A morpholino phenocopy of the cyclops mutation.

Abstract

Mutations of the cyclops locus produce a characteristic pattern of abnormalities, including prechordal plate and notochord defects, loss of ventral forebrain tissue with cyclopia, deletion of the medial floor plate cells of the spinal cord, and malformation of the tail (“curly tail down morphology”) (Hatta et al., 1991; Thisse et al., 1994; Yan et al., 1995; Odenthal et al., 2000). The various cyclops alleles can be classified according to the severity of the central nervous system (CNS) and eye defects (Brand et al., 1996). However, cyclops mutations have little or no effect on cardiac left–right asymmetry, which stands in sharp contrast to the strong laterality phenotype of many other midline or curly tail down mutations (Chen et al., 1997; Bisgrove et al., 2000; Chin et al., 2000). The cyclops locus itself encodes a member of the nodal-related subclass of the TGF-b superfamily (Rebagliati et al., 1998; Sampath et al., 1998). Molecular and/or genetic data suggest that the weak and strong cyclops mutations are hypomorphic or null alleles, respectively (Hatta et al., 1991; Halpern et al., 1997; Rebagliati et al., 1998; Sampath et al., 1998; Talbot et al., 1998; Gritsman et al., 1999; Miller and Rebagliati, unpublished data). Consistent with this, we report here that injection of an inhibitory cyclops morpholino (CycMO: 59-GCGACTCCGAGCGTGTGCATGATG-39) reproduces the range of phenotypes seen in existing weak and strong cyclops alleles in a dose-dependent fashion (Table 1). Cyc-MO-injected embryos could be placed into two phenotypic categories: a weak class [cyclopia and loss of ventral forebrain similar to that caused by the weak cylops allele, cyc (Brand et al., 1996); Fig. 1B] and a strong class [more severe cyclopia and more extensive ventral forebrain loss, resembling that of the strong cyclops alleles, cyc (Hatta et al., 1991), cyc (Brand et al., 1996) and cyc (Schier et al., 1996), Fig. 1C–E]. These classes correlated well with the dose of injected morpholino (Table 1). A high dose (8 ng) of a negative control morpholino (GeneTool negative control MO: 59-CCTCTTACCTCAGTTACAATTTATA-39) had no effect (n 5 69, data not shown). Variable CNS necrosis was seen, and was most frequent at the two highest Cyc-MO doses (4 and 8 ng). All cyclopic embryos from the weak and strong classes also showed the curly tail down morphology (Fig. 1G). To determine the occurrence of floor plate defects, Cyc-MO-injected embryos were analyzed by in situ hybridization for netrin expression, which marks the medial floor plate and parts of the brain (Strahle et al., 1997). The cyclops morpholino produced the expected floor plate deficit, as evidenced by the partial or complete loss of netrin staining within the embryonic spinal cord [compare Fig. 1H (uninjected) versus Fig. 1I (Cyc-MO-injected)]. Mutations in squint, another zebrafish nodal-related gene, also cause cyclopia and midline defects. This raises the question as to whether the Cyc-MO could also be