Mechanisms of Development 115 (2002) 113–116 www.elsevier.com/locate/modo
Gene expression pattern
cDNA cloning, sequence comparison, and developmental expression of Xenopus rac1 Jennifer M. Lucas, Ivana Nikolic, Mark D. Hens* Department of Biology, University of North Carolina at Greensboro, P.O. Box 26170, Greensboro, NC 27402, USA Received 16 January 2002; received in revised form 11 March 2002; accepted 11 March 2002
Abstract The Rho family of small GTP-binding proteins are important signaling molecules that regulate the dynamics of the actin cytoskeleton and mediate changes in cell morphology and motility. Here, we describe the temporal and spatial patterns of expression of the Rho family member, rac, during the development of the amphibian, Xenopus laevis. We also present the deduced amino acid sequence of Xenopus rac (Xrac). At the amino acid level, Xrac is highly conserved relative to previously characterized rac homologs, and is nearly identical to human rac1. RNase protection assays and Western blot analysis indicate that Xrac mRNA and protein are present from fertilization through tailbud stages of development. Whole-mount in situ hybridizations show that Xrac transcripts are especially abundant in cells of the involuting marginal zone, and later, in the cranial neural crest, the developing central nervous system, and in the somites. The remarkable degree of evolutionary conservation observed in the Xrac primary structure together with its high level of expression in cells and structures critical to morphogenesis suggest a functionally important role for this Rho family member in early vertebrate development. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Xenopus laevis; rac; Rho GTPase; Embryo; Expression; Mesoderm; Cranial neural crest; Cranial nerve; Branchial arch; Somite; In situ hybridization; Western blot
1. Results During early amphibian development, the morphogenetic movements of cells in the embryo and subsequent tissue formation come about as the result of several cellular behaviors, including changes in cell shape, radial and mediolateral intercalation, as well as cell migration (Keller and Winklbauer, 1992). Often, these movements are triggered by extracellular cues, including growth factors and extracellular matrix, and are dependent on the actin cytoskeleton (Tamai et al., 1999). Rho family GTPases regulate actin cytoskeleton dynamics, and consequently, changes in cell morphology, motility, and adhesive behaviors in cultured mammalian cells (reviewed by Van Aelst and D’SouzaSchorey, 1997). A number of recent studies have shown that these regulatory proteins are pivotal components of signaling pathways that control the movements of embryonic cells during the development of many organisms, including vertebrates (Sugihara et al., 1998; WunnenbergStapleton et al., 1999; reviewed by Settleman, 1999). We have isolated the Xenopus homolog of the small GTPase, * Corresponding author. Tel.: 11-336-334-4945; fax: 11-336-334-5839. E-mail address:
[email protected] (M.D. Hens).
Fig. 1. Complete amino acid sequence of Xrac1 deduced from nucleotide sequence analysis of the pXrac7B cDNA. This sequence was aligned with rac homologs from other species for comparison using the ‘Pileup’ program included in the GCG software package (Devereux et al., 1984). Dashes (‘-’) indicate identical residues. Dots (‘.’) indicate missing residues. The positions of the forward and reverse primers used in the amplification of XPrac are indicated by the overlined residues. Deduced amino acid sequences are from Xenopus laevis (Accession number: AF174644), human (Didsbury et al., 1989; Accession number: M29870), Drosophila melanogaster (Hariharan et al., 1995; Accession number: L38309), Caenorhabditis elegans (Chen et al., 1993; Accession number: L03711), and Dictyostelium discoideum (Bush et al., 1993; Accession number: L11589).
0925-4773/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(02)00117-X
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Table 1 Pairwise comparisons of rac homologs (% identity/% similarity) compiled using the ‘Bestfit’ program included in the GCG software package a
Xenopus Dictyostelium Caenorhabditis elegans Drosophila a
Human
Drosophila
Caenorhabditis elegans
Dictyostelium
99/100 81/89 83/90 90/92
89/92 79/87 80/86
82/90 78/85
80/89
Deduced amino acid sequences were translated from nucleotide data in GenBank using the accession numbers indicated in Fig. 1.
rac1, and have characterized the expression of this Rho family member during early Xenopus development.
2. Xrac1 sequence Approximately 10 5 plaques from a gastrula-stage Xenopus cDNA library were screened for Xenopus rac (Xrac)-containing cDNAs. Complete nucleotide sequence analysis of a novel cDNA designated pXrac7B revealed the presence of a 579 base pair (bp) open reading frame encoding Xrac (GenBank Accession number: AF174644; Fig. 1). The deduced amino acid sequence indicates a polypeptide of 192 residues that shares remarkably high sequence identity with previously described homologs (Fig. 1). Xrac shares between 80 and 99% sequence identity with rac homologs characterized from Dictyostelium, Caenorhabditis elegans, Drosophila, and human (Table 1). The designation of our Xenopus cDNA as Xrac1 is based on the greater structural similarity to the human rac1 polypeptide (99% identical) compared to the slightly lower degrees of identity shared with the human rac2 (91%; Didsbury et al., 1989; Accession number: M29871) and human rac3 (93%; Haataja et al., 1997; Accession number: AF008591) amino acid sequences.
Fig. 2. Time course RNase protection analyses of total embryonic RNA indicate that Xrac is an abundant maternal message present at fertilization (stage 1), and during blastula (stage 8) and gastrula stages (stages 10 and 12). Xrac mRNA is observed at similar levels at neurulation (stage 17), hatching (stage 25), and tailbud (stage 33/34) stages of development. A probe for the elongation factor, Ef-1a, served as a control for mRNA loading. Torula RNA (c) was included as a control for nonspecific hybridization. Protected fragments are smaller than the undigested probe (upper arrowhead) due to short stretches of vector sequence mismatch at the ends of the probe transcript.
3. Xrac expression RNase protection analyses of total RNA prepared from fertilized eggs, blastula-, gastrula-, neurula-, and tailbudstage embryos were performed to determine Xrac mRNA levels during early development (Fig. 2). Xrac mRNA is an abundant maternal message present at high levels at the time of fertilization as well as at blastula stage 8. Consistently high transcript levels persist during gastrulation and continue through tailbud stages of development. Western blot analysis of embryos collected at the same stages of development indicates that rac protein is present uniformly from fertilization through stage 25, and that rac protein levels increase somewhat during tailbud stages of development (Fig. 3).
Fig. 3. Analysis of rac protein expression in early Xenopus development. Detergent lysates of ten embryos from several stages of development were prepared. Approximately one embryo equivalent was loaded per lane, electrophoresed on a 15% acrylamide gel, and transferred to nitrocellulose. Blots were probed with anti-rac antibodies (BD Transduction Laboratories, catalog number R56220) to detect total rac protein (A), or incubated with anti-a actin antibodies (Sigma catalog number A2066) as a control for protein loading (B). Rac protein is present in the embryo at fertilization and throughout blastula, gastrula, and neurula stages. Protein levels increase slightly by tailbud stage 33/34. (B) Actin (lower band) is present in equal amounts at all stages examined.
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Fig. 4. Localization of rac mRNA by whole-mount in situ hybridization. Xrac mRNA is detected in cells of the marginal zone during early and late gastrulation (A,B, stage 10.5, vegetal view; C, stage 12, lateral view). ‘d’ indicates the position of the dorsal blastopore lip, and ‘y’ indicates the yolk plug of the blastopore. The embryo in (A) is uncleared. Xbra expression in an early gastrula-stage embryo indicates the location of presumptive mesodermal cells for comparison (D). Sagittal paraffin sections of hybridized gastrula-stage embryos (F,G). At stage 10.5, rac mRNA is abundant in the presumptive ectoderm and is also detected at elevated levels in cells of the dorsal involuting marginal zone (F). ‘d’ marks the dorsal blastopore groove. In (G), a sagittal section of the stage 12 embryo shown in whole-mount in (C) reveals rac transcripts specifically in cells of the dorsal (dim) and ventral (vim) involuting mesoderm, the prechordal plate (p), and the sensorial layer of the neuroectoderm (n). The archenteron (a) and blastocoel (b) are indicated. In late neurula-stage embryos, Xrac is expressed in the anterior neural plate (anp) and the notochord (nc) (H,I). At stage 25, Xrac is expressed in the cement gland (cg), the eye (e), the brain (the rhombencephalon (rh) is indicated), the notochord (nc), and somites (so), and is also expressed in the cranial neural crest (cnc), including cells of the hyoid (hy), and anterior (ab) and posterior (pb) branchial streams (J,K). At tailbud stage 33/34, Xrac expression persists in the eye and central nervous system, and is evident in the branchial arches (ba), the otic vesicle (ov), and in the trigeminal (t), facial (f), glossopharyngeal (g), and vagus (v) cranial nerves (M,N). Gastrula-, hatching-, and tailbudstage embryos were incubated with Xrac sense probe to control for nonspecific hybridization (E,L,O). Scale bar equals 0.5 mm in (A–E,H–J,L,M,O), and 0.2 mm in (F,G,K,N).
Whole-mount in situ hybridizations were carried out to determine the spatial patterns of Xrac expression (Fig. 4). During gastrulation, Xrac mRNAs are detected in virtually all cells of the animal hemisphere and marginal zone. At the beginning of gastrulation, Xrac transcripts are abundant in the animal pole ectoderm and in the marginal zone, including dorsal involuting cells of the organizer region (Fig. 4A,B,F). Later in gastrulation, Xrac mRNA is abundant in
the cells of the dorsal and ventral involuting mesoderm (Fig. 4C,G). This pattern of expression resembles that of the mesoderm-specific transcription factor, brachyury (Xbra; Smith et al., 1991), which is expressed in presumptive mesodermal cells around the blastopore (Fig. 4D). At stage 12, Xrac transcripts are also present in the prechordal plate mesoderm, and the sensorial layer of the neuroepithelium (Fig. 4G). In neurula-stage embryos, Xrac mRNAs are
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observed in the anterior neural plate and in the notochord (Fig. 4H,I). At stage 25, Xrac transcripts are evident in the developing brain and neural tube, in the notochord and somites, and in the cement gland (Fig. 4J,K). Xrac is also clearly expressed in the hyoid stream and in the anterior and posterior branchial streams of cranial neural crest cells (Fig. 4J,K). At tailbud stage 33/34, Xrac transcripts are present in the brain, neural tube, somites, and the branchial arches (Fig. 4M). Xrac mRNA is also visible in the eye, otic vesicle, and in several cranial nerves (Fig. 4N). Close inspection of hybridized embryos shows some level of rac expression in all cells of embryos examined from fertilization through tailbud stages. This is not surprising considering the importance of this small GTPase in so many different cellular processes. In this report, we provide evidence for tissue-specific, elevated rac expression in several regions of the embryo where epiboly, convergence and extension, cell migration, and changes in cell shape are taking place. These data suggest an important role for rac in a number of morphogenetic events in early vertebrate development. 4. Methods Xrac-specific probes were synthesized using a 390 bp Xrac cDNA obtained by reverse transcriptase polymerase chain reaction (RT-PCR). pXPrac (Xenopus PCR-derived rac) was amplified using degenerate primers and cDNA prepared from stage 35 embryos, cloned into pBluescript S/K 2 (Stratagene, Inc.), and used as a template for probe synthesis in library screens and in situ hybridizations. DNA sequence analysis of the cloned PCR product confirmed its identity as a rac cDNA and indicated the orientation of the insert in the plasmid. Degenerate primers used in the amplification of the XPrac cDNA are as follows: forward, 5 0 -GAGGATTCATGCA(G/A)GCIAT(A/T/C)AA(G/A)TG(T/C)GT-3 0 ; and reverse, 5 0 -GAGGATTCTT(C/T)TC(T/A/G)ATIGT(G/ A)T(T/C)(C/T)TT(A/G)TC(A/G)TC-3 0 . A gastrula stage 11 lZAP cDNA library was kindly provided by D.W. DeSimone (University of Virginia). The RT-PCR, library screen, in situ hybridizations, and Western blots were performed as described (Hens and DeSimone, 1995). Embryos were staged according to Nieuwkoop and Faber (1967). Acknowledgements We gratefully acknowledge Ms Rongqin Ren for techni-
cal assistance with the in situ hybridizations. This work was supported in part by a Regular Faculty Grant to M.D.H. from the Office of the Provost at the University of North Carolina at Greensboro.
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