The exit of axons and glial membrane from the developing Drosophila retina requires integrins

Coordinated development of neurons and glia is essential for the establishment of neuronal circuits during embryonic development. In the developing Drosophila visual system, photoreceptor (R cell) axons and wrapping glial (WG) membrane extend from the eye disc through the optic stalk into the optic lobe. Extensive studies have identified a number of genes that control the establishment of R-cell axonal projection pattern in the optic lobe. The molecular mechanisms directing the exit of R-cell axons and WG membrane from the eye disc, however, remain unknown. In this study, we show that integrins are required in R cells for the extension of R-cell axons and WG membrane from the eye disc into the optic stalk. Knockdown of integrins in R cells but not WG caused the stalling of both R-cell axons and WG membrane in the eye disc. Interfering with the function of Rhea (i.e. the Drosophila ortholog of vertebrate talin and a key player of integrin-mediated adhesion), caused an identical stalling phenotype. These results support a key role for integrins on R-cell axons in directing R-cell axons and WG membrane to exit the eye disc.

Six homeobox 6 (SIX6) is differentially expressed in the primary tumors of breast cancer patients treated with trastuzumab.

Trastuzumab, a monoclonal antibody targeted against the human epidermal growth factor receptor 2 (HER2) is utilized for the treatment of human breast cancer (1, 2), but a complete understanding of how tumor signal transduction is modulated by trastuzumab treatment is lacking. By mining published and public microarray and gene expression data (3, 4) from the primary tumors of patients treated with trastuzumab, we found that the six homeobox 6, SIX6, was among the genes most differentially expressed in the primary tumors of patients treated with trastuzumab, and expressed at higher levels in the tumors of patients treated with trastuzumab. Thus, the use of trastuzumab in patients with breast cancer is associated with activation of a gene important for maintenance of multipotent retinal progenitors and expressed in regions of the central nervous system involved in visual processing including the optic stalk and retina (5-8).

Early lineage segregation of the retinal basal glia in the Drosophila eye disc

The retinal basal glia (RBG) is a group of glia that migrates from the optic stalk into the third instar larval eye disc while the photoreceptor cells (PR) are differentiating. The RBGs are grouped into three major classes based on molecular and morphological characteristics: surface glia (SG), wrapping glia (WG) and carpet glia (CG). The SGs migrate and divide. The WGs are postmitotic and wraps PR axons. The CGs have giant nucleus and extensive membrane extension that each covers half of the eye disc. In this study, we used lineage tracing methods to determine the lineage relationships among these glia subtypes and the temporal profile of the lineage decisions for RBG development. We found that the CG lineage segregated from the other RBG very early in the embryonic stage. It has been proposed that the SGs migrate under the CG membrane, which prevented SGs from contacting with the PR axons lying above the CG membrane. Upon passing the front of the CG membrane, which is slightly behind the morphogenetic furrow that marks the front of PR differentiation, the migrating SG contact the nascent PR axon, which in turn release FGF to induce SGs’ differentiation into WG. Interestingly, we found that SGs are equally distributed apical and basal to the CG membrane, so that the apical SGs are not prevented from contacting PR axons by CG membrane. Clonal analysis reveals that the apical and basal RBG are derived from distinct lineages determined before they enter the eye disc. Moreover, the basal SG lack the competence to respond to FGFR signaling, preventing its differentiation into WG. Our findings suggest that this novel glia-to-glia differentiation is both dependent on early lineage decision and on a yet unidentified regulatory mechanism, which can provide spatiotemporal coordination of WG differentiation with the progressive differentiation of photoreceptor neurons.

Inverse oculomotor responses of achiasmatic mice expressing a transfer-defective Vax1 mutant

ABSTRACTIn animals that exhibit stereoscopic visual responses, the axons of retinal ganglion cells (RGCs) connect to brain areas bilaterally by forming a commissure called the optic chiasm (OC). Ventral anterior homeobox 1 (Vax1) contributes to formation of the OC, acting endogenously in optic pathway cells and exogenously in growing RGC axons. Here, we generated Vax1AA/AA mice expressing the Vax1AA mutant, which is selectively incapable of intercellular transfer. We found that RGC axons cannot take up Vax1AA protein from Vax1AA/AA mouse optic stalk (OS) cells, of which maturation is delayed, and fail to access the midline. Consequently, RGC axons of Vax1AA/AA mice connect exclusively to ipsilateral brain areas, resulting in the loss of stereoscopic vision and the inversed oculomotor responses. Together, our study provides physiological evidence for the necessity of intercellular transfer of Vax1 and the importance of the OC in binocular visual responses.

A Micro-Optic Stalk (μOS) System to Model the Collective Migration of Retinal Neuroblasts

Contemporary regenerative therapies have introduced stem-like cells to replace damaged neurons in the visual system by recapitulating critical processes of eye development. The collective migration of neural stem cells is fundamental to retinogenesis and has been exceptionally well-studied using the fruit fly model of Drosophila Melanogaster. However, the migratory behavior of its retinal neuroblasts (RNBs) has been surprisingly understudied, despite being critical to retinal development in this invertebrate model. The current project developed a new microfluidic system to examine the collective migration of RNBs extracted from the developing visual system of Drosophila as a model for the collective motile processes of replacement neural stem cells. The system scales with the microstructure of the Drosophila optic stalk, which is a pre-cursor to the optic nerve, to produce signaling fields spatially comparable to in vivo RNB stimuli. Experiments used the micro-optic stalk system, or μOS, to demonstrate the preferred sizing and directional migration of collective, motile RNB groups in response to changes in exogenous concentrations of fibroblast growth factor (FGF), which is a key factor in development. Our data highlight the importance of cell-to-cell contacts in enabling cell cohesion during collective RNB migration and point to the unexplored synergy of invertebrate cell study and microfluidic platforms to advance regenerative strategies.

The Pebble/Rho1/Anillin pathway controls polyploidization and axonal wrapping activity in the glial cells of the Drosophila eye

During development glial cell are crucially important for the establishment of neuronal networks. Proliferation and migration of glial cells can be modulated by neurons, and in turn glial cells can differentiate to assume key roles such as axonal wrapping and targeting. To explore the roles of actin cytoskeletal rearrangements in glial cells, we studied the function of Rho1 in Drosophila developing visual system. We show that the Pebble (RhoGEF)/Rho1/Anillin pathway is required for glia proliferation and to prevent the formation of large polyploid perineurial glial cells, which can still migrate into the eye disc if generated. Surprisingly, this Rho1 pathway is not necessary to establish the total glial membrane area or for the differentiation of the polyploid perineurial cells. The resulting polyploid wrapping glial cells are able to initiate wrapping of axons in the basal eye disc, however the arrangement and density of glia nuclei and membrane processes in the optic stalk are altered and the ensheathing of the photoreceptor axonal fascicles is reduced.

Patterning the Vertebrate Retina with Morphogenetic Signaling Pathways

The primordium of the vertebrate eye is composed of a pseudostratified and apparently homogeneous neuroepithelium, which folds inward to generate a bilayered optic cup. During these early morphogenetic events, the optic vesicle is patterned along three different axes—proximo-distal, dorso-ventral, and naso-temporal—and three major domains: the neural retina, the retinal pigment epithelium (RPE), and the optic stalk. These fundamental steps that enable the subsequent development of a functional eye, entail the precise coordination among genetic programs. These programs are driven by the interplay of signaling pathways and transcription factors, which progressively dictate how each tissue should evolve. Here, we discuss the contribution of the Hh, Wnt, FGF, and BMP signaling pathways to the early patterning of the retina. Comparative studies in different vertebrate species have shown that their morphogenetic activity is repetitively used to orchestrate the progressive specification of the eye with evolutionary conserved mechanisms that have been adapted to match the specific need of a given species.

Visualisation of ribosomes in Drosophila axons using Ribo-BiFC

Rates of protein synthesis and the number of translating ribosomes vary greatly between different cells in various cell states. The distribution of assembled, and potentially translating, ribosomes within cells can be visualised in Drosophila by using Bimolecular Fluorescence Complementation (BiFC) to monitor the interaction between tagged pairs of 40S and 60S ribosomal proteins (RPs) that are close neighbours across inter-subunit junctions in the assembled 80S ribosome. Here we describe transgenes that express two novel RP pairs tagged with Venus-based BiFC fragments that considerably increase the sensitivity of this technique that we termed Ribo-BiFC. This improved method should provide a convenient way of monitoring the local distribution of ribosomes in most Drosophila cells and we suggest that could be implemented in other organisms. We visualized 80S ribosomes in larval photoreceptors and in other neurons. Assembled ribosomes are most abundant in the various neuronal cell bodies, but they are also present along the lengths of axons and are concentrated in growth cones of larval and pupal photoreceptors. Surprisingly, there is relatively less puromycin incorporation in the distal portion of axons in the optic stalk, suggesting that some of the ribosomes that have started translation may not be engaged in elongation in axons that are still growing.

Morphogenetic and patterning defects explain the coloboma phenotype of the eye in the Mexican cavefish

The morphogenesis of the vertebrate eye consists of a complex choreography of cell movements, tightly coupled to axial regionalization and cell type specification processes. Any disturbance in these events can lead to developmental defects and blindness. Here we have deciphered the sequence of defective events leading to coloboma phenotype in the embryonic eye of the blind cavefish of the species Astyanax mexicanus. Using comparative live imaging on targeted enhancer-trap Zic1:hsp70:GFP reporter lines of both the normal, river-dwelling morph and the cave morph of the species, we identified major defects in initial optic vesicle size and optic cup invagination in cavefish. Combining these results with gene expression analyses, we also discovered defects in axial patterning affecting mainly the temporal retina, in optic stalk tissue specification, and in the spreading processes involving the retinal pigmented epithelium cells. Based on these results, we propose a developmental scenario to explain the cavefish phenotype and discuss developmental constraints to morphological evolution. The cavefish eye appears as an outstanding natural mutant model to study molecular and cellular processes involved in optic region morphogenesis.