Zolessi Elizalde, Flavio Rafael

Research interests

Role of cell polarity in the process of neural differentiation in vertebrates, mainly focusing in two aspects: neural tube formation and neuronal differentiation in the retina.

Working area

The central problem that I am interested in addressing, and to which I have dedicated myself since my doctoral thesis until now, together with the Developmental Neural Cell Biology (BCDN) group that I direct, is that of neural differentiation in the development of vertebrates. To do this, at the BCDN we use numerous experimental tools that allow us to analyze the process of establishing the primordium of the central nervous system on the one hand, and the differentiation of neurons on the other, in vertebrate embryos. We mainly use zebrafish (Danio rerio) or chicken embryos for this, and techniques of pharmacological or genetic manipulation of biological functions, as well as microscopy and various molecular methods to evaluate the results.
For example, in one of the lines, we have characterized different aspects of the participation of actin-modulating proteins of the MARCKS family in the development of the nervous system. First, following the discovery and characterization of a stably phosphorylated form of MARCKS in differentiating neurons, and then following the discovery that MARCKS accumulates in the apical region of the neuroepithelium of the neural plate during neural tube closure, in both cases in chick embryos (Zolessi and Arruti, 2001a; Zolessi and Arruti, 2001b; Toledo et al., 2013). Other groups had previously shown an essential role in neural tube closure of the two members of the family in mice (MARCKS and MARCKS Like-1), so we were interested in characterizing the possible functions of these proteins in neurulation in chick and zebrafish embryos. In the chick, we found that reduced MARCKS expression, as well as its phosphorylation in the effector domain by PKC, causes a generalized loss of apico-basal polarity of neural plate cells, with massive apical extrusion and failure of neural tube closure (Aparicio et al., 2018). More recently, we have addressed the understanding of this phenomenon by computational modelling of neuroepithelium dynamics (S. Bosch, 2024, Master's thesis).
We have also analysed the functions of the four genes encoding MARCKS proteins in zebrafish, finding that all are expressed and have particular functions during early embryonic development, but particularly two, MARCKSB and MARCKSL1A, seem to be more important in neural tube morphogenesis (Prieto and Zolessi, 2017). We are currently trying to better understand these two proteins, having found that they have different subcellular localizations, and that the reduction of MARCKSL1A expression causes a phenotype almost identical to the mutation of the N-cadherin gene. We have found that both genes effectively interact in the process of neural tube generation (L. Veloz, PhD thesis in progress).
Furthermore, we have been interested in how the polarity of the neuroepithelium influences the localization and polarization of neurons, particularly in the retina of the zebrafish. We have characterized in detail the differentiation process of retinal ganglion cells in vivo, by means of time-lapse experiments, finding a highly stereotyped behavior in all the cells analyzed. Furthermore, we show that in mutants in which the polarity of the neuroepithelium is altered, neurons polarize, but they do so in a disordered manner, and even with their orientation inverted (Zolessi et al., 2006). Polarized signals, such as Laminin A1 from the basal lamina, appear to be essential in determining the correct orientation of these neurons (Randlett et al., 2011). In addition, other signals such as the secreted factor Slit1b, or the presence of the primary cilia, also participate in the early differentiation process of these neurons, although not in their orientation or polarization (Zolessi et al., 2006; Lepanto et al., 2016). We have also found complementary functions of the factors Slit2 and Slit3 in guiding the growth of the axons of these neurons, from the retinal interior to the synaptic target in the optic tectum (Davison and Zolessi, 2021; Davison et al., 2022).
Another type of retinal neuron of great interest, due to its combined characteristics between epithelial cells and neurons, are photoreceptors. We recorded the differentiation of photoreceptor progenitors using time-lapse confocal microscopy, thus being able to determine a process that is "inverse" to that of ganglion cells: they retract a basal process to then accommodate their body in the outer nuclear layer, where they remain tightly bound. In that position, these progenitors, already committed to their differentiation destiny, undergo up to two more cell divisions (Aparicio et al., 2021). In the same work, we showed that the primary cilium is necessary in photoreceptor progenitors for the correct retraction of the basal process. At that stage, in addition, these cells showed great cortical activity in control embryos, dynamically extending filopodium-type cellular processes, which we call tangential processes (Aparicio et al., 2021). More recently, we have found that tangential processes are present, although gradually decreasing in frequency, until post-mitotic stages, around the time of formation of the internal segments of the photoreceptors, while other types of processes, more rigid, the calyceal processes, begin to appear (Sharkova et al., 2024).
In our current projects, and near future perspectives, we are interested in better understanding: a) the mechanisms of establishment and maintenance of the polarity and integrity of the neuroepithelium, b) the mechanisms of organization of neural progenitors and differentiating neurons, both in the apico-basal and planar aspects with respect to the neuroepithelium, and c) how these processes potentially affect the functioning and survival of neurons. This is particularly important to understand several neurodegenerative diseases of the retina and the central nervous system in general, where neurons die in post-embryonic stages due to defects in the mechanisms of maintenance of cell polarity generated during development. Many of the new approaches we are considering are being carried out within the framework of national and international collaborations, some of them funded (ICGEB; ECOS Sud; ACIP-Pasteur), with researchers from Peru, Brazil, Canada, France and Italy.