Our group is interested in the developmental biology of higher vertebrates. We would like to understand how the vertebrate body is generated during embryogenesis. Starting off as a single cell, the embryo acquires progressively complex traits, including multiple cell types, multiple tissue types, and their species-specific arrangement in functional organs which cooperate to form the developing organism. We are investigating the cellular and molecular mechanisms regulating this fascinating process, including cell fate choice, cell migration, pattern formation and morphogenesis. Beyond embryogenesis we are searching for clues from developmental processes to understand the evolution of vertebrate anatomy.
Model systems and techniques
We mainly use the chicken embryo as a model system, as it allows us to combine classical techniques like microsurgery and immunohistochemistry with modern molecular approaches like local transfection of embryonic cells by in ovo electroporation and retroviral gene delivery, and visualization of the expression of developmentally relevant genes by in situ hybridization.
1. 2,5 days old chicken embryo in ovo (unpublished original)
2. 4 days old chicken embryo stained for muscle specific gene expression (MyoD) in the myotomes by in situ hybridization (unpublished original)
3. Muscle precursor cells emigrating from the somites into limb bud labelled by GFP electroporation, visualized by confocal microscopy (Scaal et al. 2004)
1. Three myotomes of a 5 days old chicken embryo labelled by GFP electroporation, visualized by confocal microscopy (unpublished original)
2. Quail-chicken-chimerization: After transplantation of a ventral somite half of a quail donor into a chicken host, donor cells (green) are found in sclerotomal anlagen of the vertebral column and in the wall of the dorsal aorta (red) (Wiegreffe et al. 2007)
Our research is focussed on the development of the somites. Somites are transient structures found in all chordate embryos. They form a series of mesodermal segments along the body axis, and give rise to a variety of mesodermal organs including the axial skeleton, skeletal muscle, connective tissue of the trunk, and blood vessels. We are studying the molecular regulatory networks and cellular processes underlying the cell fate decisions within the somites, and the subsequent developmental steps leading to the differentiation of somite-derived cells into tissues and organs.
Presently, we are investigating the following questions:
What is the role of signalling filopodia in somite cell fate choice and differentiation?
Somites are initially formed as epithelial spheres, which undergo epithelial-mesenchymal transition (EMT) in the ventral subcompartment to form the sclerotome, which gives rise to the vertebral column. The dorsal somite compartment remains an epithelial cell sheet, the dermomyotome, with its basal pole on the dorsal side facing the ectoderm, and its apical pole on the ventral side facing the sclerotome. Due to individual cell fate decisions which are still not well understood, a fraction of dermomyotomal cells undergo EMT and migrate ventrally between dermomyotome and sclerotome, where they form the primitive musculature of the embryo, the myotome. Another fraction of dermomyotomal cells also undergo EMT, but migrate dorsally to from the dermal and subcutaneous connective tissue of the back.
On a molecular level, it is known that EMT in the dermomyotome is regulated by Wnt signaling from the neural tube and the ectoderm (Geetha-Loganathan et al. 2006, Krück and Scaal 2012).
We are interested in the role of long filopodia-like protrusions, or signaling filopodia, in long range signaling events during dermomyotomal differentiation. Interestingly, we could show that these signaling filopodia show retrograde membrane flow and retrograde transport of the Wnt-receptor Frizzled7 (Sagar et al. 2015, Pröls et al. 2016). We are presently characterizing the cytoskeletal architecture, molecular regulation, and function of these signaling filopodia during somite development.
Epithelial somite showing filopodia-like protrusions extending towards the ectoderm. Green: Actin-GFP, Blue: DAPI-stained nuclei. (unpublished original).
Close-up of somite cells carrying filopodia-like protrusions after transfection with Tubulin-GFP (green). Blue: DAPI stained nuclei. (Sagar et al. 2015)
What are the regional specifics of somite development?
Most studies on somite development are referring to the somites at the level of the trunk, i.e. at thoracolumbar levels, which are most amenable for experimental manipulation. However, the developmental program of somites in other regions along the body axis may be different. We showed that at occipital and cervical level, the sequence of developmental events leading to somite compartment formation differs from thoracic and lumbar levels (Maschner et al 2016). We are also studying somite development at caudal levels, and want to find out potential molecular specifics of the regulation of somite development at occipitocervical and caudal levels.
Sagittal section through all somites of a 11 somite stage chicken embryo, showing somites of occipital (1-4) and cervical levels. Cranial is to the right. (Maschner et al. 2016)
What determines the number of somites in different vertebrates?
Even closely related vertebrate species show different numbers of body segments, namely of caudal vertebrae, i.e. a different length of the tail. Within archosaurs, crocodyles have long, muscular tails, whereas birds have only rudimentary tails. In order to understand how the different numbers of tail segments in birds and reptiles is determined during development, we are comparing the developmental program of tail somite formation and somite differentiation in crocodile and chicken embryos.
FGF8 expression in the tail of Crocodylus niloticus embros of different developmental stages (unpublished original).
How do somite cells form the ventrolateral body wall?
In vertebrate embryos, the ventrolateral body wall, i.e. the thorax and the abdominal wall, are formed by immigration of somite cells into the mesenchyme of the lateral plate mesoderm. Specifically, intercostal and abdominal muscle cells are formed from the hypaxial myotomes which are thought to extend laterally into the resident mesenchyme, and the anlagen of the ribs are extensions of the lateral sclerotome domain which converge at the sternal anlage in the ventral midline. The molecular and morphogenetic events guiding these processes are largely unknown. We investigate the lateral extension movements of rib, intercostal muscle and abdominal muscle anlagen to provide a basis for studies on the molecular regulation of these morphogenetic steps in embryogenesis.
Trunk region of a 6 days old chicken embryo showing intercostal muscles extending into the ventral body wall. Immunohistochemistry showing Myosin Heavy Chain (MF20)in red. (unpublished original).
Does modulation of the ER-Stress Response affect the formation of vessels?
The formation of blood vessels is an essential process for embryonic development, for wound healing but also for tumor growth and the spreading of metastasis. The formation and regression of vessels is therefore of broad biological and medical interest.
We searched for genes that are specifically upregulated during angiogenesis and focused on members of the chaperone family localized in the endoplasmic reticulum (ER). Chaperones in the ER are essential for the correct folding of proteins that are secreted in the extracellular space (as e.g. ligands but also proteins of the extracellular matrix) or integrated into the cellular membrane (as e.g. receptors). The chaperone machinery in the ER also allocates the quality control system in the ER and proteins that are not refoldable are released from the folding circle and retrogradely transported to the cytosol where they are subjected to proteasome-mediated degradation. Changes in the cellular microenvironment such as nutrient deficiency, hypoxia or ionic changes challenge the folding and quality control machinery in the ER and elicit stress, which is sensed by three integral membrane receptors (IRE1a, PERK and ATF6) and transmitted to the nucleus via the socalled unfolded protein response (UPR).
The UPR signaling pathways not only control the ER-stress associated cell metabolism. Novel insights reveal that the protein components of the UPR signaling cascades also control stress-independent cellular responses such as deepitheliazation, cell migration, proliferation and tube formation.
Our studies show that chaperones of the ER are highly elevated in chronic wounds (Fig. 1).
Fig. 1: Immunohistochemical staining of BiP, Mdg1 and Grp94 in dermal chronic wound tissue. (a) Endothelial cells, characterized by factor VIII staining, are positive for BiP, Mdg1/ERdJ4 and Grp94 in inflamed tissue (Tsaryk et al. 2015).
We further elaborated that BiP is essential for migration and the tube forming ability of endothelial cells, which we tested in a tube forming assay where endothelial cells are embedded in a three dimensional matrix (Fig. 2; Tsaryk et al. 2015).
Fig. 2: Downregulation of BiP and of Mdg1/ERdJ4 by transfection of siRNAs into endothelial cells. The cells were subsequently transferred to three dimensional fibrin/collagen gels. Endothelial cells (green) are visualized by their calcein-AM uptake and their tube forming ability was determined.
Presently we are interested in investigating, which signaling pathways are controlling migration and terminal differentiation of endothelial cells, which are essential for physiological and pathological processes, such as vessel formation during embryonic development and growth on one hand and revascularization of wounded tissue and tumorangiogenesis on the other.
This work is largely based on the cooperation with Dr. Roman Tsaryk, Max-Planck-Institut für Molekulare Biomedizin in Münster.
In collaboration with the Department of Traumatic and Orthopedic Surgery in Cologne, the Department of Pediatrics and other clinical departments of the University of Cologne we are engaged in biomechanical studies and investigations of novel surgical techniques with a special focus on surgery in the locomotory system, as well as intensive care preocedures in newborns. Thanks to the body donation program of the Cologne Center of Anatomy, we are able to perform these studies using authentic human material, which enables us to acquire results of high clinical relevance and applicability. We are grateful to to our body donators who thus allow us to contribute to the scientific progress in m sciences to the benefit of patients.
Testing setup to measure peri-implant failures after plate application to a human humerus. Collaboration with Department of Traumatic and Orthopedic Surgery (Hackl et al. 2015).