Nestin is essential for zebrafish brain and eye development through control of progenitor cell apoptosis.
BACKGROUND
Nestin is expressed in neural progenitor cells (NPC) of developing brain. Despite its wide use as an NPC marker, the function of nestin in embryo development is unclear.


RESULTS
As nestin is conserved in zebrafish and its predicted sequence is clustered with the mammalian nestin orthologue, we used zebrafish as a model to investigate its role in embryogenesis. Injection of nestin morpholino (MO) into fertilized eggs induced time- and dose-dependent brain and eye developmental defects. Nestin morphants exhibited characteristic morphological changes including small head, small eyes and hydrocephalus. Histological examinations show reduced hind- and mid-brain size, dilated ventricle, poorly organized retina and underdeveloped lens. Injection of control nestin MO did not induce brain or eye changes. Nestin MO injection reduced expression of ascl1b (achaete-scute complex-like 1b), a marker of NPCs, without affecting its distribution. Nestin MO did not influence Elavl3/4 (Embryonic lethal, abnormal vision, Drosophila-like 3/4) (a neuronal marker), or otx2 (a midbrain neuronal marker), but severely perturbed cranial motor nerve development and axon distribution. To determine whether the developmental defects are due to excessive NPC apoptosis and/or reduced NPC proliferation, we analyzed apoptosis by TUNEL assay and acridine orange staining and proliferation by BrdU incorporation, pcna and mcm5 expressions. Excessive apoptosis was noted in hindbrain and midbrain cells. Apoptotic signals were colocalized with ascl1b. Proliferation markers were not significantly altered by nestin MO.


CONCLUSIONS
These results suggest that nestin is essential for zebrafish brain and eye development probably through control of progenitor cell apoptosis.
Chen
Hua-Ling
HL
Institute of Cellular and System Medicine, National Health Research Institutes, Zhunan, Taiwan.
Yuh
Chiou-Hwa
CH
Wu
Kenneth K
KK
D000818:Animals; D030801:Animals, Genetically Modified; D017209:Apoptosis; D001921:Brain / Q000166:cytology / Q000196:embryology* / Q000378:metabolism; D049109:Cell Proliferation; D004625:Embryo, Nonmammalian / Q000166:cytology / Q000196:embryology / Q000378:metabolism; D005123:Eye / Q000166:cytology / Q000196:embryology* / Q000378:metabolism; D005260:Female; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D020287:In Situ Nick-End Labeling; D007381:Intermediate Filament Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D008297:Male; D018613:Microscopy, Confocal; D009025:Morpholines / Q000008:administration & dosage / Q000737:chemistry; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D064231:Nestin; D016376:Oligonucleotides, Antisense / Q000008:administration & dosage / Q000737:chemistry; D013234:Stem Cells / Q000166:cytology / Q000502:physiology; D015027:Zebrafish / Q000196:embryology* / Q000378:metabolism; D029961:Zebrafish Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*
D007381:Intermediate Filament Proteins; D009025:Morpholines; D009419:Nerve Tissue Proteins; D064231:Nestin; D016376:Oligonucleotides, Antisense; D029961:Zebrafish Proteins; D049452:Green Fluorescent Proteins