Senescence is a developmental mechanism that contributes to embryonic growth and patterning.

Senescence is a form of cell-cycle arrest linked to tumor suppression and aging. However, it remains controversial and has not been documented in nonpathologic states. Here we describe senescence as a normal developmental mechanism found throughout the embryo, including the apical ectodermal ridge (AER) and the neural roof plate, two signaling centers in embryonic patterning. Embryonic senescent cells are nonproliferative and share features with oncogene-induced senescence (OIS), including expression of p21, p15, and mediators of the senescence-associated secretory phenotype (SASP). Interestingly, mice deficient in p21 have defects in embryonic senescence, AER maintenance, and patterning. Surprisingly, the underlying mesenchyme was identified as a source for senescence instruction in the AER, whereas the ultimate fate of these senescent cells is apoptosis and macrophage-mediated clearance. We propose that senescence is a normal programmed mechanism that plays instructive roles in development, and that OIS is an evolutionarily adapted reactivation of a developmental process.

A quantitative method for staging mouse embryos based on limb morphometry.

To determine the developmental stage of embryonic mice, we apply a geometric morphometric approach to the changing shape of the mouse limb bud as it grows from embryonic day 10 to embryonic day 15 post-conception. As the ontogenetic sequence results in the de novo emergence of shape features not present in the early stages, we have created a standard ontogenetic trajectory for limb bud development - a quantitative characterization of shape change during limb morphogenesis. This trajectory of form as a function of time also gives us the reverse function: the ability to infer developmental stage from form, with a typical uncertainty of 2 h. We introduce eMOSS (embryonic mouse ontogenetic staging system) as a fast, reliable, convenient and freely available online tool for staging embryos from two-dimensional images of their limb buds, and illustrate its use in phenotyping early limb abnormalities.

OPTiSPIM: integrating optical projection tomography in light sheet microscopy extends specimen characterization to nonfluorescent contrasts.

Mesoscopic 3D imaging has become a widely used optical imaging technique to visualize intact biological specimens. Selective plane illumination microscopy (SPIM) visualizes samples up to a centimeter in size with micrometer resolution by 3D data stitching but is limited to fluorescent contrast. Optical projection tomography (OPT) works with fluorescent and nonfluorescent contrasts, but its resolution is limited in large samples. We present a hybrid setup (OPTiSPIM) combining the advantages of each technique. The combination of fluorescent and nonfluorescent high-resolution 3D data into integrated datasets enables a more extensive representation of mesoscopic biological samples. The modular concept of the OPTiSPIM facilitates incorporation of the transmission OPT modality into already established light sheet based imaging setups.

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.

Attenuation artifacts in light sheet fluorescence microscopy corrected by OPTiSPIM.

Light sheet fluorescence microscopy (LSFM) is rapidly becoming an essential technology for mesoscopic imaging of samples such as embryos and adult mouse organs. However, LSFM can suffer from optical artifacts for which there is no intrinsic solution. The attenuation of light due to absorbing material causes "shadow" artifacts along both the illumination and detection paths. Several approaches have been introduced to reduce this problem, including scanning illumination and multi-view imaging. However, neither of these approaches completely eliminates the problem. If the distribution of the absorbing material is complex, shadows cannot be avoided. We introduce a new approach that relies on multi-modal integration of two very different mesoscopic techniques. Unlike LSFM, optical projection tomography (OPT) can operate in transmission mode to create a voxel map of the 3D distribution of the sample's optical attenuation. Here, we demonstrate a hybrid instrument (OPTiSPIM) that can quantify this attenuation and use the information to correct the shadow artifacts of LSFM.

Quantification of gene expression patterns to reveal the origins of abnormal morphogenesis.

The earliest developmental origins of dysmorphologies are poorly understood in many congenital diseases. They often remain elusive because the first signs of genetic misregulation may initiate as subtle changes in gene expression, which are hard to detect and can be obscured later in development by secondary effects. Here, we develop a method to trace back the origins of phenotypic abnormalities by accurately quantifying the 3D spatial distribution of gene expression domains in developing organs. By applying Geometric Morphometrics to 3D gene expression data obtained by Optical Projection Tomography, we determined that our approach is sensitive enough to find regulatory abnormalities that have never been detected previously. We identified subtle but significant differences in the gene expression of a downstream target of a Fgfr2 mutation associated with Apert syndrome, demonstrating that these mouse models can further our understanding of limb defects in the human condition. Our method can be applied to different organ systems and models to investigate the etiology of malformations.

Migratory appendicular muscles precursor cells in the common ancestor to all vertebrates.

In amniote embryos, skeletal muscles in the trunk are derived from epithelial dermomyotomes, the ventral margin of which extends ventrally to form body wall muscles. At limb levels, ventral dermomyotomes also generate limb-muscle precursors, an Lbx1-positive cell population that originates from the dermomyotome and migrates distally into the limb bud. In elasmobranchs, however, muscles in the paired fins were believed to be formed by direct somitic extension, a developmental pattern used by the amniote body wall muscles. Here we re-examined the development of pectoral fin muscles in catsharks, Scyliorhinus, and found that chondrichthyan fin muscles are indeed formed from Lbx-positive muscle precursors. Furthermore, these precursors originate from the ventral edge of the dermomyotome, the rest of which extends towards the ventral midline to form body wall muscles. Therefore, the Lbx1-positive, de-epithelialized appendicular muscle precursors appear to have been established in the body plan before the divergence of Chondrichthyes and Osteichthyes.

Publisher Correction: Migratory appendicular muscles precursor cells in the common ancestor to all vertebrates.

In Fig. 2 of this Article originally published, some erroneous lines appeared on the left side of the images in panels c, e and g. The figure should have appeared as shown below. These errors have now been corrected in all versions of the Article.

A Local, Self-Organizing Reaction-Diffusion Model Can Explain Somite Patterning in Embryos.

During somitogenesis in embryos, a posteriorly moving differentiation front arrests the oscillations of "segmentation clock" genes, leaving behind a frozen, periodic pattern of expression stripes. Both mathematical theories and experimental observations have invoked a "clock and wavefront" model to explain this phenomenon, in which long-range molecular gradients control the movement of the front and therefore the placement of the stripes in the embryo. Here, we develop a fundamentally different model-a progressive oscillatory reaction-diffusion (PORD) system driven by short-range interactions. In this model, posterior movement of the front is a local, emergent phenomenon that, in contrast to the clock and wavefront model, is not controlled by global positional information. The PORD model explains important features of somitogenesis, such as size regulation, that previous reaction-diffusion models could not explain. Moreover, the PORD and clock and wavefront models make different predictions about the results of FGF-inhibition and tissue-cutting experiments, and we demonstrate that the results of these experiments favor the PORD model.