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.     

Cell segregation in the vertebrate hindbrain relies on actomyosin cables located at the interhombomeric boundaries

Segregating cells into compartments during embryonic development is essential for growth and pattern formation. Physical mechanisms shaping compartment boundaries were recently explored in Drosophila, where actomyosin‐based barriers were revealed to be important for keeping cells apart. In vertebrates, interhombomeric boundaries are straight interfaces, which often serve as signaling centers that pattern the surrounding tissue. Here, we demonstrate that in the hindbrain of zebrafish embryos cell sorting sharpens the molecular boundaries and, once borders are straight, actomyosin barriers are key to keeping rhombomeric cells segregated. Actomyosin cytoskeletal components are enriched at interhombomeric boundaries, forming cable‐like structures in the apical side of the neuroepithelial cells by the time morphological boundaries are visible. When myosin II function is inhibited, cable structures do not form, leading to rhombomeric cell mixing. Downregulation of EphA4a compromises actomyosin cables and cells with different rhombomeric identity intermingle, and the phenotype is rescued enhancing myosin II activity. Moreover, enrichment of actomyosin structures is obtained when EphA4 is ectopically expressed in even‐numbered rhombomeres. These findings suggest that mechanical barriers act downstream of EphA/ephrin signaling to segregate cells from different rhombomeres.     

Epifluorescence Microscopy of Surface Domain Microheterogeneity in Myelin Monolayers at the Air-Water Interface

Myelin lipids form liquid-expanded monolayers at the air-water interface, with no evidence of surface pressure-induced two-dimensional phase transition. However, the film doped with 2 mole % of the fluorescent probe N-(7-nitro-2-1,3-benzoxadiazol-4-yl) Diacyl Phosphatidyl-ethanolamine (NBD-PE) shows an irregular pattern of coexisting laterally segregated surface domains with diffuse boundaries that change from smooth patterns to fractal-like structures depending on surface pressure. Successive expansion-recompression cycles lead to more defined domains, with a general reorganization occurring at surface pressures of about 20 mN/m. At least two coexisting phases occur over almost all the compression isotherms. The presence of proteins in whole myelin monolayers induces defined domain textures with relatively sharp boundaries. The patterns during compression and expansion are quite similar and, after the first cycle, little changes occur under recompression. The patterns observed provide topographical evidence for the existence of dynamic domain microheterogeneity in the surface of myelin interfaces.     

Fluorescence methods to detect phase boundaries in lipid bilayer mixtures

Phase diagrams of lipid mixtures can show several different regions of phase coexistence, which include liquid-disordered, liquid-ordered, and gel phases. Some phase regions are small, and some have sharp boundaries. The identity of the phases, their location in composition space, and the nature of the transitions between the phases are important for understanding the behavior of lipid mixtures. High fidelity phase boundary detection requires high compositional resolution, on the order of 2% compositional increments. Sample artifacts, especially the precipitation of crystals of anhydrous cholesterol, can occur at higher cholesterol concentrations unless precautions are taken. Fluorescence resonance energy transfer (FRET) can be used quantitatively to find the phase boundaries and even partition coefficients of the dyes between coexisting phases, but only if data are properly corrected for non-FRET contributions. Self-quenching of the dye fluorescence can be significant, distorting the data at dye concentrations that intuitively might be considered acceptable. Even more simple than FRET experiments, measurements of single-dye fluorescence can be used to find phase boundaries. Both FRET and single-dye fluorescence readily detect the formation of phase domains that are much smaller than the wavelength of light, i.e. "nanoscopic" domains.     

Molecular mechanisms of cell segregation and boundary formation in development and tumorigenesis

The establishment and maintenance of precisely organized tissues requires the formation of sharp borders between distinct cell populations. The maintenance of segregated cell populations is also required for tissue homeostasis in the adult, and deficiencies in segregation underlie the metastatic spreading of tumor cells. Three classes of mechanisms that underlie cell segregation and border formation have been uncovered. The first involves differences in cadherin-mediated cell-cell adhesion that establishes interfacial tension at the border between distinct cell populations. A second mechanism involves the induction of actomyosin-mediated contraction by intercellular signaling, such that cortical tension is generated at the border. Third, activation of Eph receptors and ephrins can lead to both decreased adhesion by triggering cleavage of E-cadherin, and to repulsion of cells by regulation of the actin cytoskeleton, thus preventing intermingling between cell populations. These mechanisms play crucial roles at distinct boundaries during development, and alterations in cadherin or Eph/ephrin expression have been implicated in tumor metastasis.     

Replication timing properties across the pseudoautosomal region boundary and cytogenetic band boundaries on human distal Xp

The establishment of human chromosomal regions as distinct and characteristic domains has been demonstrated by the reproducible banding patterns observed on metaphase chromosomes as a result of various staining techniques. Although the exact molecular properties responsible for the patterns are not well understood, a general correlation has been established between the time of replication of a particular region of DNA and its banding characteristics. Using a replication timing assay based on fluorescence in situ hybridization patterns, we investigated replication timing properties across chromosomal regions with potentially distinct chromatin properties. Relative replication timing values were determined using cosmid DNA probes around the pseudoautosomal region boundary in Xp22.3 and the cytogenetic band boundary regions surrounding Xp22.2. Although we observed replication timing domains that were generally consistent with cytogenetic banding patterns, we did not find sharp replication timing boundaries at either the pseudoautosomal region boundary or at the cytogenetic band boundaries. Received: 6 September 1997; in revised form: 16 December 1997 / Accepted: 5 January 1998     

Regulatory systems of genome domains with vague boundaries

The specific features of genome domains lacking distinct boundaries are considered. These domains cannot be mapped by testing extended genome regions for nuclease sensitivity and thereby differ from structural domains determined at the level of DNA folding in chromatin. Yet they possess the properties of typical functional domains, containing a gene or several coordinated genes along with a complex of cis-regulatory elements, which control these genes. Domains with vague boundaries may be mapped with certain structural tests, e.g., by assessing histone acetylation or the distribution of tissue-specific DNase I-hypersensitive sites through extended genome regions. The mechanisms are described in detail that regulate the function of genes in domains with vague boundaries, including overlapping domains with genes differing in tissue specificity of expression.     

Regulatory Systems of Genome Domains with Vague Boundaries

The specific features of genome domains lacking distinct boundaries are considered. These domains cannot be mapped by testing extended genome regions for nuclease sensitivity and thereby differ from structural domains determined at the level of DNA folding in chromatin. Yet they possess the properties of typical functional domains, containing a gene or several coordinated genes along with a complex of cis-regulatory elements, which control these genes. Domains with vague boundaries may be mapped with certain structural tests, e.g., by assessing histone acetylation or the distribution of tissue-specific DNase I-hypersensitive sites through extended genome regions. The mechanisms are described in detail that regulate the function of genes in domains with vague boundaries, including overlapping domains with genes differing in tissue specificity of expression.     

High-resolution analysis of DNA replication domain organization across an R/G-band boundary.

Establishing how mammalian chromosome replication is regulated and how groups of replication origins are organized into replication bands will significantly increase our understanding of chromosome organization. Replication time bands in mammalian chromosomes show overall congruency with structural R- and G-banding patterns as revealed by different chromosome banding techniques. Thus, chromosome bands reflect variations in the longitudinal structure and function of the chromosome, but little is known about the structural basis of the metaphase chromosome banding pattern. At the microscopic level, both structural R and G bands and replication bands occupy discrete domains along chromosomes, suggesting separation by distinct boundaries. The purpose of this study was to determine replication timing differences encompassing a boundary between differentially replicating chromosomal bands. Using competitive PCR on replicated DNA from flow-sorted cell cycle fractions, we have analyzed the replication timing of markers spanning roughly 5 Mb of human chromosome 13q14.3/q21.1. This is only the second report of high-resolution analysis of replication timing differences across an R/G-band boundary. In contrast to previous work, however, we find that band boundaries are defined by a gradient in replication timing rather than by a sharp boundary separating R and G bands into functionally distinct chromatin compartments. These findings indicate that topographical band boundaries are not defined by specific sequences or structures.     

Dynamic domains of gene expression in the early avian forebrain.

The expression domains of genes implicated in forebrain patterning often share borders at specific anteroposterior positions. This observation lies at the heart of the prosomeric model, which proposes that such shared borders coincide with proposed compartment boundaries and that specific combinations of genes expressed within each compartment are responsible for its patterning. Thus, genes such as Emx1, Emx2, Pax6, and qin (Bf1) are seen as being responsible for specifying different regions in the forebrain (diencephalon and telencephalon). However, the early expression of these genes, before the appearance of putative compartment boundaries, has not been characterized. In order to determine whether they have stable expression domains before this stage, we have compared mRNA expression of each of the above genes, relative both to one another and to morphological landmarks, in closely staged chick embryos. We find that, between HH stage 8 and HH stage 13, each of the genes has a dynamic spatial and temporal expression pattern. To test for autonomy of gene expression in the prosencephalon, we grafted tissue from this region to more caudal positions in the neural tube and analyzed for expression of Emx1, Emx2, qin, or Pax6. We find that gene expression is autonomous in prosencephalic tissue from as early as HH stage 8. In the case of Emx1, our data suggest that, from as early stage 8, presumptive telencephalic tissue also is committed to express this gene. We propose that early patterning along the anteroposterior axis of the presumptive telencephalon occurs across a field that is subdivided by different combinations of genes, with some overlapping areas, but without either sharp boundaries or stable interfaces between expression domains.     

Petal Development in Lotus japonicus

Previous studies have demonstrated that petal shape and size in legume flowers are determined by two separate mechanisms, dorsoventral (DV) and organ internal (IN) asymmetric mechanisms, respectively. However, little is known about the molecular mechanisms controlling petal development in legumes. To address this question, we investigated petal development along the floral DV axis in Lotus japonicus with respect to cell and developmental biology by comparing wild‐type legumes to mutants. Based on morphological markers, the entire course of petal development, from initiation to maturity, was grouped to define 3 phases or 13 stages. In terms of epidermal micromorphology from adaxial surface, mature petals were divided into several distinct domains, and characteristic epidermal cells of each petal differentiated at stage 9, while epidermal cells of all domains were observed until stage 12. TCP and MIXTA‐like genes were found to be differentially expressed in various domains of petals at stages 9 and 12. Our results suggest that DV and IN mechanisms interplay at different stages of petal development, and their interaction at the cellular and molecular level guides the elaboration of domains within petals to achieve their ideal shape, and further suggest that TCP genes determine petal identity along the DV axis by regulating MIXTA‐like gene expression.     

Rethinking Indigenous Place: Igorot Identity and Locality in the Philippines

Spanish and American colonisers ascribed the identity ‘Igorot’ to the peoples of the northern Philippine mountains, positioning them in the ‘tribal slot’, somewhere between ordinary peasants and ‘backward’ primitives. From this marginal position, contemporary Igorot communities have been comparatively successful in formalising their entitlements to land and resources in their dealings with the Philippine State. This success depends on a discourse tying indigenous or ‘tribal’ culture to particular places. Colonial and, now, local anthropology has been recruited to this process through the mapping of community boundaries. This has allowed groups to secure official status as ‘cultural communities' and gain legal recognition of their ancestral domains. Ironically, even as ancestral domains are recognised, the municipalities that hold such domains have ceased to be bounded containers for Igorot localities, if they ever were. Participation in global indigenous networks, circular migration, and ongoing relations with emigrants overseas blur the spatial, temporal, and social boundaries of Igorot communities. Transnational flows of people, information, and value are recruited to support the essentialised versions of indigenous identity necessary for negotiations with the state. Here, I show how the specific history of the Igorot ‘tribal slot’ enables communities to perform essentialised indigeneity and simultaneously enact highly translocal modes of cultural reproduction.     

Cell specification in the Arabidopsis root epidermis requires the activity of ECTOPIC ROOT HAIR 3--a katanin-p60 protein.

The Arabidopsis root is composed of radial cell layers, each with distinct identities. The epidermal layer is composed of rows of hair cells flanked on either side by rows of non-hair epidermal cells. The development of hair and non-hair cells is dependent on domains of positional information with strict boundaries. The pattern of cell differentiation and the expression of molecular markers of cell fate is altered in the ectopic root hair 3 (erh3) mutant epidermis indicating that ERH3 is required for the specification of cell fates from early in development (in the meristem) through differentiation. Furthermore the expression of molecular markers indicates that the specification of cell identities is defective within other radial cell layers. ERH3 encodes a p60 katanin protein that is expressed throughout the plant. Katanin proteins are known to sever microtubules, and have a role in the organisation of the plant cell wall since mutants with decreased katanin activity have been shown to have defective walls. We suggest that microtubules are involved in the specification of cell identities in cells of the Arabidopsis root. Microtubules may be required for the localization of positional cues in the wall that have previously been shown to operate in the development of the root epidermis. Alternatively microtubules may be involved in another as yet undefined process required for the specification of cell identity in plants.