Proceedings 9th International Coral Reef Symposium, Bali, Indonesia 23-27 October 2000 Molecular control of development in the reef coral, Acropora millepora E. E. Ball1, D. C. Hayward1, J. Catmull1,2, J. S. Reece-Hoyes1,2, N. R. Hislop1,2, P. L. Harrison3 and D. J. Miller2 ABSTRACT A brief overview of the embryonic and larval development of Acropora, including some previously unpublished data, provides the background for this review of our rapidly expanding knowledge of the genes that control early development in corals, with particular emphasis on Hox and Hox-like genes. Since the Phylum Cnidaria is widely accepted to be an ancient group of organisms, genes, and motifs within genes, that are shared by corals and higher metazoans are presumably ancient. Thus, shared genes allow us to study how gene structure and function have changed with time, while genes specific to higher metazoans have, presumably, evolved more recently. Anatomically, corals have many fewer cell types than higher metazoans, but it is not clear that this apparent simplicity will be reflected at the molecular level. We have already found Acropora representatives of structural genes, housekeeping genes, nuclear receptors, Hox-like genes, Pax genes and components of the dpp signalling pathway. However, thus far there is no unequivocal evidence for the cluster of Hox genes, known as the zootype genes, that is otherwise widespread among the Metazoa. As more data become available, the Cnidaria are making an increasing contribution to our knowledge of the evolution of gene structure, function, and regulation. We here illustrate the evolutionary approach that we are taking to the characterisation of coral genes with a review of our work on the Acropora Hox-like gene, cnox2-Am. Keywords Coral, Acropora, Cnox2, Hox nisms which had reached the indicated level of development. Introduction way of approaching this problem is to attempt to establish Phylogenies based on morphological data (e.g. Fig. 1) the presence, structure and probable function in are in agreement with those based on molecular data that cnidarians of genes that are important in controlling the the Cnidaria are primitive and can therefore be regarded early development of higher animals. Although the as an outgroup for the higher Metazoa (Adoutte et al. freshwater cnidarian, Hydra, has been the subject of 1999). For this reason the Cnidaria are particularly useful many molecular studies, developmental stages are not for understanding the evolution of gene structure and easily obtainable. In contrast, broadcast spawning function and the extent to which molecular mechanisms scleractinian corals present an opportunity to obtain large governing development are common to all animals. numbers of nearly syn-chronous developmental stages One (e.g. Harrison et al. 1984, Babcock et al. 1986, Harrison and Wallace 1990). We here first present a simplified description of the em-bryonic development of the branching reef coral, Acro-pora millepora, including some new anatomical ob-servations. Then we review knowledge of Hox-like genes in Acropora and other cnidarians, illustrating our ap-proach to analysis of their structure and function from our published work on the gene cnox2–Am. Finally, we discuss possible future techniques for studying gene function in Acropora and summarise conclusions from the work thus far. Methods Technical details of molecular techniques discussed in this paper are given in Hayward et al. (2001). For ana- tomical studies, embryos were fixed in 2.5% glutaral- dehyde in 0.1M sodium cacodylate buffer (pH 8.0) made Fig. 1 A traditional version of the family tree of o metazoan life (Adoutte et al. 1999), showing that the up in filtered (0.45 μm) sea water for 2-8 hours at 4 C Phylum Cnidaria, which contains the corals, separated (Harrison 1988). Embryos were then rinsed for an extended period in buffer, followed by post-fixation for 1 from the ancestors of the higher Metazoa quite early in o evolution. To the left of the tree are listed some of the hour at 4 C in 1% OsO4 in sodium cacodylate buffered morphological features probably associated with orga- seawater. This was followed by dehydration and embed- 1 Research School of Biological Sciences, Australian National University, P.O. Box 475, Canberra, ACT 2601, Australia e-mail: [email protected] Tel: +61-2-61254496; Fax: +61-2-61258294 2 Biochemistry and Molecular Biology, James Cook University, Townsville, Queensland 4811, Australia 3 School of Resource Science and Management, Southern Cross University, PO Box 157, Lismore, NSW 2480, Australia ding in Epon 812 or LR White resins using standard High magnification videography of Acropora techniques. planulae has shown that ciliary movement along the Serial sections (2-5 μm) of material embedded in planula creates strong, local currents capable of Epon 812 were cut, floated onto microscope slides, and transporting small particles towards the oral pore dried down. As we wished to correlate stages of (Harrison unpublished). This morphology and ciliary differentiation and the appearance of new cell types with movement raises the possibility that planulae may be gene ex-pression, we experimented to find a histological supplementing the energy obtained from yolk absorption stain that would maximise our recognition of newly with nutrition derived from small particulate matter or the appearing cell types. Best results, in comparison to capture of small prey, which are carried to the oral pore toluidene blue, Harris hematoxylin, basic fuchsin or by the beating cilia as the planula swims along, aboral Alsop's (1974) stain, were obtained with the polychrome end first. The particulate matter or prey may then be stain of Van Reempts and Borgers (1975). Celestine blue digested extracellularly within the oral cavity. Thus, our staining was for two hours at 60oC, followed by anatomical studies on Acropora are consistent with counterstaining with Cason’s stain for 15 min at 60oC. previous behavioural observations of particle feeding in planulae of Caryophyllia (Tranter et al. 1982), Results and Discussion Cyphastrea (Wright 1986), and Fungia (Schwarz et al. 1999). More detailed research on feeding behaviour in Morphological development coral planulae is needed. A schematic diagram of the embryonic and larval development of Acropora millepora is presented in Figure 2, while scanning electron micrographs of selected stages are presented in Miller and Ball (2000). Recent papers on the embryology of other species of Acropora containing light micrographs or scanning electron micrographs of developmental stages include Hayashibara et al. (1997), Gilmour (1999), containing micrographs from Heyward (1987) and Wallace (1999). As described for other species (reviewed in Harrison and Wallace 1990, Hayashibara et al. 1997), observations of living A. millepora planula larvae reveal them to be highly active swimmers. The planulae are initially spheroidal and spin slowly about their axis near the water surface as cilia develop over the epidermis and begin to beat synchronously (Harrison unpublished). With subsequent development, the larvae become pear-shaped and then elongated, and are in-creasingly active, swimming throughout the water column aboral end foremost while rotating about their longitudinal axis. Elongated, motile planulae with a well developed, semi- translucent epidermis exhibit a range of swimming behaviours including rapid, directed straight swimming, spiralling and spinning. As planulae become competent to settle, they begin to swim lower in the water column and initiate demersal swimming and benthic searching and substratum testing behaviour. Preliminary analysis of our sectioned material shows that there are marked differences between the ectoderm covering most of the planula and that lining the oral cavity (Fig. 3). The former (Fig. 3A) has a diversity of Fig. 2 Schematic summary of the embryonic cell types including nematocysts (n), cnidocytes, and development of Acropora millepora. Times shown are several types of cells of a secretory appearance (gc#1, from the time of fertilization and are typical for this gc#2), similar to those described in planulae of other species in the vicinity of Townsville, Queensland, coral species (reviewed in Harrison and Wallace 1990, Australia. Colloquial terms for some of the stages are Hayashibara et al. 1997). The oral ectoderm, in contrast, also shown. 72 hr and 96 hr planulae are all in the same appears to consist of a single type of secretory cell (Fig. orientation: or=oral end, ab=aboral end. Modified from 3B). Cilia line the oral cavity, and appear to be densely Miller and Ball (2000). packed (Fig 3B), although the apparently greater density may only be due to the limited space within which they are confined. provide positional inform-ation along the primary axis of metazoan body plans, could serve to define an animal (the "zootype concept"). The genes of the zootype take the names of the corres-ponding genes of the fruitfly, Drosophila melanogaster, the organism in which they were first described and in which their function was deciphered. A subset of the true Hox genes forms the core of the zootype, while other genes defining position nearer the anterior and posterior ends of the embryo (i.e. empty spiracles (ems), orthodenticle (otd) and even- skipped (eve) are also in-cluded. Several Hox-like sequences have been isolated from cnidarians, (Gauchat et al. 2000, for review see Ferrier and Holland 2001) as well as orthologs of ems, otd and eve (Mokady et al. 1998, Smith et al. 1999, Miller and Miles 1993, Miller et al. unpublished). The Hox-like sequences can be placed into five groups on the basis of sequence similarity (Ferrier and Holland 2001, and references therein), but their relationships to genes from other animals are not always clear. Even within the Cni- daria the assignment of genes as orthologous can be difficult, probably reflecting the diversity of the phylum. The cnox-2 class, however, does form an unambiguous orthologous group, and representatives have been isolated from three cnidarian classes, the Anthozoa, Hydrozoa, and Scyphozoa.