Co-factors of LIM-HD transcription factors in neural development and axon pathfinding in zebrafish
The zebrafish neuromuscular system is an elegant model to study neural development. To reveal a specific programme for zebrafish motor axon pathfinding I established a method to selectively block motor axon pathfinding by interfering with LIM domain transcription factor signaling. LIM homeodomain proteins (LIM-HDs) are an important class of transcriptional regulators and involved in neural development as well as neuron fate decision in vertebrates. DD domain dimerization of CLIM (cofactor of LIM-HDs) can activate LIM-HDs and downstream gene transcription while over-expression of dominant-negative CLIM (DN-CLIM), which lacks the DD domain, blocks LIM-HD activity. Motor neurons fluoresce in HB9:GFP transgenic zebrafish as the promoter of the motor neuron specific gene Hb9 drives expression of GFP. Motor axons in DN-CLIM injected HB9:GFP zebrafish are unable to exit the spinal cord, instead they grow inside the spinal cord. Thus axon pathfinding, but not general growth appears to be impaired in these neurons. This provides an excellent research model to find genes involved in motor axon pathfinding downstream of LIM-HDs. Gene array expression profiling was carried out on GFP+ motor neurons by fluorescence-activated flow sorting (FACS) with and without prior injection of DN-CLIM mRNA to elucidate the potential genes relevant to motor axon pathfinding. Genes that were most strongly down-regulated in DN-CLIM injected embryos were considered to belong to a motor axon specific guidance programme. Calca, tac-1 and chodl genes, retrieved from the gene array data, showed specific expression pattern in motor neuron and obvious down-regulation after DN-CLIM injection by in situ hybridization. This validated the array results. Chodl contains a C-type lectin domain representing a potential cell surface receptor for guidance factors. Gene knock-down experiments with two independent morpholinos led to stalling of CaP motor axons at the horizontal myoseptum, a pivotal choice point for axon pathfinding. This suggests that this novel gene specifically affects motor axon pathfinding in zebrafish. Single stranded DNA binding protein 1 (SSDP1) functions as an activator of SSDP1/CLIM/LIM-HD complex which involved in the transcriptional control of embryonic development. To verify how SSDP1 function in neural development in zebrafish, I have cloned Zebrafish SSDP1a and SSDP1b, which are most closely related to mouse and human SSDP1. SSDP1a is widely expressed during zebrafish development while SSDP1b is specifically expressed in sensory trigeminal and Rohon-Beard neurons. Over-expression of the N-terminal portion of SSDP1 (N-SSDP1) increases endogenous CLIM protein levels in vivo and impairs the formation of eyes and midbrain-hindbrain boundary. In addition, SSDP1b knock down impairs trigeminal and Rohon-Beard sensory axon growth. N-SSDP1 can partially rescue the inhibition of axon growth induced by DN-CLIM. These results reveal specific functions of SSDP1 in neural patterning and sensory axon growth which are in part due to the stabilization of LIM-HD/CLIM complexes. In summary, co-factors of LIM-HDs play important roles in neural development, cell fate specification as well as axon pathfinding.