Wu Sheng, Adams Bruce A, Fradinger Erica A, Sherwood Nancy M
Department of Biology, University of Victoria, 3800 Finnerty Road, Victoria, B.C. V8W 3N5, Canada.
Ann N Y Acad Sci. 2006 Jul;1070:602-21. doi: 10.1196/annals.1317.091.
To study the role of pituitary adenylate cyclase-activating polypeptide (PACAP) in early brain development, we examined PACAP and its receptors for first expression and then separately knocked down the two forms of PACAP in zebrafish where development is rapid and observable. We injected morpholinos (antisense oligonucleotides) into fertilized eggs to block PACAP. Morphological changes in the brain were observed in embryos at 27 h post fertilization (hpf). Using in situ hybridization of early brain marker genes, we found that the most striking effects were an increase in pax2.1 expression in eye stalks associated with absence of either form of PACAP or an increase in eng2 and fgf8 in the midbrain-hindbrain boundary after loss of PACAP2. These marker genes are among the earliest factors in the formation of the midbrain-hindbrain boundary, an early organizing center. We suggest that PACAP is a target gene with feedback inhibition on pax2.1, eng2, or fgf8 in specific brain areas. In the hindbrain, the absence of either form of PACAP had little effect, as shown by expression of ephA4 and meis1.1. During midbrain development, our evidence suggests that PACAP1 can activate mbx. In both the diencephalon and/or forebrain, lack of PACAP1 or PACAP2 led to an increase in fgf8, again suggesting a suppressive effect of PACAP during development on these important genes that help to define cells in the forebrain. The early expression of transcripts for PACAP and its receptors by 0.5-6 hpf make both PACAP1 and PACAP2 candidates for factors that influence brain development.
为了研究垂体腺苷酸环化酶激活多肽（PACAP）在早期脑发育中的作用，我们首先检测了PACAP及其受体的首次表达，然后分别在发育迅速且可观察的斑马鱼中敲低两种形式的PACAP。我们将吗啉代（反义寡核苷酸）注入受精卵中以阻断PACAP。在受精后27小时（hpf）观察胚胎大脑的形态变化。通过早期脑标记基因的原位杂交，我们发现最显著的影响是，在缺乏任何一种形式的PACAP时，眼柄中pax2.1表达增加；或者在PACAP2缺失后，中脑 - 后脑边界处eng2和fgf8表达增加。这些标记基因是中脑 - 后脑边界形成过程中最早的因素之一，而中脑 - 后脑边界是一个早期的组织中心。我们认为PACAP是一个对特定脑区中pax2.1、eng2或fgf8具有反馈抑制作用的靶基因。在后脑中，如ephA4和meis1.1的表达所示，缺乏任何一种形式的PACAP影响很小。在中脑发育过程中，我们的证据表明PACAP1可以激活mbx。在间脑和/或前脑中，缺乏PACAP1或PACAP2会导致fgf8增加，这再次表明PACAP在发育过程中对这些有助于定义前脑细胞的重要基因具有抑制作用。PACAP及其受体的转录本在0.5 - 6 hpf时的早期表达，使PACAP1和PACAP2都成为影响脑发育的候选因子。
