[Show abstract][Hide abstract] ABSTRACT: 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 non-anatomical 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 post-fertilization 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.
[Show abstract][Hide abstract] ABSTRACT: One of the most fundamental questions in biology is that of biological pattern: how do the structures and shapes of organisms arise? Undoubtedly, the two most influential ideas in this area are those of Alan Turing's 'reaction-diffusion' and Lewis Wolpert's 'positional information'. Much has been written about these two concepts but some confusion still remains, in particular about the relationship between them. Here, we address this relationship and propose a scheme of three distinct ways in which these two ideas work together to shape biological form.
Development 03/2015; 142(7):1203-1211. DOI:10.1242/dev.114991 · 6.46 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To what extent does the dynamical mechanism producing a specific biological phenotype bias the ability to evolve into novel phenotypes? We use the interpretation of a morphogen gradient into a single stripe of gene expression as a model phenotype. Although there are thousands of three-gene circuit topologies that can robustly develop a stripe of gene expression, the vast majority of these circuits use one of just six fundamentally different dynamical mechanisms. Here we explore the potential for gene circuits that use each of these six mechanisms to evolve novel phenotypes such as multiple stripes, inverted stripes, and gradients of gene expression. Through a comprehensive and systematic analysis, we find that circuits that use alternative mechanisms differ in the likelihood of reaching novel phenotypes through mutation. We characterize the phenotypic transitions and identify key ingredients of the evolutionary potential, such as sensitive interactions and phenotypic hubs. Finally, we provide an intuitive understanding on how the modular design of a particular mechanism favors the access to novel phenotypes. Our work illustrates how the dynamical mechanism by which an organism develops constrains how it can evolve. It is striking that these dynamical mechanisms and their impact on evolvability can be observed even for such an apparently simple patterning task, performed by just three-node circuits.
Proceedings of the National Academy of Sciences 02/2015; 112(7). DOI:10.1073/pnas.1411065112 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Gradients of fibroblast growth factors often induce cells to adopt different fates. A study in zebrafish embryos reveals another, unexpected role when the factors are trapped in small spaces by a special arrangement of cells. SEE LETTER P.120
[Show abstract][Hide abstract] ABSTRACT: Highlights ENU-induced mutation of mouse ESCRT-II/Vps25 causes polydactyly Vps25 hypomorphic mutants survive until late gestation unlike ESCRT LOF embryos ESCRT-II constrains digit number by endosome-mediated mod-ulation of FGF signaling Mutations in ESCRT reveal a mechanism underlying congenital limb defects In Brief Using a polydactylous mouse line car-rying a hypomorphic mutation in the Vps25 subunit of the ESCRT-II complex, Handschuh et al. now establish that ubiq-uitously expressed machineries that sort signaling proteins preferentially regulate, or are rate limiting for, select signaling pathways in different contexts of the developing embryo.
[Show abstract][Hide abstract] ABSTRACT: Apert syndrome is a rare congenital disorder characterized by cranial, neural, limb and visceral malformations. Over 98% of Apert cases are caused by two FGFR2 mutations, Ser252Trp and Pro253Arg, which alter the ligand-binding specificity of the receptors. Patients carrying the P253R mutation show more severe limb malformations, such as syndactyly and symphalangism. Here we explored limb morphogenesis in early development using an inbred mouse model of Apert syndrome, Fgfr2+/P253R, to assess whether the P253R mutation induces changes in the expression pattern of Dusp6, a downstream gene of the FGF/FGFR signaling pathway, and whether these genetic changes can be associated with limb malformations in mutant littermates. We performed Geometric Morphometric (GM) analyses of 3D landmark-based data recorded on optical projection tomography (OPT) images of 11.5 embryonic day (E11.5) embryos labeled for Dusp6 expression using whole-mount in situ hybridization. Comparative quantitative analysis of large samples of limbs and their corresponding Dusp6 gene expression pattern show differences in limb size and shape between mutant and unaffected littermates. At E11.5, mutant mice are significantly smaller and appear underdeveloped in comparison with unaffected littermates. Results show that size explains more than 80% of limb shape variation, but when this allometric effect is minimized the limbs of mutant mice, especially hindlimbs, remain different from the limbs of unaffected littermates. Shape differences are also found at the gene expression level, suggesting that altered FGF/FGFR signaling due to the FGFR2 mutation alters Dusp6 gene expression patterns and contributes to limb malformations as early as E11.5. Our morphometric assessment of gene expression patterns by combining OPT and GM is a novel and useful tool to compare normal and disease-altered patterns of variation. Precise embryo phenotyping of Apert syndrome mice will allow us to identify the origins of abnormal processes that can have a damaging effect on the developing limbs.
X Meeting Spanish Society for Develpmental Biology, Madrid; 10/2014
[Show abstract][Hide abstract] ABSTRACT: Synthetic biology is a promising tool to study the function and properties of gene regulatory networks. Gene circuits with predefined behaviours have been successfully built and modelled, but largely on a case-by-case basis. Here we go beyond individual networks and explore both computationally and synthetically the design space of possible dynamical mechanisms for 3-node stripe-forming networks. First, we computationally test every possible 3-node network for stripe formation in a morphogen gradient. We discover four different dynamical mechanisms to form a stripe and identify the minimal network of each group. Next, with the help of newly established engineering criteria we build these four networks synthetically and show that they indeed operate with four fundamentally distinct mechanisms. Finally, this close match between theory and experiment allows us to infer and subsequently build a 2-node network that represents the archetype of the explored design space.
[Show abstract][Hide abstract] ABSTRACT: During limb development, digits emerge from the undifferentiated mesenchymal tissue that constitutes the limb bud. It has
been proposed that this process is controlled by a self-organizing Turing mechanism, whereby diffusible molecules interact
to produce a periodic pattern of digital and interdigital fates. However, the identities of the molecules remain unknown.
By combining experiments and modeling, we reveal evidence that a Turing network implemented by Bmp, Sox9, and Wnt drives digit
specification. We develop a realistic two-dimensional simulation of digit patterning and show that this network, when modulated
by morphogen gradients, recapitulates the expression patterns of Sox9 in the wild type and in perturbation experiments. Our systems biology approach reveals how a combination of growth, morphogen
gradients, and a self-organizing Turing network can achieve robust and reproducible pattern formation.
[Show abstract][Hide abstract] ABSTRACT: Interpreting a morphogen gradient into a single stripe of gene-expression is a fundamental unit of patterning in early embryogenesis. From both experimental data and computational studies the feed-forward motifs stand out as minimal networks capable of this patterning function. Positive feedback within gene networks has been hypothesised to enhance the sharpness and precision of gene-expression borders, however a systematic analysis has not yet been reported. Here we set out to assess this hypothesis, and find an unexpected result. The addition of positive-feedback can have different effects on two different designs of feed-forward motif- it increases the parametric robustness of one design, while being neutral or detrimental to the other. These results shed light on the abundance of the former motif and especially of mutual-inhibition positive feedback in developmental networks.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: The biology and mechanobiology of joint cavitation have undergone extensive investigation, but we have almost no understanding of the development of joint shape. Joint morphogenesis, the development of shape, has been identified as the 'least understood aspect of joint formation' (2005, Birth Defects Res C Embryo Today 75, 237), despite the clinical relevance of shape morphogenesis to postnatal skeletal malformations such as developmental dysplasia of the hip. In this study, we characterise development of early hip joint shape in the embryonic chick using direct capture 3D imaging. Contrary to formerly held assumptions that cavitation precedes morphogenesis in joint development, we have found that the major anatomical features of the adult hip are present at Hamburger Hamilton (HH)32, a full day prior to cavitation of the joint at HH34. We also reveal that the pelvis undergoes significant changes in orientation with respect to the femur, despite the lack of a joint cavity between the rudiments. Furthermore, we have identified the appearance of the ischium and pubis several developmental stages earlier than was previously reported, illustrating the value and importance of direct capture 3D imaging.
Journal of Anatomy 11/2013; 224(4). DOI:10.1111/joa.12143 · 2.10 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Direct videomicroscopic visualization of organ formation and regeneration in toto is a powerful strategy to study cellular processes that often cannot be replicated in vitro. Intravital imaging aims at quantifying changes in tissue architecture or subcellular organization over time during organ development, regeneration or degeneration. A general feature of this approach is its reliance on the optical isolation of defined cell types in the whole animals by transgenic expression of fluorescent markers. Here we describe a simple and robust method to analyze sensory hair-cell development and regeneration in the zebrafish lateral line by high-resolution intravital imaging using laser-scanning confocal microscopy (LSCM) and selective plane illumination microscopy (SPIM). The main advantage of studying hair-cell regeneration in the lateral line is that it occurs throughout the life of the animal, which allows its study in the most natural context. We detail protocols to achieve continuous videomicroscopy for up to 68 hours, enabling direct observation of cellular behavior, which can provide a sensitive assay for the quantitative classification of cellular phenotypes and cell-lineage reconstruction. Modifications to this protocol should facilitate pharmacogenetic assays to identify or validate otoprotective or reparative drugs for future clinical strategies aimed at preserving aural function in humans.
Frontiers in Neuroanatomy 10/2013; 7:33. DOI:10.3389/fnana.2013.00033 · 3.54 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Growth and Differentiation Factor 5 (GDF5) is a secreted growth factor that belongs to the Bone Morphogenetic Protein (BMP) family and plays a pivotal role during limb development. GDF5 is a susceptibility gene for osteoarthritis (OA) and mutations in GDF5 are associated with a wide variety of skeletal malformations ranging from complex syndromes such as acromesomelic chondrodysplasias to isolated forms of brachydactylies or multiple synostoses syndrome 2 (SYNS2). Here, we report on a family with an autosomal dominant inherited combination of SYNS2 and additional brachydactyly type A1 (BDA1) caused by a single point mutation in GDF5 (p.W414R). Functional studies, including chondrogenesis assays with primary mesenchymal cells, luciferase reporter gene assays and Surface Plasmon Resonance analysis, of the GDF5(W414R) variant in comparison to other GDF5 mutations associated with isolated BDA1 (p.R399C) or SYNS2 (p.E491K) revealed a dual pathomechanism characterized by a gain- and loss-of-function at the same time. On the one hand insensitivity to the main GDF5 antagonist NOGGIN (NOG) leads to a GDF5 gain of function and subsequent SYNS2 phenotype. Whereas on the other hand, a reduced signaling activity, specifically via the BMP receptor type IA (BMPR1A), is likely responsible for the BDA1 phenotype. These results demonstrate that one mutation in the overlapping interface of antagonist and receptor binding site in GDF5 can lead to a GDF5 variant with pathophysiological relevance for both, BDA1 and SYNS2 development. Consequently, our study assembles another part of the molecular puzzle of how loss and gain of function mutations in GDF5 affect bone development in hands and feet resulting in specific types of brachydactyly and SYNS2. These novel insights into the biology of GDF5 might also provide further clues on the pathophysiology of OA.