Claudio Stern - US grants

The vertebral column and axial skeletal muscles develop from paired structures in the embryo, called somites. They arise from loosely packed cells which come from the primitive streak at very early stages of development. Despite considerable interest for more than a century in understanding how the segmented organization of the somites, vertebrae and axial muscles is established, the processes that control this organization are still unknown at any level: tissue, cellular or molecular. The broad aim of this research is to elucidate these control processes at all levels. This project investigates the cellular aspects, to address four fundamental questions: (a) do early somite progenitors behave as stem cells? (b) which cell population is responsible for setting up the segmental pattern? (c) is segmentation cell autonomous and/or does it involve cell sorting? and (d) does each vertebra arise from the caudal half of one somite and the rostral half of the adjacent somite, as is widely believed? To answer them, a combination of recently developed techniques will be used. They include labeling of small groups of cells with carbocyanine dyes, single cell lineage analysis by intracellular injection of a tracer, following marked cells with time-lapse video microscopy and microsurgical operations to challenge the fates of small groups of embryonic cells. Without the information that the research will yield, it will be difficult to provide direction to a search for the molecules that control these important processes. The knowledge gained will help understand developmental mutations affecting the vertebral column as well as the etiology of some frequent and severe congenital abnormalities, including scoliosis, kyphosis, lordosis, Klippel-Feil syndrome and many others.

Hensen's node, the amniote equivalent of the amphibian "Spemann's organizer", is the most important region of the very early, gastrulating embryo. Not only does it generate the midline organs of the body (notochord, somites, gut, floor plate of the neural tube), but is also responsible for inducing and patterning the whole of the central nervous system. Classical studies have claimed, based on morphological criteria, that when Hensen's node is ablated, notochord and somites form normally. This project consists of new investigation of this problem, taking advantage of the many molecular markers now available, to reveal the conditions that specify this unique region of the embryo as "the organizer" and set its cells aside from the rest of the primitive streak. The study will help to define the organizer in both molecular and embryological terms and to identify the conditions that lead to the regulation of expression of several genes in this region. Finally, the results will reveal which properties of the organizer are under independent control at very early stages of embryonic development. Using transplantation experiments combined with in situ hybridization and retroviral vectors to cause misexpression of specific genes, the following specific questions will be addressed: Do the notochord and somites form after node ablation? Does the stump of the primitive streak express node- specific markers (e.g. goosecoid, sonic hedgehog) after node ablation? After ablation of the node, can the stump of the primitive streak acquire neural inducing ability and/or the ability to induce extra digits in a host limb bud? Is the regenerated node left/right asymmetric (like the normal node)? Is the ability to regenerate node properties restricted to a specific portion of the primitive streak? If any of the above properties cannot be acquired by the primitive streak, can it be made to regenerate these properties if made to express appropriate node marker genes?

During the early stages on embryonic development, the formation of the embryonic axis is initiated at one pole of the embryo. Most of what is known about the molecular mechanisms underlying this process is based on amphibian embryos, where maternally-derived determinants control this polarity. In amniotes (birds and mammals) this is unlikely because embryos at much later stages can initiate axis formation at any position. Here, the mechanisms that determine embryonic polarity in the early stages of development will be studied using the chick embryo as a model, because of their ease of manipulation and culture. First, the role of the hypoblast (which had been suggested to be responsible for polarity determination) will be tested directly. Then, the region homologous to the amphibian "Nieuwkoop center" (which has been shown in frogs to determine the position of future "organizer" and axial cells) will be located. Finally, a number of genes already found to be expressed in a way that predicts the future polarity of the embryo will be placed in hierarchial order, taking advantage of the fact that portions of the embryo will initiate axis formation when isolated from the rest of the embryo. The results of this project will be of value in suggesting how the molecular cascades that have been implicated in the early development of lower vertebrates (fish, amphibians) can be applied to higher vertebrates (birds and mammals). Potentially, knowledge of these cascades could lead to the development of new molecular diagnostic tools for some of the many serious congenital disorders that affect the early development of the embryonic axis, and may help determine whether monozygotic and conjoined twinning are likely to have a molecular basis.

neural plate /tube

DESCRIPTION (provided by applicant): The vertebral column and skeletal musculature derive from embryonic structures called somites, which form sequentially from head to tail. Normal musculoskeletal development requires the correct number of cells in each somite and that each somite acquires its correct axial address. Perturbations can lead to malformations of the spine ranging from complete disarray of the vertebral elements (e.g. spondylocostal and spondylothoracic dysostoses and dysplasias) to deviations of the spine (lordosis, kyphosis, and scoliosis) and misspecification of the regional identity of skeletal elements (e.g. Klippel-Feil syndrome). Research in the last two decades has uncovered molecular oscillators and gradients of growth factors hypothesized to control somite size and identity. However, little is known about how (or if) these molecular players relate to cell behaviors like cell adhesion, proliferation and migration that result in somite patterning. In part this uncertainty is due to the spatiotemporal complexity of somitogenesis, the number of mechanisms involved and the relative lack of cross-talk between model and experiment in the past. This project undertakes a multiscale approach to address all three issues. The NIH-led Interagency Modeling and Analysis Group (IMAG) has identified as key goals the development of open-source, multi-scale biological simulation environments and the deployment of demonstration projects that integrate models operating at different scales. This project will build comprehensive 3D multiscale predictive models of vertebrate somitogenesis able to generate and test specific hypotheses concerning the mechanisms of interspecies differences (as a model of individual to individual variability and robustness) and perturbations. It will refine a tissue simulation environment (CC3D) to improve its usability to the community, perform new biological experiments to collect data as inputs for 3D somitogenesis models and to test model predictions, and deploy models and experimental data using emerging standards for sharing of multicellular information (CBO, CBMSL). Specifically, it will: 1) develop new 3D models to integrate behaviors at molecular, cellular and tissue scales to reproduce the normal dynamics of segmentation and test them by quantitative measurements using advanced time-lapse fluorescence microscopy and microfluidics-based gradient-cell technology; 2) use a novel experimental paradigm that allows segmentation to be studied independently of the molecular segmentation clock, for challenging and validating the segmentation models; 3) extend the models and experiments to understand how somites acquire positional identities and 4) open-source release data and models in sharable formats. In addition to generating a predictive model for vertebral column development and its anomalies, this project should enable future studies of the development of other organs and establish the role of multi-scale modeling in biomedical science. Its emphasis on model and data share ability will promote efficient sharing of resources, tools and models among biomodelers and experimentalists, significantly reducing duplication of effort.