Abstract
Insulinoma-associated1a (insm1a) is a zinc-finger transcription factor playing a series of functions in cell formation and differentiation of vertebrate central and peripheral nervous systems and neuroendocrine system. However, its roles on the development of motor neuron have still remained uncovered. Here, we provided evidences that insm1a was a vital regulator of motor neuron development, and provided a mechanistic understanding of how it contributes to this process. Firstly, we showed the localization of insm1a in spinal cord, and primary motor neurons (PMNs) of zebrafish embryos by in situ hybridization, and imaging analysis of transgenic reporter line Tg(insm1a: mCherry)ntu805. Then we demonstrated that the deficiency of insm1a in zebrafish larvae lead to the defects of PMNs development, including the reduction of caudal primary motor neurons (CaP), and middle primary motor neurons (MiP), the excessive branching of motor axons, and the disorganized distance between adjacent CaPs. Additionally, knockout of insm1 impaired motor neuron differentiation in the spinal cord. Locomotion analysis showed that swimming activity was significantly reduced in the insm1a-null zebrafish. Furthermore, we showed that the insm1a loss of function significantly decreased the transcript levels of both olig2 and nkx6.1. Microinjection of olig2 and nkx6.1 mRNA rescued the motor neuron defects in insm1a deficient embryos. Taken together, these data indicated that insm1a regulated the motor neuron development, at least in part, through modulation of the expressions of olig2 and nkx6.1.
Highlights
In vertebrates, motor neurons have precise subtype identities that characterized by a number of morphological criteria, such as soma location, and shape, axon path, and target muscle innervation (Shirasaki and Pfaff, 2002; Lewis and Eisen, 2003)
To confirm phenotypic specificity induced by the insm1a MO injection, we performed rescue experiment by co-injection of 50 ng of insm1a mRNA with insm1a MO into zebrafish embryos, and the results showed that the co-injection significantly decreased the loss, and premature branching of primary motor neurons (PMNs) (Supplementary Figure 4E)
Previous studies have identified its role in regulating the endocrine cells divisions of the pancreas, the neuroendocrine development, the differentiation of retina progenitors and neurogenesis of nervous system (Gierl et al, 2006; Duggan et al, 2008; Farkas et al, 2008; Jacob et al, 2009; FIGURE 3 | Insm1a deficiency suppressed neuronal cells differentiation. (A) Confocal imaging analysis of primary motor neuron in control group, insm1a mutant group and morphant group at 30 and 48 hpf Tg(mnx1:GFP)ml2
Summary
Motor neurons have precise subtype identities that characterized by a number of morphological criteria, such as soma location, and shape, axon path, and target muscle innervation (Shirasaki and Pfaff, 2002; Lewis and Eisen, 2003). The PMNs can be further classified into three groups, caudal primary motor neurons (CaP), middle primary motor neurons (MiP), and rostral primary motor neurons (RoP), by the positions of somata in the spinal cord, and the trajectory of neuron axons (Myers et al, 1986; Westerfield et al, 1986). CaPs, whose somata locate in the middle of each spinal cord hemisegment, can innervate ventral axial muscle, and have been well-studied because of their easy observation and distinct axon projection (Myers et al, 1986; Rodino-Klapac and Beattie, 2004). It was reported that during the development of zebrafish retina, insm could regulate cell cycle kinetics and differentiation of the progenitor cells by acting the upstream of the basic helix-loop-helix (bHLH) transcription factors, and the photoreceptor specification genes (Forbes-Osborne et al, 2013). We examined the function of insm1a in the primary motor neurons development by CRISPR/ Cas9-mediated knockout in the Tg(mnx1:GFP)ml transgenic zebrafish and investigated the possible transcriptional network during this process
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