The LRR proteins capricious and Tartan mediate cell interactions during DV boundary formation in the Drosophila wing

Mechanisms to segregate cell populations play important roles in tissue patterning during animal development. Rhombomeres and compartments in the ectoderm and imaginal discs of Drosophila are examples in which initially homogenous populations of cells come to be separated by boundaries of lineage restriction. Boundary formation depends in part on signaling between the distinctly specified cell populations that comprise compartments and in part on formation of affinity boundaries that prevent intermingling of these cell populations. Here, we present evidence that two transmembrane proteins with leucine-rich repeats, known as Capricious and Tartan, contribute to formation of the affinity boundary between dorsal and ventral compartments during Drosophila wing development.     

Two linked hairy/Enhancer of split-related zebrafish genes,her1 and her7, function together to refine alternating somite boundaries

The formation of somites, reiterated structures that will give rise to vertebrae and muscles, is thought to be dependent upon a molecular oscillator that may involve the Notch pathway. hairy/Enhancer of split related [E(spl)]-related (her or hes) genes, potential targets of Notch signaling, have been implicated as an output of the molecular oscillator. We have isolated a zebrafish deficiency, b567, that deletes two linked her genes, her1 and her7. Homozygous b567 mutants have defective somites along the entire embryonic axis. Injection of a combination of her1 and her7 (her1+7) morpholino modified antisense oligonucleotides (MOs) phenocopies the b567 mutant somitic phenotype, indicating that her1 and her7 are necessary for normal somite formation and that defective somitogenesis in b567 mutant embryos is due to deletion of her1 and her7. Analysis at the cellular level indicates that somites in her1+7-deficient embryos are enlarged in the anterior-posterior dimension. Weak somite boundaries are often found within these enlarged somites which are delineated by stronger, but imperfect, boundaries. In addition, the anterior-posterior polarity of these enlarged somites is disorganized. Analysis of her1 MO-injected embryos and her7 MO-injected embryos indicates that although these genes have partially redundant functions in most of the trunk region, her1 is necessary for proper formation of the anteriormost somites and her7 is necessary for proper formation of somites posterior to somite 11. By following somite development over time, we demonstrate that her genes are necessary for the formation of alternating strong somite boundaries. Thus, even though two potential downstream components of Notch signaling are lacking in her1+7-deficient embryos, somite boundaries form, but do so with a one and a half to two segment periodicity.     

Compartments and their boundaries in vertebrate brain development

Fifteen years ago, cell lineage restriction boundaries were discovered in the embryonic vertebrate hindbrain, subdividing it into a series of cell-tight compartments (known as rhombomeres). Compartition, together with segmentally reiterative neuronal architecture and the nested expression of Hox genes, indicates that the hindbrain has a truly metameric organization. This finding initiated a search for compartments in other regions of the developing brain. The results of recent studies have clarified where compartment boundaries exist, have shed light on molecular mechanisms that underlie their formation and have revealed an important function of these boundaries: the positioning and stabilization of local signalling centres.     

Notch activation regulates the segregation and differentiation of rhombomere boundary cells in the zebrafish hindbrain

During segmentation of the vertebrate hindbrain, a distinct population of boundary cells forms at the interface between each segment. Little is known regarding mechanisms that regulate the formation or functions of these cells. We have investigated a potential role of Notch signaling and find that in the zebrafish hindbrain, radical fringe is expressed in boundary cells and delta genes are expressed adjacent to boundaries, consistent with a sustained activation of Notch in boundary cells. Mosaic expression experiments reveal that activation of the Notch/Su(H) pathway regulates cell affinity properties that segregate cells to boundaries. In addition, Notch signaling correlates with a delayed neurogenesis at hindbrain boundaries and is required to inhibit premature neuronal differentiation of boundary cells. These findings reveal that Notch activation couples the regulation of location and differentiation in hindbrain boundary cells. Such coupling may be important for these cells to act as a stable signaling center.     

Somitogenesis as a model to study the formation of morphological boundaries and cell epithelialization

The formation of morphological boundaries between developing tissues is an integral mechanism for generating body forms and functions. For the molecular and cellular studies of how such morphological boundaries form, somitogenesis serves as a particularly good model. When an intersomitic boundary forms in the anterior end of the presomitic mesoderm, cells undergo dynamic behaviors including a separation of tissues and changes in cell shape from mesenchymal to epithelial. Moreover, these events occur repeatedly and periodically. We here overview the inductive events that have recently been shown to play important roles in the formation of the intersomitic fissures. We then discuss molecular mechanisms underlying these inductive actions, and also discuss how the fissure formation is interpreted by the subsequent morphogenesis, including cell epithelialization and the acquisition of anterior–posterior identities in the newly formed somite. Thus, somitogenesis provides a unique model to understand how sequentially occurring processes of morphogenesis are coordinated in a 3-D environment.     

Boundaries of belief: religious practices and the construction of ethnic identity in Hui Muslim communities†

Though scholars of ethnicity remark that religion is an important qualifying attribute for membership in an ethnic group, the nature of the relationship between religious faith and ethnic identity requires further exploration. An approach that emphasizes the importance of religious practices in forming and maintaining ethnic boundaries may offer a more complete explanation of the relationship between religion and ethnicity. This article proposes a framework for understanding how religious practices influence ethnic boundary formation and maintenance processes. I propose that religious practices may play a universalizing, negotiating, or differentiating role in influencing the formation and maintenance of ethnic boundaries. To illustrate these various roles played by religious processes, the article presents a heuristic case study of Islamic faith in boundary setting processes in Hui Muslim communities in China.     

Multiple morphogens and rapid elongation promote segmental patterning during development

The vertebrate hindbrain is segmented into rhombomeres (r) initially defined by distinct domains of gene expression. Previous studies have shown that noise-induced gene regulation and cell sorting are critical for the sharpening of rhombomere boundaries, which start out rough in the forming neural plate (NP) and sharpen over time. However, the mechanisms controlling simultaneous formation of multiple rhombomeres and accuracy in their sizes are unclear. We have developed a stochastic multiscale cell-based model that explicitly incorporates dynamic morphogenetic changes (i.e. convergent-extension of the NP), multiple morphogens, and gene regulatory networks to investigate the formation of rhombomeres and their corresponding boundaries in the zebrafish hindbrain. During pattern initiation, the short-range signal, fibroblast growth factor (FGF), works together with the longer-range morphogen, retinoic acid (RA), to specify all of these boundaries and maintain accurately sized segments with sharp boundaries. At later stages of patterning, we show a nonlinear change in the shape of rhombomeres with rapid left-right narrowing of the NP followed by slower dynamics. Rapid initial convergence improves boundary sharpness and segment size by regulating cell sorting and cell fate both independently and coordinately. Overall, multiple morphogens and tissue dynamics synergize to regulate the sizes and boundaries of multiple segments during development.     

Neural fold formation at newly created boundaries between neural plate and epidermis in the axolotl

According to a recent model, the cortical tractor model, neural fold and neural crest formation occurs at the boundary between neural plate and epidermis because random cell movements become organized at this site. If this is correct, then a fold should form at any boundary between epidermis and neural plate. To test that proposition, we created new boundaries in axolotl embryos by juxtaposing pieces of neural plate and epidermis that would not normally participate in fold formation. These boundaries were examined superficially and histologically for the presence of folds, permitting the following observations. Folds form at each newly created boundary, and as many folds form as there are boundaries. When two folds meet they fuse into a hollow "tube" of neural tissue covered by epidermis. Sections reveal that these ectopic folds and "tubes" are morphologically similar to their natural counterparts. Transplanting neural plate into epidermis produces nodules of neural tissue with central lumens and peripheral nerve fibers, and transplanting epidermis into neural plate causes the neural tube and the dorsal fin to bifurcate in the region of the graft. Tissue transplanted homotypically as a control integrates into the host tissue without forming folds. When tissue from a pigmented embryo is transplanted into an albino host, the presence of pigment allows the donor cells to be distinguished from those of the host. Mesenchymal cells and melanocytes originating from neural plate transplants indicate that neural crest cells form at these new boundaries. Thus, any boundary between neural plate and epidermis denotes the site of a neural fold, and the behavior of cells at this boundary appears to help fold the epithelium. Since folds can form in ectopic locations on an embryo, local interactions rather than classical neural induction appear to be responsible for the formation of neural folds and neural crest.     

Common mechanisms for boundary formation in somitogenesis and brain development: shaping the 'chic' chick

When organs and tissues acquire their characteristic shapes and functions during early development, boundaries are established that distinguish between and delimit distinct areas. Such boundaries are not mere edges, but also play important roles as secondary signaling centers in subsequent morphogenesis. Following on pioneering findings provided by studies in Drosophila, the mechanisms underlying boundary formation in vertebrate embryogenesis have attracted the interest of an increasing number of researchers. Somitogenesis and brain development, in particular, serve as model systems for the study of the molecular and cellular events occurring at developing boundaries. Recent findings allow us to draw some general pictures concerning the shared mechanisms that participate in these processes of organogenesis, in which Notch, Eph/ephrin and cadherin-mediated signaling are among the main key regulators.     

Methylation-mediated silencing of TMS1/ASC is accompanied by histone hypoacetylation and CpG island-localized changes in chromatin architecture.

Aberrant methylation of CpG-dense islands in the promoter regions of genes is an acquired epigenetic alteration associated with the silencing of tumor suppressor genes in human cancers. In a screen for endogenous targets of methylation-mediated gene silencing, we identified a novel CpG island-associated gene, TMS1, which is aberrantly methylated and silenced in response to the ectopic expression of DNA methyltransferase-1. TMS1 functions in the regulation of apoptosis and is frequently methylated and silenced in human breast cancers. In this study, we characterized the methylation pattern and chromatin architecture of the TMS1 locus in normal fibroblasts and determined the changes associated with its progressive methylation. In normal fibroblasts expressing TMS1, the CpG island is defined by an unmethylated domain that is separated from densely methylated flanking DNA by distinct 5' and 3' boundaries. Analysis of the nucleoprotein architecture of the locus in intact nuclei revealed three DNase I-hypersensitive sites that map within the CpG island. Strikingly, two of these sites coincided with the 5'- and 3'-methylation boundaries. Methylation of the TMS1 CpG island was accompanied by loss of hypersensitive site formation, hypoacetylation of histones H3 and H4, and gene silencing. This altered chromatin structure was confined to the CpG island and occurred without significant changes in methylation, histone acetylation, or hypersensitive site formation at a fourth DNase I-hypersensitive site 2 kb downstream of the TMS1 CpG island. The data indicate that there are sites of protein binding and/or structural transitions that define the boundaries of the unmethylated CpG island in normal cells and that aberrant methylation overcomes these boundaries to direct a local change in chromatin structure, resulting in gene silencing.     

Regulation of proximal-distal intercalation during limb regeneration in the axolotl (Ambystoma mexicanum)

Intercalation is the process whereby cells located at the boundary of a wound interact to stimulate proliferation and the restoration of the structures between the boundaries that were lost during wounding. Thus, intercalation is widely considered to be the mechanism of regeneration. When a salamander limb is amputated, the entire cascade of regeneration events is activated, and the missing limb segments and their boundaries (joints) as well as the structures within each segment are regenerated. Therefore, in an amputated limb it is not possible to distinguish between intersegmental regeneration (formation of new segments/joints) and intrasegmental regeneration (formation of structures within a given segment), and it is not possible to study the differential regulation of these two processes. We have used two models for regeneration that allow us to study these two processes independently, and report that inter- and intrasegmental regeneration are different processes regulated by different signaling pathways. New limb segments/joints can be regenerated from cells that dedifferentiate to form blastema cells in response to signaling that is mediated in part by fibroblast growth factor.     

Anteroposterior patterning is required within segments for somite boundary formation in developing zebrafish

Somite formation involves the establishment of a segmental prepattern in the presomitic mesoderm, anteroposterior patterning of each segmental primordium and formation of boundaries between adjacent segments. How these events are co-ordinated remains uncertain. In this study, analysis of expression of zebrafish mesp-a reveals that each segment acquires anteroposterior regionalisation when located in the anterior presomitic mesoderm. Thus anteroposterior patterning is occurring after the establishment of a segmental prepattern in the paraxial mesoderm and prior to somite boundary formation. Zebrafish fss(-), bea(-), des(-) and aei(-) embryos all fail to form somites, yet we demonstrate that a segmental prepattern is established in the presomitic mesoderm of all these mutants and hox gene expression shows that overall anteroposterior patterning of the mesoderm is also normal. However, analysis of various molecular markers reveals that anteroposterior regionalisation within each segment is disturbed in the mutants. In fss(-), there is a loss of anterior segment markers, such that all segments appear posteriorized, whereas in bea(-), des(-) and aei(-), anterior and posterior markers are expressed throughout each segment. Since somite formation is disrupted in these mutants, correct anteroposterior patterning within segments may be a prerequisite for somite boundary formation. In support of this hypothesis, we show that it is possible to rescue boundary formation in fss(-) through the ectopic expression of EphA4, an anterior segment marker, in the paraxial mesoderm. These observations indicate that a key consequence of the anteroposterior regionalisation of segments may be the induction of Eph and ephrin expression at segment interfaces and that Eph/ephrin signalling subsequently contributes to the formation of somite boundaries.     

Integrinalpha5-dependent fibronectin accumulation for maintenance of somite boundaries in zebrafish embryos

Boundary formation and epithelialization are crucial processes in the morphological segmentation of vertebrate somites. By a genetic screening procedure with zebrafish, we identified two genes, integrinalpha5 (itga5) and fibronectin (fn), required for these processes. Fibronectin proteins accumulate at somite boundaries in accordance with epithelialization of the somites. Both Fibronectin accumulation and the epithelialization are dependent on itga5, which is expressed in the most medial part of somites. Although somite boundaries are initially formed, but not maintained, in the anterior trunk of the mutant embryos deficient in either gene, their maintenance is defective at all axial levels of embryos deficient for both of these genes. Therefore, Integrinalpha5-directed assembly of Fibronectin appears critical for epithelialization and boundary maintenance of somites. Furthermore, with an additional deficiency in ephrin-B2a, the segmental defect in itga5 or fn mutant embryos is expanded posteriorly, indicating that both Integrin-Fibronectin and Eph-Ephrin systems function cooperatively in maintaining somite boundaries.     

Making connections: boundaries and insulators in Drosophila

In eukaryotes, enhancers must often exert their effect over many tens of kilobases of DNA with a choice between many different promoters. Given this situation, elements known as chromatin boundaries have evolved to prevent adventitious interactions between enhancers and promoters. The amenability of Drosophila to molecular genetics has been crucial to the discovery and analysis of these elements. Since these elements are involved in such diverse processes and show little or no sequence similarity between them, no single molecular mechanism has been identified that accounts for their activity. However, over the past approximately 5 years, evidence has accumulated suggesting that boundaries probably function through the formation of long-distance chromatin loops. These loops have been proposed to play a crucial role in both controlling enhancer-promoter interactions and packing DNA.     

Communication compartments in mixed cell cultures

Mixed cultures of epithelial (BRL) cells and fibroblasts (BHK), which sort themselves out into separate domains of each cell type, form communication compartments. Electrical coupling, dye coupling and metabolic coupling measurements have been used to show that small ions and molecules can move freely via intercellular junctions between all the cells in a domain, while their movement across the boundaries between domains is severely restricted. Metabolic coupling is the most sensitive method for detecting trans-boundary communication but the results obtained from all three methods are compatible. The data suggest the reduced transfer across the boundaries is due to fewer channels, resulting from a lower frequency of junction formation between heterologous cells, rather than to channels of smaller diameter. Concentration gradients of small cytoplasmic molecules can be established within these communication compartments which are similar to those predicted to explain pattern formation in developing systems. It is suggested that the cell surface features which cause this sorting out are also responsible for the reduced frequency of heterologous junction formation and hence for compartmentalization.     

Interactions between muscle fibers and segment boundaries in zebrafish

The most obvious segmental structures in the vertebrate embryo are somites: transient structures that give rise to vertebrae and much of the musculature. In zebrafish, most somitic cells give rise to long muscle fibers that are anchored to intersegmental boundaries. Therefore, this boundary is analogous to the mammalian tendon in that it transduces muscle-generated force to the skeletal system. We have investigated interactions between somite boundaries and muscle fibers. We define three stages of segment boundary formation. The first stage is the formation of the initial epithelial somite boundary. The second "transition" stage involves both the elongation of initially round muscle precursor cells and somite boundary maturation. The third stage is myotome boundary formation, where the boundary becomes rich in extracellular matrix and all muscle precursor cells have elongated to form long muscle fibers. It is known that formation of the initial epithelial somite boundary requires Notch signaling; vertebrate Notch pathway mutants show severe defects in somitogenesis. However, many zebrafish Notch pathway mutants are homozygous viable suggesting that segmentation of their larval and adult body plans at least partially recovers. We show that epithelial somite boundary formation and slow-twitch muscle morphogenesis are initially disrupted in after eight (aei) mutant embryos (which lack function of the Notch ligand, DeltaD); however, myotome boundaries form later ("recover") in a Hedgehog-dependent fashion. Inhibition of Hedgehog-induced slow muscle induction in aei/deltaD and deadly seven (des)/notch1a mutant embryos suggests that slow muscle is necessary for myotome boundary recovery in the absence of initial epithelial somite boundary formation. Because we have previously demonstrated that slow muscle migration triggers fast muscle cell elongation in zebrafish, we hypothesize that migrating slow muscle facilitates myotome boundary formation in aei/deltaD mutant embryos by patterning coordinated fast muscle cell elongation. In addition, we utilized genetic mosaic analysis to show that somite boundaries also function to limit the extent to which fast muscle cells can elongate. Combined, our results indicate that multiple interactions between somite boundaries and muscle fibers mediate zebrafish segmentation.     

An RNA Interference Screen for Genes Required to Shape the Anteroposterior Compartment Boundary in Drosophila Identifies the Eph Receptor

The formation of straight compartment boundaries separating groups of cells with distinct fates and functions is an evolutionarily conserved strategy during animal development. The physical mechanisms that shape compartment boundaries have recently been further elucidated, however, the molecular mechanisms that underlie compartment boundary formation and maintenance remain poorly understood. Here, we report on the outcome of an RNA interference screen aimed at identifying novel genes involved in maintaining the straight shape of the anteroposterior compartment boundary in Drosophila wing imaginal discs. Out of screening 3114 transgenic RNA interference lines targeting a total of 2863 genes, we identified a single novel candidate that interfered with the formation of a straight anteroposterior compartment boundary. Interestingly, the targeted gene encodes for the Eph receptor tyrosine kinase, an evolutionarily conserved family of signal transducers that has previously been shown to be important for maintaining straight compartment boundaries in vertebrate embryos. Our results identify a hitherto unknown role of the Eph receptor tyrosine kinase in Drosophila and suggest that Eph receptors have important functions in shaping compartment boundaries in both vertebrate and insect development.     

Impact of Nanoscale Elemental Distribution in High‐Performance Kesterite Solar Cells

The effort to develop earth‐abundant kesterite solar cells has led to an approximate doubling of the power conversion efficiency over the past five years to 12.6%, primarily due to increases in short‐circuit current and fill factor; open‐circuit voltage has resisted similar change, limiting further efficiency improvement. In the present investigation, Auger nanoprobe spectroscopy, X‐ray/ultraviolet photoelectron spectroscopy, and device characterization are used to provide a comprehensive understanding of the role of grain boundaries and interfaces in limiting performance in kesterite‐based devices. High photovoltaic performance is found to correlate with grain boundaries that are Cu‐depleted and enriched with SnOx. The formation of this bulk‐like oxide at grain boundaries with type I band offset provides a unique effective passivation that limits electron‐hole recombination. Building on these new insights, photovoltaic device simulations are performed that show optimized electrostatic designs can compensate for bulk defects, allowing efficiencies closer to the theoretical limit.