Manipulating Robo ExpressionIn VivoPerturbs Commissural Axon Pathfinding in the Chick Spinal Cord

Abstract

In vertebrate embryos, most spinal commissural axons cross the ventral midline (VM) and project either alongside or significant distances away from the floor plate (FP). The upregulation of repulsive Robo1/2 receptors on postcrossing commissural axons, in mammals, presumably allows these axons to respond to the midline-associated repellents, Slit1-3, facilitating their expulsion from, and prohibiting their reentry into, the FP. Compelling data suggest that Robo3 represses Robo1/2 function on precrossing axons and that Robo1/2 inhibit attractive guidance receptors on postcrossing axons, thereby ensuring that decussated axons are selectively responsive to midline Slits. However, whether Robo1/2 expel decussated commissural axons from the VM and/or prevent their reentry into the FP has not been explicitly established in vivo. Furthermore, some commissural axons do not require Robo1/2 to elaborate appropriate contralateral projections in the mouse spinal cord. Here, we use unilateral in ovo electroporation together with Atoh1 and Neurog1 enhancer elements to visualize, and assess the consequences of manipulating Robo expression on, dl1 and dl2 chick commissural axons. In response to misexpressing a cytoplasmic truncation of Robo1 and/or Robo2, which should block all Robo-ligand interactions, postcrossing commissural axons extend alongside, but do not project away from or reenter the FP. In contrast, misexpression of full-length Robo2 prevents many commissural axons from crossing the VM. Together, these findings support key and selective in vivo roles for Robo receptors in presumably altering the responsiveness of decussated commissural axons and facilitating their expulsion from the VM within the chick spinal cord.

Axonal Patterns and Targets of dA1 Interneurons in the Chick Hindbrain

“…We have previously used transient-transgenic chick screen of highly conserved human noncoding sequences and identified numerous enhancer elements that are expressed in dI1-dI3 of the chick spinal cord (Pennacchio et al, 2006;Visel et al, 2007;Avraham et al, 2009Avraham et al, , 2010a. Unilateral electroporation of these elements enabled us to direct the expression of a reporter gene in specific neurons in a spatio-temporal controlled manner, and to follow or manipulate axonal projections on both sides of spinal cord (Reeber et al, 2008;Avraham et al, 2009Avraham et al, , 2010b. This strategy circumvented the difficulty of tracing axonal projections in mice, where germ-line insertion of enhancer elements drives robust bilateral symmetric labeling of axons that may lead to masking of ascending/descending and ipsilateral/contralateral axonal tracts.…”

“…To trace hindbrain dA1 interneurons, we used two enhancer elements that have previously been characterized as specific for spinal dI1 neurons (Helms et al, 2000;Lumpkin et al, 2003;Reeber et al, 2008;Avraham et al, 2009) and analyzed their expression in the chick hindbrain. The tested enhancers were the mouse Atoh1 (Helms et al, 2000;Ebert et al, 2003;Lai et al, 2011) and edI1 enhancer element (Avraham et al, 2009).…”

Collaborative and Specialized Functions of Robo1 and Robo2 in Spinal Commissural Axon Guidance

“…Robo-dependent sorting of axons into different parts of the VLF Ectopic expression of a dominant-negative Robo1 in chick commissural neurons leads to a medial shift of these axons after floor plate crossing (Reeber et al, 2008), indicating that interfering with Robo function can disrupt mediolateral sorting of commissural axons. However, these results did not address which Robo family member(s) is required for correct axonal positioning after midline crossing.…”

Netrin-1-Dependent Spinal Interneuron Subtypes Are Required for the Formation of Left-Right Alternating Locomotor Circuitry

“…CINs choose different trajectories after crossing the midline (Imondi and Kaprielian, 2001;Kadison and Kaprielian, 2004), which could influence the efficiency of our tracing technique. For example, some CINs originating from the dI1 and dI2 domains have been shown to diverge directly to the lateral funiculus while others project several segments alongside the floor plate (Reeber et al,2008;Avraham et al, 2009). However, using our approach we detected at least part of these dorsal subpopulations (supplemental Fig.…”