The anatomy of organogenesis: novel solutions to old problems

Understanding anatomical aspects of mammalian organ development, in both normal and mutant animals, is important for basic biology and also for regenerative medicine and tissue engineering. The size and complexity of developing organs, together with variations in their detailed anatomy, has made the obtaining of high-resolution time-courses of anatomical change difficult to obtain. The fact that organ development tends to use the same genes as early embryogenesis also makes genetic manipulation difficult, as so many mutant embryos die before organogenesis begins. These problems have seriously hampered the study of organogenesis. Here, we describe three significant advances that promise solutions: (1) the production of GFP-reporter mice that can be used for high-resolution live-imaging of small tissues as they grow, (2) RNA interference, which allows the manipulation of specific genes at any stage of organ development, and (3) optical projection tomography, which allows medium-resolution three-dimensional images of complete embryos to be obtained easily. We finish by looking ahead to the prospects of uniting these three technologies to allow accurate, high-throughput screening of mutants and automated comparison of biological data with computer predictions.     

siRNA as a tool for investigating organogenesis: The pitfalls and the promises

Removing the function of a specific gene from a developing organ, by making a ‘knockout’ mouse, is a powerful method for analyzing the molecular pathways that control organogenesis. The technique is expensive, though, in terms of time and money, and complex strategies for producing conditional knockouts are needed for genes that are essential for early development of the embryo, for which an unconditional knockout would be lethal before the organ of interest begins to form. Small interfering RNAs (siRNAs) offer a method of knocking down the expression of specific genes with no need for genomic manipulation. Almost as soon as they had been discovered, siRNAs began to be used to explore the molecular biology of mammalian cells in conventional, two-dimensional culture. They have now also been applied successfully, by several groups, to knock down specific genes in various organ rudiments developing in organ culture. This article reviews the basic technique of siRNA-mediated gene knockdown and how it is being applied to organ culture. It also reviews some of the current problems and challenges in the field, and the ways in which these problems are likely to be overcome.Key words: siRNA, RNAi, organ culture, organogenesis, organ development, 3D culture     

Arabidopsis mutants with increased organ regeneration in tissue culture are more competent to respond to hormonal signals

Shoots and roots can be regenerated through organogenesis in tissue culture by subjecting plant explants to the appropriate regime of hormone treatments. In an effort to understand the control of shoot organogenesis, we screened for mutants in Arabidopsis thaliana (L.) Heynh. Columbia ecotype for enhanced shoot development at sub-optimal concentrations of cytokinin. Mutants in four different complementation groups were identified, one of which represents a new locus named increased organ regeneration1 (ire1) and another that is allelic to the previously identified pom1/erh2 mutant. Although the mutants were selected for their response to cytokinin, they were neither hypersensitive to, nor were they over-producers of cytokinins. The mutations identified in this study not only promote more robust shoot production in tissue culture, but also enhance green-callus and root formation. We interpret this to mean that, in tissue culture, IRE genes act before organ specification during the time when root explants acquire the competency to respond to organ formation signals. In normal plant development, IRE genes may down-regulate the competency of vegetative tissue to respond to hormonal signals involved in shoot and root organogenesis.     

A systematic genome-wide screen for mutations affecting organogenesis in Medaka, Oryzias latipes

A large-scale mutagenesis screen was performed in Medaka to identify genes acting in diverse developmental processes. Mutations were identified in homozygous F3 progeny derived from ENU-treated founder males. In addition to the morphological inspection of live embryos, other approaches were used to detect abnormalities in organogenesis and in specific cellular processes, including germ cell migration, nerve tract formation, sensory organ differentiation and DNA repair. Among 2031 embryonic lethal mutations identified, 312 causing defects in organogenesis were selected for further analyses. From these, 126 mutations were characterized genetically and assigned to 105 genes. The similarity of the development of Medaka and zebrafish facilitated the comparison of mutant phenotypes, which indicated that many mutations in Medaka cause unique phenotypes so far unrecorded in zebrafish. Even when mutations of the two fish species cause a similar phenotype such as one-eyed-pinhead or parachute, more genes were found in Medaka than in zebrafish that produced the same phenotype when mutated. These observations suggest that many Medaka mutants represent new genes and, therefore, are important complements to the collection of zebrafish mutants that have proven so valuable for exploring genomic function in development.     

Leaf phenomics: a systematic reverse genetic screen for Arabidopsis leaf mutants

The study and eventual manipulation of leaf development in plants requires a thorough understanding of the genetic basis of leaf organogenesis. Forward genetic screens have identified hundreds of Arabidopsis mutants with altered leaf development, but the genome has not yet been saturated. To identify genes required for leaf development we are screening the Arabidopsis Salk Unimutant collection. We have identified 608 lines that exhibit a leaf phenotype with full penetrance and almost constant expressivity and 98 additional lines with segregating mutant phenotypes. To allow indexing and integration with other mutants, the mutant phenotypes were described using a custom leaf phenotype ontology. We found that the indexed mutation is present in the annotated locus for 78% of the 553 mutants genotyped, and that in half of these the annotated T‐DNA is responsible for the phenotype. To quickly map non‐annotated T‐DNA insertions, we developed a reliable, cost‐effective and easy method based on whole‐genome sequencing. To enable comprehensive access to our data, we implemented a public web application named PhenoLeaf ( http://genetics.umh.es/phenoleaf ) that allows researchers to query the results of our screen, including text and visual phenotype information. We demonstrated how this new resource can facilitate gene function discovery by identifying and characterizing At1g77600, which we found to be required for proximal–distal cell cycle‐driven leaf growth, and At3g62870, which encodes a ribosomal protein needed for cell proliferation and chloroplast function. This collection provides a valuable tool for the study of leaf development, characterization of biomass feedstocks and examination of other traits in this fundamental photosynthetic organ.     

Patterning of the third pharyngeal pouch into thymus/parathyroid by Six and Eya1

Previous studies have suggested a role of the homeodomain Six family proteins in patterning the developing vertebrate head that involves appropriate segmentation of three tissue layers, the endoderm, the paraxial mesoderm and the neural crest cells; however, the developmental programs and mechanisms by which the Six genes act in the pharyngeal endoderm remain largely unknown. Here, we examined their roles in pharyngeal pouch development. Six1-/- mice lack thymus and parathyroid and analysis of Six1-/- third pouch endoderm demonstrated that the patterning of the third pouch into thymus/parathyroid primordia is initiated. However, the endodermal cells of the thymus/parathyroid rudiments fail to maintain the expression of the parathyroid-specific gene Gcm2 and the thymus-specific gene Foxn1 and subsequently undergo abnormal apoptosis, leading to a complete disappearance of organ primordia by E12.5. This thus defines the thymus/parathyroid defects present in the Six1 mutant. Analyses of the thymus/parathyroid development in Six1-/-;Six4-/- double mutant show that both Six1 and Six4 act synergistically to control morphogenetic movements of early thymus/parathyroid tissues, and the threshold of Six1/Six4 appears to be crucial for the regulation of the organ primordia-specific gene expression. Previous studies in flies and mice suggested that Eya and Six genes may function downstream of Pax genes. Our data clearly show that Eya1 and Six1 expression in the pouches does not require Pax1/Pax9 function, suggesting that they may function independently from Pax1/Pax9. In contrast, Pax1 expression in all pharyngeal pouches requires both Eya1 and Six1 function. Moreover, we show that the expression of Tbx1, Fgf8 and Wnt5b in the pouch endoderm was normal in Six1-/- embryos and slightly reduced in Six1-/-;Six4-/- double mutant, but was largely reduced in Eya1-/- embryos. These results indicate that Eya1 appears to be upstream of very early events in the initiation of thymus/parathyroid organogenesis, while Six genes appear to act in an early differentiation step during thymus/parathyroid morphogenesis. Together, these analyses establish an essential role for Eya1 and Six genes in patterning the third pouch into organ-specific primordia.     

Recalcitrance in clonal propagation,in particular of conifers

Despite major advances in forest biotechnology, clonal regeneration by somatic embryogenesis or organogenesis is still difficult for many woody species and is often limited to the use of juvenile explants. Adventitious regeneration of plants from gymnosperms older than zygotic embryos, and frequently even from highly immature zygotic embryos, is often difficult or has not yet been achieved. A number of experimental approaches that could eventually lead to overcoming recalcitrance are suggested in this review. When cloning trees of various ages, it is important to determine first which part of the individual contains the most responsive cells and at what time of the year these cells are in the most responsive state. This allows selection of the most useful explants. In hardwood trees and a few gymnosperms, responsive tissues are found in root or stump sprouts and in tissues near the site of meiosis at about the time that meiosis takes place. Another potentially active area is the shoot apex with most or all of its leaf or needle primordia removed. Apomixis is a natural form of clonal regeneration but occurs naturally in only one gymnosperm species. As the genetic mechanism of apomixis has been in part elucidated, the induction of apomixis by experimental means may soon be possible. The cytoplasm plays a major role in the expression or repression of nuclear genes that control embryogenesis. Expression of nuclear genes can be manipulated by nuclear transfer into de-nucleated cells (e.g., the cytoplasm of egg cells). Cytoplasmic control also plays a role in regeneration by androgenesis, asymmetric cell division and cell isolation. A short overview is presented of the genetic mechanisms involved in embryo initiation, maturation and germination and how manipulation of these mechanisms by genetic transformation could help in overcoming recalcitrance. It is expected that rapid development in the fields of research areas discussed in this review will over time eliminate the problem of recalcitrance in many instances where it is currently prevalent.     

Global gene expression during the human organogenesis: from transcription profiles to function predictions

Human embryogenesis includes an integrated set of complex yet coordinated development of different organs and tissues, which is regulated by the spatiotemporal expression of many genes. Deciphering the gene regulation profile is essential for understanding the molecular basis of human embryo development. While molecular and genetic studies in mouse have served as a valuable tool to understand mammalian development, significant differences exists in human and mouse development at morphological and genomic levels. Thus it is important to carry out research directly on human embryonic development. Here we will review some recent studies on gene regulation during human embryogenesis with particular focus on the period of organogenesis, which had not been well studied previously. We will highlight a gene expression database of human embryos from the 4(th) to the 9(th) week. The analysis of gene regulation during this period reveals that genes functioning in a given developmental process tend to be coordinately regulated during human embryogenesis. This feature allows us to use this database to identify new genes important for a particular developmental process/pathway and deduce the potential function of a novel gene during organogenesis. Such a gene expression atlas should serve as an important resource for molecular study of human development and pathogenesis.     

MACCHI-BOU 2 is required for early embryo patterning and cotyledon organogenesis in Arabidopsis

The phytohormone auxin is a key regulator of organogenesis in plants and is distributed asymmetrically via polar transport. However, the precise mechanisms underlying auxin-mediated organogenesis remain elusive. Here, we have analyzed the macchi-bou 2 (mab2) mutant identified in a pinoid (pid) enhancer mutant screen. Seedlings homozygous for either mab2 or pid showed only mild phenotypic effects on cotyledon positions and/or numbers. In contrast, mab2 pid double mutant seedlings completely lacked cotyledons, indicating a synergistic interaction. We found that mab2 homozygous embryos had defective patterns of cell division and showed aberrant cotyledon organogenesis. Further analysis revealed that the mab2 mutation affected auxin response but not auxin transport in the embryos, suggesting the involvement of MAB2 in auxin response during embryogenesis. MAB2 encodes an Arabidopsis ortholog of MED13, a putative regulatory module component of the Mediator complex. Mediator is a multicomponent complex that is evolutionarily conserved in eukaryotes and its regulatory module associates with Mediator to control the interaction of Mediator and RNA polymerase II. MAB2 interacts with a regulatory module component in yeast cells. Taken together, our data suggest that MAB2 plays a crucial role in embryo patterning and cotyledon organogenesis, possibly through modulating expression of specific genes such as auxin-responsive genes.     

Two SERK genes are markers of pluripotency in Cyclamen persicum Mill

The genetic basis of stem cell specification in somatic embryogenesis and organogenesis is still obscure. SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) genes are involved in embryogenesis and organogenesis in numerous species. In vitro culture of Cyclamen persicum immature ovules provides a system for investigating stem cell formation and maintenance, because lines forming either organs or embryos or callus without organs/embryos are available for the same cultivar and plant growth regulator conditions. The present aim was to exploit this property of cyclamen cultures to understand the role of SERK(s) in stem cell formation and maintenance in somatic embryogenesis and organogenesis in vitro, in comparison with expression in planta. CpSERK1 and CpSERK2 were isolated from embryogenic callus. CpSERK1 and CpSERK2 levels by RT-PCR showed that expression is high in embryogenic, moderate in organogenic, and null in recalcitrant calli. in situ hybridizations showed that the expression of both genes started in clumps of pluripotent stem cells, from which both pre-embryogenic aggregates and organ meristemoids derived, and continued in their trans-amplifying, meristem-like, derivatives. Expression declined in organ meristemoids, in parallel with a partial loss of meristematization. In mature somatic embryos, and in shoot and root primordia, CpSERK1 and CpSERK2 were expressed in meristems, and similar patterns occurred in zygotic embryo and primary meristems in planta. The results point to SERK1 and SERK2 as markers of pluripotency in cyclamen. It is proposed that the high expression of these genes in the trans-amplifying derivatives of the stem cells maintains a pluripotent condition leading to totipotency and, consequently, somatic embryogenesis.     

Normal epithelial branching morphogenesis in the absence of collagen I

Interstitial collagens are thought to mediate epithelial-mesenchymal interactions during organogenesis. We have used the collagen I-deficient mouse mutant Mov13 to directly investigate the role of this major representative of the interstitial collagens in epithelial branching morphogenesis. Since homozygous embryos die at midgestation, we have studied the development of organ rudiments from Mov13 homozygous (i.e., collagen I-deficient), heterozygous, and wild-type embryos in culture. Development of all explants, including lung, kidney, salivary glands, pancreas, and skin, was normal by light and electron microscopic criteria and was independent of the genotype of the donor embryo. Metabolic labeling and immune staining verified the complete absence of collagen I in homozygous explants while revealing substantial production of collagens III and V in explants of all three genotypes. These results indicate either that collagen I has no role in the morphogenesis of these organs, or that its function is shared, or can be substituted for, by other fibrillar collagens.     

A novel 3D mouse embryo atlas based on micro-CT

The goal of the International Mouse Phenotyping Consortium (IMPC) is to phenotype targeted knockout mouse strains throughout the whole mouse genome (23,000 genes) by 2021. A significant percentage of the generated mice will be embryonic lethal; therefore, phenotyping methods tuned to the mouse embryo are needed. Methods that are robust, quantitative, automated and high-throughput are attractive owing to the numbers of mice involved. Three-dimensional (3D) imaging is a useful method for characterizing morphological phenotypes. However, tools to automatically quantify morphological information of mouse embryos from 3D imaging have not been fully developed. We present a representative mouse embryo average 3D atlas comprising micro-CT images of 35 individual C57BL/6J mouse embryos at 15.5 days post-coitum. The 35 micro-CT images were registered into a consensus average image with our automated image registration software and 48 anatomical structures were segmented manually. We report the mean and variation in volumes for each of the 48 segmented structures. Mouse organ volumes vary by 2.6-4.2% on a linear scale when normalized to whole body volume. A power analysis of the volume data reports that a 9-14% volume difference can be detected between two classes of mice with sample sizes of eight. This resource will be crucial in establishing baseline anatomical phenotypic measurements for the assessment of mutant mouse phenotypes, as any future mutant embryo image can be registered to the atlas and subsequent organ volumes calculated automatically.     

Tissue-specific RNA interference in post-implantation mouse embryos using directional electroporation and whole embryo culture

In mammals, embryonic development is more difficult to analyze than in non-mammalian species because this development occurs in utero. Interestingly, whole embryo culture allows the normal development of mouse post-implantation embryos for up to 2 days in vitro. One limitation of this technology has been the difficulty of performing loss-of-gene function studies in this system. RNA interference (RNAi), whereby double-stranded RNA molecules suppress the expression of complementary genes, has rapidly become a widely used tool for gene function analyses. We have combined the technologies of mouse whole embryo culture and RNAi to allow the molecular dissection of developmental processes. Here, we review the manipulation by topical injection followed by directional electroporation of endoribonuclease-prepared siRNA to demonstrate that this technology may be useful to knock down genes in a tissue- and region-specific manner in several organs of the developing mouse embryo.