Development 123, 117-128 117 Printed in Great Britain © The Company of Biologists Limited 1996 DEV3340 Mutations affecting development of the notochord in zebrafish Derek L. Stemple*, Lilianna Solnica-Krezel*, Fried Zwartkruis1, Stephan C. F. Neuhauss, Alexander F. Schier2, Jarema Malicki, Didier Y. R. Stainier3, Salim Abdelilah, Zehava Rangini4, Elizabeth Mountcastle-Shah and Wolfgang Driever† Cardiovascular Research Center, Massachusetts General Hospital and Harvard Medical School, 149 13th Street, 4th Floor, Charlestown, MA 02129, USA *Contributed equally to the work 1Present address: Laboratory for Physiological Chemistry, Utrecht University, 3584 CG Utrecht, The Netherlands 2Present address: Skirball Institute of Biomolecular Medicine, New York University Medical Center, 550 First Avenue, New York, NY 10016, USA 3Present address: Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, CA 94143-0554, USA 4Present address: Department of Oncology, Sharett Institute, Hadassah Hospital, Jerusalem 91120, Israel †Author for correspondence (e-mail: [email protected]) SUMMARY The notochord is critical for the normal development of phenotypic pathway, which in turn suggests a genetic vertebrate embryos. It serves both as the major skeletal pathway for the development of the notochord. Perturba- element of the embryo and as a signaling source for the tions within adjacent tissues in mutant embryos further establishment of pattern within the neurectoderm, the indicate the importance of notochord-derived signals for paraxial mesoderm and other tissues. In a large-scale sys- patterning within the embryo and suggest that these tematic screen of mutations affecting embryogenesis in mutations will yield additional insight into the cues that zebrafish we identified 65 mutations that fall into 29 com- regulate these patterning processes. plementation groups, each leading to a defect in the formation and/or maintenance of the notochord. These mutations produce phenotypic abnormalities at numerous Key words: zebrafish, notochord, floor plate, mesoderm, stages of notochord development, thereby establishing a embryogenesis INTRODUCTION also involved in dermomyotome patterning. In avian experi- ments, notochord extirpation leads to a complete loss of The notochord is a structure common to all members of the epaxial muscles, i.e. deep back muscles, whereas hypaxial phylum Chordata. It serves as the major skeletal element for muscles, i.e. limb and ventrolateral body wall muscles, are lower chordates and as such provides an important mechanical unaffected (Christ et al., 1992; Rong et al., 1992). Muscle element for the locomotion of the individual. In lower ver- specification occurs in the absence of notochord, but a tebrates at larval stages of development the notochord plays a notochord derived signal is required to maintain the epaxial similar role serving as the major skeletal element in the body muscle fate (Bober et al., 1994). Mosaic analysis of the apparently necessary for coordinated movement. Perhaps more zebrafish mutation no tail (ntl), a mutation in a gene homolo- importantly, in all vertebrates, the notochord is required for the gous to the mouse Brachyury gene, indicates that differentiated proper patterning of adjacent tissues, including the neurecto- notochord is also required for the proper patterning of muscle derm, paraxial mesoderm and the heart. In the neurectoderm, pioneers in the developing myotome (Halpern et al., 1993; the notochord functions to signal the formation of the floor Herrmann et al., 1990; Schulte-Merker et al., 1992). Interest- plate, and, independently, can signal the formation of motoneu- ingly, mutants of the ntl locus form a floor plate (Halpern et rons (Placzek et al., 1993; van Straaten et al., 1988; Yamada al., 1993), suggesting that not all of the notochord signaling et al., 1993). In addition to specifying ventral fates the activities are disrupted by the mutation. In addition to its role notochord suppresses the expression of dorsal fates in the in patterning the CNS and somites, the notochord may also neurectoderm (Bovolenta and Dodd, 1991; Goulding et al., serve to specify other tissues such as sympathoadrenal prog- 1993). The notochord appears to play several roles in the pat- enitors (Stern et al., 1991), the left-right asymmetry of the heart terning of somitic tissue. First, based on experimental manip- tube (Danos and Yost, 1995), and the differentiation of certain ulation of avians as well as analysis of several mouse gut derivatives (Wiertz-Hoessels et al., 1987). There is mutations, the notochord is responsible for the specification evidence that specification of the floor plate and patterning of and maintenance of ventral, i.e. sclerotome, fates in the somite the somite are both mediated at least in part by Sonic hedgehog (Dietrich et al., 1993; Pourquie et al., 1993). The notochord is (shh) (Fan et al., 1995; Fan and Tessier-Lavigne, 1994; 118 D. L. Stemple and others Johnson et al., 1994a; Roelink et al., 1994). Other peptide (Theiler, 1959). In addition, several targeted gene disruptions, growth factors, such as bone morphogenic proteins BMP-2 and such as in the HNF-3β gene (Ang and Rossant, 1994; BMP-7 have also been found to be expressed in the notochord Weinstein et al., 1994), or in the fibronectin gene (George et (Lyons et al., 1995). Taken together these data point to the al., 1993) produce notochord phenotypes in homozygous importance of the notochord, not only for the structural mutant mice. integrity and locomotion of the embryo, but also for the estab- In order to identify genes necessary for normal embryogen- lishment of axial pattern within the mesoderm, neurectoderm esis of vertebrates we have performed a systematic large-scale and other tissues. Additional mutations affecting notochord genetic screen. The details of the screen are described development would therefore provide insight into the mecha- elsewhere (Driever et al., 1996). In this report the isolation and nisms governing notochord differentiation, the tissue interac- the genetic and phenotypic analysis of mutations affecting the tions underlying the establishment of axial patterning, and ulti- formation and maintenance of the notochord are described. mately, to the molecular determinants mediating these events. These mutations provide a genetic framework for dissecting The entire axial mesoderm, that is the prechordal plate and the process of notochord specification and differentiation as the notochord are derived from the dorsal organizer. The well as further defining the role of the notochord in the devel- organizer was originally identified by its ability to induce the opment of other embryonic tissues. ectopic formation of dorsal-anterior structures when trans- planted into embryonic regions that would otherwise give rise only to more ventral and lateral structures (Spemann, 1938). MATERIALS AND METHODS The organizer, originally described for amphibia, is function- ally equivalent to the embryonic shield of teleost fish (Ho, Animals 1992; Oppenheimer, 1936). In normal development the Fish were maintained in the zebrafish facility at the Cardiovascular organizer provides an activity primarily responsible for the Research Center, of Massachusetts General Hospital (Driever et al., induction of the axis of the embryo. Importantly, transplanted 1996; Solnica-Krezel et al., 1994). Heterozygous stocks were organizer will cause host tissue to acquire novel fate while con- generated by mating the originally identified heterozygous F2 fish tributing predominantly to the prechordal plate, notochord and with wild-type AB strain (Johnson et al., 1994b), Hong-Kong strain, partially to somites and neurectoderm. India strain (Knapik, 1996), or Tü strain (Mullins and Nüsslein- Volhard, 1993) partners; growing the outcrossed lines, then reidenti- Morphologically distinguishable stages are apparent during fying heterozygous fish by sibling matings. Mutant and wild-type notochord development in zebrafish (Kimmel et al., 1995). siblings for analysis were obtained by mating a male and female het- Soon after the onset of the gastrulation movements, the direct erozygous for the given mutation. Embryos were raised at a constant antecedent of the notochord, the chordamesoderm, can be dis- 28°C and approximate stages are given in hours postfertilization at tinguished from the paraxial mesoderm by virtue of its 28°C (hpf). Once a fish was identified as a heterozygous carrier it was continued expression of the Brachyury gene (Herrmann et al., crossed again for confirmation before being added to a pool of het- 1990; Schulte-Merker et al., 1992) and by the expression of a erozygous fish. Four or more fish of a given genotype were maintained number of other molecular markers including Shh, and later in 5 liter tanks as a pool of no more than 30 adult fish. Single fish α1-collagen Type II (col2a1) (Yan et al., 1995). Prior to seg- were maintained in 1 liter boxes. All fish were raised and maintained mentation the chordamesoderm becomes morphologically as described by Solnica-Krezel et al. (1994). distinct from the paraxial mesoderm. During the segmentation Complementation testing period of development, central cells of the notochord differen- Allelism was tested first between mutations producing similar phe- tiate and acquire a large vacuole. The entire notochord notypes. This defined a smaller number of complementation groups becomes surrounded by a sheath of tissue, which in combina- between which complementation