Early Commissural Diencephalic Neurons Control Habenular Axon Extension and Targeting

Abstract

Most neuronal populations form on both the left and right sides of the brain. Their efferent axons appear to grow synchronously along similar pathways on each side, although the neurons or their environment often differ between the two hemispheres [1-4]. How this coordination is controlled has received little attention. Frequently, neurons establish interhemispheric connections, which can function to integrate information between brain hemispheres (e.g., [5]). Such commissures form very early, suggesting their potential developmental role in coordinating ipsilateral axon navigation during embryonic development [4]. To address the temporal-spatial control of bilateral axon growth, we applied long-term time-lapse imaging to visualize the formation of the conserved left-right asymmetric habenular neural circuit in the developing zebrafish embryo [6]. Although habenular neurons are born at different times across brain hemispheres [7], we found that elongation of habenular axons occurs synchronously. The initiation of axon extension is not controlled within the habenular network itself but through an early developing proximal diencephalic network. The commissural neurons of this network influence habenular axons both ipsilaterally and contralaterally. Their unilateral absence impairs commissure formation and coordinated habenular axon elongation and causes their subsequent arrest on both sides of the brain. Thus, habenular neural circuit formation depends on a second intersecting commissural network, which facilitates the exchange of information between hemispheres required for ipsilaterally projecting habenular axons. This mechanism of network formation may well apply to other circuits, and has only remained undiscovered due to technical limitations.

Patterns of spon1b:GFP expression during early zebrafish brain development

“…1g). These two populations correspond to early expression in the habenula (Hb), consistent with other markers of habenular complex development with onset at ~ 32 hpf [13].…”

“…Transmitted and fluorescence images were overlaid for anatomical context. A color coded MIP Fiji macro developed by Beretta et al [13] was applied to code depth with color. Stacks were aligned with the FiJi plugin Linear Stack Alignment with SIFT [20].…”

Temporal control of Wnt signaling is required for habenular neuron diversity and brain asymmetry

“…To assess the importance of early Wnt signaling suppression in nascent habenular neurons, we transiently activated the pathway for short periods between 22 hpf and 32 hpf. As a readout for habenular neuron development and innervation of the main target of habenular efferent axons, the interpeduncular nucleus (IPN), we used Et(-1.0otpa: mmGFP) transgenic embryos (Beretta et al, 2012(Beretta et al, , 2013(Beretta et al, , 2017. In these embryos, GFP expression in dHb neurons is initiated at 43 hpf.…”

Brain and Behavioral Asymmetry: A Lesson From Fish

“…For example, Gamse et al (2003Gamse et al ( , 2005 defined six molecular distinct domains in the zebrafish larval habenula using a combinatorial expression of potassium channel tetramerization domain containing genes (kctd12.1, kctd12.2, and kctd8). A combined approach that implies the use of a transgenic line [Tg(brn3a-hsp70:GFP)] and an expression marker (kctd12.1) helped to clarify the boundaries between the medial (brn3a) and lateral (kctd12.1) habenula (Aizawa et al, 2005), the neurotransmitters map of the asymmetric dorsal habenular nuclei (deCarvalho et al, 2014), and the extension of axons and asymmetric connections of the habenular compartments toward zebrafish telencephalic hemispheres and ventral midbrain (Beretta et al, 2017). Another important tool to study brain asymmetry and laterality was represented by Tg(foxD3:GFP) fishes that express the green fluorescent protein (GFP) under the control of the foxd3 promoter, a marker of pineal and parapineal precursors and neurons.…”