Role of hunchback in segment patterning of Locusta migratoria manilensis revealed by parental RNAi

In long germ embryos, all body segments are specified simultaneously during the blastoderm stage. In contrast, in short germ embryos, only the anterior segments are specified during the blastoderm stage, leaving the rest of the body plan to be specified later. The striking embryological differences between short and long germ segmentation imply fundamental differences in patterning at the molecular level. To gain insights into the segmentation mechanisms of short germ insects, we have investigated the role of the homologue of the Drosophila gap gene hunchback (hb) in a short germ insect Locusta migratoria manilensi by paternal RNA interference (RNAi). Phenotypes resulting from hb knockdown were categorized into three classes based on severity. In the most extreme case, embryos developed the most anterior structures only, including the labrum, antennae and eyes. The following conclusions were drawn: (i) L. migratoria manilensis hb (Lmm'hb) controls germ band morphogenesis and segmentation in the anterior region; (ii) Lmm'hb may function as a gap gene in a wide domain including the entire gnathum and thorax; and (iii) Lmm'hb is required for proper growth of the posterior germ band. These findings suggest a more extensive role for L. migratoria manilensis hunchback in anterior patterning than those described in Drosophila.     

Absence of Nodal signaling promotes precocious neural differentiation in the mouse embryo

After implantation, mouse embryos deficient for the activity of the transforming growth factor-beta member Nodal fail to form both the mesoderm and the definitive endoderm. They also fail to specify the anterior visceral endoderm, a specialized signaling center which has been shown to be required for the establishment of anterior identity in the epiblast. Our study reveals that Nodal-/- epiblast cells nevertheless express prematurely and ectopically molecular markers specific of anterior fate. Our analysis shows that neural specification occurs and regional identities characteristic of the forebrain are established precociously in the Nodal-/- mutant with a sequential progression equivalent to that of wild-type embryo. When explanted and cultured in vitro, Nodal-/- epiblast cells readily differentiate into neurons. Genes normally transcribed in organizer-derived tissues, such as Gsc and Foxa2, are also expressed in Nodal-/- epiblast. The analysis of Nodal-/-;Gsc-/- compound mutant embryos shows that Gsc activity plays no critical role in the acquisition of forebrain characters by Nodal-deficient cells. This study suggests that the initial steps of neural specification and forebrain development may take place well before gastrulation in the mouse and highlights a possible role for Nodal, at pregastrula stages, in the inhibition of anterior and neural fate determination.     

A Gbx homeobox gene in amphioxus: insights into ancestry of the ANTP class and evolution of the midbrain/hindbrain boundary

In the vertebrate central nervous system (CNS), mutual antagonism between posteriorly expressed Gbx2 and anteriorly expressed Otx2 positions the midbrain/hindbrain boundary (MHB), but does not induce MHB organizer genes such as En, Pax2/5/8 and Wnt1. In the CNS of the cephalochordate amphioxus, Otx is also expressed anteriorly, but En, Pax2/5/8 and Wnt1 are not expressed near the caudal limit of Otx, raising questions about the existence of an MHB organizer in amphioxus. To investigate the evolutionary origins of the MHB, we cloned the single amphioxus Gbx gene. Fluorescence in situ hybridization showed that, as in vertebrates, amphioxus Gbx and the Hox cluster are on the same chromosome. From analysis of linked genes, we argue that during evolution a single ancestral Gbx gene duplicated fourfold in vertebrates, with subsequent loss of two duplicates. Amphioxus Gbx is expressed in all germ layers in the posterior 75% of the embryo, and in the CNS, the Gbx and Otx domains abut at the boundary between the cerebral vesicle (forebrain/midbrain) and the hindbrain. Thus, the genetic machinery to position the MHB was present in the protochordate ancestors of the vertebrates, but is insufficient for induction of organizer genes. Comparison with hemichordates suggests that anterior Otx and posterior Gbx domains were probably overlapping in the ancestral deuterostome and came to abut at the MHB early in the chordate lineage before MHB organizer properties evolved.     

Heterotypic cell interactions on a dually patterned surface

It is worth investigating heterotypic cell-cell interactions by mimicking their in vivo structures and environment. In the present study, physiological cellular response and behavior of hepatocytes and endothelial cells were investigated by controlling their contact periphery in a new co-culture system. Rat primary hepatocytes and bovine endothelial cells were co-cultured on a dually patterned surface. Hepatic physiological functions such as albumin secretion and ammonium metabolism were enhanced by increasing heterotypic cell-cell interactions in a patterned co-culture. Furthermore, enhanced hepatic functions through heterotypic interactions are effective within a limited area apart from endothelial cells as evidenced by immunofluorescence staining of hepatic intracellular albumin, indicating that heterotypic interactions act in a paracrine manner. Thus, heterotypic cell communications that play indispensable roles in increasing hepatic physiological functions should be obtained with an increasing periphery of two-cell domains. These findings are important for the reconstruction of complex tissues such as liver and pancreas.     

Scale-dependent interactions and community structure on cobble beaches

Recent theory suggests that scale-dependent interaction between facilitation and competition can generate spatial structure in ecological communities. The application of this hypothesis, however, has been limited to systems with little underlying heterogeneity. We evaluated this prediction in a plant community along an intertidal stress gradient on cobble beaches in Rhode Island, USA. Prior studies have shown that Spartina alterniflora facilitates a forb-dominated community higher in the intertidal by modifying the shoreline environment. We tested the hypothesis that, at a smaller scale, Spartina competitively excludes forb species, explaining their marked absence within the lower Spartina zone. Transplant experiments showed forb species grow significantly better in the Spartina zone when neighbours were removed. Removal of the Spartina canopy led to a massive emergence of annual forbs, showing that competition limits local occupation. These findings indicate that interaction of large-scale facilitation and small-scale competition drives plant zonation on cobble beaches. This study is the first to provide empirical evidence of scale-dependent interactions between facilitation and competition spatially structuring communities in heterogeneous environments.     

Silk Film Culture System for in vitro Analysis and Biomaterial Design

Silk films are promising protein-based biomaterials that can be fabricated with high fidelity and economically within a research laboratory environment ^1,2 . These materials are desirable because they possess highly controllable dimensional and material characteristics, are biocompatible and promote cell adhesion, can be modified through topographic patterning or by chemically altering the surface, and can be used as a depot for biologically active molecules for drug delivery related applications ^3-8 . In addition, silk films are relatively straightforward to custom design, can be designed to dissolve within minutes or degrade over years in vitro or in vivo, and are produce with the added benefit of being transparent in nature and therefore highly suitable for imaging applications ^9-13. The culture system methodology presented here represents a scalable approach for rapid assessments of cell-silk film surface interactions. Of particular interest is the use of surface patterned silk films to study differences in cell proliferation and responses of cells for alignment ^12,14 . The seeded cultures were cultured on both micro-patterned and flat silk film substrates, and then assessed through time-lapse phase-contrast imaging, scanning electron microscopy, and biochemical assessment of metabolic activity and nucleic acid content. In summary, the silk film in vitro culture system offers a customizable experimental setup suitable to the study of cell-surface interactions on a biomaterial substrate, which can then be optimized and then translated to in vivo models. Observations using the culture system presented here are currently being used to aid in applications ranging from basic cell interactions to medical device design, and thus are relevant to a broad range of biomedical fields.