Shape Of Somite Research Articles

An emerging role for tissue plasticity in developmental precision.

Reproducible tissue morphology is a fundamental feature of embryonic development. To ensure such robustness during tissue morphogenesis, inherent noise in biological processes must be buffered. While redundant genes, parallel signaling pathways and intricate network topologies are known to reduce noise, over the last few years, mechanical properties of tissues have been shown to play a vital role. Here, taking the example of somite shape changes, I will discuss how tissues are highly plastic in their ability to change shapes leading to increased precision and reproducibility.

Left-right symmetry of zebrafish embryos requires somite surface tension.

The body axis of vertebrate embryos is periodically segmented into bilaterally symmetric pairs of somites1,2. The anteroposterior length of somites, their position and left-right symmetry are thought to be molecularly determined before somite morphogenesis3,4. Here we show that, in zebrafish embryos, initial somite anteroposterior lengths and positions are imprecise and, consequently, many somite pairs form left-right asymmetrically. Notably, these imprecisions are not left unchecked and we find that anteroposterior lengths adjust within an hour after somite formation, thereby increasing morphological symmetry. We find that anteroposterior length adjustments result entirely from changes in somite shape without change in somite volume, with changes in anteroposterior length being compensated by corresponding changes in mediolateral length. The anteroposterior adjustment mechanism is facilitated by somite surface tension, which we show by comparing in vivo experiments and in vitro single-somite explant cultures using a mechanical model. Length adjustment is inhibited by perturbation of molecules involved in surface tension, such as integrin and fibronectin. By contrast, the adjustment mechanism is unaffected by perturbations to the segmentation clock, therefore revealing a distinct process that influences morphological segment lengths. We propose that tissue surface tension provides a general mechanism to adjust shapes and ensure precision and symmetry of tissues in developing embryos.

Vangl2-environment interaction causes severe neural tube defects, without abnormal neuroepithelial convergent extension.

ABSTRACTPlanar cell polarity (PCP) signalling is vital for initiation of mouse neurulation, with diminished convergent extension (CE) cell movements leading to craniorachischisis, a severe neural tube defect (NTD). Some humans with NTDs also have PCP gene mutations but these are heterozygous, not homozygous as in mice. Other genetic or environmental factors may interact with partial loss of PCP function in human NTDs. We found that reduced sulfation of glycosaminoglycans interacts with heterozygosity for the Lp allele of Vangl2 (a core PCP gene), to cause craniorachischisis in cultured mouse embryos, with rescue by exogenous sulphate. We hypothesized that this glycosaminoglycan–PCP interaction may regulate CE, but, surprisingly, DiO labelling of the embryonic node demonstrates no abnormality of midline axial extension in sulfation-depleted Lp/+ embryos. Positive-control Lp/Lp embryos show severe CE defects. Abnormalities were detected in the size and shape of somites that flank the closing neural tube in sulfation-depleted Lp/+ embryos. We conclude that failure of closure initiation can arise by a mechanism other than faulty neuroepithelial CE, with possible involvement of matrix-mediated somite expansion, adjacent to the closing neural tube.

A new species ofLyphiraGalil, 2009 (Crustacea, Decapoda, Leucosiidae) from Gujarat, India

A new species of crab belonging to the genus Lyphira Galil, 2009 of the family Leucosiidae Samouelle, 1819, is described from Gujarat state, India. Lyphira georgei n. sp. is distinguished from the related species L. perplexa Galil, 2009, in the shape of main shaft and apical lobe of male first gonopod, shape and size of the male telson as well as size and shape of somite 2 of male abdomen. The details of morphological differences are presented in the report.http://zoobank.org/urn:lsid:zoobank.org:pub:8DC6CACE-4BF9-45F4-B151-CFB2BAF4D82B

Zebrafish Dkk3a Protein Regulates the Activity of myf5 Promoter through Interaction with Membrane Receptor Integrin α6b

Myogenic regulatory factor Myf5 plays important roles in muscle development. In zebrafish myf5, a microRNA (miR), termed miR-3906 or miR-In300, was reported to silence dickkopf-3-related gene (dkk3r or dkk3a), resulting in repression of myf5 promoter activity. However, the membrane receptor that interacts with ligand Dkk3a to control myf5 expression through signal transduction remains unknown. To address this question, we applied immunoprecipitation and LC-MS/MS to screen putative membrane receptors of Dkk3a, and Integrin α6b (Itgα6b) was finally identified. To further confirm this, we used cell surface binding assays, which showed that Dkk3a and Itgα6b were co-expressed at the cell membrane of HEK-293T cells. Cross-linking immunoprecipitation data also showed high affinity of Itgα6b for Dkk3a. We further proved that the β-propeller repeat domains of Itgα6b are key segments bound by Dkk3a. Moreover, when dkk3a and itgα6b mRNAs were co-injected into embryos, luciferase activity was up-regulated 4-fold greater than that of control embryos. In contrast, the luciferase activities of dkk3a knockdown embryos co-injected with itgα6b mRNA and itgα6b knockdown embryos co-injected with dkk3a mRNA were decreased in a manner similar to that in control embryos, respectively. Knockdown of itgα6b resulted in abnormal somite shape, fewer somitic cells, weaker or absent myf5 expression, and reduced the protein level of phosphorylated p38a in somites. These defective phenotypes of trunk muscular development were similar to those of dkk3a knockdown embryos. We demonstrated that the secreted ligand Dkk3a binds to the membrane receptor Itgα6b, which increases the protein level of phosphorylated p38a and activates myf5 promoter activity of zebrafish embryos during myogenesis.

March 2012: This Month in JoVE

The March issue of the Journal of Visualized Experiments (JoVE) begins with an article that focuses on three fundamentally important assays for measuring Drosophila locomotor behavior. Specifically, these assays include the larval crawling assay (for larval motor function), the RING assay (for adult motor function), and the courtship assay (to assess coordination and sensory abilities). The larval crawling assay involves collection of larva and exposure of the juvenile flies to an experimental drug of interest. Animals are then transferred to a petri dish laid atop a sheet of graph paper, and the number of gridlines crossed per minute can be calculated. The Rapid Iterative Negative Geotaxis, or RING, protocol, involves placing adult flies in specialized vials where they are forced to the bottom by gentle tapping. Fruit flies instinctively will crawl upward, against gravity. Through image analysis, the extent of adult locomotor ability can be determined by measuring how far upward the flies can climb in a short period of time. To assess a more complex sensory-guided behavior, the courtship assay is used and begins by placing sexually-naive males and females into a specialized apparatus known as the mating wheel. During experimental sessions, investigators record the amount of time in which the male fly exhibits several characteristic courtship behaviors. Impressively, within a matter of minutes, the male fly will orient himself toward the female, tap her abdomen, generate a song with his wing, lick the female and ultimately copulate. Taken together, these assays represent a series of powerful behavioral tests that can help elucidate the genetic and environmental factors that contribute to drosophila behavior. Later in the month, JoVE shifts its focus to exercise physiology, where investigators from the University of Sau Paulo present a method designed to uncover the contribution of the various energy systems to complex exercise, like sports. As an example, our authors have chosen judo for their demonstration. Using a carefully positioned portable gas analyzer, oxygen consumption can be measured during judo exercise under controlled conditions. By comparing oxygen consumption before, during, and after exercise, the energy contribution of aerobic and alactic anaerobic metabolism can be determined. Blood collection before and after exercise, followed by electrochemical analysis is used to determine the energy contribution of lactic anaerobic metabolism to the sport of judo. When combined, oxygen consumption and plasma lactate concentration data, can be used to determine the relative contributions of the different energy systems to specific aspects of complex exercise, like one's favorite judo throw. Also in March, JoVE plans the release of a developmental study involving alcohol exposure in zebrafish. Once embryos develop to the desired timepoint, the effects of alcohol exposure on gene expression can be assessed by purifying mRNA and performing qPCR and by whole mount in situ hybridization. Using light microscopy, morphological changes such as somite shape can also be assessed. If a gene of interest is observed to decrease or increase by ethanol exposure, either capped mRNA or morpholino can be injected into the embryo, somewhere around the two-cell stage, in order to rescue the alcohol-induced phenotype. Our authors show that alcohol exposure in the developing zebrafish results in similar morphological phenotypes and gene expression profiles to that observed in mammals, and they also demonstrate that mRNA injection can reverse the effects of alcohol on developmental abnormalities. In Immunology and Infection, JoVE presents an experiment in plant parasitology, from UC Riverside. In particular, our authors are interested in the root knot nematode - an obligate plant parasite that moves freely through wet soil to infect the roots of plants, and feed. First, methods are described for processing infected plants and collection of root knot nematodes. Once juvenile worms develop to a stage with high infectivity, soil can be inoculated. Later, roots can be stained and assessed for the presence of the characteristic root knots generated by these parasites. In addition to experiments with potted plants, our authors show how root knot assays can be conducted with plants grown in clear pouches, allowing for easy viewing of roots, and facilitating high throughput screening for the presence of this devastating infection that accounts for 5% of global crop loss, yearly. This summary of JoVE's content for March highlights four of over fifty articles scheduled for release in the month. Other notable experiments involve methods for imaging odor-evoked responses in the antenna of fruit flies, using an electronic nose to analyze the volatile compounds from fruit, and the repurposing of ordinary office equipment for the delivery of macromolecules into living cells.

Deciphering the role of Shh signaling in axial defects produced by ethanol exposure

The phenotype of embryos exposed to ethanol is complex and likely due to multiple alterations in developmental pathways. We have previously demonstrated that Sonic hedgehog signaling (Shh-s) was reduced in both chicken and zebrafish embryos when exposed to ethanol. There are many tissues affected by embryonic ethanol exposure, and in this article we explore the development of axial tissues, using zebrafish embryos. We then compare these effects to the phenotypes produced by exposure to two drugs that also inhibit Shh-s: cyclopamine and forskolin. We found alterations in the development of the notochord and somites produced by all three compounds, although only ethanol produced developmental delay of epiboly. Upon observation of early developing embryos, muscle pioneer cells were completely lost in cyclopamine-treated embryos, and reduced, but less so, in embryos treated with forskolin and ethanol. Ethanol treatment produced a dose-dependent reduction in total body length that may be linked to epiboly delay seen earlier during development. Despite the differences between cyclopamine and forskolin, we found that shh mRNA injection rescued the short body length, the alteration in somite shape, and the cyclopia produced by ethanol exposure. Taken together, each teratogen produced a unique set of phenotypic changes in the body axis, suggesting that each compound affects Shh-s and also produces a distinctive set of molecular alterations. However, addition of exogenous Shh to ethanol treated zebrafish prevented many of the gross physical phenotypes, suggesting that the suppression of Shh-s is one of the major effects of ethanol exposure.

The you Gene Encodes an EGF-CUB Protein Essential for Hedgehog Signaling in Zebrafish

Hedgehog signaling is required for many aspects of development in vertebrates and invertebrates. Misregulation of the Hedgehog pathway causes developmental abnormalities and has been implicated in certain types of cancer. Large-scale genetic screens in zebrafish have identified a group of mutations, termed you-class mutations, that share common defects in somite shape and in most cases disrupt Hedgehog signaling. These mutant embryos exhibit U-shaped somites characteristic of defects in slow muscle development. In addition, Hedgehog pathway mutations disrupt spinal cord patterning. We report the positional cloning of you, one of the original you-class mutations, and show that it is required for Hedgehog signaling in the development of slow muscle and in the specification of ventral fates in the spinal cord. The you gene encodes a novel protein with conserved EGF and CUB domains and a secretory pathway signal sequence. Epistasis experiments support an extracellular role for You upstream of the Hedgehog response mechanism. Analysis of chimeras indicates that you mutant cells can appropriately respond to Hedgehog signaling in a wild-type environment. Additional chimera analysis indicates that wild-type you gene function is not required in axial Hedgehog-producing cells, suggesting that You is essential for transport or stability of Hedgehog signals in the extracellular environment. Our positional cloning and functional studies demonstrate that You is a novel extracellular component of the Hedgehog pathway in vertebrates.

Analysis of the physical forces that influence the shape of chick somites

AbstractWith experiments and measurements we support the hypothesis that chick somites are shaped by packing forces produced by increases in somite volumes. Volume measurements show that somites expand considerably during the period from initial segmentation to acquisition of the mature rectangular shape (as viewed from above). Anterior somites begin as roundish objects. Somite expansion flattens the intersomitic faces. Posterior somites emerge from a compressed segmental plate with the interface between somite and segmental plate already planar. In all somites, subsequent increase in volume causes expansion dorsally, ventrally and laterally. The dorso‐ventral expansion accompanies the increase in height of the neural tube. When a single somite is excised, neighboring somites or segmental plate fill in the available space. The bulging of somites into vacated spaces suggests the presence of compression which could be the product of previous increase in somite volumes. The pliability of somites is demonstrated with experiments that bend the trunk of the embryo to the left and right reversibly changing the shapes of the somites. Carbon marks placed in pairs in somites and in adjacent neural tube stay together; this suggests that somites do not move relative to the neural tube in response to the compressive forces. Segmental plates isolated without neural tube or notochord form long oval somites. In order to be shaped properly, somites must be in intimate association with, and probably bound to, the neural tube and also most likely the notochord.

On the Relation between Movements and the Shape of Somites in Early Embryos of the Teleost Brachydanio Rerio

As a working hypothesis it is supposed that in the teleost Brachydanio rerio, the muscle contractions, the growth, and probably some other factors, cause the first changes in the shape of the somites. Furthermore, the movements of the embryo could yield the forces by which the somites are brought to their theoretically optimal shape. In order to test this hypothesis, we investigated the somite shape and structure of spontaneously immobile embryos. Although the results are difficult to interpret, they certainly do not contradict the hypothesis. For further analysis we applied two kinds of lesions in order to immobilize early embryos: removal of the brain, and damage to the midbody somites. The results of these experiments indicate that both the development of the shape of the somite and the arrangement of the muscle fibres are dependent on movements of the embryo.

Somite segmentation in amphibian embryos: is there a transmitted control mechanism?

ABSTRACT In all vertebrate embryos, somite segmentation begins in the occipital region and proceeds from here caudally with remarkable regularity. The segmentation pattern and the shape, size and number of somites formed are constant for each species, and it appears as if some quite complex mechanism must control their development. That this control mechanism must be versatile as well as precise is shown, for instance, by the fact that haploid newts have a larger number of cells per somite than the normal diploid, while polyploids have smaller numbers of somite cells than normal, so that the somite sizes are very nearly the same in all cases: the number of somites per embryo is also always the same as in the diploid (Fankhauser, 1945). Further, when mesoderm is either added to or excised from the somite region of the archenteron roof experimentally (Waddington & Deuchar, 1953), there is regulation so that the overall shape of the somites remains nearly normal, and again, their number per embryo is unchanged from that typical of the species.