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Keywords:

  • protocadherin 10;
  • paraxial protocadherin;
  • paraxial mesoderm;
  • somitogenesis;
  • adaxial cells;
  • Danio rerio;
  • molecular cloning;
  • in situ hybridization;
  • morpholino

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Here, we present the first report of the molecular cloning of zebrafish protocadherin 10 (Pcdh10, OL-protocadherin) and describe its functional analyses in the development of segmental plate. Epitope-tagged Pcdh10 expressed in embryos was localized on cell peripheries of adjacent cells. In situ hybridization showed that pcdh10 was expressed in the paraxial mesoderm (PAM) and developing somites, and in the pineal body, the diencephalon, and the vicinity of otocysts. Expression in PAM increased in the last few presumptive somites, reached the maximum level in the latest segmenting somites, and decreased thereafter during somite maturation. These expression patterns suggested that Pcdh10 is involved in development of PAM and somites. This was confirmed by morpholino knockdown and dominant-negative inhibition of Pcdh10 in embryos, which disturbed movements of PAM cells and somite segmentation. Comparative studies showed that pcdh10 expression lasted up to approximately three times longer in maturing somites than that of paraxial protocadherin (pcdh8). They also indicated that the adaxial cells expressed pcdh8 but not pcdh10. We propose that Pcdh10 is involved in the morphogenic movements of PAM cells and somite segmentation and that differential adhesion of Pcdh8 and Pcdh10 plays a role in the morphogenic machinery of somites and adaxial cells. Developmental Dynamics 235:506–514, 2006. © 2005 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Cell adhesion is regarded as a pivotal part of the machinery of morphogenic events in developing embryos, including gastrulation, germ layer formation, domain segmentation, tissue segregation, cellular path finding, and epithelization (for review, see Steinberg, 1996). The significance of cell coherence in animal development was first corroborated by tissue recombination experiments in the mid-twentieth century, where amphibian cells and tissues of different origins associated in various combinations in culture segregated themselves and formed structures mimicking the natural tissues and organs. These findings led to formulation of the “selective adhesion” (Townes and Holtfreter, 1955) and “differential adhesion” hypotheses (Steinberg, 1964).

The discovery of the cell-to-cell adhesion molecules, namely, CAMs (Rutishauser et al., 1976) and cadherins (Takeichi, 1977), provided a molecular basis to explain these formulations. Evidences showed that the boundaries of tissues are formed by different classes or amounts of cell adhesion molecules on different types of cells. Although several classes of molecule are involved in morphogenic events, cadherins appear to be the major adhesion molecule mediating differential cell adhesion and tissue formation.

The cadherins are transmembrane glycoproteins responsible for calcium-dependent homophilic cell-to-cell adhesion (Thiery et al., 1988; Yagi and Takeichi, 2000; Wheelock and Johnson, 2003). The cadherins constitute a large superfamily that includes the classic cadherins, desmosomal cadherins, protocadherins, and atypical cadherins. The classic cadherins share conserved structures, including multiple extracellular repeats called cadherin repeats, a single-pass transmembrane domain, and a cytoplasmic domain. The specific recognition site responsible for homophilic cell adhesion is located in the first cadherin repeat. The cytoplasmic domain of the classic cadherins binds to the catenin complex, which interacts with the actin cytoskeleton and relocates cadherin molecules to the cell adhesion sites. Such cadherin accumulation on both of the apposing plasma membranes of adjacent cells forms a zipper-like anti-parallel array of the extracellular domains, which enhances the adhesion of the molecules in a synergistic manner. The desmosomal cadherins interact with intermediate filaments constituting transmembrane bridges of desmosomes. Although the protocadherins share the extracellular structures of cadherins, their cytoplasmic domains are very diverse and have no well-characterized motifs of subcellular functions (Suzuki, 1996; Frank and Kemler, 2002).

The cadherins have been shown to play roles in somitogenesis as in other developmental events (Dale and Pourquie, 2000; Stickney et al., 2000; Pourquie, 2001; Saga and Takeda, 2001). Cadherin 2 (Cdh2, N-cadherin) in developing somites has been studied in different animals. This molecule was localized to developing somites in chick and zebrafish embryos (Duband et al., 1987; Radice et al., 1997; Crawford et al., 2003). Administration of antibodies that perturb Cdh2-mediated adhesion to chick embryos resulted in disarranged or anomalous somites (Linask et al., 1998). Mice lacking cdh2 had small, irregular, and less-cohesive somites (Radice et al., 1997). A zebrafish cdh2 mutant, glass onion, showed disorganized somite boundaries (Pujic and Malicki, 2001) as well as neural and retinal malformations, although another cdh2 mutant, parachute, had no apparent somite defects (Lele et al., 2002).

Recently, protocadherin 8 (Pcdh8, paraxial protocadherin, arcadlin) was proposed to be involved in the morphogenic movements of paraxial mesoderm (segmental plate, PAM) and the establishment of somitic segmental boundaries. Pcdh8 is expressed in Spemann's organizer, PAM, and developing somites in zebrafish (Yamamoto et al., 1998), Xenopus (Kim et al., 1998, 2000; Wessely et al., 2004), and mouse embryos (Yamamoto et al., 2000; Rhee et al., 2003). Pcdh8 is a downstream target of a T-box gene spadetail that encodes a transcription factor required for mesodermal cell movements in morphogenic events (Yamamoto et al., 1998). Administration of a dominant-negative form of Pcdh8 disturbed the development of the segmental plate in zebrafish (Yamamoto et al., 1998), the activin-induced elongation of Xenopus animal cap explants (Kim et al., 1998), and the epithelial organization of cells in developing somites of mouse embryos (Rhee et al., 2003). Paradoxically, mice homozygous for a null allele of pcdh8 showed no somite defects (Yamamoto et al., 2000). This finding suggests the existence of additional cell adhesion molecule(s) that act concurrently or differentially with Pcdh8 in somitogenic functions. Here, we report that another member of the protocadherins, protocadherin 10, is involved in the development of PAM and the somitogenesis in zebrafish embryos.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

cDNA Cloning of a Protocadherin Expressed in the Developing Somites

Our cDNA library screening yielded a putative morphogenic protocadhrin expressed in the segmental plate. We screened a zebrafish cDNA library for cadherin superfamily molecules possibly involved in morphogenesis of specific tissues or organs (see Experimental Procedures section for details). Two of clones with cadherin homologies, 6-4 and 10-5, showed virtually identical expression patterns revealed by whole-mount in situ hybridization (ISH), with specific staining of the presomitic and somitic regions as well as some rostral parts of the embryo (details described later).

Clone 10-5 contained an insert of 4,743 bases including the complete coding sequence of a putative protocadherin. Although it had no typical Kozak consensus as in many protocadherin genes, the ATG at position 402 aligned well to the initiation site of known protocadherins so was considered the most likely initiation site. Clone 6-4 was found to be a partial sequence of 10-5. We decided to study clone 10-5 further as another putative morphogenic adhesion factor of somitogenesis.

Primary Structure of Zebrafish Protocadherin 10

We concluded that clone 10-5 encodes the zebrafish ortholog of protocadherin 10 (Pcdh10, OL-protocadherin, KIAA1400; DDBJ accession no. AB086625) based on the sequence comparisons described below.

The predicted protein of clone 10-5 had a conserved structure shared with other protocadherins, including a signal sequence (S), six extracellular cadherin repeats (EC1–EC6), a single-pass transmembrane domain (TM), and a cytoplasmic domain (CP; Fig. 1). It consisted of 1,026 residues. BLAST searches indicated that the deduced protein of 10-5 showed the highest degrees of similarity to chicken, mouse, and human Pcdh10. It scored ∼1,200–1,450 bits of similarities with protocadherin 10s (the self-alignment of 10-5 protein scored ∼1,900 bits), and lower degrees of similarity, ∼650 bits or less, to other known protocadherins.

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Figure 1. Structure of zebrafish Pcdh10 and experimental constructs. Colored bars show the structure of zebrafish Pcdh10 and experimental constructs. The chart above the bars shows identity of the each domain of zebrafish Pchd10 with the mouse counterpart. Pcdh10, wild-type protocadherin 10. Pcdh10G, green fluorescent protein (GFP) fusion construct. Pcdh10F, Flag-tagged construct. Pcdh10ecF, dominant-negative, Flag-tagged construct missing transmembrane and cytoplasmic domains. Boxes indicate domains and tags. Orange box, signal sequence; light blue, extracellular cadherin repeats EC1–6; red, transmembrane domain; light purple, cytoplasmic domain; dark purple, CM-1 and -2 conserved motifs found in the cytoplasmic domains of Pcdh8, 10, 18 and 19; green, GFP; yellow, Flag tag. x-axis, protein length in number of amino acids (A.A.).

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Multiple alignments of the 10-5 protein with the chicken, mouse, and human counterparts indicated that the most conserved domain was EC1, showing 81–82% identity, which was supposed to define specific homophilic adhesion of cadherins, leaving TM with 100% identity. The cytoplasmic domain of 10-5 protein showed significantly high degrees of similarity only to Pcdh10 (75–76% identity) but not to other classes of protocadherins. Protocadherins of different subclasses are known to show little, if any, similarity in their cytoplasmic domains. The 10-5 protein shared the cytoplasmic motifs of unknown function, CM-1 and 2, conserved among human protocadherin 8 (paraxial protocadherin, arcadlin), 10, 18, and 19 (Wolverton and Lalande, 2001).

Ectopically Expressed Pcdh10 Behaved as a Cell Adhesion Molecule in Zebrafish Cells In Situ

To verify the cell adhesion properties of Pcdh10, we performed expression studies of green fluorescent protein (GFP) -tagged or Flag-tagged constructs in zebrafish embryos. Mis-expressed Pcdh10 constructs localized to cell peripheries and maintain cell coherence during gastrulation.

Complimentary DNAs encoding Pcdh10-GFP fusion (pcdh10G) and a Flag-tagged Pcdh10 (pcdh10F) were constructed (Fig. 1), and capped RNAs were synthesized in vitro from the constructs. The RNAs were injected into a single blastomere of eight-cell embryos. Embryos were fixed at 9 or 11 hours postfertilization (hpf) for confocal microscopy. GFP fluorescence of the embryos injected with pcdh10G localized on apposing peripheries of adjacent cells (Fig. 2c). Cells with GFP fluorescence remained coherent during gastrulation, despite generalized cell mixing (Ho, 1992; Wilson et al., 1995; Woo et al., 1995), to form patches of fluorescent cells (Fig. 2a). Embryos injected with GFP RNA showed fluorescent cells dispersed over the whole embryo (Fig. 2b). Injection of pcdh10F RNA produced virtually the same localization to the cell peripheries in immunofluorescence (IF) -stained embryos as pcdh10G (data not shown).

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Figure 2. Ectopic expression of tagged Pcdh10 in zebrafish embryos. Single blastomeres of eight-cell embryos were injected with an RNA of GFP-tagged Pcdh10 construct, Pcdh10G. a: Confocal microscopy of 11 hours postfertilization (hpf) embryos expressing Pcdh10G revealed that fluorescent cells remained coherent during gastrulation to form patches (arrowheads) on the embryo. In this particular embryo, the left segmental plate was split from the notochord (arrow) at such a patch of green cells. GFP image (green) was superimposed on a phalloidin image (red). Left-side oblique view with rostral to the top. b: Confocal image of a 11 hpf embryo injected with GFP control RNA showed green cells dispersed over the embryo. Left-side view with rostral to the top. c: Detailed confocal scan of a 9 hpf embryo showing Pcdh10G localized to the cell peripheries (green, arrowheads). Red, phalloidin; blue, 4,6-diamidino-2-phenylindole (DAPI). The animal pole is to the top, dorsal to the right. a and b, Z projections of serial optical sections. c, a single section. Scale bars = 50 μm.

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Expression of Zebrafish pcdh10 and pcdh8 in PAM and Somites

We investigated detailed expression patterns of pcdh10 and pcdh8 in zebrafish embryos by whole-mount ISH. The pcdh10 transcript appeared in the domains fated to the paraxial mesoderm (PAM) of 8 hpf embryos but was excluded from the dorsal midline (data not shown). pcdh10 continued to be expressed in PAM. An increase in the expression appeared in the first two presumptive somites (presomites) at 10 hpf when myoD, used here as a somite marker, was expressed only in adaxial cells (Fig. 3). During somitogenesis, pcdh10 expression reached its peak in the visually segmenting somites and faded out in maturing somites thereafter. This wave of expression progressed caudally along with the timing of somitogenesis. The somitic expression of pcdh10 disappeared by 22 hpf except in the last two to three somites in the tail.

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Figure 3. Expression of pcdh10 in zebrafish embryos. Zebrafish embryos were stained by whole-mount in situ hybridization for pcdh10 or myoD. a–f:myoD. g–r:pcdh10. a–l: Dorsal views of the developing somites. m–r: Lateral views. a,g,m, 10 hours postfertilization (hpf); b,h,n, 11.5 hpf; c,i,o, 14 hpf; d,j,p, 16 hpf; e,k,q, 19 hpf; f,l,r, 22 hpf. Closed arrowheads indicate the latest visually segmenting somites. Closed arrows, epiphysis. Open arrowheads, otic vesicles. Open arrows, head mesoderm. Rostral is to the top. Scale bar = 100 μm.

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We compared the somitic expression of pcdh10 and pcdh8 by whole-mount ISH performed concurrently with age-matched embryos. Both pcdh10 and pcdh8 transcripts increased in the rostral few presumptive somites and reached the maxima of their expression in the latest visually segmenting somites (Fig. 4). The pcdh8 transcript faded out in two to three somites after segmentation, whereas pcdh10 expression continued longer than pcdh8 depending on embryonic age, in approximately three somites in 12 hpf, four in 14 hpf, and eight in 16 hpf embryos. Both protocadherins were expressed in the whole of the forming and newly formed somites but later became localized to the rostral part of somites as they matured. The pcdh8 expression was limited to the very front row of cells in the most rostral pcdh8-expressing somites, whereas pcdh10 expression remained in the rostral two to three rows of in the last pcdh10-expressing somites. The transcripts of pcdh8 but no pcdh10 were expressed in the adaxial cells (arrowheads in Fig. 4).

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Figure 4. Comparison of expressions of pcdh10 and pcdh8 in paraxial mesoderm. Embryos were stained by whole-mount ISH for pcdh10 or pcdh8, manually sectioned, and flat-mounted for microscopy. a,b: 12 hours postfertilization (hpf). c,d: 14 hpf. a,c: pcdh10. b,d: pcdh8. Arrows indicate the latest visually segmenting somites. Asterisks indicate the older somites with positive pcdh10 or pcdh8 staining. Plus signs indicate the latest presumptive somites. Opposing arrowheads, adaxial cells; N, notochords. Dorsal views with the rostral to the left. Scale bar = 50 μm.

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Dominant-Negative Form and Antisense Morpholino of pcdh10 Perturbed PAM and Somite Development

We then investigated the functional significance of Pcdh10 in the development of PAM and somitogenesis using a dominant-negative form of the protein and an antisense morpholino oligonucleotide (MO) directed against pcdh10. Protocadherin constructs without the transmembrane and cytoplasmic domains are known to be secreted extracellularly and to bind to their intrinsic wild-type form. Consequently, such constructs dominantly inhibit the wild-type form to function when overexpressed in cultured cells and in developing embryos (Yamamoto et al., 1998). According to this experimental design, we constructed a cDNA encoding a dominant-negative, secreted form of Pcdh10, Pcdh10ecF, which is truncated at the end of the extracellular domain and linked in-frame to a Flag expression tag. A capped RNA was then synthesized in vitro from the pcdh10ecF cDNA. An MO was designed complementary to the translation initiation site of pcdh10 to knockdown pcdh10 messages (pcdh10mo). pcdh10ecF RNA or pchd10mo was microinjected into embryos. The embryos at 14–15 hpf were fixed for morphological analyses of somitogenesis using ISH for myoD.

pcdh10ecF RNA and pcdh10mo resulted in PAM and somite malformations, although to various degrees (Fig. 5a–f). For statistical analyses, PAM or somite development was regarded as disturbed when myoD pattern matched one or more of following criteria: (1) right–left asymmetry of somites (class AS); (2) bridges of myoD-expressing cells between adjacent somites, compression (narrowing of intervals or fusion) of adjacent somites, or blurred somite boundaries (class BR); (3) lack or misplacement of some somites or generally widened, disarranged midline structures (class MS).

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Figure 5. Antisense morpholino and dominant-negative construct of Pcdh10 disturbed somitogenesis. b–f: Embryos were injected with pcdh10 antisense morpholino oligonucleotide (MO), pcdh10mo, fixed at 14–15 hours postfertilization (hpf), and stained by in situ hybridization (ISH) for MyoD to show somite phenotypes. Somitogenesis was scored as affected when (AS) left–right asymmetry was found in some somites (arrowheads in b); (BR) there were bridges (right arrowhead in c), compression of adjacent somites (arrowhead in d) or blurred somite boundaries (left arrowhead in c); or (MS) some somites were missing or misplaced (arrowheads in e), or the midline structures were widened and disorganized (f). a: Embryo injected with negative control MO with normal morphologies. g,h: Embryos were injected with Pcdh10ecF RNA at the four-cell stage. Embryos with unilateral expression of GFP as a tracking dye (arrowheads in g) were fixed for MyoD ISH. Note the deformation of somites (arrowheads in h) ipsilateral to GFP expression. Dorsal views with rostral to the top. Scale bars = 100 μm.

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pcdh10ecF expressed in the whole embryo affected somitogenesis in 61% of embryos, whereas 5–9% of control embryos with no injection or injected with β-gal RNA or negative control MO had disturbed somites (Table 1). Injection of pcdh10ecF RNA resulted in class MS in 27% of embryos, suggesting its disturbances to morphogenic movements of PAM cells. It also caused class BR in 14% embryos, indicating affected somitic segmentation. pcdh10mo affected 78% of embryos (12% AS, 18% BR, and 43% MS). We also injected pcdh10ecF RNA into a single blastomere of four-cell embryos to obtain chimeric expression of the construct in a limited part of the embryo (Fig. 5g,h). Some of these injected embryos expressed the foreign material marked by coinjected GFP RNA unilaterally in their left or right side. Approximately 60% of such embryos had ipsilateral malformed, misplaced, or missed somites, whereas none had contralateral disturbed somites.

Table 1. Suppression of Somitogenesis by pcdh10 Antisense MO and Dominant-negative Pcdh10a
Injection% Affected embryosNo. of embryos studied
  • a

    Class AS, right–left asymmetry; BR, bridging or compression; MS, misplacement or absence.

  • *

    Significant difference at P < 0.5%.

No injectionTotal:5.7261
  Class AS:0.4 
  Class MS:5.3 
β-gal RNATotal:8.7150
  Class MS:8.7 
Negative control MOTotal:5.654
  Class AS:1.9 
  Class MS:3.7 
pcdh10ecF RNA, expressed in the whole embryoTotal:*61101
  Class AS:*21 
  Class BR:*14 
  Class MS:*27 
pchd10ecF RNA, unilateral expressionIpsilateral:*6216
 Contralateral:0 
pchd10mo antisense MOTotal:*7895
  Class AS:*12 
  Class BR:*18 
  Class MS:*43 

pcdh10 Expression in the Head and Central Nervous System

The rostroventral tip of the body expressed pcdh10 strongly from 10–12 hpf, and the level of expression decreased by 14 hpf when the tip formed the rostroventral mesoderm. Expression of pcdh10 appeared in the epiphysis (arrows in Fig. 3) and the mesodermal tissue by the ear vesicles (open arrowheads in Fig. 3) at approximately 12 hpf and reached peaks around 16 hpf. pcdh10 was expressed in the head mesoderm surrounding the telencephalon and eyes in embryos at 14 hpf and later. By 22 hpf, pcdh10 began to be expressed in the diencephalon, part of the hindbrain, and the epithelial cells of the otocysts as well as in the eye muscles and the pectoral fins (Fig. 6).

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Figure 6. pchd10 expression in the rostral structures. pcdh10 was expressed in the epiphysis (double arrowheads), diencephalon (arrowheads), myelencephalon (open arrows), otocyst (asterisk), eye muscles (closed arrow), and fin buds (closed arrows). a–c: Dorsal (a), lateral (b), and frontal (c) views of 33-hours postfertilization (hpf) embryos stained by whole-mount in situ hybridization for pcdh10. The dashed line in b indicates the focal plane of c. The focal plane of the inset in b is lateral to that outside the inset. Scale bar = 100 μm.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Functional Significance of Zebrafish Pcdh10

We cloned a zebrafish ortholog of Pcdh10, which showed a high degree of similarity (73–74% identity) to the known counterparts in mouse (Hirano et al., 1999), human (Wolverton and Lalande, 2001), and chicken (accession no. NP 999837). Chicken Pcdh10 is 88–89% identical to mouse and human counterparts; and mouse Pcdh10 is 97% identical to human counterpart.

It is suggested that zebrafish Pcdh10 is involved in the morphogenic machinery of the movements of PAM cells and somite segmentation by the following results. It showed cell adhesion properties in situ when misexpressed in zebrafish embryos. It was expressed in PAM and the developing somites. The expression surged at segmentation of the latest presumptive somites and somites, continued during somite maturation and eventually faded out in matured somites. The antisense MO against pcdh10 and the dominant-negative form of Pcdh10 disturbed the movements of PAM cells and somite segmentation when introduced in embryos.

We presume that Pcdh10 might serve as another cell adhesion factor in PAM and somite development based on its expression and functions analogous to those of Pcdh8. The concurrent action of Pcdh8 and Pcdh10 is thought to be complementary, not simply redundant, in zebrafish because inhibition of either Pcdh8 (Yamamoto et al., 1998) or Pcdh10 (as shown in this article) resulted in disturbed somites. As pcdh10 was expressed in maturing somites for longer than pcdh8, Pcdh10 might take over from Pcdh8 and maintain cell coherence of older somites. Pcdh8 and Pcdh10 may also account for the differential establishment of the adaxial cells from somitic cells. This was inferred from the observation that the adaxial cells expressed pcdh8 but no pcdh10. pchd8 was shown to be a downstream target of spadetail (Griffin et al., 1998; Yamamoto et al., 1998). We are investigating the upstream regulation of pcdh10, including spadetail as a putative regulatory factor.

Hypothetical Pcdh10-Associated Factors

The structural and functional similarities of Pcdh8 and Pcdh10 suggest the presence of a factor or factors associated with the cytoplasmic motifs of these protocadherins, although only little is known about the protocadherin-associated cytoplasmic factors. This postulation is supported by the conserved cytoplasmic motifs CP-1 and 2, which are shared by a protocadherin subclass consisting of Pcdh8, Pcdh10, Pcdh18, and Pcdh19 (Wolverton and Lalande, 2001). We currently are investigating such Pcdh10-associated factors involved in the molecular architecture of the cell adhesion machinery in somitogenic cells. This knowledge would also contribute to a comprehensive understanding of biological significance of the protocadherin subclass with Pcdh8-type cytoplasmic motifs.

Pcdh10 in Head and Central Nervous System

pcdh10 was also expressed in some parts of the rostral structures of embryos, including the epiphysis, the head mesoderm, and mesoderm around the ear. In 1-day and older embryos, it was also expressed in the diencephalon, the hindbrain, the otocyst epithelium, the eye muscles, and the fin buds. pcdh10 expression in the central nervous system (CNS) was described previously in mouse late embryos and neonates (Hirano et al., 1999; Aoki et al., 2003), although they used an isoform with a shorter cytoplasmic domain lacking the conserved motifs CM-1 and 2, in contrast to the present study in which the longer isoform was examined. Our preliminary investigations have indicated neither the presence nor absence of the shorter variant of Pcdh10 in zebrafish. Detailed comparative studies are required to elucidate the functions of Pcdh10 isoforms in the head and CNS. We currently are investigating Pcdh10 expression in the zebrafish CNS in greater detail.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Fish Care

Fish were purchased from a local pet shop. Fish of the golden strain were used as wild-type in some of the experiments because of their attenuated pigment production suited for microscopy and otherwise normal development (Weinberg et al., 1996; Bellipanni et al., 2000). Fish were maintained and embryos were obtained as described elsewhere (Westerfield, 2000). Embryos were staged in hours postfertilization at the standard temperature of 28.5°C.

Oligonucleotides for Cadherins and Protocadherins

Degenerate primers for cadherin polymerase chain reaction (PCR), cad-f1.1, f3.1b, f3.1c (forward primers) and r2.1 (reverse primer), were designed as described previously (Suzuki, 1996). Their sequences were 5′-CGG AAT TCA CNG CNC CNC CNT AYG A-3′, 5′-CGG AAT TCG ARG ARG AYA CNC ARG MNT WYG A-3′, 5′-CGG AAT TCG ARG ARG AYC ARG MNT WYG A-3′, and 5′-TAT CCG GAG CTC NTC NGC NAR YTT YTT RAA-3′, respectively (B = C/G/T, D = A/G/T, H = A/C/T, K = G/T, M = A/C, N = any base, R = A/G, S = C/G, V = A/C/G, W = A/T, Y = C/T). Primers for protocadherin, pc-f2 (forward) and r2 (reverse), were virtually identical to those described previously (Sano et al., 1993). The sequences were 5′-CCG AAT TCA ARS SNN TNG AYT WYG A-3′ and 5′-CCG GAT CCN NNG GNG CRT TRT CRT T-3′, respectively.

PCR Amplification and Cloning of Cadherin and Protocadherin Fragments

Zebrafish genomic DNA extracted from a female and a male adult zebrafish (Westerfield, 2000) was used as the template for PCR using Pyrobest DNA polymerase (Takara Bio, Shiga, Japan) and the cadherin primers cad-f1.1, f3.1b, f3.1c, and r2.1. The protocadherin primers, pc-f2 and r2, were used for PCR with a zebrafish embryonic cDNA library (Uni-ZAP XR library, Stratagene, La Jolla, CA) as the template. The products were cloned into the pCR-Blunt vector (Invitrogen, Carlsbad, CA) and sequenced as described below. Three clones with homology to cadherin and another three with homology to protocadherin were used as templates for cDNA library screening.

cDNA Cloning

Digoxigenin (DIG) -labeled probes were made from each of the three cadherin and three protocadherin clones using a PCR DIG Probe Synthesis Kit (Roche Diagnostics, Tokyo, Japan). The Zebrafish cDNA library was screened according to the manufacturer's instructions with a mixture of the six probes. Thirteen of the positive clones were sequenced from 5′ and 3′ ends for Blast homology searches (http://www.ncbi.nlm.nih.gov/BLAST/). The clones were also evaluated by their expression patterns in zebrafish embryos using in situ hybridization.

DNA Sequencing and Data Processing

Plasmid clones were sequenced with a BigDye Terminator Kit and ABI Prism 310 and 3100 Genetic Analyzers (Applied Biosystems Japan, Tokyo, Japan). The sequence data were processed with DNA Strider (C. Marck, CEA, Cedex, France) running on a Macintosh computer (Apple Japan, Tokyo, Japan). The sequence was submitted to DNA Data Bank of Japan (DDBJ; National Institute of Genetics, Shizuoka, Japan) in June 2002 and published in February 2003 with accession no. AB 086625. GenBank lists it as protocadherin 10b with accession no. NP 878305. Homology searches were performed with Translated Blast. Multiple alignments of protein sequences were performed using Parallel PRRN (Yasushi Totoki, University of Tokyo, Tokyo, Japan. http://prrn.ims.u-tokyo.ac.jp/).

cDNA Modifications

The pcdh10 cDNA was recloned into the pCS2+ vector (Dave Turner, University of Michigan, MI. http://sitemaker.umich.edu/dlturner.vectors) for in vitro capped RNA synthesis as follows. Fragments of pcdh10 cDNA flanking the 5′-untranslated region to the stop codon or to the end of the extracellular domain were amplified by PCR with T3 primer (forward) and 5′-CGG AAG GTC GAC GCA TAT CCT TTT CCG CGA-3′ (reverse), or with T3 and 5′-GAG CGT GTC GAC CAG AGA CGC GTC TTT GGC-3′, respectively. Such fragments were cloned into the pCMV-Tag4A vector (Stratagene, La Jolla, CA) to add a Flag tag. The Flag-tagged constructs (Pcdh10F and Pcdh10ecF) were recloned into pCS2+ by PCR using primers flanking the insert and the Flag tag, T3 and 5′-CGG TCT AGA CTT ATC GTC GTC ATC CTT GTA ATC CTC GA-3′. Another fragment of pcdh10, amplified by PCR with T3 and 5′-AGT GTC GGA TCC GGA GCC ACC ATG TTT GTG TTT TTG CTC CTG CTG-3′, was cloned into pCS2p+GFPbgl2 (D. Turner) to construct a Pcdh10-GFP fusion (Pcdh10G).

Morpholinos

Fluorescein-labeled MOs were purchased from Gene Tools (Philomath, OR). An MO was designed complimentary to the 5′ sequence near the translation initiation site of pcdh10 (pcdh10mo). The sequence was 5′-CAG CAG GAG CAA AAA CAC AAA CAT C-3′. The negative control MO was 5′-CCT CTT ACC TCA GTT ACA ATT TAT A-3′.

Microinjection

Capped mRNAs were synthesized from the pCS2+ vector clones as described previously (Bellipanni et al., 2000). For the Flag-tagged dominant-negative construct, Pcdh10ecF, a mixture of RNAs of GFP and the construct, each at 100 μg/ml, were injected into embryos by using an IM-300 microinjector (Narishige, Tokyo, Japan). To have the construct expressed in the whole embryo, 4 nl of the RNA mixture was injected to the blastomere of one-cell embryos or to the yolk just below the blastomeres of two-cell embryos. For unilateral expression, 1 nl of the RNA mixture was injected to a single blastomere of four-cell embryos. RNA of the tagged construct, Pcdh10G or Pcdh10F, at 25 μg/ml was injected into a single blastomere of eight-cell embryos in a volume of 1 nl. The morpholinos were dissolved in 1× Danieau solution (Nasevicius and Ekker, 2000) at 0.25 mM and 1 nl of the solution was injected to the yolk of two- to eight-cell embryos. Successful injections were monitored by GFP fluorescence for RNAs and fluorescein fluorescence for morpholinos.

ISH and IF Staining

Zebrafish MyoD (Weinberg et al., 1996) and paraxial protocadherin (Yamamoto et al., 1998) cDNAs were gifts from Dr. Eric. S. Weinberg and Dr. Akihito Yamamoto, respectively. DIG-labeled RNA probes were synthesized using the cDNA clones as templates. Staged zebrafish embryos were stained by whole-mount ISH as described elsewhere (Weinberg et al., 1996; Bellipanni et al., 2000). Embryos expressing a Flag-tagged construct were stained with rabbit polyclonal anti-Flag antibody (Sigma-Aldrich Japan, Tokyo, Japan) and Alexa Fluor 594–labeled anti-rabbit IgG (H+L) antibody (Molecular Probes, Eugene, OR). Alexa Fluor–labeled phalloidins and 4,6-diamidino-2-phenylindole (DAPI; Molecular Probes) were used as counter stains for some embryos.

Microscopy and Image Processing

Embryos stained by whole-mount ISH were mounted on 1.5% agarose plates with pits cast with 0.5-mm glass beads, and viewed with an Olympus SZX-12 dissection microscope (Olympus, Tokyo, Japan). A Nikon E-1000 compound microscope (Nikon, Tokyo, Japan) was used for manually sectioned embryos and fluorescent embryos. Embryos or sections were mounted between a glass slide and a coverslip with plastic adhesive tape as a spacer (Weinberg et al., 1996; Kozlowski et al., 1997; Bellipanni et al., 2000).

Microscopic images were recorded with a C5810 chilled 3CCD camera (Hamamatsu Photonics, Shizuoka, Japan) or a Spot RT SE6 Monochrome cooled CCD camera (Diagnostic Instruments, Sterling Heights, MI) and processed with Adobe Photoshop (Adobe Systems, Tokyo, Japan) on a Macintosh to match colors to the real images. “Extended-focus” pictures were composed for thick specimens of whole- and flat-mount embryos. To make an extended-focus picture, a series of two to five images of a specimen was taken by manually shifting the focus. Each image was placed as an individual layer on a single Photoshop picture. Out-of-focus areas of each layer were masked out so that regions of interest of the specimen were revealed in-focus in the Photoshop image.

Serial optical section images of whole-mount fluorescent embryos were obtained with a Bio-Rad MRC-1024 confocal system (Carl Zeiss Japan, Tokyo, Japan) fitted on a Zeiss Axioplan microscope (Zeiss), or a Nikon C1si spectral imaging confocal system (Nikon, Tokyo, Japan). The Bio-Rad files were processed with ImageJ software (Wayne Rasband, NIH, Bethesda, MD. http://rsb.info.nih.gov/nih-image/) for three-dimensional projections. Nikon spectral images were processed with Nikon's proper software to unmix fluorescence signals.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

We thank Dr. Eric S. Weinberg at the University of Pennsylvania, Dr. Akihito Yamamoto at RIKEN, and Dr. Dave Turner at the University of Michigan for providing zebrafish MyoD cDNA, zebrafish Pcdh8 cDNA, and pCS2+ vectors, respectively. T.M. thanks E.S.W. for his generous support in his zebrafish studies. The Spot RT SE6 camera was received as a giveaway by Microscopy and Analysis. T.M. was funded by the Ministry of Education, Culture, Sports, Science, and Technology of Japan and support from Life Science Foundation of Japan.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES