Resumen de Aislamientos y caracterización de nuevos genes implicados en el desarrollo de la extremidad de vertebrados
Clara García Andrés
Developmental biology is the discipline that studies the mechanisms that transform a single cell known as the zigote in a multicellular complex organism.
Identifying such mechanisms in the whole embryo is quite complicated, so wechose the vertebrate limb as a model to look into the molecules that run this process.
The appendages have several advantages in comparison with other structures: they can be manipulated without compromising embryo viability; its signalling events are similar to the ones that operate in the rest of the body and are conserved through evolution.
In this thesis we have used mice and chicken embryos to look for novel genes implicated in vertebrate limb patterning.
For that purpose, we performed a microarray experiment with mesenchyme cells taken from the distal part of a mouse limb bud at different stages. This part of the mesenchyme is composed of undifferentiated cells and gives rise to all the skeletal elements and connective tissue of the limb; it is known that some proximo-distal markers (patterning genes) are differentially expressed in this region at different developmental points.
The results of the experiment revealed that 250 genes have a significant expression in the limb among which we selected 167 candidates with a more dynamic profile; from this group we picked 30 to analyse them by in situ Hybridization. All this genes showed a remarkable and dynamic expression in the vertebrate limb and, in most of the cases, their function had not been described in the literature previously, so we decided to choose two candidates to carry out a functional study.
The first gene we wanted to investigate encodes a secreted protein called CXCL14, which belong to the chemokine family. These set of proteins enclose a group of small basic signalling factors of the Immune system classically associated with leukocyte trafficking.
In the recent years just the chemokine SDF1 and its receptor CXCR4 has been known to have a role in different processes during embryogenesis in addition of their function in the immune System. Furthermore, both, ligand and receptor are expressed in the limb bud mesenchyme and are important for the patterning and survival of muscle precursors.
Interestingly, our experiments showed that SDF1 and CXCL14 have complementary expression domains, since the first one is found in the central mesenchyme of the limb bud, while CXCL14 is expressed in the ectoderm and in two subectodermic mesenchyme populations. Furthermore, in the chicken embryo the expression of these chemokines is complementary not only in the limb, but also in many other tissues. Its high conservation along evolution, its specific expression in the embryo, and a putative functional interaction with SDF1, prompted us to study CXCL14 during limb development.
8 Previous studies had demonstrated an essential role of the ectoderm in the maintenance of limb bud mesenchyme populations. To address CXCL14 function in limb ectoderm we made gain of function studies in the chicken embryo. For this purpose we electroporated a plasmid with CXCL14 coding sequence in the limb mesenchyme and analysed mesenchyme populations like muscle and tendon precursors. The conclusions are that CXCL14 by itself it is not able to modify muscle and tendon markers, such as Pax3 and Scleraxis, respectively.
The ectoderm has also been involved in the maintenance of the avascular zone, which lies right underneath it. Taking into account that CXCL14 can function as an angiogenesis inhibitor in other systems, we decided to look for any change of the vasculature after the electroporation, using the technique of ink injection in the viteline arteries. In spite of good electroporation efficiency, we could not see a significant difference between the specimens electroporated with the plasmid carrying the chemokine and the electroporation with the empty plasmid.
The second gene that we selected from the Microarray experiment encoded a transcription factor called Prdm16, which contains a PR-protein-protein interaction domain homologue to the SET domain of Drosophila melanogaster, involved in chromatin remodelling. We demonstrated that this gene is expressed specifically throughout the whole limb mesenchyme and its expression starts before limb induction in the lateral plate mesoderm.
To test the function of this gene in the vertebrate limb, we analysed the phenotype of a Prdm16 gene-trap line which abolishes protein production. These mice die at birth and present different skeletal defects and defects in the aortic arch arterial tree. Among the skeletal anomalies, we focused our attention on the clavicle truncation. This bone has some peculiarities that make it really interesting: first, it is the first to ossify along with the dentary of the mandible, and second, it contains the two types of ossification, membranous in the lateral part and endochondral in its medial region. Skeletal staining of cartilage and bone using Alcian blue and Alizarin red reagents, respectively, and cartilage and bone riboprobes, allowed us to study the histogenesis of this poorly studied bone. Strikingly, staining of the mutant embryos showed that Prdm16-/- are not able to develop the medial part of the clavicle, which grows by endochondral ossification, while the membranous part of the clavicle is spared. This phenotype is unique and will help to understand the developmental origins of the clavicle and the understanding of congenital clavicle hypoplasias in man.
