Let's go for more!

Patterns are orders embedded in randomness. They may appear as spatial arrangements or temporal series, and the elements may appear identical or with variations. Patterns exist in the physical world as well as in living systems. In the biological world, patterns can range from simple to complex, forming the basic building blocks of life. The process which generates this ordering in the biological world was termed pattern formation. Since Wolpert promoted this concept four decades ago, scientists from molecular biology, developmental biology, stem cell biology, tissue engineering, theoretical modeling and other disciplines have made remarkable progress towards understanding its mechanisms. It is time to review and re-integrate our understanding. Here, we explore the origin of pattern formation, how the genetic code is translated into biological form, and how complex phenotypes are selected over evolutionary time. We present four topics: Principles, Evolution, Development, and Stem Cells and Regeneration. We have interviewed several leaders in the field to gain insight into how their research and the field of pattern formation have shaped each other. We have learned that both molecular process and physico-chemical principles are important for biological pattern formation. New understanding will emerge through integration of the analytical approach of molecular-genetic manipulation and the systemic approach of model simulation. We regret that we could not include every major investigator in the field, but hope that this Special Issue of the Int. J. Dev. Biol. represents a sample of our knowledge of pattern formation today, which will help to stimulate more research on this fundamental process.

The fish egg is surrounded by a thick envelope called the chorion. The fertilizing spermatozoon enters the egg through a canal-like structure in the chorion, the micropyle. Examination of micropyle at fertilization is difficult if eggs are large and have no distinct landmarks surrounding the micropyle, or if they are positively buoyant in water. Eggs of many commercially important fishes (e.g., flounder, sea bream and eel) are buoyant in water or only slightly adhere to solid objects (e.g., sands, rock and water plants), which makes observation of spermatozoa at fertilization difficult. Here, we report that such eggs can be firmly attached to plastic and glass dishes that have been previously coated with poly-L-lysine. These adhering eggs can be fertilized and develop normally on the dishes. Observations of micropyles of three fish species, before and after sperm entry are presented.

A complexity imposed on the events of conception during the evolution of eutherian mammals is reflected not only in the placenta but also in a series of gamete-related novelties that involve the design of sperm and eggs, their interactions leading to fertilization, epididymal function, and even the scrotal state. While their functional significance has been difficult to determine, I suggest on the basis of present evidence that the genesis of these novel features relates ultimately to changes in the egg associated with nidation, that they imply a new fertilization strategy, and that most are causally linked--a major first 'domino' being the formidable zona pellucida. The eutherian zona pellucida stands out by virtue of an elastic resilience and thickness which allow it to stretch around the expanding blastocyst. However, this quality of the zona together with its relative protease-insensitivity creates a physical challenge which appears to have determined the design of the sperm head, its behaviour at fertilization, and indirectly even the pattern of sperm maturation in the epididymis. In turn, that pattern appears to have evoked the development of the sperm storage function of the cauda region, with both sperm capacitation and the scrotal state as ultimate legacies of this. Coincidentally, the vulnerability of the small yolkless egg to polyspermy appears to have selected for a unique parsimonious pattern of sperm transport in the Fallopian tube, and possibly for the role of the cumulus oophorus as a sperm sequestering device. These ideas are supported generally by evidence from marsupials, as well as by the deviant patterns seen in some insectivorous mammals.

Proteins involved in reproduction often evolve rapidly, raising the possibility that changes in these proteins contribute to reproductive isolation between species. We review the evidence for rapid and adaptive change in reproductive proteins in animals, focusing on studies in recently diverged vertebrates. We identify common patterns and point out promising directions for future research. In particular, we highlight the ways that integrating the different but complementary approaches of evolutionary and developmental biology will provide new insights into fertilization processes.

Cnidaria are the most basal phylum containing a well-developed visual system located on specialized sensory structures (rhopalia) with eyes and statocyts. We have been exploring the cubozoan jellyfish, Tripedalia cystophora. In addition to containing simple photoreceptive ocelli, each rhopalium in Tridedalia has a large and small complex, camera-type eye with a cellular lens containing three distinct families of crystallins which apparently serve non-lenticular functions. Thus, Tridpedalia recruited crystallins by a gene sharing strategy as have mollusks and vertebrates. Tripedalia has a single Pax gene, PaxB, which encodes a structural and functional Pax 2/5/8-like paired domain as well as an octapeptide and Pax6-like homeodomain. PaxB binds to and activates Tripedalia crystallin promoters (especially J3-crystallin) and the Drosophila rhodopsin rh6 gene in transfection tests and induces ectopic eyes in Drosophila. In situ hybridization showed that PaxB and crystallin genes are expressed in the lens, retina and statocysts. We suggest from these results that an ancestral PaxB gene was a primordial gene in eye evolution and that eyes and ears (mechanoreceptors) may have had a common evolutionary origin. Thus, the numerous structural and molecular features of Tridpalia rhopalia indicate that ancient cubozoan jellyfish are fascinating models for evo/devo insights into eyes and other sensory systems.

A history of embryological studies of lens development and regeneration is sketched, paying special attention to the contribution of these studies to the conceptual aspect of embryology and later developmental biology. Emphasis is made on the fact that the interaction of different tissues during development, namely embryonic induction, was first discovered during studies of the lens and that the degree of the dependence of lens development on the inductive effect of the eye-cup varies among different species. Studies along the line of comparative-experimental embryology at the species level should be informative, in particular in combination with the recent trend of evo-devo studies. The processes of lens regeneration and in vitro transdifferentiation indicate the existence of multiple cell lineages with the potential of lens development in one animal. The occurrence of Wolffian lens regeneration in nature can be reconsidered from the new "eco-devo" viewpoint.

Molecular and cellular analysis of early mammalian development is compromised by the experimental inaccessibility of the embryo. Pluripotent embryonic stem (ES) cells are derived from and retain many properties of the pluripotent founder population of the embryo, the inner cell mass. Experimental manipulation of these cells and their environment in vitro provides an opportunity for the development of differentiation systems which can be used for analysis of the molecular and cellular basis of embryogenesis. In this review we discuss strengths and weaknesses of the available ES cell differentiation methodologies and their relationship to events in vivo. Exploitation of these systems is providing novel insight into embryonic processes as diverse as cell lineage establishment, cell progression during differentiation, patterning, morphogenesis and the molecular basis for cell properties in the early mammalian embryo.

This paper discusses current efforts to produce databases of gene expression for the major model embryos used in developmental biology. The efforts to build these resources were motivated by the need for immediate internet access to all types of research data, and the production of these databases is a major and new challenge for bioinformatics. Thus far bioinformatics has mainly been concerned with textually oriented resources and data, much of it concerned with gene and protein sequences. Because the genetic basis of developmental biology is integrated with developmental anatomy, these databases require the use of images to link molecular data with spatial information. In order to standardise database formats, digital atlases of some model systems are being produced that include integrated anatomical descriptions and these are being linked to appropriate genetic data. Integrating such image-based, searchable data into databases makes new demands on the field of bioinformatics and we consider here the imaging modalities that are used to obtain information and we discuss in particular the production of 3D images from serial sections. Next, we consider how to integrate textual and spatial descriptions of gene expression and the key tool needed to make this possible, i.e. anatomical nomenclature. A short review of internet resources on developmental biology is also given and future prospects for the development of these databases are discussed.

Direct development is a widespread alternate reproductive mode in living amphibians that is characterized by evolutionary loss of the free-living, aquatic larval stage. Courtship, mating, and oviposition occur on land, and the terrestrial egg hatches as a fully formed, miniature adult. While it is the most common reproductive mode in urodeles, development outside the reproductive tract of the female that proceeds directly to a terrestrial hatchling occurs in only a single lineage, the lungless salamanders of the family Plethodontidae. Evolution of direct development in plethodontids has contributed importantly to the extraordinary evolutionary success of this speciose, geographically widespread, and morphologically and ecologically diverse taxon. Developmental consequences and correlates include increased egg size and embryonic development time, loss of larval structures and ontogenetic repatterning, and altered pattern formation in organogenesis. Evolutionary and phylogenetic consequences and correlates include the loss of larval constraints and origin of morphological novelty, and frequent homoplasy. Analysis of direct development in an evolutionary context illustrates the complex interplay between processes of phylogenetic divergence and developmental biology, and substantiates the prominent role of developmental processes in both constraining phenotypic variation and promoting phenotypic diversity. Despite the proven suitability of direct-developing plethodontid salamanders for laboratory and field study, knowledge of basic features of their developmental biology remains far below that available for many other urodeles. Examination of such features of these "non-model" organisms is an appropriate and deserving goal of future research.