Toward Molecular Understanding of In Vitro and In Planta Shoot Organogenesis

Shoot organogenesis, one of the in vitro plant regeneration processes that occur during in vitro micropropagation, is used in the study of plant development. Morphological, physiological, and molecular aspects of in vitro shoot organogenesis have been extensively studied for over 50 years. Because of the research progress in plant genetics and molecular biology, our understanding of in planta and in vitro shoot meristem development, the cell cycle and cytokinin signal transduction has advanced significantly. These research advances provide useful information as well as molecular tools to study further the genetic and molecular aspects of shoot organogenesis. A number of key molecular markers, genes, and pathways have been shown to play a critical role in the process of in vitro shoot organogenesis. Furthermore, these studies reveal that in vitro shoot organogenesis, as with in planta shoot development, is a complex, well-coordinated developmental process, given that the induction of a single molecular event is likely to be insufficient to induce the entire process. Continued study is required to identify additional molecular events that trigger dedifferentiation and act as developmental switches for de novo shoot development.     

Molecular Analysis of In Vitro Shoot Organogenesis

Shoot organogenesis is one of the in vitro plant regeneration pathways. It has been widely employed in plant biotechnology for in vitro micropropagation and genetic transformation, as well as in study of plant development. Morphological and physiological aspects of in vitro shoot organogenesis have already been extensively studied in plant tissue culture for more than 50 years. Within the last ten years, given the research progress in plant genetics and molecular biology, our understanding of in vivo plant shoot meristem development, plant cell cycle, and cytokinin signal transduction has advanced significantly. These research advances have provided useful molecular tools and resources for the recent studies on the genetic and molecular aspects of in vitro shoot organogenesis. A few key molecular markers, genes, and probable pathways have been identified from these studies that are shown to be critically involved in in vitro shoot organogenesis. Furthermore, these studies have also indicated that in vitro shoot organogenesis, just as in in vivo shoot development, is a complex, well-coordinated developmental process, and induction of a single molecular event may not be sufficient to induce the occurrence of the entire process. Further study is needed to identify the early molecular event(s) that triggers dedifferentiation of somatic cells and serves as the developmental switch for de novo shoot development.     

Dynamic expression of the LAP family of genes during early development of <Emphasis Type="Italic">Xenopus tropicalis</Emphasis>

The leucine-rich repeats and PDZ (LAP) family of genes are crucial for the maintenance of cell polarity as well as for epithelial homeostasis and tumor suppression in both vertebrates and invertebrates. Four members of this gene family are known: densin, erbin, scribble and lano. Here, we identified the four members of the LAP gene family in Xenopus tropicalis and studied their expression patterns during embryonic development. The Xenopus LAP proteins show a conserved domain structure that is similar to their homologs in other vertebrates. In Xenopus embryos, these genes were detected in animal cap cells at the early gastrula stage. At later stages of development, they were widely expressed in epithelial tissues that are highly polar in nature, including the neural epithelia, optic and otic vesicles, and in the pronephros. These data suggest that the roles of the Xenopus LAP genes in the control of cell polarity and morphogenesis are conserved during early development. Erbin and lano show similar expression patterns in the developing head, suggesting potential functional interactions between the two molecules in vivo.     

Using Xenopus to analyze neurocristopathies like Kabuki syndrome

Neurocristopathies are human congenital syndromes that arise from defects in neural crest (NC) development and are typically associated with malformations of the craniofacial skeleton. Genetic analyses have been very successful in identifying pathogenic mutations, however, model organisms are required to characterize how these mutations affect embryonic development thereby leading to complex clinical conditions. The African clawed frog Xenopus laevis provides a broad range of in vivo and in vitro tools allowing for a detailed characterization of NC development. Due to the conserved nature of craniofacial morphogenesis in vertebrates, Xenopus is an efficient and versatile system to dissect the morphological and cellular phenotypes as well as the signaling events leading to NC defects. Here, we review a set of techniques and resources how Xenopus can be used as a disease model to investigate the pathogenesis of Kabuki syndrome and neurocristopathies in a wider sense.     

An ontology for <Emphasis Type="Italic">Xenopus</Emphasis> anatomy and development

Background  

The frogs Xenopus laevis and Xenopus (Silurana) tropicalis are model systems that have produced a wealth of genetic, genomic, and developmental information. Xenbase is a model organism database that provides centralized access to this information, including gene function data from high-throughput screens and the scientific literature. A controlled, structured vocabulary for Xenopus anatomy and development is essential for organizing these data.     

An amphibian with ambition: a new role for Xenopus in the 21st century

Much of our knowledge about the mechanisms of vertebrate early development comes from studies using Xenopus laevis. The recent development of a remarkably efficient method for generating transgenic embryos is now allowing study of late development and organogenesis in Xenopus embryos. Possibilities are also emerging for genomic studies using the closely related diploid frog Xenopus tropicalis.     

Studies on the function of TM20, a transmembrane protein present in cereal embryos

The possible function of the maize transmembrane protein TM20 in hormone transport has been investigated using immunological methods and by microinjection of TM20 cRNA in Xenopus oocytes. The existence of a similar gene in rice indicates that the overall structure of the deduced protein is conserved between these two cereals. An antibody raised against a conserved motif, in a loop between two transmembrane domains, locates the protein (TM20) in differentiating provascular cells in maize embryo. The protein has a polarized distribution within the cell in the most differentiated stages of development. Xenopus laevis oocytes microinjected with TM20 appear to modify transport activities across the plasma membrane. These results are discussed in relation to other transport proteins that influence plant development.     

Xenopus research: metamorphosed by genetics and genomics

Research using Xenopus takes advantage of large, abundant eggs and readily manipulated embryos in addition to conserved cellular, developmental and genomic organization with mammals. Research on Xenopus has defined key principles of gene regulation and signal transduction, embryonic induction, morphogenesis and patterning as well as cell cycle regulation. Genomic and genetic advances in this system, including the development of Xenopus tropicalis as a genetically tractable complement to the widely used Xenopus laevis, capitalize on the classical strengths and wealth of achievements. These attributes provide the tools to tackle the complex biological problems of the new century, including cellular reprogramming, organogenesis, regeneration, gene regulatory networks and protein interactions controlling growth and development, all of which provide insights into a multitude of human diseases and their potential treatments.     

Xenopus radial spoke protein 3 gene is expressed in the multiciliated cells of epidermis and otic vesicles and sequentially in the nephrostomes

We describe the phylogenetic analysis and expression pattern of the Xenopus radial spoke protein 3 (RSP3) gene during early development. The Xenopus RSP3 protein presents characteristic features of the RSP3 family. It contains a radial spoke domain, which is 75 and 72 % identical to the corresponding region of human and Chlamydomonas RSP3 proteins, respectively. Examination of the phylogenetic relationship between the Xenopus RSP3 protein and its known homologues from different deuterostomes indicates that the RSP3 proteins are highly conserved among deuterostomes. Whole-mount in situ hybridization analyses show that Xenopus RSP3 is a maternal mRNA enriched in the animal hemisphere during cleavage stages. The expression is detected in the dorsal region of the embryo during gastrulation, then in the presumptive neuroectoderm at the end of gastrulation. During neurulation and at the subsequent stages, the expression of RSP3 mRNA is detected in the entire multiciliated cells of epidermis. At tail-bud stages, it is progressively expressed in the otic vesicles and sequentially expressed in the nephrostomes. Expression could be also detected in the floor plate of the neural tube. This expression pattern persists until at least late tail-bud stages.     

Maturation of neurites in mixed cultures of spinal cord neurons and muscle cells from Xenopus laevis embryos followed with antibodies to neurofilament proteins

Dissociated cell cultures of Xeopus laevis embryonic spinal cord have proved useful for studying the differentiation of neuronal ionic channel and membrane properties and for examining the dynamics of microtubules in developing neurons. To examine their usefulness for studying neurofilaments in developing neurites, we prepared similar cultures from stage 22 embryos. Between 3 and 55 h after plating, these cultures were fixed and immunostained with antibodies directed against various epitopes of neurofilament proteins from X. Laevis. These antibodies were specific for nonphosphorylated epitopes of the two low molecular weight Xenopus neurofilament proteins (Xenopus NF-L and the Xenopus neuronal intermediate filament protein, XNIF), both phosphorylated and nonphosphorylated epitopes of the Xenopus middle molecular weight neurofilament protein (NF-M), and a nonphosphorylated epitope of the Xenopus high molecular weight neurofilament protein (NF-H). The emergence of these neurofilament proteins in culture was compared to the time course previously reported for them in Xenopus spinal cord neurons in situ. To facilitate the comparison of times in culture to developmental stages, the age of cultured neurons was converted to an equivalent Nieuwkoop and Faber normal stage using data presented here on the effect of changing temperature on developmental rates of X. laevis. With the exception of the nonphosphorylated epitope of NF-H, which is indicative of the most mature axons found in situ. the emergence of the other neurofilament protein antibody epitopes closely paralleled that previously reported for these antibodies in situ. Thus, with respect to XNIF, NF-M, and NF-L, the neurities of cultured neurons were typical of young embryonic Xenopus laevis spinal cord axons. This system should prove useful for studying both the function of these neurofilament proteins during the early stages of axonal development and the dynamics of their transport. 1994 John Wiley & Sons, Inc.     

Identification of genes associated with regenerative success of <Emphasis Type="Italic">Xenopus laevis</Emphasis>hindlimbs

Background  

Epimorphic regeneration is the process by which complete regeneration of a complex structure such as a limb occurs through production of a proliferating blastema. This type of regeneration is rare among vertebrates but does occur in the African clawed frog Xenopus laevis, traditionally a model organism for the study of early development. Xenopus tadpoles can regenerate their tails, limb buds and the lens of the eye, although the ability of the latter two organs to regenerate diminishes with advancing developmental stage. Using a heat shock inducible transgene that remains silent unless activated, we have established a stable line of transgenic Xenopus (strain N1) in which the BMP inhibitor Noggin can be over-expressed at any time during development. Activation of this transgene blocks regeneration of the tail and limb of Xenopus tadpoles.     

Engineered telomeres in transgenic Xenopus laevis

The expanding roles of telomeres in epigenetic gene regulation, nuclear organization, and human disease have necessitated the establishment of model organisms in which to study telomere function under normal developmental conditions. We present an efficient system for generating numerous vertebrate animals containing engineered telomeres using a Xenopus laevis transgenesis technique. Our results indicate Xenopus zygotes efficiently recognize telomeric repeats at chromosome break points and form telomeric complexes thus generating a new telomere. The resulting transgenic animals progress through normal development and successfully metamorphose into froglets despite the chromosome breakage. Overall, this presents an efficient mechanism for generating engineered telomeres in a vertebrate system and provides an opportunity to investigate epigenetic aspects of telomere function during normal vertebrate development.     

Xenopus to the rescue: A model to validate and characterize candidate ciliopathy genes

Cilia are present on most vertebrate cells and play a central role in development, growth, and homeostasis. Thus, cilia dysfunction can manifest into an array of diseases, collectively termed ciliopathies, affecting millions of lives worldwide. Yet, our understanding of the gene regulatory networks that control cilia assembly and functions remain incomplete. With the advances in next‐generation sequencing technologies, we can now rapidly predict pathogenic variants from hundreds of ciliopathy patients. While the pace of candidate gene discovery is exciting, most of these genes have never been previously implicated in cilia assembly or function. This makes assigning the disease causality difficult. This review discusses how Xenopus, a genetically tractable and high‐throughput vertebrate model, has played a central role in identifying, validating, and characterizing candidate ciliopathy genes. The review is focused on multiciliated cells (MCCs) and diseases associated with MCC dysfunction. MCCs harbor multiple motile cilia on their apical surface to generate extracellular fluid flow inside the airway, the brain ventricles, and the oviduct. In Xenopus, these cells are external and present on the embryonic epidermal epithelia, facilitating candidate genes analysis in MCC development in vivo. The ability to introduce patient variants to study their effects on disease progression makes Xenopus a powerful model to improve our understanding of the underlying disease mechanisms and explain the patient phenotype.     

Closing the MCM cycle at replication termination sites

The initiation of eukaryotic DNA replication is a highly regulated process conserved from yeast to human. The past decade has seen significant advances in understanding how the CMG (Cdc45‐MCM‐GINS) replicative helicase is loaded onto DNA. However, very little was known on how this complex is removed from chromatin at the end of S phase. Two papers in a recent issue of Science 1 2 show that in yeast and in Xenopus, the CMG complex is unloaded at replication termination sites by an active mechanism involving the polyubiquitylation of Mcm7.     

Modulation of Protein Phosphorylation by M r 25,000 Protein Partially Overlapping Phosvitin and Lipovitellin 2 in Xenopus laevis Vitellogenin B1 Protein

A phosphorylated protein with molecular mass of 25,000 (pp25) is a component of Xenopus laevis vitellogenin B1. In an attempt to elucidate the physiological role of pp25, its effect on protein phosphorylation was studied. In vitro phosphorylation of some endogenous proteins from the cytoplasm and membrane fraction of Xenopus oocytes by casein kinase II and protein kinase C (PKC) was inhibited by increasing the concentration of pp25. By Western blot analysis using an antibody against phospho-(Ser/Thr) PKC substrate, phosphorylation of some endogenous proteins, especially in the cytoplasm, of Xenopus embryos was seen to increase when pp25 disappeared during developmental stages 35–45. These results suggest that pp25 may have a role as an inhibitory modulator of some protein phosphorylation in Xenopus oocytes and embryos.     

FishNet: an online database of zebrafish anatomy

Background  

Over the last two decades, zebrafish have been established as a genetically versatile model system for investigating many different aspects of vertebrate developmental biology. With the credentials of zebrafish as a developmental model now well recognized, the emerging new opportunity is the wider application of zebrafish biology to aspects of human disease modelling. This rapidly increasing use of zebrafish as a model for human disease has necessarily generated interest in the anatomy of later developmental phases such as the larval, juvenile, and adult stages, during which many of the key aspects of organ morphogenesis and maturation take place. Anatomical resources and references that encompass these stages are non-existent in zebrafish and there is therefore an urgent need to understand how different organ systems and anatomical structures develop throughout the life of the fish.