Experimental study of fgf8 morphogenetic activity in the establishment of neuroepithelial positional information in mouse brain development

Resumen

Early brain patterning depends on proper establishment of positional information along the neuroepithelium. That information is given by gradients of secreted signaling molecules (morphogens) read by individual cells within a tissue, leading to specific fate decisions. How morphogen gradients are established and are actually translated in distinct transcriptional activity in the target tissue is a topic, which is of long standing interest in the field of developmental biology. Morphogens form long-range concentration gradients emanating from discrete sources and diffuse across a target tissue determining its developmental fate/response according to the molecular concentration of these extracellular factors the cells in the tissue are exposed. The process of neurulation in vertebrates implies a major morphogenetic step for the initiation of brain regionalization. During its course localized neural centers along the neural tube (called secondary organizers) and more precissely the morphogens emanating from them dictate the proper regionalization and patterning of the surrounding neuroepithelium refining the multiple subdivisions of the embryonic neural tube. FGF8 secreted from the Isthmic Organiser cells influences cell specification and anterior-posterior patterning of the entire mesencephalic and anterior rhombencephalic territories. Understanding these processes are crucial to understand the increasing number of newly classified malformations and anomalies of the cerebellum and brainstem development. Three main objectives were clearly defined for the global achievments of the present Thesis Dissertation: I) To define the molecular pattern of the Isthmic organizer by analysing the expression range of Fgf8 and isthmic Fgf8 downstream genes: the search for direct read-out molecules of FGF8 signalling activity; II) To study the molecular dynamics and biological mechanisms of the FGF8 signalling at the initial steps of its receptor activation in dissociated cell culture system and in anterior neural tube explants (ONTCs); III) To study the specification and maintenance properties of FGF8-dependent positional information along the neural tube by analysing the endocytosis and exocytosis mechanisms in FGF8 producing cells and in FGF8 target cells both in dissociated cell culture system and in anterior neural tube explants (ONTCs). These subjects concerning the temporal and spatial aspects of FGF morphogen gradient formation, signal propagation, patterning and tissue specification are of common interest in the field of embryonic development. Moreover, addressing this objectives and understanding the general mechanisms of FGF8b morphogen dynamics is also of importance for the adult organism comprehension where FGF8b plays a determinant role e.g. for adult neurogenesis, tissue homeostasis and tumor formation. The model of study exploited during my Thesis was the mouse anterior neural tissue at the embryonic stage E9.5 of mouse development. The secondary organizers but particularly, the Isthmic Organizer (IsO) was the context and the neuroepithelial region in which the FGF8 morphogen was analysed. During the experimental approach, I have combined genetic manipulation of the neural tissue using hypomorphic mice for the Fgf8 gene, in vitro cell cultures, ex vivo classical embryology manipulation in explants of the anterior neural tissue (ONTCs-organotypic neurat tube tissue cultures-), and pharmacological blocking of endocytotic and exocytotic pathways both ex vivo and in vitro. Analytical tools included mRNA detection by in situ hybridization techniques, protein immunodetection by Western blots, fluorescence, or biochemistry staining, both on whole mount embryonic tissue, cryostat sections and cell culture samples. More important, during the years of my PhD Studentship, a new molecular tool was created and improved for farther experimental works: a fusion protein between the mouse FGF8b protein and the enhanced GFP protein. The FGF8b-eGFP fusion protein was transfected on HEK293 cells and different new cloned cell lines were created that served to the in vitro study of the FGF8b protein and also to the extrapolation and analysis in the ex vivo system. On this Thesis Dissertation, we report that the morphogen FGF8 exerts initially a differential propagation signal activity effect along the E9.5 mouse neural tube. FGF8 downstream genes at E9.5 isthmic organizer show a gradiental distribution being the En2 gene expression pattern the longest range of expansion observed. However attending at the intracellular triggering cascade, the detection of the phosphorylated forms of ERK1/2 is the most expanded FGF8-target (readout) reaching the mesencephalic/diencephalic boundary and the limit between rhombomeres1/2 at this stage of development. Interestingly, we have found that ERK1/2 enzymes present dynamic patterns of phosphorylation/non-phosphorylation states over the mesencephalic and anterior rhombencephalic territories from E8.5 to E9.5 stages of development. Those patterns of activity depend directly on the presence and combination of the different gradiental expression of the negative modulators of FGF8 and hence on the strength of the extracellular FGF8 signal itself. At E9.5 mouse neuroepithelium ERK1/2 activity is directly dependent and very sensitive to the FGF8 dosage concentration. The phosphorylation of ERK1/2 forms triggered by the FGF8 morphogenetic signal activity is the earliest and fastest effector of this signal making its detection the best readout to study FGF8 function. At mouse E9.5 developmental stage the FGF8 morphogenetic signal coming from the secondary organizers provides crucial positional information to the neuroepithelial cells along the anterior-posterior axis of the anterior neural tube. We demonstrate that this polarizing effect of FGF8 signal activity is driven by the differential activation of RAS-regulated ERK1/2 enzymes and depends directly from the distance to the topographical location of FGF8-related secondary organizers. The levels of ERK1/2 activity and hence the positional information of a given cell are controlled mainly by the presence of the negative modulators giving robustness to the morphogen gradiental signal. The induction of FGF8 negative modulators genes in the target neuroepithelial cells of the mesencephalic and rhombomere 1 territories of the mouse neural tube depends directly on both the concentration of the extracellular molecular gradient of FGF8 that the cells receive and the duration of the intracellular signal triggered by this morphogen (differential signal strength). Moreover, the differential endocytosis rates of the FGFR/FGF8 complexes modulate the actual spread of the morphogen through the extracellular compartments. The differential sorting mechanisms of FGFR/FGF8 complexes to degradation or recycling pathways are also implicated on the FGF8 intracellular signalling duration. Finally, we have demonstrated that over the E9.5 mouse embryo neuroepithelium exist an extracellular FGF8b molecular gradient that spreads from the IsO region towards mesencephalic and rhombencephalic territories. Besides that molecular gradient diffuses through both apical and basal lamina compartments, the FGF8b exocytosis is only taken on at the ventricular (luminal) side of the IsO cells. FGF8b protein extracellular diffusion from its source (Isthmic Organizer) and the sink function exerted by the target cells modulates the final formation and shape of the FGF8 morphogen gradient through the mouse neuroepithelium at E9.5 stages. Additional intracellular mechanisms of endocytosis and recycling sorting of the FGF8-FGFR complexes should be implicated in the posterior translocation of the FGF8 protein from the ventricular compartment to the basal lamina of the pseudostratified neuroepithelium at E9.5.