Insm1a Regulates Motor Neuron Development in Zebrafish

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.

Keywords: development; differentiation; insm1a; motor neuron; zebrafish.

Figure 1

Insm1a expression in embryonic zebrafish spinal cord and primary motor neurons. (A) At 24 hpf, the in situ hybridization signal of insm1a is localized in the spinal cord., Scale bar = 200 μm. (B) The confocal imaging analysis of the transgene insm1a:mCherry expression at 30 hpf. Square in dash line indicates the magnified region in (B') Scale bar = 50 μm. (C,C',C”). Confocal imaging analysis of Tg(mnx1:GFP)^ml2 × Tg(insm1a: mCherry)^ntu805 transgenic line.

Figure 2

Primary motor neuron morphogenesis defects in the insm1a mutant zebrafish embryos. (A) Confocal imaging analysis of primary motor neuron in control group and insm1a mutant groups at 30 hpf and 48 hpf Tg(mnx1:GFP)^ml2. Caps in dash line are showed in diagrams. Scale bar = 50 μm. (B) Quantification of zebrafish embryos with abnormal Caps. The zebrafish embryos are classified into three categories according to its loss degree: severe group with over 80% loss of Cap primary motor neuron, moderate group with <80% loss, and normal group with <20% loss. (C) Quantification of distance between adjacent motor neurons (μm) in control group and insm1a mutant groups at 30 hpf (n = 33 and 41 respectively) and 48 hpf (n = 27 and 76 respectively). (D,E) The length and branching number of Cap axons in control group and insm1a mutant groups at 30 and 48 hpf. Asterisks above the bars indicate significant differences (^**P < 0.01). (F) Quantification of zebrafish embryos with abnormal Caps at 30 and 48 hpf.

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. Phenotypes of neuronal cells in the spinal cord in control group, morphant group, and insm1a mutant groups at 30 hpf and 48 hpf. Asterisks indicate undifferentiated neuronal cells. Scale bar = 50 μm. (B) Quantification of the undifferentiated neuronal cell in the insm1a different treatment zebrafish. Asterisks above the bars indicate significant differences (^*P < 0.05, ^**P < 0.01). (C) Time-lapse imaging analysis of the primary motor neuron in control group and insm1a mutant groups. Asterisks represent undifferentiated neuronal cells. Scale bar = 50 μm

Figure 4

The swimming behavior analysis of control and insm1a mutant zebrafish embryos at 7 and 10 dpf. (A,C). The swimming trajectory of the control and insm1a mutant zebrafish embryos at 7 and 10 dpf. (B,D). Quantification of the swimming distance of control and insm1a mutant zebrafish embryos at 7 and 10 dpf per 5 mins (n = 36 in each group). Asterisks indicate the significant difference (^*P < 0.05, ^**P < 0.01).

Figure 5

Over expressions of nkx6.1 and olig2 rescued the motor neuron defects in insm1a deficient embryos. (A,B). Effects of insm1a knockdown on the expressions of nkx6.1 and olig2 at 19, 24, 36, and 48 hpf. Asterisks indicate significant differences (^*P < 0.05). (C) Abnormal Caps in insm1a knockdown zebrafish embryos were restored by co-injection of nkx6.1 or olig2 mRNA. Diagrams of Caps in dash line are displayed near the corresponding confocal image. Scale bar = 50 μm. (D) Quantification of zebrafish embryos with abnormal Cap primary motor neuron. Asterisks indicate significant differences (^**P < 0.01).