4D reconstruction of murine developmental trajectories using spherical harmonics.

Normal organogenesis cannot be recapitulated in vitro for mammalian organs, unlike in species including Drosophila and zebrafish. Available 3D data in the form of ex vivo images only provide discrete snapshots of the development of an organ morphology. Here, we propose a computer-based approach to recreate its continuous evolution in time and space from a set of 3D volumetric images. Our method is based on the remapping of shape data into the space of the coefficients of a spherical harmonics expansion where a smooth interpolation over time is simpler. We tested our approach on mouse limb buds and embryonic hearts. A key advantage of this method is that the resulting 4D trajectory can take advantage of all the available data while also being able to interpolate well through time intervals for which there are little or no data. This allows for a quantitative, data-driven 4D description of mouse limb morphogenesis.

Geometric Morphometrics on Gene Expression Patterns Within Phenotypes: A Case Example on Limb Development.

How the genotype translates into the phenotype through development is critical to fully understand the evolution of phenotypes. We propose a novel approach to directly assess how changes in gene expression patterns are associated with changes in morphology using the limb as a case example. Our method combines molecular biology techniques, such as whole-mount in situ hybridization, with image and shape analysis, extending the use of Geometric Morphometrics to the analysis of nonanatomical shapes, such as gene expression domains. Elliptical Fourier and Procrustes-based semilandmark analyses were used to analyze the variation and covariation patterns of the limb bud shape with the expression patterns of two relevant genes for limb morphogenesis, Hoxa11 and Hoxa13. We devised a multiple thresholding method to semiautomatically segment gene domains at several expression levels in large samples of limb buds from C57Bl6 mouse embryos between 10 and 12 postfertilization days. Besides providing an accurate phenotyping tool to quantify the spatiotemporal dynamics of gene expression patterns within developing structures, our morphometric analyses revealed high, non-random, and gene-specific variation undergoing canalization during limb development. Our results demonstrate that Hoxa11 and Hoxa13, despite being paralogs with analogous functions in limb patterning, show clearly distinct dynamic patterns, both in shape and size, and are associated differently with the limb bud shape. The correspondence between our results and already well-established molecular processes underlying limb development confirms that this morphometric approach is a powerful tool to extract features of development regulating morphogenesis. Such multilevel analyses are promising in systems where not so much molecular information is available and will advance our understanding of the genotype-phenotype map. In systematics, this knowledge will increase our ability to infer how evolution modified a common developmental pattern to generate a wide diversity of morphologies, as in the vertebrate limb.

The Wilms' tumour protein (WT1) shuttles between nucleus and cytoplasm and is present in functional polysomes.

Mutations of the Wilms' tumour-1 (WT1) gene in humans can lead to childhood kidney cancer, life-threatening glomerular nephropathy and gonadal dysgenesis. The WT1 protein is normally expressed in the developing genitourinary tract, heart, spleen and adrenal glands and is crucial for their development, however it's function at the molecular level is yet to be fully understood. The protein is predominantly nuclear and there is evidence that the two different isoforms of WT1 (-KTS and +KTS) are involved in two different steps of gene expression control: transcription and RNA processing. In this study we report a novel property of WT1, namely that it shuttles between the nucleus and cytoplasm. Moreover, western blot analysis showed that between 10 and 50% of total cellular WT1 can be detected in the cytoplasm depending on the cell type. A significant proportion of cytoplasmic WT1 is in association with ribonucleoprotein particles (RNPs), which strengthens the idea of its involvement in RNA metabolism. Furthermore, we report that WT1 is associated with actively translating polysomes, extending even further the potential roles of WT1 and opening the possibility that it is involved in the regulation of translation. Interestingly, despite the functional differences between two of the WT1 isoforms (+/-KTS) within the nucleus, both isoforms share the shuttling property and are found in translating polysomes.

Faithful expression of a tagged Fugu WT1 protein from a genomic transgene in zebrafish: efficient splicing of pufferfish genes in zebrafish but not mice.

The teleost fish are widely used as model organisms in vertebrate biology. The compact genome of the pufferfish, Fugu rubripes, has proven a valuable tool in comparative genome analyses, aiding the annotation of mammalian genomes and the identification of conserved regulatory elements, whilst the zebrafish is particularly suited to genetic and developmental studies. We demonstrate that a pufferfish WT1 transgene can be expressed and spliced appropriately in transgenic zebrafish, contrasting with the situation in transgenic mice. By creating both transgenic mice and transgenic zebrafish with the same construct, we show that Fugu RNA is processed correctly in zebrafish but not in mice. Furthermore, we show for the first time that a Fugu genomic construct can produce protein in transgenic zebrafish: a full-length Fugu WT1 transgene with a C-terminal beta-galactosidase fusion is spliced and translated correctly in zebrafish, mimicking the expression of the endogenous WT1 gene. These data demonstrate that the zebrafish:Fugu system is a powerful and convenient tool for dissecting both vertebrate gene regulation and gene function in vivo.

Biochemical function of female-lethal (2)D/Wilms' tumor suppressor-1-associated proteins in alternative pre-mRNA splicing.

Genetic and molecular data have implicated the Drosophila gene female-lethal (2)d (fl (2)d) in alternative splicing regulation of genes involved in sexual determination. Sex-specific splicing is under the control of the female-specific regulatory protein sex-lethal (SXL). Co-immunoprecipitation and mass spectrometry results indicate that SXL and FL (2)D form a complex and that the protein VIRILIZER and a Ran-binding protein implicated in protein nuclear import are also present in complexes containing FL (2)D. A human homolog of FL (2)D was identified and cloned. Interestingly, this gene encodes a protein (WTAP) that was previously found to interact with the Wilms' tumor suppressor-1 (WT1), an isoform of which binds to and co-localizes with splicing factors. Alternative splicing of transformer pre-mRNA, a target of SXL regulation, was affected by immunodepletion of hFL (2)D/WTAP from HeLa nuclear extracts, thus arguing for a biochemical function of FL (2)D/WTAP proteins in splicing regulation.