Expression of Distal-Less in Molluscan Eggs, Embryos, and Larvae

EVOLUTION & DEVELOPMENT 1:3, 172–179 (1999) Expression of Distal-less in molluscan eggs, embryos, and larvae Shannon E. Lee* and David K. Jacobs Department of Organismic Biology, Ecology, and Evolution, University of California at Los Angeles, 621 Charles E. Young Drive, Los Angeles, CA 90095-1606 USA *Author for correspondence (email: [email protected]) SUMMARY Distal-less (Dll ) is best known as a transcrip- in all cells of the pregastrulae, and predominant expression in tion factor involved with “limb patterning” in Drosophila mela- the ectoderm of postgastrulae and early veliger larvae. Com- nogaster. Observations of both deuterostome and protostome parison of our observations on spiralian taxa, thought to have phyla have led to the suggestion that some aspect of this conservative development with previous work, primarily on gene’s function in “appendage” or proximal–distal “out- deuterostomes, suggests the possibility of an ancient role(s) growth” development is conserved. Here we explore the pos- for DLL in early development. Possible functions appear to sibility of other conserved roles operating earlier in include maternal and zygotic involvement in the establish- development. We examine the expression of DLL protein ment of embryonic polarity, involvement in the process of during the early development of two molluscan classes, Poly- germ layer formation, and a role in the specification and/or placophora (chiton) and Gastropoda (snail). Using an anti- differentiation of ectoderm/epithelia. We note that the explo- body approach, we find DLL expression in the oocytes of a ration of conserved gene function in early development may chiton (Mopalia muscosa) and in the pregastrulae through be clarified by examining taxa whose early development has early veliger larvae of a marine snail (Kelletia kelletii ). We ob- putatively not been subject to dramatic evolutionary change. serve antibody localization in the oocyte, nuclear expression INTRODUCTION inson and Mahon 1994; Simeone et al. 1994; Weiss et al. 1994; Morasso et al. 1995; Nakamura et al. 1996; Liu et al. The Distal-less (Dll) gene from Drosophila melanogaster is 1997; Qiu et al. 1997). Most work has focused on this asso- best known for its involvement in limb patterning. The ex- ciation with limb development and differentiation of tissues pression of this homeodomain-containing gene is regulated late in development. Less attention has been given to DLL by the interactions and activity of other developmental expression in early development. In this study, we document genes, such as decapentaplegic, wingless, and hedgehog, early developmental expression of DLL in the phylum Mol- and is an early marker of the “distal” fated central region of lusca. Drosophila imaginal discs (Cohen and Jürgens 1989; Cohen Previous observations of DLL expression in early devel- 1990, 1991; Campbell et al. 1993; Diaz-Benjumea et al. opment can be divided into several categories. First, an ex- 1994; Held et al. 1994; Lecuit and Cohen 1997). “Ho- tra-embryonic or maternal DLL role is suggested by verte- mologs” of this gene (hereafter refered to as DLL genes) brate expression studies. In Xenopus laevis, DLL (Xdll) have been found in a handful of invertebrate taxa (Pangani- transcripts were isolated from an ovary cDNA library and ban et al. 1995), and multiple copies are present in the verte- this “maternal” contribution continues in the oocytes, unfer- brate lineages (see Stock et al. 1996 for review). tilized eggs, and cleavage-stage embryos (Asano et al. Through the use of a polyclonal antibody (Panganiban et 1992). Similarly, DLL (Dlx4) is expressed in the human pla- al. 1995), expression patterns have been documented for a centa (Quinn et al. 1997). number of invertebrate taxa and suggest that this gene has Second, there are reports of DLL activity very early in de- some conserved evolutionary role in the proximal–distal pat- velopment, specifically associated with the animal pole and/ terning of “outgrowths” (Panganiban et al. 1997). In verte- or anterior portions of the embryo. In Drosophila, DLL (Dll) brates, the expression of DLL gene family members marks is first expressed as a stripe during cellularization of the blas- limb buds as well as whiskers, teeth, and branchial arches toderm. This region corresponds to the future anterior seg- (Price et al. 1991; Robinson et al. 1991; Dollé et al. 1992; ments and is localized to the dorsal third of the embryo (Co- Özçelik et al. 1992; Bulfone et al. 1993; Dirksen et al. 1993, hen 1990). In the basal chordate, Amphioxus, Holland et al. 1994; Papalopulu and Kitner 1993; Porteus et al. 1994; Rob- (1996) report the expression of DLL (AmphiDll) in the ani- © BLACKWELL SCIENCE, INC. 172 Lee and Jacobs Expression of Distal-less 173 mal hemisphere of the gastrula. Asano et al. (1992) find that Adults were collected by hand from the intertidal at Palos Verdes zygotic transcription of Xdll increases during Xenopus em- Peninsula, Los Angeles County, California. We isolated gametes bryogenesis and suggest that this gene may play a role in es- and gonadal tissue upon dissection of these specimens. Although tablishing the anterior structures of the embryo. eggs and sperm were allowed to mix, it is unlikely that fertilization Third, DLL genes are involved with ectoderm or epithe- occurred since chiton eggs must pass through the gonopore to be properly activated for fertilization (Strathmann 1987). Oocytes lial patterning. The cephalochordate gastrula (Holland et al. (nonmature unfertilized eggs) were separated and used for this 1996) and the zebrafish gastrula (Akimenko et al. 1994) ex- study. press DLL (AmphiDll and Dlx3, respectively) in the regions Kelletia kelletii, the Kellet’s whelk, is a subtidal predatory ma- of presumptive ectoderm. Likewise, Morasso et al. (1993) rine snail (class Gastropoda) that reproduces through copulation report DLL (Xdll-2) expression in the epidermis of the devel- and then deposition of egg capsules (Fig. 1B). Each capsule con- oping Xenopus embryo. DLL (Dlx3) is expressed in the sur- tains several hundred fertilized eggs that slowly develop through to face epithelium of various murine structures (Morasso et al. a veliger larval stage. Specimens of K. kelletii were collected at Cat- 1995), and another mouse study suggests that DLL (specifi- alina Island, Los Angeles County, California, by hand, using self- cally, Dlx2) may be a regulator of epidermal differentiation contained underwater breathing apparatus (SCUBA). Individuals (Porteus et al. 1994). In Xenopus, DLL (xDll-2) is expressed held in aquaria were observed copulating and laying eggs. Egg cap- in the embryonic ectoderm, persists in the epidermis, and sules were separated from the adult snails and fixed daily. Embryos and eggs were fixed in 3.7% formaldehyde in phos- then appears to have a continuous requirement in adult skin phate-buffered saline (PBS), dehydrated sequentially into ethanol, tissue (Dirksen et al. 1994). and then stored in methanol at 2208C. Expression of DLL was vi- Most of the information regarding early DLL function sualized using a polyclonal antibody (graciously supplied by G. comes from studies of deuterostomes. The early expression Panganiban) that has been shown to work for a wide variety of in- of DLL in the fly blastoderm (Cohen 1990) is difficult to invertebrate taxa (Panganiban et al. 1997). terpret evolutionarily in light of this organism’s derived Our antibody protocol consisted of treatment with 3% hydrogen synctial early development. In the protostome phylum Ar- peroxide (15 min), rehydration, primary block (2 h) in PBS with thropoda, the outgrowth role of DLL has been emphasized. Triton-X, and bovine serum albumin (PBT) plus 5% dimethyl sul- Understudied protostome (Panganiban et al. 1994, 1997; Po- foxide (DMSO), overnight incubation in DLL antibody (1 mg/ml in padic et al. 1996; Grenier et al. 1997; Niwa et al. 1997; Wil- PBT plus 1% DMSO), washes with PBS plus Triton-X (10 times liams 1998) and deuterostome (Holland et al. 1996; Lowe over 1 h), incubation (1 h) in anti-rabbit secondary antibody conju- gated to horseradish peroxidase (1:400 with PBT plus 5% DMSO), and Wray 1997; Panganiban et al. 1997) taxa have received repeat washes, and then 393-diaminobenzidine HCl (DAB) color re- scant attention on both fronts. To examine early protostome development for comparison with deuterostomous taxa, less derived systems must be considered. Many marine molluscs undergo a form of larval development thought to be little modified from the ancestral condition in spiralians (see Ver- donk et al. 1983; Collier 1997). We focus on this taxa as a representative of classic spiralian early development. In this study, we examine species from two molluscan classes, Polyplacophora and Gastropoda, and with differing life history strategies, broadcast spawning versus copulation. Using an antibody approach, we report the expression of a DLL gene in oocytes, embryos, and larvae. Examination of maternal influences and earliest development is more difficult in the gastropod due to internal fertilization. Thus, the observation on chiton eggs supplements the gastropod result. Our observations document expression patterns comparable to the maternal, animal pole/anterior, and ectodermal expression patterns documented in other taxa and suggest the possibility of conserved roles for DLL in early development. MATERIALS AND METHODS Fig. 1. Photographs of the study organisms. (A) Mopalia muscosa, an intertidal polyplacophoran mollusc. (B) Kelletia

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