The Role of Tumor Necrosis Factor-alpha on the Neurogenesis in Zebrafish Retina

Abstract

 

Purpose

 

To investigate the role of tumor necrosis factor-alpha (TNF-α) in the developing zebrafish retina.To investigate the role of tumor necrosis factor-alpha (TNF-α) in the developing zebrafish retina.

 

Methods

 

Wild-type embryonic zebrafish were used in this study. Morpholino oligonucleotides, complementary to the translation start site of zebrafish TNF-α mRNA sequence, were synthesized and injected into embryos at 1-4-cell stage. The specificity of the translation-blocking was verified by western blot with an anti-TNF-α antibody and whole mount in situ hybridization with a hepatocyte-specific mRNA probe ceruloplasmin. The retinal neurodifferentiation was analyzed by immunohistochemistry with antibodies Zn12, Zpr1 and Zpr-3 which labelled ganglion cells, cones and rods, respectively. An fms mRNA probe and the 4C4 antibody were used to examine the distribution of microglia in the brain and retina.

 

Results

 

Targeted knockdown of TNF-α resulted in specific suppression of TNF-α expression and caused a reduction in liver size. In TNF-α morphants, the neurogenesis in was initiated on time, and ganglion cells, cones and rods were well differentiated at 72 hours postfertilization (hpf).

 

Conclusions

 

TNF-α is not a key regulator for normal retinal growth and neurogenesis.  

 

Figure 1. Tnf-α-targeted morpholino inhibits TNF-α protein synthesis.<br /> Panel A illustrates the gross development of uninjected (UI), mismatch control (MM) and tnf-α-morphant (MO) larvae at 72hpf. No apparent malformation is observed in tnf-α-morphant. Panel B illustrates sequences of the morpholino oligonucleotides and the respective mismatch control used in this study. Panel C is a Western blot of proteins from whole embryos at 72hpf. Note the decrease in the levels of TNF-α in morphants.

 

 

Figure 2. Tnf-α knockdown does not affect neuronal differentiation.<br /> <br /> Panels A-I illustrate sections taken through the retinas from uninjected (UI), mismatch control (MM) and tnf-α morphant (MO) at 72 hpf. Panels A-C illustrate Zn12 staining; panels D-F illustrate Zpr1 staining; panels G-I illustrate Zpr3 staining. Note that in the fms morphant, the retinas are well laminated and differentiated, showing strong expression of Zn12, Zpr1 and Zpr3. L, lens; gcl, ganglion cell layer; inl, inner nuclear layer; onl, outer nuclear layer; ON, optic nerve. Scale bar: A-I, 50μm.