Zebrafish Activate-Dependent Neuroprotective Protein (ADNP) Plays Important Roles in Brain Development

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We cloned zebrafish ADNP and sequence comparison showed that zebrafish ADNP shared 74% amino acid sequence similarity with that of both human and mouse ADNP. Like mammalian ADNP, zebrafish ADNP possesses nine zinc-fingers DNA binding motifs and one homeobox domain. Whole mount in situ hybridization showed that ADNP mRNA is a maternal transcript and is ubiquitously expressed during gastrulation and somite stages. In 24 and 48 hpf (hours post fertilization) embryos, ADNP is specifically expressed in the brain region. Decreased expression in the brain region is observed in 72 and 96 hpf embryos. In order to test the function of ADNP, we designed two antisense morpholino oligonucleotides to inhibit ADNP protein synthesis. Knockdown ADNP function caused indented midbrain-hindbrain boundary at 24hpf. High level expression of p53 in the brain and spinal cord regions was identified in ADNP morphants. Apoptosis was identified in the brain and spinal cord region of 24 hpf morphants by acridine orange staining and apoptosis can be prevented by co-injection with p53 spMO. This result suggests that absence of ADNP induces p53-dependent apoptosis in zebrafish embryos. We then analyzed the effect on brain formation in ADNP morphants using different marker genes. We detected decreased expression of p57 and ngn 1 in the whole brain region, and decreased expression of pax6a in the telencephalon, dorsal diencephalon and retina regions in 24 hpf ADNP morphants. Similar phenomenon was also seen with the lower expression level of cerebellum marker, atoh1a and ptf1α, in ADNP morphants at 24 hpf. In addition, co-injection of ADNP MO with p53 spMO can rescue decreased expression of p57 and ngn 1 in the brain region at 24 hpf. Surprisingly, the expression of p57 strongly up-regulated in the brain of 48 hpf ADNP morphants. We also detected increased expression of ngn1 in the midbrain and hindbrain and increased expression of atoh1a and ptf1α in the URL and LRL of 48 hpf ADNP morphants. We also analyzed expression levels of neuronal progenitor, ascl1b and terminal differentiation marker, huC. Similarly, we detected decreased ascl1b and huC expressions in 24 hpf ADNP morphants while observed an increased expression of these two genes in morphants at 48 hpf. Co-injection with p53 spMO can partially rescue the alteration of expression level of ascl1b and huC. Overall, our results suggest that zebrafish ADNP does not function as a regulator for brain morphogenesis. However, like its mammalian counterparts, zebrafish ADNP functions as a neuroprotector to against physiological stresses and environmental insults.