Structural and Functional Organization of Visual Responses in the Inferior Olive of Larval Zebrafish.

The olivo-cerebellar system plays an important role in vertebrate sensorimotor control. Here, we investigate sensory representations in the inferior olive (IO) of larval zebrafish and their spatial organization. Using single-cell labeling of genetically identified IO neurons, we find that they can be divided into at least two distinct groups based on their spatial location, dendritic morphology, and axonal projection patterns. In the same genetically targeted population, we recorded calcium activity in response to a set of visual stimuli using two-photon imaging. We found that most IO neurons showed direction-selective and binocular responses to visual stimuli and that the functional properties were spatially organized within the IO. Light-sheet functional imaging that allowed for simultaneous activity recordings at the soma and axonal level revealed tight coupling between functional properties, soma location, and axonal projection patterns of IO neurons. Taken together, our results suggest that anatomically defined classes of IO neurons correspond to distinct functional types, and that topographic connections between IO and cerebellum contribute to organization of the cerebellum into distinct functional zones.

Recent advances in the study of limb development: the emergence and function of the apical ectodermal ridge.

Vertebrate extremities develop from limb buds, which emerge as paired protrusions in the lateral plate mesoderm. Forelimb buds are located anteriorly and hindlimb buds are positioned posteriorly. The morphogenesis of the limb requires coordinated actions of several organizing centers, among which the apical ectodermal ridge (AER) plays crucial roles in limb development. Recent studies have shown how the life of the AER (induction, maturation, maintenance and regression) is regulated. This regulation includes cell type- and process- specific roles of previously identified molecules, such as fibroblast growth factors (FGFs), Wnts and bone morphogenetic proteins (BMPs). The studies have also revealed several new players, such as Arid3b, R-Spondin 2 and Flrt3. These advances have enhanced the understanding of how the AER is regulated from its emergence to its regression. Progress has also been made in understanding AER function in relation to processes critical for limb development: proximal-distal patterning, anterior-posterior patterning, chondrogenesis and apoptosis. By focusing on two major model systems, chick and mouse embryos, we will review recent advances in combination with relevant previous studies in the development and function of the AER.

FLRT3 as a key player on chick limb development.

Limb outgrowth is maintained by a specialized group of cells, the apical ectodermal ridge (AER), a thickening of the limb epithelium at its distal tip. It has been shown that fibroblast growth factor (FGF) activity and activation of the Erk pathway are crucial for AER function. Recently, FLRT3, a transmembrane protein able to interact with FGF receptors, has been implicated in the activation of ERK by FGFs. In this study, we show that flrt3 expression is restricted to the AER, co-localizing its expression with fgf8 and pERK activity. Loss-of-function studies have shown that silencing of flrt3 affects the integrity of the AER and, subsequently, its proper function during limb bud outgrowth. Our data also indicate that flrt3 expression is not regulated by FGF activity in the AER, whereas ectopic WNT3A is able to induce flrt3 expression. Overall, our findings show that flrt3 is a key player during chicken limb development, being necessary but not sufficient for proper AER formation and maintenance under the control of BMP and WNT signalling.

[Determination of HER2 by fluorescence in situ hybridization (FISH) in breast cancer. Experience of the Reference Laboratory of Lisbon]. / Carcinoma da mama determinação da amplificação do HER2 por hibridação in situ de fluorescência (FISH).

The knowledgement of HER2/c-erbB-2 status in breast cancer is essential for the eligibility of the patients for therapy with monoclonal antibody anti-HER-2/trastuzumab. From the various existent techniques for the determination of HER-2, the most widely used are: immunohistochemistry (IHC), that estimates the protein expression, and in situ hybridization (FISH), that evaluates its amplification. FISH is essential as complementary of IHQ in tumors with equivocal or non interpreted staining, and also in those with complete weak to moderate membrane staining in >10 % of neoplastic cells (score 2+) because, of these, only cases with gene amplification (FISH positive) respond to therapy. Recent studies also support that FISH should be performed in cases with score 3+, because 10-12% of them don't have amplification. The expensive equipment and reagents for standard FISH method and the need of extensive training and expertise for both the technique and the evaluation, justify a centralized testing in Reference Laboratories. Our aim was to describe the work of the Department of Pathology of the Portuguese Institute of Oncology of Lisbon as a Reference Laboratory in the execution of FISH technique and to present the results obtained from May 2001 to August 2004. FISH was performed with Kit INFORM HER2/neu Gene Detection System, using the BenckMark system of Ventana. We evaluated a series of 4499 invasive carcinoma primary of the breast, that included 587 cases of the Portuguese Institute of Oncology of Lisboa and 3912 cases from different Hospitals of Lisboa, Cascais, Almada, Barreiro, Santarém, Evora, Faro, Portimão, Guimarães, Funchal and Angra do Heroísmo. We verified that 591 cases (13.5%) had HER2 gene amplification, being those patients eligible for therapy with trastuzumab.