Specific subjects

  1. Role of cell adhesion-mediated signalling in craniofacial malformations: an integrated molecular and morphological study
  2. Continuous tooth replacement in non-mammalian vertebrates: the possible involvement of epithelial stem cells
  3. Normal and pathological fusion of vertebrae in bony fish: an “evo-devo” study
  4. Individualisation of cartilaginous elements in the zebrafish skeleton
  5. The role of cell adhesion molecules in tooth development in the zebrafish
  6. Signalling pathways involved in tooth development and replacement: a developmental approach using mutants

Role of cell adhesion-mediated signalling in craniofacial malformations:
an integrated molecular and morphological study

Promotor: Prof. Dr. F. Van Roy

Research GOA

Co-promotors:

  • Prof. Dr. K. Vleminckx
  • Prof. Dr. A. Huysseune
  • Prof. Dr. D. Adriaens
  • Dr. J. Van Hengel

Additional investigators

  • Drs. B. Verstraeten
  • E. Sanders

Funding : Special Research Fund (BOF) – Ghent University


Context

The head is anatomically the most complex part of the body and besides the brain it contains the primordial sensory organs and important portals to the outside world. Craniofacial malformations generally have far reaching consequences, both in humans and in other craniates. Given the complexity of the craniofacial structures, many genes are known or presumed to be involved in their ontogeny. This implies that many craniofacial defects may originate from various mutations. Although multiple genetic alterations are known to cause several syndromes with craniofacial malformations, the genetic causes of many other human craniofacial abnormalities await identification. There is strong evidence that various cell-cell adhesion molecules play essential roles in morphogenetic processes. In particular, members of the cadherin protein family are involved in the ‘physical sorting out’ of similar cells. In addition, classic cadherins are also involved in extremely important signalling pathways by binding to cytoplasmic catenin proteins, of which β-catenin (β-ctn) and p120ctn are the best studied. On the other hand, protocadherins display a much higher isoform diversity than classical cadherins, and their functions and signalling association partners remain largely unknown. Evidence for the involvement of cell adhesion-mediated signalling in craniofacial development is steadily growing. Studies have shown that inactivation of particular cadherins, protocadherins or catenins in the head region leads to striking abnormalities in the brain and in craniofacial structures, including teeth.

Aims
The aim of the consortium to which our research group belongs, is to investigate the extent of involvement of particular cell-cell adhesion molecules, namely classical cadherins and delta-protocadherins, and the coupled intracellular signalling processes, in the normal development of complex craniofacial structures. The focus in our research group lies on the teeth. We hypothesize that mutations or abnormal regulation of cell adhesion molecules or associated signals underlie several craniofacial and dental malformations of unknown genetic origin. To test this hypothesis, we use Xenopus laevis and X. tropicalis as well as zebrafish (Danio rerio) for analysis of signalling pathways, to test that cell adhesion mediated signalling is required for tooth development, and for the discovery and validation of candidate key genes.

Continuous tooth replacement in non-mammalian vertebrates:
the possible involvement of epithelial stem cells

Stem cells 1

Promotor: Prof. Dr. A. Huysseune

Co-promotors:

  • Prof. Dr. K. Vleminckx
  • Dr. J.-Y. Sire

Additional investigators:

Stem cells 2
  • Dr. M. Willems
  • Drs. S. Vandenplas
  • E. Sanders

Funding: Fund for Scientific Research Flanders (FWO Vlaanderen)


Context

Several tissues and organs in the vertebrate body are maintained by adult stem cells. These are characterized by their capacity to divide to give rise to a new stem cell and a cell that goes on differentiating.
The existence of stem cells for repair and renewal of epithelial tissues has been demonstrated for many years in different epithelia (e.g. epidermis, cornea epithelium, intestinal crypt epithelium, or in organs with an epithelial component (e.g. liver, pancreas, lungs, mammary glands and hairs). Epithelial stem cells rely on mesenchymal signals for their survival and differentiation. In addition, they function in a specific micro-environment, called the “stem cell niche”.
Teeth, like many other organs in the vertebrate body, develop as a result of epithelio-mesenchymal interactions. In all vertebrates, tooth development starts with the formation of an epithelial thickening which subsequently invaginates into the underlying mesenchyme to form a bud. The process is next carried through several steps, which can differ according to the vertebrate group considered.

Aims
In this project we test the hypothesis (1) that

Kop xenopus

epithelial stem cells underlie the process of continuous tooth replacement in non-mammalian vertebrates, and (2) that these stem cells are under control of the Wnt signalling pathway. We use two model organisms: the zebrafish (Danio rerio) and the clawed toad (Xenopus tropicalis).

Methodology
The first hypothesis is tested by collecting morphological evidence for the presence of epithelial stem cells, such as ultrastructural indications, as well as patterns of proliferation in the odontogenic epithelium.
We collect molecular evidence for the involvement of the Wnt signalling pathway in the continuous formation of new tooth germs,  via two approaches. We study the spatio-temporal pattern of expression of genes involved in the Wnt signalling pathway, either at the level of the transcripts (by in situ hybridisation), or at the level of their proteins (via immunolocalisation). Furthermore we collect direct evidence for the activation of the Wnt-signalling pathway by taking advantage of transgenic Xenopus lines (Wnt-reporter line and lines with  Wnt-activating and Wnt-repressing constructs; see Denayer et al., 2006, FEBS Lett. 580: 393-398).

Normal and pathological fusion of vertebrae in bony fish: an “evo-devo” study

Promotor: Prof. Dr. A. Huysseune

Fusing vertebrae

Co-promotor: Prof. Dr. P.E. Witten

Additional investigators:

  • Drs. A. Bensimon-Brito
  • T. D’Heuvaert

Funding: Fund for Scientific Research Flanders (FWO Vlaanderen)

Context
Skeletal malformations negatively influence animal growth, welfare, and represent an important economical loss to the fish farmers. Clearly, there is an urgent need for research into the aetiology and mechanisms of vertebral malformations, in particular fusions. Based on previous work, and on our knowledge on the role of the notochord (= the future intervertebral tissue) in vertebral differentiation, it is clear that not just the cartilage and the bone of the vertebral centra, but also the intervertebral tissue is involved in the fusion process.
In this project, we use an evolutionary developmental (‘evo-devo’) approach to attempt to understand why vertebrae sometimes fuse, or otherwise can persist as separate entities. This approach is based on the knowledge that the fusion of vertebrae does not need to be pathological in all cases.

Aims
Our working hypothesis states that ‘normality’ of vertebral fusion in evolution (at the level of the caudal vertebrae) can provide us with insights into the pathological process of vertebral fusion in (late) development (at the level of the trunk vertebrae).
To test this hypothesis we seek to answer the following questions, using two species of teleosts:
(1) how does the normal fusion process of caudal vertebrae in Atlantic salmon differ from the pathological fusion of trunk vertebrae?
(2) how does the normal fusion process of caudal vertebrae in zebrafish differ from the persistence of individualised trunk vertebrae?
(3) can we induce the fusion of zebrafish trunk vertebrae or, conversely, prevent the fusion of caudal vertebrae in vitro (and thus elicit an atavism)?

Methodology
The first two questions are addressed through (i) an in-depth histo-morphological study of timing and progression of the fusion process in the tail versus the trunk through X-rays, clearing and staining of material, and through analysis of serial sections; and (ii) a study of spatial and temporal patterns of gene expression of a number of key genes involved in cartilage and bone formation, in both regions (tail versus trunk). 
The third question is addressed using in vitro organ cultures. By changing the culture conditions we attempt to prevent fusion of caudal vertebrae, or, conversely, stimulate fusion of trunk vertebrae.

Individualisation of cartilaginous elements in the zebrafish skeleton

Research Jasper Dewit

Promotor: Prof. Dr. A. Huysseune

Co-promotor: Prof. Dr. P.E. Witten

Additional investigator: Drs. J. Dewit
    

Funding: Agency for Innovation by Science and Technology (IWT)


Context
The vertebrate cartilaginous skeleton develops early in embryonic development, as it has to fulfil a number of vital functions. Recent studies, mostly on zebrafish, have yielded already important insights on the specification and determination of mesenchymal cells into chondrogenic cells, as well as on the genetic mechanisms that determine the morphological pattern of a cartilaginous element. Yet, the developmental processes situated more downstream, and the mechanisms that control these, remain largely unknown.
In the zebrafish, as in other teleosts, many cartilaginous elements which exist as separate entities in the adult, develop as part of one continuous element, and are only later separated by joints or sutures.

Aims
This study aims at investigating how initially continuous chondrogenic condensations or early chondrification centers divide into separate elements. Three different anatomical regions of the zebrafish skeleton are compared: left and right hemimandibula, which divide and make a suture, the lower jaw joint, which develops an articulation, and the radials in the pectoral fin, which develop into independent elements.

Methodology
For each of the anatomical regions we test which one(s) of the following mechanisms contribute to the division of the initially continuous element:
- resorption of the extracellular matrix and the cells present;
- apoptosis followed by resorption of the extracellular matrix (ECM);
- transdifferentiation or metaplasia of the cells and resorption or remodelling of the ECM.

Techniques used include detailed light and transmission electron microscopic observations, immunolocalisation and confocal laser scanning microscopy..

The role of cell adhesion molecules in tooth development in the zebrafish

Promotor: Prof. Dr. A. Huysseune

E-cadherin expression (brown) in the pharyngeal epithelium and the developing zebrafish teeth at 52 hours post fertilization

Additional investigators:

  • Drs. B. Verstraeten
  • E. Sanders
  • M. Soenens

Funding: Agency for Innovation by Science and Technology (IWT)


Context
Although the importance of cell adhesion in morphogenesis is known for quite some time, remarkably few studies have focused on the presence and function of cell adhesion molecules (CAMs) during tooth development. Cell junctions form solid connections between cells. They allow communication between cells and the elaboration of tissues. The most important cell adhesion molecules belong to the cadherin subfamilies. Cadherin-dependent signalling also influences cellular processes such as proliferation, survival, polarisation, differentiation, shaping and migration. These processes are of vital importance during embryogenesis and organogenesis.

Aims
In this project we test whether spatial and temporal patterns of distribution of certain adhesion molecules correlate with specific developmental events during tooth development. Second, we wish to experimentally test the role of certain cell adhesion molecules.

Methodology

We use in situ hybridisation and immunocytochemistry on developmental stages of zebrafish (Danio rerio) to study the distribution of adhesion molecules at the mRNA and protein level, respectively.
For a functional analysis, we examine zebrafish mutants exhibiting a defect in a specific cell adhesion molecule, and we use morpholino-knockdown technology to prevent translation of the adhesion molecule selected.
 

Signalling pathways involved in tooth development and replacement:
a developmental approach using mutants

Promotor: Prof. Dr. A. Huysseune

Additional investigators:

  • Dr. M. Willems
  • Drs. S. Vandenplas
        

Funding: Fund for Scientific Research Flanders (KaN, FWO Vlaanderen)


Context

Coupe gebit

Teeth develop as a result of a cascade of epithelio-mesenchymal interactions, involving signaling molecules, their receptors, intracellular molecules and transcription factors.

A considerable amount of data has been acquired regarding the molecular control of tooth development in mammals (mostly the mouse, see http://bite-it.helsinki.fi/). There is however a serious backlog regarding the study of the molecular control of tooth development and replacement in non-mammalians.

Kop zebravis

Aims

We use zebrafish with mutations in genes belonging to one of the major families of paracrine factors, their receptors and/or downstream signalling partners, to identify the role of a given gene during the development and/or replacement of teeth.

Kop mutante zebravis

Methodology
To reveal all details of tooth development and patterning (number, position, developmental stage of the teeth) we use serial 1µm thick sections and 3D-reconstructions of the dentition.