Organ Primordia Research Articles

A Proteoglycan-Like Molecule Offers Insights Into Ground Substance Changes During Holothurian Intestinal Regeneration.

Extracellular matrix remodeling is an essential component of regenerative processes in metazoans. Among these animals, holothurians (sea cucumbers) are distinguished by their great regenerative capacities. We have previously shown that fibrous collagen as well as other fibrous components disappear from the connective tissue (CT) early during intestinal regeneration, and later return as the organ primordia form. We now report on changes of the nonfibrous component of the CT. We have used Alcian Blue staining and an antibody, Proteoglycan Like-1 (PGL-1), that recognizes a proteoglycan-like antigen to identify the presence of proteoglycans in normal and regenerating intestines. Our results show that early in regeneration, the ground substance resembles that of the mesentery, the structure from where the new intestine originates. As regeneration proceeds, Alcian Blue staining and PGL-1 labeling reorganize, so that by 4 weeks the normal intestinal CT pattern is achieved. Together with our previous findings, the data suggest that CT components that might be detrimental to regeneration disappear early on, while those that might be beneficial to regeneration, such as proteoglycans, are present throughout the regenerative process.

Regulation of cell protrusions by small GTPases during fusion of the neural folds.

Epithelial fusion is a crucial process in embryonic development, and its failure underlies several clinically important birth defects. For example, failure of neural fold fusion during neurulation leads to open neural tube defects including spina bifida. Using mouse embryos, we show that cell protrusions emanating from the apposed neural fold tips, at the interface between the neuroepithelium and the surface ectoderm, are required for completion of neural tube closure. By genetically ablating the cytoskeletal regulators Rac1 or Cdc42 in the dorsal neuroepithelium, or in the surface ectoderm, we show that these protrusions originate from surface ectodermal cells and that Rac1 is necessary for the formation of membrane ruffles which typify late closure stages, whereas Cdc42 is required for the predominance of filopodia in early neurulation. This study provides evidence for the essential role and molecular regulation of membrane protrusions prior to fusion of a key organ primordium in mammalian development.

Expression of grapevine AINTEGUMENTA-like genes is associated with variation in ovary and berry size.

Fruit size is a highly important trait for most fruit and vegetable crops. This trait has been a main selection target and could be involved in divergent selection processes leading to the differentiation between modern table and wine cultivars. Even though its determination is highly influenced by cultural practices, several regions within the grapevine genome have been identified affecting berry size, either directly or indirectly through their effect on seed content. Using grapevine seeded cultivars, we have analyzed the relationship between ovary cell number and the final size of ovaries and berry fruits. We also performed the characterization of the grapevine AINTEGUMENTA-LIKE family, since it is well reported in Arabidopsis that AINTEGUMENTA (ANT) regulates cell proliferation and organ growth in flower organ primordia by maintaining the meristematic competence of cells during organogenesis. Here we show that orthologous grapevine gene expression associate with flower developmental stages suggesting a similar biological role for this gene family in this species. Moreover, we detected a correlation between those organs size and the level of expression of VviANT1 the grapevine homolog of AtANT. This grapevine gene also co-localizes in linkage group 18 with the confidence interval of a previously detected QTL for berry size. Thus our results suggest the involvement of ANT in the regulation of berry size in grapevine.

Avian dendritic cells: Phenotype and ontogeny in lymphoid organs

Dendritic cells (DC) are critically important accessory cells in the innate and adaptive immune systems. Avian DCs were originally identified in primary and secondary lymphoid organs by their typical morphology, displaying long cell processes with cytoplasmic granules. Several subtypes are known. Bursal secretory dendritic cells (BSDC) are elongated cells which express vimentin intermediate filaments, MHC II molecules, macrophage colony-stimulating factor 1 receptor (CSF1R), and produce 74.3+ secretory granules. Avian follicular dendritic cells (FDC) highly resemble BSDC, express the CD83, 74.3 and CSF1R molecules, and present antigen in germinal centers. Thymic dendritic cells (TDC), which express 74.3 and CD83, are concentrated in thymic medulla while interdigitating DC are found in T cell-rich areas of secondary lymphoid organs. Avian Langerhans cells are a specialized 74.3−/MHC II+ cell population found in stratified squamous epithelium and are capable of differentiating into 74.3+ migratory DCs. During organogenesis hematopoietic precursors of DC colonize the developing lymphoid organ primordia prior to immigration of lymphoid precursor cells. This review summarizes our current understanding of the ontogeny, cytoarchitecture, and immunophenotype of avian DC, and offers an antibody panel for the in vitro and in vivo identification of these heterogeneous cell types.

Differential regulation of meristem size, morphology and organization by the ERECTA, CLAVATA and class III HD-ZIP pathways.

ABSTRACTThe shoot apical meristem (SAM) of angiosperm plants is a small, highly organized structure that gives rise to all above-ground organs. The SAM is divided into three functional domains: the central zone (CZ) at the SAM tip harbors the self-renewing pluripotent stem cells and the organizing center, providing daughter cells that are continuously displaced into the interior rib zone (RZ) or the surrounding peripheral zone (PZ), from which organ primordia are initiated. Despite the constant flow of cells from the CZ into the RZ or PZ, and cell recruitment for primordium formation, a stable balance is maintained between the distinct cell populations in the SAM. Here we combined an in-depth phenotypic analysis with a comparative RNA-Seq approach to characterize meristems from selected combinations of clavata3 (clv3), jabba-1D (jba-1D) and erecta (er) mutants of Arabidopsis thaliana. We demonstrate that CLV3 restricts meristem expansion along the apical-basal axis, whereas class III HD-ZIP and ER pathways restrict meristem expansion laterally, but in distinct and possibly perpendicular orientations. Our k-means analysis reveals that clv3, jba-1D/+ and er lead to meristem enlargement by affecting different aspects of meristem function; for example, clv3 displays an increase in the stem cell population, whereas jba-1D/+ er exhibits an increase in mitotic activity and in the meristematic cell population. Our analyses demonstrate that a combined genetic and mRNA-Seq comparative approach provides a precise and sensitive method to identify cell type-specific transcriptomes in a small structure, such as the SAM.

Organotypic Culture of the Mouse Cochlea from Embryonic Day 12 to the Neonate.

The development of the mammalian cochlea is a complex process involving several intersecting signaling pathways to ultimately generate its highly organized cellular architecture. In humans, and in the mouse, there is one row of inner hair cells aligned next to three rows of outer hair cells. The support cells intercalate between the hair cells to create a cellular mosaic across the organ of Corti (OC). Organotypic culture of the cochlea is a valuable technique for investigating the early stages of OC development. Cultures can be established at proliferative stages and maintained in vitro until cellular differentiation commences. It is straightforward to monitor differentiation in response to perturbations of key signaling pathways using pharmacological agents. While postnatal cochlea organ cultures have already been adapted for in vitro studies, it is more challenging to establish cultures at early embryonic stages due to the small size of the organ primordium. The protocol described in this chapter is permissive for all stages of development from E12 cochleas to day 5 neonatal murine cochleas, allowing culture survival up to 2 weeks in vitro.

Migration of Founder Epithelial Cells Drives Proper Molar Tooth Positioning and Morphogenesis

The proper positioning of organs during development is essential, yet little is known about the regulation of this process in mammals. Using murine tooth development as a model, we have found that cell migration plays a central role in positioning of the organ primordium. By combining lineage tracing, genetic cell ablation, and confocal live imaging, we identified a migratory population of Fgf8-expressing epithelial cells in the embryonic mandible. These Fgf8-expressing progenitors furnish the epithelial cells required for tooth development, and the progenitor population migrates toward a Shh-expressing region in the mandible, where the tooth placode will initiate. Inhibition of Fgf and Shh signaling disrupted the oriented migration of cells, leading to a failure of tooth development. These results demonstrate the importance of intraepithelial cell migration in proper positioning of an initiating organ.

Active Control of Cell Size Generates Spatial Detail during Plant Organogenesis

SummaryHow cells regulate their dimensions is a long-standing question [1, 2]. In fission and budding yeast, cell-cycle progression depends on cell size, although it is still unclear how size is assessed [3, 4, 5]. In animals, it has been suggested that cell size is modulated primarily by the balance of external signals controlling growth and the cell cycle [1], although there is evidence of cell-autonomous control in cell cultures [6, 7, 8, 9]. Regardless of whether regulation is external or cell autonomous, the role of cell-size control in the development of multicellular organisms remains unclear. Plants are a convenient system to study this question: the shoot meristem, which continuously provides new cells to form new organs, maintains a population of actively dividing and characteristically small cells for extended periods [10]. Here, we used live imaging and quantitative, 4D image analysis to measure the sources of cell-size variability in the meristem and then used these measurements in computer simulations to show that the uniform cell sizes seen in the meristem likely require coordinated control of cell growth and cell cycle in individual cells. A genetically induced transient increase in cell size was quickly corrected by more frequent cell division, showing that the cell cycle was adjusted to maintain cell-size homeostasis. Genetically altered cell sizes had little effect on tissue growth but perturbed the establishment of organ boundaries and the emergence of organ primordia. We conclude that meristem cells actively control their sizes to achieve the resolution required to pattern small-scale structures.

Connecting physical cues and tissue patterning.

Several signaling pathways work together, via a protein called Amotl2a, to establish the size and shape of a zebrafish sense organ primordium.

Intronic sequences are required for AINTEGUMENTA-LIKE6 expression in Arabidopsis flowers.

BackgroundThe AINTEGUMENTA-LIKE6/PLETHORA3 (AIL6/PLT3) gene of Arabidopsis thaliana is a key regulator of growth and patterning in both shoots and roots. AIL6 encodes an AINTEGUMENTA-LIKE/PLETHORA (AIL/PLT) transcription factor that is expressed in the root stem cell niche, the peripheral region of the shoot apical meristem and young lateral organ primordia. In flowers, AIL6 acts redundantly with AINTEGUMENTA (ANT) to regulate floral organ positioning, growth, identity and patterning. Experiments were undertaken to define the genomic regions required for AIL6 function and expression in flowers.ResultsTransgenic plants expressing a copy of the coding region of AIL6 in the context of 7.7 kb of 5′ sequence and 919 bp of 3′ sequence (AIL6:cAIL6-3′) fail to fully complement AIL6 function when assayed in the ant-4 ail6-2 double mutant background. In contrast, a genomic copy of AIL6 with the same amount of 5′ and 3′ sequence (AIL6:gAIL6-3′) can fully complement ant-4 ail6-2. In addition, a genomic copy of AIL6 with 590 bp of 5′ sequence and 919 bp of 3′ sequence (AIL6m:gAIL6-3′) complements ant-4 ail6-2 and contains all regulatory elements needed to confer normal AIL6 expression in inflorescences. Efforts to map cis-regulatory elements reveal that the third intron of AIL6 contains enhancer elements that confer expression in young flowers but in a broader pattern than that of AIL6 mRNA in wild-type flowers. Some AIL6:gAIL6-3′ and AIL6m:gAIL6-3′ lines confer an over-rescue phenotype in the ant-4 ail6-2 background that is correlated with higher levels of AIL6 mRNA accumulation.ConclusionsThe results presented here indicate that AIL6 intronic sequences serve as transcriptional enhancer elements. In addition, the results show that increased expression of AIL6 can partially compensate for loss of ANT function in flowers.

HANABA TARANU (HAN) Bridges Meristem and Organ Primordia Boundaries through PINHEAD, JAGGED, BLADE-ON-PETIOLE2 and CYTOKININ OXIDASE 3 during Flower Development in Arabidopsis.

Shoot organ primordia are initiated from the shoot apical meristem and develop into leaves during the vegetative stage, and into flowers during the reproductive phase. Between the meristem and the newly formed organ primordia, a boundary with specialized cells is formed that separates meristematic activity from determinate organ growth. Despite interactions that have been found between boundary regulators with genes controlling meristem maintenance or primordial development, most boundary studies were performed during embryogenesis or vegetative growth, hence little is known about whether and how boundaries communicate with meristem and organ primordia during the reproductive stage. We combined genetic, molecular and biochemical tools to explore interactions between the boundary gene HANABA TARANU (HAN) and two meristem regulators BREVIPEDICELLUS (BP) and PINHEAD (PNH), and three primordia-specific genes PETAL LOSS (PTL), JAGGED (JAG) and BLADE-ON-PETIOLE (BOP) during flower development. We demonstrated the key role of HAN in determining petal number, as part of a set of complex genetic interactions. HAN and PNH transcriptionally promote each other, and biochemically interact to regulate meristem organization. HAN physically interacts with JAG, and directly stimulates the expression of JAG and BOP2 to regulate floral organ development. Further, HAN directly binds to the promoter and intron of CYTOKININ OXIDASE 3 (CKX3) to modulate cytokinin homeostasis in the boundary. Our data suggest that boundary-expressing HAN communicates with the meristem through the PNH, regulates floral organ development via JAG and BOP2, and maintains boundary morphology through CKX3 during flower development in Arabidopsis.

JAK/STAT and Hox Dynamic Interactions in an Organogenetic Gene Cascade.

Organogenesis is controlled by gene networks activated by upstream selector genes. During development the gene network is activated stepwise, with a sequential deployment of successive transcription factors and signalling molecules that modify the interaction of the elements of the network as the organ forms. Very little is known about the steps leading from the early specification of the cells that form the organ primordium to the moment when a robust gene network is in place. Here we study in detail how a Hox protein induces during early embryogenesis a simple organogenetic cascade that matures into a complex gene network through the activation of feedback and feed forward interaction loops. To address how the network organization changes during development and how the target genes integrate the genetic information it provides, we analyze in Drosophila the induction of posterior spiracle organogenesis by the Hox gene Abdominal-B (Abd-B). Initially, Abd-B activates in the spiracle primordium a cascade of transcription factors and signalling molecules including the JAK/STAT signalling pathway. We find that at later stages STAT activity feeds back directly into Abd-B, initiating the transformation of the Hox cascade into a gene-network. Focusing on crumbs, a spiracle downstream target gene of Abd-B, we analyze how a modular cis regulatory element integrates the dynamic network information set by Abd-B and the JAK/STAT signalling pathway during development. We describe how a Hox induced genetic cascade transforms into a robust gene network during organogenesis due to the repeated interaction of Abd-B and one of its targets, the JAK/STAT signalling cascade. Our results show that in this network STAT functions not just as a direct transcription factor, but also acts as a "counter-repressor", uncovering a novel mode for STAT directed transcriptional regulation.

Deciphering principles of morphogenesis from temporal and spatial patterns on the integument.

How tissue patterns form in development and regeneration is a fundamental issue remaining to be fully understood. The integument often forms repetitive units in space (periodic patterning) and time (cyclic renewal), such as feathers and hairs. Integument patterns are visible and experimentally manipulatable, helping us reveal pattern formative processes. Variability is seen in regional phenotypic specificities and temporal cycling at different physiological stages. Here we show some cellular/molecular bases revealed by analyzing integument patterns. (1) Localized cellular activity (proliferation, rearrangement, apoptosis, differentiation) transforms prototypic organ primordia into specific shapes. Combinatorial positioning of different localized activity zones generates diverse and complex organ forms. (2) Competitive equilibrium between activators and inhibitors regulates stem cells through cyclic quiescence and activation. Dynamic interactions between stem cells and their adjacent niche regulate regenerative behavior, modulated by multi-layers of macro-environmental factors (dermis, body hormone status, and external environment). Genomics studies may reveal how positional information of localized cellular activity is stored. In vivo skin imaging and lineage tracing unveils new insights into stem cell plasticity. Principles of self-assembly obtained from the integumentary organ model can be applied to help restore damaged patterns during regenerative wound healing and for tissue engineering to rebuild tissues. Developmental Dynamics 244:905-920, 2015. © 2015 Wiley Periodicals, Inc.

Cell walls as a stage for intercellular communication regulating shoot meristem development.

Aboveground organs of plants are ultimately derived/generated from the shoot apical meristem (SAM), which is a proliferative tissue located at the apex of the stem. The SAM contains a population of stem cells that provide new cells for organ/tissue formation. The SAM is composed of distinct cell layers and zones with different properties. Primordia of lateral organs develop at the periphery of the SAM. The shoot apex is a dynamic and complex tissue, and as such intercellular communications among cells, layers and zones play significant roles in the coordination of cell proliferation, growth and differentiation to achieve elaborate morphogenesis. Recent findings have highlighted the importance of a number of signaling molecules acting in the cell wall space for the intercellular communication, including classic phytohormones and secretory peptides. Moreover, accumulating evidence has revealed that cell wall properties and their modifying enzymes modulate hormone actions. In this review, we outline how behaviors of signaling molecules and changes of cell wall properties are integrated for the shoot meristem regulation.

Links between genome replication and chromatin landscapes.

Post-embryonic organogenesis in plants requires the continuous production of cells in the organ primordia, their expansion and a coordinated exit to differentiation. Genome replication is one of the most important processes that occur during the cell cycle, as the maintenance of genomic integrity is of primary relevance for development. As it is chromatin that must be duplicated, a strict coordination occurs between DNA replication, the deposition of new histones, and the introduction of histone modifications and variants. In turn, the chromatin landscape affects several stages during genome replication. Thus, chromatin accessibility is crucial for the initial stages and to specify the location of DNA replication origins with different chromatin signatures. The chromatin landscape also determines the timing of activation during the S phase. Genome replication must occur fully, but only once during each cell cycle. The re-replication avoidance mechanisms rely primarily on restricting the availability of certain replication factors; however, the presence of specific histone modifications are also revealed as contributing to the mechanisms that avoid re-replication, in particular for heterochromatin replication. We provide here an update of genome replication mostly focused on data from Arabidopsis, and the advances that genomic approaches are likely to provide in the coming years. The data available, both in plants and animals, point to the relevance of the chromatin landscape in genome replication, and require a critical evaluation of the existing views about the nature of replication origins, the mechanisms of origin specification and the relevance of epigenetic modifications for genome replication.

Editorial introductions

Editorial introductions

Plant-specific CC-type glutaredoxins: functions in developmental processes and stress responses.

Glutaredoxins (GRXs) are small oxidoreductases of the thioredoxin family proteins that can either regulate the thiol redox state of proteins or are linked to iron metabolism because of their ability to incorporate iron-sulfur [2Fe-2S] clusters. Here we review recent research on a land plant-specific class of GRX-like proteins, which are characterized by the conserved CC motif in the active centre. Loss-of-function mutants of CC-type GRXs in Arabidopsis (also named ROXYs), maize, and rice have unraveled a role in floral development, including regulation of organ primordia initiation, control of organ identity gene expression, and progression into meiosis in the male germ line. Other CC-type GRXs play a role in stress responses, most likely through their capacity to regulate nuclear gene expression. Consistently, CC-type GRXs, physically and genetically interact with individual members of the TGA transcription factor family. One of the challenges in the future is to unravel whether ROXYs control the redox state of TGA factors or other yet unknown target proteins or whether they regulate gene expression through other processes. Other intriguing questions concern the original function of the first CC-type GRXs in basal land plants and their potential contribution to the extremely successful radiation of angiosperms.

Co-ordination of Flower Development Through Epigenetic Regulation in Two Model Species: Rice and Arabidopsis.

Angiosperms produce flowers for reproduction. Flower development is a multistep developmental process, beginning with the initiation of the floral meristems, followed by floral meristem identity specification and maintenance, organ primordia initiation, floral organ identity specification, floral stem cell termination and finally floral organ maturation. During flower development, each of a large number of genes is expressed in a spatiotemporally regulated manner. Underlying these molecular and phenotypic events are various genetic and epigenetic pathways, consisting of diverse transcription factors, chromatin-remodeling factors and signaling molecules. Over the past 30 years, genetic, biochemical and genomic assays have revealed the underlying genetic frameworks that control flower development. Here, we will review the transcriptional regulation of flower development in two model species: Arabidopsis thaliana and rice (Oryza sativa). We focus on epigenetic regulation that functions to co-ordinate transcription pathways in flower development.

ALTERED MERISTEM PROGRAM1 suppresses ectopic stem cell niche formation in the shoot apical meristem in a largely cytokinin-independent manner.

Plants are able to reiteratively form new organs in an environmentally adaptive manner during postembryonic development. Organ formation in plants is dependent on stem cell niches (SCNs), which are located in the so-called meristems. Meristems show a functional zonation along the apical-basal axis and the radial axis. Shoot apical meristems of higher plants are dome-like structures, which contain a central SCN that consists of an apical stem cell pool and an underlying organizing center. Organ primordia are formed in the circular peripheral zone (PZ) from stem cell descendants in which differentiation programs are activated. One mechanism to keep this radial symmetry integrated is that the existing SCN actively suppresses stem cell identity in the PZ. However, how this lateral inhibition system works at the molecular level is far from understood. Here, we show that a defect in the putative carboxypeptidase ALTERED MERISTEM PROGRAM1 (AMP1) causes the formation of extra SCNs in the presence of an intact primary shoot apical meristem, which at least partially contributes to the enhanced shoot meristem size and leaf initiation rate found in the mutant. This defect appears to be neither a specific consequence of the altered cytokinin levels in amp1 nor directly mediated by the WUSCHEL/CLAVATA feedback loop. De novo formation of supernumerary stem cell pools was further enhanced in plants mutated in both AMP1 and its paralog LIKE AMP1, indicating that they exhibit partially overlapping roles to suppress SCN respecification in the PZ.

Histone Acetylation Accompanied with Promoter Sequences Displaying Differential Expression Profiles of B-Class MADS-Box Genes for Phalaenopsis Floral Morphogenesis

Five B-class MADS-box genes, including four APETALA3 (AP3)-like PeMADS2∼5 and one PISTILLATA (PI)-like PeMADS6, specify the spectacular flower morphology in orchids. The PI-like PeMADS6 ubiquitously expresses in all floral organs. The four AP3-like genes, resulted from two duplication events, express ubiquitously at floral primordia and early floral organ stages, but show distinct expression profiles at late floral organ primordia and floral bud stages. Here, we isolated the upstream sequences of PeMADS2∼6 and studied the regulatory mechanism for their distinct gene expression. Phylogenetic footprinting analysis of the 1.3-kb upstream sequences of AP3-like PeMADS2∼5 showed that their promoter regions have sufficiently diverged and contributed to their subfunctionalization. The amplified promoter sequences of PeMADS2∼6 could drive beta-glucuronidase (GUS) gene expression in all floral organs, similar to their expression at the floral primordia stage. The promoter sequence of PeMADS4, exclusively expressed in lip and column, showed a 1.6∼3-fold higher expression in lip/column than in sepal/petal. Furthermore, we noted a 4.9-fold increase in histone acetylation (H3K9K14ac) in the translation start region of PeMADS4 in lip as compared in petal. All these results suggest that the regulation via the upstream sequences and increased H3K9K14ac level may act synergistically to display distinct expression profiles of the AP3-like genes at late floral organ primordia stage for Phalaenopsis floral morphogenesis.