Sim1a and Arnt2 contribute to hypothalamo-spinal axon guidance by regulating Robo2 activity via a Robo3-dependent mechanism

Abstract

SUMMARYPrecise spatiotemporal control of axon guidance factor expression is a prerequisite for formation of functional neuronal connections. Although Netrin/Dcc-and Robo/Slit-mediated attractive and repulsive guidance of commissural axons have been extensively studied, little is known about mechanisms controlling mediolateral positioning of longitudinal axons in vertebrates. Here, we use a genetic approach in zebrafish embryos to study pathfinding mechanisms of dopaminergic and neuroendocrine longitudinal axons projecting from the hypothalamus into hindbrain and spinal cord. The transcription factors Sim1a and Arnt2 contribute to differentiation of a defined population of dopaminergic and neuroendocrine neurons. We show that both factors also control aspects of axon guidance: Sim1a or Arnt2 depletion results in displacement of hypothalamo-spinal longitudinal axons towards the midline. This phenotype is suppressed in robo3 guidance receptor mutant embryos. In the absence of Sim1a and Arnt2, expression of the robo3 splice isoform robo3a.1 is increased in the hypothalamus, indicating negative control of robo3a.1 transcription by these factors. We further provide evidence that increased Robo3a.1 levels interfere with Robo2-mediated repulsive axon guidance. Finally, we show that the N-terminal domain unique to Robo3a.1 mediates the block of Robo2 repulsive activity. Therefore, Sim1a and Arnt2 contribute to control of lateral positioning of longitudinal hypothalamic-spinal axons by negative regulation of robo3a.1 expression, which in turn attenuates the repulsive activity of Robo2.

Transcription factors and effectors that regulate neuronal morphology

“…In a subset of zebrafish hypothalamic neurons that project to the hindbrain and spinal cord, the bHLH-PAS transcription factors Sim1a and Arnt2 regulate lateral position by downregulating Robo3 (Schweitzer et al, 2013 …”

Role of Slit and Robo Proteins in the Development of Dopaminergic Neurons

“…To date 7 studies have specifically investigated the role that Slit proteins, through their interaction with Robo receptors, play in the growth and guidance of dopaminergic neurites [10,11,12,13,14,15,16] (table 1); 2 of these studies, Lin et al [11] and Lin and Isacson [12], reported in vitro studies using primary cultures of the ventral midbrain of rats and murine embryonic stem (ES) cell-derived dopaminergic neurons, and 1 study has reported how Slit2 affects human ES cell-derived dopaminergic neurons in vitro [14]. The other 4 studies have investigated the role of Slits and Robos in the growth of longitudinal dopaminergic projections in vivo (mouse: [10,15] zebrafish: [13,16]).…”

“…The other 4 studies have investigated the role of Slits and Robos in the growth of longitudinal dopaminergic projections in vivo (mouse: [10,15] zebrafish: [13,16]). In addition, a recent study has also reported an increase in the number of tyrosine hydroxylase (TH, the rate-limiting enzyme for dopamine synthesis) immunoreactive (TH-ir) profiles in the adult zebrafish optic tectum in Robo2 astray mutants [17] (astray means ‘away from the correct path'; this is a mutant zebrafish line identified due to defects in their retinotectal pathfinding [18]).…”

“…Finally, a recent study has shown that the lateral positioning of the descending dopaminergic axons in zebrafish is also regulated by the transcription factor Sim1a [16]. Sim1a negatively regulates Robo3a.1 transcription, which in turn attenuates the repulsive action of Robo2 from the midline.…”

“…The authors of this study showed that in Sim1a morphants Robo3a.1 expression increases and TH+ descending axons display a medial displacement. In Sim1a morphants of Robo3 twitch twice mutant genotype this medial displacement was attenuated, which indicates that the negative control of Robo3a.1 by Sim1a is required for the proper lateral positioning of dopaminergic descending axons in zebrafish [16]. …”