Translation of mRNA injected into Xenopus oocytes is specifically inhibited by antisense RNA.

The bacteriophage SP6 promoter and RNA polymerase were used to synthesize sense and antisense RNAs coding for the enzymes thymidine kinase (TK) and chloramphenicol acetyl transferase (CAT). Injection of antisense CAT RNA into frog oocytes inhibited expression of sense CAT mRNA. Similarly, antisense TK RNA inhibited expression of sense TK mRNA. Antisense RNAs were stable in oocytes and had no detectable effect on either the expression of endogenous proteins or on the expression of nonhomologous RNA transcripts. CAT activity expressed from a plasmid transcribed in the oocyte nucleus was also inhibited by antisense RNA injected into the oocyte cytoplasm. The data suggest that antisense RNA will be useful in identifying the function of specific mRNA sequences during early development of the frog

3D-printable tools for developmental biology: Improving embryo injection and screening techniques through 3D-printing technology

Developmental biology requires rapid embryo injections and screening. We applied new affordable high-resolution 3D-printing to create five easily modifiable stamp-mold tools that greatly increase injection and screening speed, while simultaneously reducing the harmful aspects of these processes. We designed two stamps that use different approaches to improve the injection efficiency for two different types of embryo, first for embryos from the snail Crepidula fornicata, and second, for those from the spider Parasteatoda tepidariorum. Both drastically improved injection speeds and embryo survival rates, even in novice hands. The other three tools were designed for rapid side-by-side organism orientating and comparison. The first screening tool allows for optimal imaging in Xenopus laevis tadpoles, while the second and third facilitate rapid high-throughput screening of Xenopus tropicalis tadpoles and Danio rerio juveniles, respectively. These designs can act as templates for many injection or screening applications

Sost and its paralog Sostdc1 coordinate digit number in a Gli3-dependent manner.

WNT signaling is critical in most aspects of skeletal development and homeostasis, and antagonists of WNT signaling are emerging as key regulatory proteins with great promise as therapeutic agents for bone disorders. Here we show that Sost and its paralog Sostdc1 emerged through ancestral genome duplication and their expression patterns have diverged to delineate non-overlapping domains in most organ systems including musculoskeletal, cardiovascular, nervous, digestive, reproductive and respiratory. In the developing limb, Sost and Sostdc1 display dynamic expression patterns with Sost being restricted to the distal ectoderm and Sostdc1 to the proximal ectoderm and the mesenchyme. While Sostdc1(-/-) mice lack any obvious limb or skeletal defects, Sost(-/-) mice recapitulate the hand defects described for Sclerosteosis patients. However, elevated WNT signaling in Sost(-/-); Sostdc1(-/-) mice causes misregulation of SHH signaling, ectopic activation of Sox9 in the digit 1 field and preaxial polydactyly in a Gli1- and Gli3-dependent manner. In addition, we show that the syndactyly documented in Sclerosteosis is present in both Sost(-/-) and Sost(-/-); Sostdc1(-/-) mice, and is driven by misregulation of Fgf8 in the AER, a region lacking Sost and Sostdc1 expression. This study highlights the complexity of WNT signaling in skeletal biology and disease and emphasizes how redundant mechanism and non-cell autonomous effects can synergize to unveil new intricate phenotypes caused by elevated WNT signaling

Calcium signaling during convergent extension in Xenopus

Background: During Xenopus gastrulation, cell intercalation drives convergent extension of dorsal tissues. This process requires the coordination of motility throughout a large population of cells. The signaling mechanisms that regulate these movements in space and time remain poorly understood. Results: To investigate the potential contribution of calcium signaling to the control of morphogenetic movements, we visualized calcium dynamics during convergent extension using a calcium-sensitive fluorescent dye and a novel confocal microscopy system. We found that dramatic intercellular waves of calcium mobilization occurred in cells undergoing convergent extension in explants of gastrulating Xenopus embryos. These waves arose stochastically with respect to timing and position within the dorsal tissues. Waves propagated quickly and were often accompanied by a wave of contraction within the tissue. Calcium waves were not observed in explants of the ventral marginal zone or prospective epidermis. Pharmacological depletion of intracellular calcium stores abolished the calcium dynamics and also inhibited convergent extension without affecting cell fate. These data indicate that calcium signaling plays a direct role in the coordination of convergent extension cell movements. Conclusions: The data presented here indicate that intercellular calcium signaling plays an important role in vertebrate convergent extension. We suggest that calcium waves may represent a widely used mechanism by which large groups of cells can coordinate complex cell movements

Genetic evidence that SOST inhibits WNT signaling in the limb

AbstractSOST is a negative regulator of bone formation, and mutations in human SOST are responsible for sclerosteosis. In addition to high bone mass, sclerosteosis patients occasionally display hand defects, suggesting that SOST may function embryonically. Here we report that overexpression of SOST leads to loss of posterior structures of the zeugopod and autopod by perturbing anterior–posterior and proximal–distal signaling centers in the developing limb. Mutant mice that overexpress SOST in combination with Grem1 and Lrp6 mutations display more severe limb defects than single mutants alone, while Sost−/− significantly rescues the Lrp6−/− skeletal phenotype, signifying that SOST gain-of-function impairs limb patterning by inhibiting the WNT signaling through LRP5/6

Ectopic Noggin Blocks Sensory and Nonsensory Organ Morphogenesis in the Chicken Inner Ear

AbstractBone morphogenetic protein 4 (Bmp4) is expressed during multiple stages of development of the chicken inner ear. At the otocyst stage, Bmp4 is expressed in each presumptive sensory organ, as well as in the mesenchymal cells surrounding the region of the otocyst that is destined to form the semicircular canals. After the formation of the gross anatomy of the inner ear, Bmp4 expression persists in some sensory organs and restricted domains of the semicircular canals. To address the role of this gene in inner ear development, we blocked BMP4 function(s) by delivering one of its antagonists, Noggin, to the developing inner ear in ovo. Exogenous Noggin was delivered to the developing otocyst by using a replication-competent avian retrovirus encoding the Noggin cDNA (RCAS-N) or implanting beads coated with Noggin protein. Noggin treatment resulted in a variety of phenotypes involving both sensory and nonsensory components of the inner ear. Among the nonsensory structures, the semicircular canals were the most sensitive and the endolymphatic duct and sac most resistant to exogenous Noggin. Noggin affected the proliferation of the primordial canal outpouch, as well as the continual outgrowth of the canal after its formation. In addition, Noggin affected the structural patterning of the cristae, possibly via a decrease of Msx1 and p75NGFR expression. These results suggest that BMP4 and possibly other BMPs are required for multiple phases of inner ear development

Osteopotentia regulates osteoblast maturation, bone formation, and skeletal integrity in mice

A component of the rough ER, SUN domain protein osteopotentia, regulates expansion of this organelle in osteoblasts during skeletal development and regeneration

Accelerated gene evolution and subfunctionalization in the pseudotetraploid frog Xenopus laevis

Ancient whole genome duplications have been implicated in the vertebrate and teleost radiations, and in the emergence of diverse angiosperm lineages, but the evolutionary response to such a perturbation is still poorly understood. The African clawed frog Xenopus laevis experienced a relatively recent tetraploidization {approx} 40 million years ago. Analysis of the considerable amount of EST sequence available for this species together with the genome sequence of the related diploid Xenopus tropicalis provides a unique opportunity to study the genomic response to whole genome duplication