Showing papers in "The International Journal of Developmental Biology in 2002"

Efficient Cre-mediated deletion in cardiac progenitor cells conferred by a 3'UTR-ires-Cre allele of the homeobox gene Nkx2-5.

Abstract: Conditional gene targeting and transgenic strategies utilizing Cre recombinase have been successfully applied to the analysis of development in mouse embryos. To create a conditional system applicable to heart progenitor cells, a Cre recombinase gene linked at its 5' end to an internal ribosome entry site (IRES) was inserted into the 3' untranslated region of the cardiac homeobox gene Nkx2-5 using gene targeting. Nkx2-5IRESCre mice were fully viable as homozygotes. We evaluated the efficacy of Cre-mediated deletion by crossing Nkx2-5IRESCre mice with the Cre-dependent R26R and Z/AP reporter strains. Efficient deletion was observed in the cardiac crescent and heart tube in both strains. However, the Z/AP locus showed transient resistance to deletion in caudal heart progenitors. Such resistance was not evident at the R26R locus, suggesting that Cre-mediated deletion in myocardium may be locus-dependent. From cardiac crescent stages, deletion was seen not only in myocardium, but also endocardium, dorsal mesocardium and pericardial mesoderm. The Cre domain apparently includes cells dorsal to the heart that have been shown to constitute a secondary heart field, contributing myocardium to the outflow tract. Other sites of Nkx2-5 expression, including pharyngeal endoderm and its derivatives, branchial arch epithelium, stomach, spleen, pancreas and liver, also showed efficient deletion. Our data suggest that the Nkx2-5IRESCre strain will be useful for genetic dissection of the multiple tiers of lineage allocation to the forming heart as well as of molecular interactions within the heart fields and heart tube.

Origin of coronary endothelial cells from epicardial mesothelium in avian embryos

Abstract: It has been established that coronary vessels develop through self-assembly of mesenchymal vascular progenitors in the subepicardium. Mesenchymal precursors of vascular smooth muscle cells and fibroblasts are known to originate from an epithelial-to-mesenchymal transformation of the epicardial mesothelium, but the origin of the coronary endothelium is still obscure. We herein report that at least part of the population of the precursors of the coronary endothelium are epicardially-derived cells (EPDCs). We have performed an EPDC lineage study through retroviral and fluorescent labelling of the proepicardial and epicardial mesothelium of avian embryos. In all the experiments onlythe surface mesothelium was labelled after 3 h of reincubation. However, endothelial cells from subepicardial vessels were labelled after 24-48 h and endothelial cells of intramyocardial vessels were also labelled after 48-96 h of reincubation. In addition, the development of the coronary vessels was studied in quail-chick chimeras, obtaining results which also support a mesothelial origin for endothelial and smooth muscle cells. Finally, quail proepicardial explants cultured on Matrigel showed colocalization of cytokeratin and QH1 (mesothelial and endothelial markers, respectively) after 24 h. These results, taken together, suggest that EPDC show similar competence to that displayed by bipotential vascular progenitor cells [Yamashita et al., Nature 408: 92-96 (2000)] which are able to differentiate into endothelium or smooth muscle depending on their exposure to VEGF or PDGF-BB. It is conceivable that the earliest EPDC differentiate into endothelial cells in response to myocardially-secreted VEGF, while further EPDC would be recruited by the nascent capillaries via PDGFR-beta signalling, giving rise to mural cells.

Vertebrate limb regeneration and the origin of limb stem cells

Abstract: The existence of multipotent cells in the adult tissues and organs of those vertebrates that are capable of regeneration has been accepted for decades. Although studies of vertebrate limb regeneration have yet to identify many of the specific molecules involved in regeneration, numerous tissue grafting experiments and studies of cell lineage have contributed significantly to an understanding of the origin, activation, proliferation and cell-cell interactions of these progenitor cells. This has allowed the development of ideas about the regulation of pattern formation to restore the structure and function of lost tissues and organs. An understanding of the molecular mechanisms controlling these processes has lagged behind the dramatic advances achieved with other model organisms. However, given the intense, new research interest in stem cells over the past few years, there is good reason to be encouraged that insights about the biology of mammalian stem cells will accelerate progress in understanding the biology of regeneration in organisms that can regenerate. Advances in regeneration research will then feed back in terms of devising new strategies for therapies to induce regeneration in organisms such as humans that have traditionally been viewed as incapable of regeneration.

Histone methylation defines epigenetic asymmetry in the mouse zygote.

Abstract: The oocyte cytoplasm regulates and enhances the epigenetic asymmetry between parental genomes and, consequently, functional differences observed between them during development in mammals. Here we demonstrate a preferential interaction of HP1beta with the maternal genome immediately after fertilisation in the mouse zygote, which also shows a high level of lysine 9-methylated histone H3. In contrast, the paternal genome has neither HP1beta binding nor methylated histone H3 at these early stages. Paternal binding of HP1beta is only detected at the pronuclear stage, prior to the appearance of lysine 9-methylated histone H3. The early recruitment of heterochromatic factors specifically to the maternal genome could explain the preferential DNA demethylation of the paternal genome in the zygote.

Limb muscle development.

Abstract: Skeletal muscle precursors for the limbs originate from the epithelial layer of the somites, the dermomyotomes. We summarize the steps of limb muscle development from the specification of precursor cells in the dermomyotome, the directed migration of these cells to and within the limb buds to muscle growth and differentiation. All steps are controlled by local signaling between embryonic structures. In dermomyotome development, signals from the neural tube, the ectoderm and the intermediate and lateral mesoderm result in a medio-lateral patterning. Only the lateral portions of the dermomyotomes give rise to muscle precursor cells destined to enter the limb buds. As a prerequisite for migration, precursor cells have to de-epithelialize as a result of interactions between SF/HGF and its receptor c-met. Precursor cells adopt a mesenchymal morphology without losing their myogenic specification. This is achieved by the expression of the transcription factors Pax3, Pax7 and myf5. During migration, premature differentiation has to be kept at bay to enable motility and proliferation. After having reached their target sites, the dorsal and ventral myogenic zones, myogenesis is initiated by the activation of the muscle determination factors MyoD, myogenin and MRF4. Finally, we briefly summarize the process of muscle hypertrophy and regeneration during which aspects of developmental processes are reinitiated.

The genetic control of eye development and its implications for the evolution of the various eye-types.

Abstract: Mutations in the Pax 6 homologs of mammals and insects prevent eye development and targeted expression of both mammal and insect Pax 6 homologs is capable of inducing functional ectopic eyes. Supported by RNA interference experiments in planarians and nemerteans, these findings indicate that Pax 6 is a universal master control gene for eye morphogenesis. Since all metazoan eyes use rhodopsin as a photoreceptor molecule and the same master control gene for eye development, we postulate a monophyletic origin of the various eye types. The finding of well developed eyes in jellyfish which essentially lack a brain, leads us to propose that the eye as a sensory organ evolved before the brain which is an information processing organ. The finding of highly developed eyes with a lens, vitreous body, stacked membranes like a retina and shielding pigment in unicellular dinoflagellates, raises the possibility that the prototypic eyes might have been acquired from symbionts.

Wnt signalling during limb development.

Abstract: Wnts control a number of processes during limb development--from initiating outgrowth and controlling patterning, to regulating cell differentiation in a number of tissues. Interactions of Wnt signalling pathway components with those of other signalling pathways have revealed new mechanisms of modulating Wnt signalling, which may explain how different responses to Wnt signalling are elicited in different cells. Given the number of Wnts that are expressed in the limb and their ability to induce differential responses, the challenge will be to dissect precisely how Wnt signalling is regulated and how it controls limb development at a cellular level, together with the other signalling pathways, to produce the functional limb capable of coordinated precise movements.

Cnidarians as a model system for understanding evolution and regeneration

Abstract: Hydra and Podocolyne are two cnidarian animals which provide complementary advantages for analysing developmental mechanisms possibly reflecting the basic developmental processes shared by most bilaterians. Interestingly, these mechanisms remain accessible all along the life of these animals, which bud and regenerate, whatever their age. The Hydra polyp permits a direct study of the molecular cascades linking amputation to regeneration. Podocoryne displays a complete life cycle, polyp and medusa stages with a fast and inducible sexual cycle and an unparalleled In vitro transdifferentiation potential. In both cases, a large number of evolutionarily conserved molecular markers are available, and analysis of their regulation highlights the molecular mechanisms which underly pattern formation in these two species.

PPAR expression and function during vertebrate development

Abstract: The peroxisome proliferator activated receptors (PPARs) are ligand activated receptors which belong to the nuclear hormone receptor family. As with other members of this superfamily, it is thought that the ability of PPAR to bind to a ligand was acquired during metazoan evolution. Three different PPAR isotypes (PPARalpha, PPARbeta, also called 6, and PPARgamma) have been identified in various species. Upon binding to an activator, these receptors stimulate the expression of target genes implicated in important metabolic pathways. The present article is a review of PPAR expression and involvement in some aspects of Xenopus laevis and rodent embryonic development. PPARalpha and beta are ubiquitously expressed in Xenopus early embryos but become more tissue restricted later in development. In rodents, PPARalpha, PPARbeta and PPARgamma show specific time- and tissue-dependent patterns of expression during fetal development and in the adult animals. PPARs are implicated in several aspects of tissue differentiation and rodent development, such as differentiation of the adipose tissue, brain, placenta and skin. Particular attention is given to studies undertaken by us and others on the implication of PPARalpha and beta in rodent epidermal differentiation.

Musca domestica, a window on the evolution of sex-determining mechanisms in insects.

Abstract: The genetic cascades regulating sex determination of the housefly, Musca domestica, and the fruitfly, Drosophila melanogaster, appear strikingly different. The bifunctional switch gene doublesex, however, is present at the bottom of the regulatory cascades of both species, and so is transformer-2, one of the genetic elements required for the sex-specific regulation of doublesex. The upstream regulators are different: Drosophila utilizes Sex-lethal to coordinate the control of sex determination and dosage compensation, i.e., the process that equilibrates the difference of two X chromosomes in females versus one X chromosome in males. In the housefly, Sex-lethal is not involved in sex determination, and dosage compensation, if existent at all, is not coupled with sexual differentiation. This allows for more adaptive plasticity in the housefly system. Accordingly, natural housefly populations can vary greatly in their mechanism of sex determination, and new types can be generated in the laboratory.

Programmed cell death in the developing limb.

Abstract: The sculpturing of shape in the developing limb together with the regression of the tail in anuran tadpoles constitute, perhaps, the most paradigmatic processes of programmed cell death. The study of these model systems has been of fundamental importance to support the idea that cell death is a physiological behavior of cells in multicellular organisms. Furthermore, different experimental approaches, including comparative analyses of the pattern of cell death in different avian species (i.e. chick interdigits versus duck interdigital webs) and in chick mutants with different limb phenotypes, provided the first evidence for the occurrence of a genetic program underlying the control of cell death. Two well known research groups in the field of limb development, the USA group headed first by John Saunders and next by John Fallon and the group of Donald Ede and Richard Hinchliffe in the U.K. provided a remarkable contribution to this topic. In spite of the historical importance of the developing limb in establishing the concept of programmed cell death, this model system of tissue regression has been largely neglected in recent studies devoted to the analysis of the molecular control of self-induced cell death (apoptosis). However, a considerable amount of information concerning this topic has been obtained in the last few years. Here we will review current information on the control of limb programmed cell death in an attempt to stimulate further molecular studies of this process of tissue regression.

Involvement of myogenic regulator factors during fusion in the cell line C2C12.

Abstract: The myogenic factors, MyoD, myogenin, Myf5 and MRF4, can activate skeletal muscle differentiation when overexpressed in non-muscular cells Gene targeting experiments have provided much insight into the in vivo functions of MRF and have defined two functional groups of MRFs MyoD and Myf5 may be necessary for myoblast determination while myogenin and MRF4 may be required later during differentiation However, the specific role of these myogenic factors has not been clearly defined during one important stage of myogenesis: the fusion of myoblasts Using cultured C2C12 mouse muscular cells, the time-course of these proteins was analyzed and a distinct expression pattern in fusing cells was revealed In an attempt to clarify the role of each of these regulators during myoblast fusion, an antisense strategy using oligonucleotides with phosphorothioate backbone modification was adoped The results showed that the inhibition of myogenin and Myf5 activity is capable of significantly preventing fusion Furthermore, the inhibition of MyoD can wholly arrest the engaged fusion process in spite of high endogenous expression of both myogenin and Myf5 Consequently, each MRF seems to have, at this defined step of myogenesis, a specific set of functions that can not be substituted for by the others and therefore may regulate a distinct subset of muscle-specific genes at the onset of fusion

Developmental biology of zebrafish myeloid cells.

Abstract: The zebrafish (Danio rerio) has emerged as an informative vertebrate model for developmental studies, particularly those employing genetic approaches such as mutagenesis and screening. Zebrafish myelopoiesis has recently been characterized, paving the way for the experimental strengths of this model organism to contribute to an improved understanding of the genetic regulation of myeloid development. Zebrafish have a multi-lineage myeloid compartment with two types of granulocyte (heterophil/neutrophil and eosinophil granulocytes), and monocyte/macrophages, each with characteristic morphological features and histochemical staining properties. Molecular markers have been characterised for various myeloid cell types and their precursor cells, for example: stem cells (scl, hhex, lmo2), myeloid lineage precursors (spi1/pu.1, c/ebp1), heterophil granulocytes (mpx/mpo), macrophages (L-plastin, fms). In zebrafish, the sites of early myeloid and erythroid commitment are anatomically separated, being located in the rostral and caudal lateral plate mesoderm respectively. Functional macrophages appear before cells displaying granulocytic markers. By the second day of life, cells expressing granulocyte- and macrophage-specific genes are scattered throughout the embryo, but tend to aggregate in the ventral venous plexus, which may be a site of their production or a preferred site for their residence. Even in early embryos, macrophages are phagocytically active, and granulocytes participate in acute inflammation. Equipped with an understanding of the developmental biology of these various myeloid cells and a set of tools for their identification and functional study, we will now be able to exploit the experimental strengths of this model organism to better understand the genetic regulation of myelopoiesis.

PAX genes in development and disease: the role of PAX2 in urogenital tract development.

Abstract: PAX genes play an important role in fetal development. Moreover, heterozygous mutations in several PAX genes cause human disease. Here we review the role of PAX2 in kidney development, focusing on the morphological effects of PAX2 mutations. We discuss the role of PAX2 in the context of an inhibitory field model of kidney branching morphogenesis and summarize recent progress in this area.

The structure and function of vertebrate fibroblast growth factor receptor 1.

Abstract: The vertebrate fibroblast growth factor receptor 1 (FGFR1) is alternatively spliced generating multiple splice variants that are differentially expressed during embryo development and in the adult body. The restricted expression patterns of FGFR1 isoforms, together with differential expression and binding of specific ligands, leads to activation of common FGFR1 signal transduction pathways, but may result in distinctively different biological responses as a result of differences in cellular context. FGFR1 isoforms are also present in the nucleus in complex with various fibroblast growth factors where they function to regulate transcription of target genes.

Interplay between the molecular signals that control vertebrate limb development.

Abstract: Vertebrate limbs display three obvious axes of asymmetry. These three axes are referred to as proximal-distal (Pr-D; shoulder to digit tips), anterior-posterior (A-P; thumb to little finger), and dorsal-ventral (D-V; back of hand to palm). At a molecular level, it is now possible to define the signals that control patterning of each of the three axes of the developing limb. These signals do not work in isolation though but rather their activity must be integrated such that the various limb elements are coordinately formed with relation to these three axes. This review will provide an overview of the intricate medley amongst the molecular signals that serve to establish and coordinate patterning information along the three primary axes of the limb.

TGF-beta3 is required for the adhesion and intercalation of medial edge epithelial cells during palate fusion.

Abstract: Transforming growth factor-beta3 (TGF-beta3) plays a critical role during palate development, since mutations of the TGF-beta3 gene give rise to cleft palate in both humans and mice. Striking alterations have been reported in the behaviour and differentiation of medial edge epithelial (MEE) cells in TGF-beta3 knockout mouse palates. In the present paper, we provide evidence of alterations in MEE intercellular adhesion in TGF-beta3 -/- mouse palates using immunohistochemistry with monoclonal antibodies to a panel of cell adhesion and cytoskeletal molecules including E-cadherin, alpha and beta catenin, beta actin, vinculin and beta2 integrin. In vitro labeling of opposing MEE with two different lipophilic markers and subsequent analysis by confocal microscopy revealed that wild type MEE cells intercalate as soon as the midline epithelial seam forms. This finding indicates that the palate may elongate in a dorso-ventral direction by means of convergent extension, as occurs in other embryonic developmental processes. In contrast, this intercalation does not occur in the TGF-beta3 -/- MEE but it can be rescued by the exogenous addition of TGF-beta3. Thus, the substantial alteration of MEE intercellular adhesion observed in TGF-beta3 -/- palates may account for the defect in palatal shelf adhesion and the formation of cleft palate.

Twist functions in mouse development.

Abstract: The remarkable similarity in the profile of genetic activity and the frequent association of developmental defects of limb and craniofacial structures in mouse mutant and hereditary disorders point to the possibility that the development of the head and limb involves common morphogenetic mechanisms. Our recent studies on the impact of the loss of Twist function has highlighted the essential role of the basic helix-loop-helix transcription factor encoded by this gene on the development of both body parts. We have summarized in this review our findings on the molecular pathways that are disrupted in Twist mutant mouse embryos. Our results revealed an evolutionarily conserved function for Twist in mesodermal differentiation, and previously unrecognised effects of the loss-of-function mutation of this gene in the outgrowth and patterning of the limb and branchial arches,and neural crest cell migration. An important outcome of our study is the demonstration of a differential requirement for Twist in forelimb versus hindlimb development, and its functional interaction with Gli3 in specifying anterior digit formation. Further evidence of the conservation of the function of Twist in different species is highlighted by similarity in the spectrum of potential downstream targets and interacting genes of Twist that have been identified by genetic, functional and microarray analysis.

Induction and patterning of the cardiac conduction system

Abstract: The cardiac conduction system (CCS) is the component of the heart that initiates and maintains a rhythmic heartbeat. As the embryonic heart forms, the CCS must continue to develop and mature in a coordinated manner to ensure that proper pace making potential and distribution of action potential is maintained at all stages. This requires not only the formation of distinct and disparate components of the CCS, but the integration of these components into a functioning whole as the heart matures. Though research in this area of development may have lagged behind other areas of heart development, in recent years there has been much progress in understanding the ontogeny of the CCS and the developmental cues that drive its formation. This is largely due to studies on the avian heart as well as the use of molecular biology approaches. This review gives a perspective on advances in understanding the development of the vertebrate CCS, and reports new data illuminating the mechanism of conduction cell determination and maintenance in the mammalian heart. As much of our knowledge about the development of the CCS has been derived from the chick embryo, one important area facing the field is the relationship and similarities between the structure and development of avian and mammalian conduction systems. Specifically, the morphology of the distal elements of the mammalian CCS and the manner in which its components are recruited from working cardiomyocytes are areas of research that will, hopefully, receive more attention in the near future. A more general and outstanding question is how the disparate components of all vertebrate conduction systems integrate into a functional entity during embryogenesis. There is mounting evidence linking the patterning and formation of the CCS to instructive cues derived from the cardiac vasculature and, more specifically, to hemodynamic-responsive factors produced by cardiac endothelia. This highlights the need for a greater understanding of the biophysical forces acting on, and created by, the cardiovascular system during embryonic development. A better understanding of these processes will be necessary if therapeutics are to be developed that allow the regeneration of damaged cardiac tissues or the construction of biologically engineered heart tissues.

The transforming growth factor-betas: multifaceted regulators of the development and maintenance of skeletal muscles, motoneurons and Schwann cells.

Abstract: This review discusses the roles of the transforming growth factor-betas (TGF-betas) as part of a complex network that regulates the development and maintenance of the neuromuscular system. The actions of the TGF-betas often vary depending on which other growth factors are present, making it difficult to extrapolate results from in vitro experiments to the in vivo situation. A new approach has therefore been needed to understand the physiological functions of the TGF-betas. The behaviours (proliferation, fusion, apoptosis) of many of the cells in the neuromuscular system have a complex pattern which varies in space and time. The actions of growth factors in this system can thus be deduced based on how well their pattern of expression correlates with known cellular behaviours. Hypotheses based on this molecular anatomical evidence can then be further tested with genetically modified mice. From this type of evidence, we suggest that: (1) TGF-beta1 is an autocrine regulator of Schwann cells; (2) maternally-derived TGF-beta1 helps to suppress self and maternal immune attack; (3) TGF-beta2 regulates when and where myoblasts fuse to myotubes; (4) motoneuron survival is regulated by multiple sources of TGF-betas, with TGF-beta2 being the more important isoform. The concept of TGF-beta1 as a regulator of secondary myotube formation is not supported by either the location of the TGF-beta1 in developing muscles or by the phenotype of TGF-beta1-/- mice. The review concludes with a discussion of whether all of these of postulated functions can occur independently of each other, within the confines of the neuromuscular system.

Ecological regulation of development: induction of marine invertebrate metamorphosis.

Abstract: In the marine environment a wide range of invertebrates have a pelagobenthic lifecycle that includes planktonic larval and benthic adult phases. Transition between these morphologically and ecologically distinct phases typically occurs when the developmentally competent larva comes into contact with a species-specific environmental cue. This cue acts as a morphogenetic signal that induces the completion of the postlarval/juvenile/adult developmental program at metamorphosis. The development of competence often occurs hours to days after the larva is morphologically mature. In the non-feeding - lecithotrophic - larvae of the ascidian Herdmania curvata and the gastropod mollusc Haliotis asinina, gene expression patterns in pre-competent and competent stages are markedly different, reflecting the different developmental states of these larval stages. For example, the expression of Hemps, an EGF-like signalling peptide required for the induction of Herdmania metamorphosis, increases in competent larvae. Induction of settlement and metamorphosis results in further changes in developmental gene expression, which apparently is necessary for the complete transformation of the larval body plan into the adult form.

Genetic studies define MAGUK proteins as regulators of epithelial cell polarity.

Abstract: Polarized epithelial cells play critical roles during early embryonic development and organogenesis. Multi-domain scaffolding proteins belonging to the membrane associated guanylate kinase (MAGUK) family are commonly found at the plasma membrane of polarized epithelial cells. Genetic studies in Drosophila melanogaster and Caenorhabditis elegans have revealed that MAGUK proteins regulate various aspects of the polarized epithelial phenotype, including cell junction assembly, targeting of proteins to the plasma membrane and the organisation of polarized signalling complexes. This review will focus on the genetic studies that have contributed to our understanding of the MAGUK family members, Dlg and Lin-2/CASK, in controlling these processes. In addition, our recent genetic analysis of mouse Dlg, in combination with genetic and biochemical studies of Lin-2/CASK by others suggests a model placing Dlg and Lin-2/CASK within the same developmental pathway.

Signalling via type IA and type IB bone morphogenetic protein receptors (BMPR) regulates intramembranous bone formation, chondrogenesis and feather formation in the chicken embryo.

Abstract: Bone morphogenetic proteins (BMPs) signal via complexes of type I and type II receptors. In this study, we mapped the expression of type IA, type IB and type II receptors during craniofacial chondrogenesis and then perturbed receptor function in vivo with retroviruses expressing dominant-negative or constitutively active type I receptors. BmprIB was the only receptor expressed within all cartilages. BmprIA was initially expressed in cartilage condensations, but later decreased within cartilage elements. BmprII was expressed at low levels in the nasal septum and prenasal cartilage and at higher levels in other craniofacial cartilages. The maxillary prominence, which gives rise to several intramembranous bones, expressed both type I receptors. Misexpression of dnBMPRIB decreased the size of cartilages and bones on the treated side. In contrast, dnBMPRIA had no effect on the skeletal phenotype. The phenotypes of caBMPRIA and caBMPRIB were similar; both led to overgrowth of cartilage elements, thinner bones with fewer trabeculae and inhibition of feather development. Infection with constitutively active viruses resulted in ectopic expression of Msx1, Msx2 and Fgfr2 throughout the maxillary mesenchyme. These data suggest that the pattern of trabeculation in membranous bones derived from the maxillary prominence was related to the change in expression pattern and that Msx and Fgfr2 genes were downstream of both type I BMP receptors. We conclude that the requirement for the type IB is greater than for the type IA receptor but, when active, both receptors play similar roles in regulating bone, cartilage and feather formation in the skull.

Differentiation and growth of kype skeletal tissues in anadromous male Atlantic salmon (Salmo salar).

Abstract: The re-initiation of bone development in adult starving Atlantic salmon (Salmo salar) during their energetically expensive upstream migration is remarkable and deserves closer examination. Dramatic alterations of the skull bones and teeth, most prominently, the development of a kype in males, are widely known but little studied or understood. We describe the microstructure and the cellular processes involved in the formation of the skeletal tissues of the kype. Fresh bone material, obtained from animals migrating upstream was subjected to radiological, histological or histochemical analysis. We show that the kype is, in part, composed of rapidly growing skeletal needles arising at the tip of the dentary. Proximally, the needles anastomose into a spongiosa-like meshwork which retains connective tissue inside bone marrow spaces. Ventrally, the needles blend into Sharpey fiber bone. Skeletal needles and Sharpey fiber bone can be distinguished from the compact bone of the dentary by radiography. Rapid formation of the skeleton of the kype is demonstrated by the presence of numerous osteoblasts, a broad distal osteoid zone, and the appearance of proteoglycans at the growth zone. The mode of bone formation in anadromous males can be described as 'making bone as fast as possible and with as little material as possible'. Unlike the normal compact bone of the dentary, the new skeletal tissue contains chondrocytes and cartilaginous extracellular matrix. Formation of the skeleton of the kype resembles antler development in deer (a form of regeneration), or hyperostotic bone formation in other teleost fishes, rather than periosteal bone growth. The type of boneformation may be understandable in the light of the animals' starvation and the energetic costs of upstream migration. However, the structured and regulated mode of bone formation suggests that the skeleton of the kype has functional relevance and is not a by-product of hormonal alterations or change of habitat.

Coral development: from classical embryology to molecular control.

Abstract: The phylum Cnidaria is the closest outgroup to the triploblastic metazoans and as such offers unique insights into evolutionary questions at several levels. In the post-genomic era, a knowledge of the gene complement of representative cnidarians will be important for understanding the relationship between the expansion of gene families and the evolution of morphological complexity among more highly evolved metazoans. Studies of cnidarian development and its molecular control will provide information about the origins of the major bilaterian body axes, the origin of the third tissue layer, the mesoderm, and the evolution of nervous system patterning. We are studying the cnidarian Acropora millepora, a reef building scleractinian coral, and a member of the basal cnidarian class, the Anthozoa. We review ourwork on descriptive embryology and studies of selected transcription factor gene families, where our knowledge from Acropora is particularly advanced relative to other cnidarians. We also describe a recent preliminary whole genome initiative, a coral EST database.

The role of the Eph-ephrin signalling system in the regulation of developmental patterning.

Abstract: The Eph and ephrin system, consisting of fourteen Eph receptor tyrosine kinase proteins and nine ephrin membrane proteins in vertebrates, has been implicated in the regulation of many critical events during development. Binding of cell surface Eph and ephrin proteins results in bi-directional signals, which regulate the cytoskeletal, adhesive and motile properties of the interacting cells. Through these signals Eph and ephrin proteins are involved in early embryonic cell movements, which establish the germ layers, cell movements involved in formation of tissue boundaries and the pathfinding of axons. This review focuses on two vertebrate models, the zebrafish and mouse, in which experimental perturbation of Eph and/or ephrin expression in vivo have provided important insights into the role and functioning of the Eph/ephrin system.

Embryonic stem cell differentiation and the analysis of mammalian development.

Abstract: 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.

Interactions between dorsal-ventral patterning genes lmx1b, engrailed-1 and wnt-7a in the vertebrate limb

Abstract: The vertebrate limb has characteristic morphological features that distinguish dorsal and ventral regions. For example, humans and most other mammals have nails on the dorsal surface of their digits, while the ventral surface is covered by skin or footpads. Internally, there is a high degree of organization along the dorsal-ventral axis. Extensor muscles are generally located dorsally while flexor muscles are generally located ventrally. The skeleton has subtle differences that allow for attachment of these muscles and distinct pools of motor neurons innervate either dorsal or ventral muscles. How is this complex arrangement of tissues generated? Recent studies have identified a molecular cascade of three factors that govern early events in dorsal-ventral limb patterning. Two of these factors, engrailed-1 and wnt-7a are expressed in the dorsal and ventral ectoderm respectively. The function of engrailed-1 is to repress the expression of wnt-7a in the ventral limb bud ectoderm. The third factor, a LIM-homeodomain transcription factor, Imx1b is induced in dorsal mesenchyme by wnt-7a and it is both necessary and sufficient to specify dorsal limb pattern. In this report, we examine genetic interactions between wnt-7a, engrailed-1, and Imx1b by analyzing the phenotypes of mice that are double mutants for Imx1b and either wnt-7a or engrailed-1. These studies indicate that Imx1b is the only target of wnt-7a and engrailed-1 that is of consequence for dorsal-ventral patterning. Moreover, this genetic analysis suggests that Imx1b plays additional roles in anterior-posterior patterning and growth that were not previously appreciated.

Physiological and induced apoptosis in sea urchin larvae undergoing metamorphosis.

Abstract: Paracentrotus lividus embryos at the early pluteus stage undergo spontaneous apoptosis. Using a TUNEL (TdT-mediated dUTP Nick-End Labelling) assay on whole mount embryos, we showed that there was a different distribution of the apoptotic cells in different optical sections. Not more than 20% of cells in plutei were spontaneously apoptotic, as confirmed by the counts of dissociated ectoderm and intestine cells. Observation of larva stages closer to metamorphosis confirmed that apoptosis is a physiological event for the development of the adult. In particular, larvae at different developmental stages showed apoptotic cells in the oral and aboral arms, intestine, ciliary band and both apical and oral ganglia. Moreover, we found that the number of apoptotic cells decreased in later larva stages, possibly because in the organism approaching metamorphosis, a smaller number of cells needs to be eliminated. Furthermore, combined phorbol ester (TPA) and heat shock treatment enhanced apoptosis by increasing the number of cells involved in the phenomenon.

Defining the cell lineages of the islets of Langerhans using transgenic mice.

Abstract: In this Special Issue of the Int. J. Dev. Biol., we summarize our own studies on the development of the mouse endocrine pancreas, with special emphasis on the cell lineage relationships between the four islet cell types. Considerable knowledge concerning the ontogeny of the endocrine pancreas has been gained in recent years, mainly through the use of two complementary genetic approaches in mice: gene inactivation and genetic labelling of precursor cells. However, neither gene inactivation in KO mice nor co-localisation of hormones in single cells during development can be taken as evidence for cell lineage relationships among different cell types. The beta-cell lineage analysis was started by selectively ablating specific islet cell types in transgenic mice. We used the diphtheria toxin A subunit coding region under the control of insulin, glucagon or pancreatic polypeptide (PP) promoters, in order to eliminate insulin-, glucagon- or PP-expressing cells, respectively. Contrary to the common view, we demonstrated that glucagon cells are not precursors of insulin-producing cells. These results were in addition the first evidence of a close ontogenetic relationship between insulin and somatostatin cells. We pursued these analyses using a novel, more subtle approach: progenitor cell labelling through the expression of Cre recombinase in doubly transgenic mice. We were able to unequivocally establish that 1) adult glucagon- and insulin-producing cells derive from precursors which have never transcribed insulin or glucagon, respectively; 2) insulin cell progenitors, but not glucagon cell progenitors transcribe the PP gene and 3) adult glucagon cells derive from progenitors which do express pdx1.