DDX24, a D-E-A-D box RNA helicase, is required for muscle fiber organization and anterior pole specification essential for head regeneration in planarians

ABSTRACTPlanarians have a remarkable ability to undergo whole-body regeneration. The timely establishment of polarity at the wound site followed by the specification of the organizing centers- the anterior pole and the posterior pole, are indispensable for successful regeneration. In planarians, polarity, pole, and positional-information determinants are predominantly expressed by muscles. The molecular toolkit that enables this functionality of planarian muscles however remains poorly understood. Here we report that SMED_DDX24, a D-E-A-D Box RNA helicase and the homolog of human DDX24, is critical for planarian head regeneration. DDX24 is enriched in muscles and its knockdown leads to defective muscle-fiber organization and failure to re-specify anterior pole/organizer. Overall, loss of DDX24 manifests into gross misregulation of many well-characterized positional-control genes and patterning-control genes, necessary for organogenesis and tissue positioning and tissue patterning. In addition, wound-induced Wnt signalling was also upregulated in ddx24 RNAi animals. Canonical WNT-βCATENIN signalling is known to suppress head identity throughout bilateria, including planarians. Modulating this Wnt activity by β-catenin-1 RNAi, the effector molecule of this pathway, partially rescues the ddx24 RNAi phenotype, implying that a high Wnt environment in ddx24 knockdown animals likely impedes their normal head regeneration. Furthermore, at a sub-cellular level, RNA helicases are known to regulate muscle mass and function by regulating their translational landscape. ddx24 knockdown leads to the downregulation of large subunit ribosomal RNA and the 80S ribosome peak, implying its role in ribosome biogenesis and thereby influencing the translational output. This aspect seems to be an evolutionarily conserved role of DDX24. In summary, our work demonstrates the role of a D-E-A-D box RNA helicase in whole-body regeneration through muscle fiber organization, and pole and positional-information re-specification, likely mediated through translation regulation.

The Microtubule Cytoskeleton during the Early Drosophila Spermiogenesis

Sperm elongation and nuclear shaping in Drosophila largely depends on the microtubule cytoskeleton that in early spermatids has centrosomal and non-centrosomal origins. We report here an additional γ-tubulin focus localized on the anterior pole of the nucleus in correspondence of the apical end of the perinuclear microtubules that run within the dense complex. The perinuclear microtubules are nucleated by the pericentriolar material, or centriole adjunct, that surrounds the basal body and are retained to play a major role in nuclear shaping. However, we found that both the perinuclear microtubules and the dense complex are present in spermatids lacking centrioles. Therefore, the basal body or the centriole adjunct seem to be dispensable for the organization and assembly of these structures. These observations shed light on a novel localization of γ-tubulin and open a new scenario on the distribution of the microtubules and the organization of the dense complex during early Drosophila spermiogenesis.

Anomalous Course of Accessory Splenic Arteries in Gastrosplenic Ligament: Case Report and Clinico-Embryological Basis

Accessory splenic arteries in the gastrosplenic ligament constitute one of the extremely sub-component of abdominal vasculature variations and it is imperative to recognize this anomaly while planning for complex surgeries in the supra-colic compartment. We report the case of accessory splenic arteries in an approximately 50-year-old male cadaver encountered during routine educational dissection. One of them arising from left gastroepiploic artery supplies the spleen in addition to splenic artery. Another variant vessel bifurcated to enter greater omentum and anterior pole of spleen, as discrete branches. The anatomical vascular variation, if recognized during the imaging work-ups for elective surgical procedures could avoid potential iatrogenic blood loss.

Detergent-extractedVolvoxmodel exhibits an anterior–posterior gradient in flagellar Ca2+sensitivity

Volvox rousseletiiis a multicellular spheroidal green alga containing ∼5,000 cells, each equipped with two flagella (cilia). This organism shows striking photobehavior without any known intercellular communication. To help understand how the behavior of flagella is regulated, we developed a method to extract the whole organism with detergent and reactivate its flagellar motility. Upon addition of ATP, demembranated flagella (axonemes) in the spheroids actively beat and the spheroids swam as if they were alive. Under Ca2+-free conditions, the axonemes assumed planar and asymmetrical waveforms and beat toward the posterior pole, as do live spheroids in the absence of light stimulation. In the presence of 10−6M Ca2+, however, most axonemes beat three-dimensionally toward the anterior pole, similar to flagella in photostimulated live spheroids. This Ca2+-dependent change in flagellar beating direction was more conspicuous near the anterior pole of the spheroid, but was not observed near the posterior pole. This anterior–posterior gradient of flagellar Ca2+sensitivity may explain the mechanism ofV. rousseletiiphotobehavior.

Geometric constraints alter cell arrangements within curved epithelial tissues

Organ and tissue formation are complex three-dimensional processes involving cell division, growth, migration, and rearrangement, all of which occur within physically constrained regions. However, analyzing such processes in three dimensions in vivo is challenging. Here, we focus on the process of cellularization in the anterior pole of the early Drosophila embryo to explore how cells compete for space under geometric constraints. Using microfluidics combined with fluorescence microscopy, we extract quantitative information on the three-dimensional epithelial cell morphology. We observed a cellular membrane rearrangement in which cells exchange neighbors along the apical-basal axis. Such apical-to-basal neighbor exchanges were observed more frequently in the anterior pole than in the embryo trunk. Furthermore, cells within the anterior pole skewed toward the trunk along their long axis relative to the embryo surface, with maximum skew on the ventral side. We constructed a vertex model for cells in a curved environment. We could reproduce the observed cellular skew in both wild-type embryos and embryos with distorted morphology. Further, such modeling showed that cell rearrangements were more likely in ellipsoidal, compared with cylindrical, geometry. Overall, we demonstrate that geometric constraints can influence three-dimensional cell morphology and packing within epithelial tissues.

The Role of Sox17 Gene Expression in Early Definition of Anterior Pole of the Rabbit Embryo at Early Pre-Gastrulation Stages

The primitive streak and notochord and previously the anterior marginal crescent (AMC), anterior visceral endoderm (AVE) and the anterior hypoblast (AHB) are embryonic entities which identify main body axes and thus establish body plan in the early stages of embryonic development. All of the anterior pre-gastrulation differentiation structures are addressed terminology as anterior pre-gastrulation differentiation (APD). These structures are defined morphologically and are called in mouse (AVE), in rabbit (AMC) and in the pig (AHB). The anterior hypoblast cells of APD are higher and denser than at the opposite pole of the embryo. Moreover, the APD stretches variously between species and has different shapes in the mammalian embryos, for example, it is crescent-like shape in the rabbit and disc-like shape in the pig. In this study, the sox17 expression patterns show that the anterior pole of rabbit is differentiated genetically prior to morphological differentiation. In Situ hybridization signals of sox17 are located in AMC area at early pre-gastrulation stages before appearance of first cellular differentiation signs. This study fixes sox17 gene as one of the important genes in definition of the polarity of the mammalian embryo before appearance morphological or axial landmarks.

Bicoid gradient formation and function in the Drosophila pre-syncytial blastoderm

Bicoid (Bcd) protein distributes in a concentration gradient that organizes the anterior/posterior axis of the Drosophila embryo. It has been understood that bcd RNA is sequestered at the anterior pole during oogenesis, is not translated until fertilization, and produces a protein gradient that functions in the syncytial blastoderm after 9–10 nuclear divisions. However, technical issues limited the sensitivity of analysis of pre-syncytial blastoderm embryos and precluded studies of oocytes after stage 13. We developed methods to analyze stage 14 oocytes and pre-syncytial blastoderm embryos, and found that stage 14 oocytes make Bcd protein, that bcd RNA and Bcd protein distribute in matching concentration gradients in the interior of nuclear cycle 2–6 embryos, and that Bcd regulation of target gene expression is apparent at nuclear cycle 7, two cycles prior to syncytial blastoderm. We discuss the implications for the generation and function of the Bcd gradient.

Optical Coherence Tomography Reveals New Insights into the Accommodation Mechanism

Purpose. To evaluate the movement of the anterior and posterior lens poles during naturally stimulated accommodation in children using anterior segment optical coherence tomography (OCT).Methods. This is a prospective, observational, noncomparative case series including 18 eyes of nine children. Analysis of the anterior segment in the accommodated and unaccommodated state (with cycloplegia) was done using anterior segment OCT. The main outcome measures were the position of the anterior and posterior lens poles (in relation to the cornea) and lens thickness (LT).Results. A Statistically significant forward movement of the anterior lens pole and backward movement of the posterior lens pole with an increase in LT were found during accommodation (P<0.001). There was no significant difference between the degree of movement of the anterior lens pole and the posterior lens pole during accommodation (P=0.944).Conclusions. Anterior segment OCT provides a rapid noncontact method for studying accommodation in children. The backward movement of the posterior lens pole during accommodation nearly equals the forward movement of its anterior pole. These data minimize the theoretical hydraulic effect of the vitreous during accommodation, adding more support to the capsular theory of Helmholtz.