Slit/Robo Signaling Regulates Multiple Stages of the Development of the Drosophila Motion Detection System

Abstract

Neurogenesis is achieved through a sequence of steps that include specification and differentiation of progenitors into mature neurons. Frequently, precursors migrate to distinct positions before terminal differentiation. The Slit-Robo pathway, formed by the secreted ligand Slit and its membrane bound receptor Robo, was first discovered as a regulator of axonal growth. However, today, it is accepted that this pathway can regulate different cellular processes even outside the nervous system. Since most of the studies performed in the nervous system have been focused on axonal and dendritic growth, it is less clear how versatile is this signaling pathway in the developing nervous system. Here we describe the participation of the Slit-Robo pathway in the development of motion sensitive neurons of the Drosophila visual system. We show that Slit and Robo receptors are expressed in different stages during the neurogenesis of motion sensitive neurons. Furthermore, we find that Slit and Robo regulate multiple aspects of their development including neuronal precursor migration, cell segregation between neural stem cells and daughter cells and formation of their connectivity pattern. Specifically, loss of function of slit or robo receptors in differentiated motion sensitive neurons impairs dendritic targeting, while knocking down robo receptors in migratory progenitors or neural stem cells leads to structural defects in the adult optic lobe neuropil, caused by migration and cell segregation defects during larval development. Thus, our work reveals the co-option of the Slit-Robo signaling pathway in distinct developmental stages of a neural lineage.

Autocrine/Paracrine Slit–Robo Signaling Controls Optic Lobe Development in Drosophila melanogaster

“…This work, as well as our previous work and reports from other labs, present several arguments in favor of this idea: 1) there is no gradient observed in the larval optic lobe; 2) the slit mutant phenotypes can be rescued by expressing Slit in various cell populations, such as medulla neurons and, partially, in glia and photoreceptor cells ( Tayler et al, 2004 ; Caipo et al, 2020 ); and 3) large loss of function clones carrying the slit 2 allele in the visual system do not produce defects in optic lobe organization ( Tayler et al, 2004 ). Indeed, Slit expression does not show a restricted source in the larval optic lobe, and previous data show that it is expressed in multiple cell types ( Tayler et al, 2004 ; Caipo et al, 2020 ; Guzman-Palma et al, 2021 ), unlike in the central brain where it is enriched in the mushroom body ( Oliva et al, 2016 ) and the VNC where it is expressed in the midline ( Kidd et al, 1999 ; Dickson and Gilestro, 2006 ). Regarding the function of Robo receptors, the knockdown of all three Robo paralogues using a general driver resulted in defects in optic lobe development ( Tayler et al, 2004 ).…”

“…It has been previously shown that Slit and the three Drosophila Robo receptors are expressed in the medulla neuropil in addition to other regions of the visual system ( Tayler et al, 2004 ; Suzuki et al, 2016 ; Caipo et al, 2020 ; Guzman-Palma et al, 2021 ). Furthermore, in our previous work, we demonstrated that Ey + medulla neurons in the optic lobe are an important source of Slit ( Caipo et al, 2020 ).…”

“…Studies in whole mutant animals showed that robo2 and robo3 mutants at the larval stage displayed similar boundary defects to those observed in slit mutants ( Suzuki et al, 2016 ). In addition, we previously assessed the phenotypes of robo2 mutants in the adult optic lobe, finding that they are subtler than the phenotypes of slit mutants in adult animals, and connected to their function in the lobula plate ( Guzman-Palma et al, 2021 ). In the case of Robo3, we examined the adult optic lobe in robo3 3 mutants ( Figures 3A–D ) and noticed defects in neuropil organization that are similar to those observed in the sli dui mutant, which is a hypomorphic allele with decreased Slit expression, especially in the optic lobe ( Figure 3E and Supplementary Figures S1E–H ).…”

“…Next, we decided to test whether Robo3 or the other Robo receptors have autonomous roles in medulla neurons. We performed knockdowns using shRNAi with the GAL4/UAS system ( Brand and Perrimon, 1993 ) in Ey + medulla neurons using the ey OK107 -GAL4 driver ( Figures 3F–J ) expressing previously tested RNAi lines in the optic lobe (( Guzman-Palma et al, 2021 ) Supplementary Figures S1I–Y ) and analyzed the morphology of the adult optic lobe. We found that only the Robo3-RNAi knockdown (KD) produced strong alterations in optic lobe morphology, using two independent lines ( Figures 3I,J ), which is a consequence of the compartmentalization defect produced during development.…”

“…One question is whether the downstream effectors are the same in these contexts. Regarding fasciculation, it is likely that the downstream effectors are adhesion molecules, such as cadherins, which are regulated by Robo in several different contexts ( Rhee et al, 2002 ; Rhee et al, 2007 ; Guzman-Palma et al, 2021 ). Here, we find that in this context, regulators of the actin cytoskeleton are likely to be the downstream effectors of Robo3 in the optic lobe, although more work is required to unravel the complete mechanism.…”