SEARCH

SEARCH BY CITATION

Keywords:

  • Fras;
  • Frem;
  • Fraser Syndrome;
  • caudal fin fold;
  • cleft cell;
  • ridge cell;
  • basement membrane;
  • extracellular matrix

Abstract

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

Mouse studies have highlighted the requirement of the extracellular matrix Fras and Frem proteins for embryonic epidermal adhesion. Mutations of the genes encoding some of these proteins underlie the blebs mouse mutants, whereas mutations in human FRAS1 and FREM2 cause Fraser syndrome, a congenital disorder characterized by embryonic blistering and renal defects. We have cloned the zebrafish homologues of these genes and characterized their evolutionary diversification and expression during development. The fish gene complement includes fras1, frem1a, frem1b, frem2a, frem2b, and frem3, which display complex overlapping and complementary expression patterns in developing tissues including the pharyngeal arches, hypochord, musculature, and otic vesicle. Expression during fin development delineates distinct populations of epidermal cells which have previously only been described at a morphological level. We detect relatively little gene expression in epidermis or pronephros, suggesting that the essential role of these proteins in mediating their development in humans and mice is recently evolved. Developmental Dynamics 237:3295–3304, 2008. © 2008 Wiley-Liss, Inc.


INTRODUCTION

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

The five mouse blebs mutants are characterized by defects in embryonic epidermal adhesion, which result in the formation of blisters or “blebs” in utero (Smyth and Scambler,2005). The subsequent disruption of epithelial/mesenchymal interactions are thought to lead to a variety of malformations including cryptophthalmos, syndactyly, and renal a/dysgenesis. Recently, the mutations in four of the five blebs loci have been cloned. Three of the phenotypes (blebbed [bl], headblebs [heb], and myelencephalicblebs [my]) are caused by mutations in proteins of the extracellular matrix known as Fras1, Frem1, and Frem2, respectively (McGregor et al.,2003; Vrontou et al.,2003; Smyth et al.,2004; Jadeja et al.,2005). Mutations in a multiple PDZ domain protein Grip1 underlie a further blebs locus (eye blebs; Takamiya et al.,2004). Subsequent studies have shown that Grip1 interacts with the intracellular C-terminus of the trans-membrane protein Fras1, and probably Frem2, and is responsible for maintaining these proteins at the cell surface, either by stabilizing them in situ or by regulating their trafficking (Takamiya et al.,2004). Frem1, on the other hand, is a secreted protein lacking a transmembrane domain or PDZ interaction motif and is therefore unlikely to interact with Grip1. The blebs mice have long been considered a model for Fraser syndrome (Winter,1990), a recessive disease with strikingly similar defects and subsequent studies have demonstrated that mutations in FRAS1 or FREM2 give rise to Fraser syndrome (McGregor et al.,2003; Jadeja et al.,2005; Slavotinek et al.,2006; Shafeghati et al.,2008).

All four of these ECM genes are expressed in a variety of tissues during mouse development. In addition to their generalized expression in the developing epidermis/dermis they are also transcribed at sites in which differentiating and proliferative epidermal components are involved in active signalling with underlying mesenchyme. These include the developing limb, hair follicle, eyelid, lung, mammary gland, and kidney (Short et al.,2007). Frem2 is also expressed in a complex pattern in the developing musculature, branchial arches, and central nervous system (Jadeja et al.,2005). In the majority of developmental contexts Fras1 and Frem2 are expressed in the epidermis whereas Frem1 is expressed in the underlying mesenchyme or dermis (Smyth et al.,2004), although this is often tissue specific and for the most part the proteins all localize to the basement membrane. Intracellular epidermal Frem1 is noted in some contexts (Chiotaki et al.,2007; Petrou et al.,2007a). These observations, along with the domain structure of the proteins, led us to speculate that the proteins interact directly to mediate epidermal adhesion (Smyth et al.,2004). Elegant biochemical studies have subsequently shown that Frem1, Frem2, and Fras1 form a ternary complex in vitro and that this complex is most probably required for normal epidermal adhesion in utero (Kiyozumi et al.,2006). It is unclear whether Frem3 is involved in this complex, but the gene's divergent expression and the normal localization of the protein in mice carrying mutations in the other three proteins suggest that it acts in an independent manner (Chiotaki et al.,2007; Petrou et al.,2007b). One way in which we can study the nature of the relationships between these genes and the pathology resulting from their loss is by comparing gene expression in mammals with other vertebrates. In this study we have cloned the homologous genes in the zebrafish and have detailed their transcription during development. Although there is relatively modest detectable expression of the genes in the epidermis or kidney primordia, the tissues affected in mice and humans, we note conserved and divergent expression of the genes in several differentiating structures which will help to elucidate the function of these developmentally important genes.

RESULTS AND DISCUSSION

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

Identification of the fras and frem Genes in Zebrafish

Mice and other mammals possess four Fras1 and Fras1 related extra-cellular matrix (Frem) genes; Frem1, -2, -3, and Fras1 (Smyth and Scambler,2005). These proteins share several different domains but are best characterized by the presence of chondroitin sulphate proteoglycan (CSPG) motifs, which we and others have shown are structurally similar to cadherin folds (Staub et al.,2002; Smyth et al.,2004). We interrogated genome assemblies from several teleosts to characterize the gene complement in fish (Fig. 1). Consistent with whole genome duplication because the teleost radiation we found two genes in the frem/fras family had been duplicated; frem1 (designated frem1a and -1b) and frem2 (designated frem2a and -2b). Assignment of orthology with respect to Frem3 was based on syntenic conservation in both the stickleback and zebrafish genomes compared with human genomes. Conservation between duplicated zebrafish genes was similarly high, with nomenclature assigned based on both protein identity and similarity with mouse Frem2 and Frem1 and developmental expression (see below).

thumbnail image

Figure 1. Evolution and domain structure of the zebrafish Frem and Fras proteins. The zebrafish genome encodes six fras and Frem proteins compared with the four commonly found in other veterbrates, the extra members being derived from duplications in frem1 and frem2. Whereas the domain organization of the proteins is predominantly conserved, zebrafish, pufferfish, stickleback, opossum, and frog Frem3 (*) includes two additional Calxβ domains, a transmembrane region and a conserved PDZ interaction motif which have been lost in mouse and human proteins (sig seq, signal sequence; CSPG, chondroitin sulfate proteoglycan domain; CalXβ, calcium exchange beta; CLect, C-type lectin; VWFC, von Willebrand Factor domain C; TM, transmembrane; PDZ i/a, PDZ interaction motif). Bootstrap analysis was conducted using gene predictions/sequences from mm, Mus musculus (mouse); fr, Takifugu rubripes (pufferfish); hs, Homo sapiens (human); dr, Danio rerio (zebrafish); gg, Gallus gallus (chicken); md, Monodelphis domestica (opossum); ga, Gasterosteus aculeatus (stickleback); xt, Xenopus tropicalis (frog). Fragmentation of genomic sequence of stickleback fras1 and Fugu frem1b precluded their inclusion in the alignment.

Download figure to PowerPoint

In mammals, the Frem3 protein comprises only three C-terminal Calxβ domains and lacks the transmembrane domain found in Frem2 and Fras1. We were therefore surprised to observe that in fish genomes the protein has the “full” complement of five Calxβ domains (Fig. 1). Additionally Frem3, like Frem2a, -b, and Fras1, has a well conserved transmembrane domain and possesses a putative C-terminal PDZ interaction signature. As in mouse, frem2a, -b, and frem3 encode very similar proteins (65, 66, and 64% identity with mouse Frem2, respectively). Interrogation of other databases showed that the “complete” structural organization of Frem3 was also apparent in the opossum and Xenopus, suggesting that the ancestral Frem3 protein had this structure and that the C-terminal domains were lost after the divergence of mammals from marsupials (∼180 million years ago) and before the eutherian radiation (∼100 million years ago). We generated in situ mRNA probes to all six zebrafish genes and detailed their expression during zebrafish development from fertilization to 72 hours postfertilization (hpf).

Expression of frem1a and frem1b

frem1a is initially expressed in the developing fin fold from its origin at the early tail bud stage. As we detail later in this report, expression begins in a medial stripe two to three cell diameters wide, which by 27 hpf becomes restricted to a single line of cells on the dorsal and ventral sides of the caudal fin fold (Fig. 2A,B). We propose that these cells correspond to the cleft cells originally described by Dane and Tucker (1985). From 24 hpf, the expression initiates strongly in medial cells of the brachial arches surrounding the differentiating otic vesicle as well as generally in the developing brain (Fig. 2C,D). Low levels of expression were noted in the vesicle itself (Fig. 2C,E). By 34 hpf, expression in the arches persists with the highest expression in the hyoid arch (Fig. 2E). Expression in derivatives of the branchial arch continued throughout the extent of our analysis up to 72 hpf when the gene is expressed in the most posterior two to three cells of the posterior ectodermal margin (PEM) of the hyoid arch (Fig. 2F,H,I). This is an actively proliferating epithelial structure which is similar to the apical ectodermal ridge of the limb bud. The PEM eventually goes on to form the operculum which covers the gills in adult fish. Expression in the pectoral fin fold was detected shortly after its emergence and persists through all of the time points studies (Fig. 2F,G). Expression in the fin fold mirrors that observed in the caudal fin as a single lines of distal cells (Fig. 2G). Despite repeated attempts with different in situ probes we were unable to detect frem1b expression at any stage, although the mRNA could be successfully amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) in adult fish and late stage (day 3) embryos. These results suggest that either the gene is expressed at very low levels during development or that it plays roles later in development or in adults.

thumbnail image

Figure 2. Expression of frem1a and frem2a during development. Expression of frem1a (A–I) and frem2a (J–Q). A,B: Lateral views of frem1a expression detail expression of the gene in the developing caudal fin fold (arrowhead). C–E: At later stages the gene is also expressed in the developing branchial arches (C and D, in section; E, lateral view with hyoid indicated (arrowhead); otic vesicle highlighted in C and E). F–I: Expression is noted in the pectoral fins (F, lateral red arrowhead; G in section) and posterior ectodermal margin (PEM) of the hyoid arch (F and H, ventral, arrowhead) and in section (I, arrowhead). The line in H is the plane of section in I. J,K: Like frem1a, frem2a is expressed in the differentiating caudal fin fold (J and K, lateral views, arrowhead). L–N: Expression in the brachial arches initiates at 24 hours postfertilization (hpf) and by 32 hpf becomes strongest in the endodermal pouches (arrowheads in L, dorsal; in M, N dorsolateral, otic vesicle highlighted). O–Q: Expression is also detected in the pectoral fin folds (O–Q, arrowheads) and persists in the caudal fin folds of older fish (P, Q). ba, branchial arch; hb, hindbrain; som, somites; pff, pectoral fin fold; ope, operculum.

Download figure to PowerPoint

Expression of frem2a and -2b

Like frem1a, expression of frem2a is first detected in the early tail bud stage during the differentiation of the caudal fin fold (Fig. 2J). Robust expression is initially detected in two stripes of cells in the fold analogous to frem3 (see below) but as the fold differentiates and emerges from the body wall (24 hpf) expression is detected in all epidermal cells of the fold including those expressing fras1, frem1a, and frem3 (Fig. 2K). frem2a expression is also initiated in the branchial arches from 24 hpf. Like fras1 and frem3 expression is detected in the differentiating endodermal pouches of the branchial arches (Fig. 2L–N). This pattern appears distinct from frem1a (Fig. 2C, D), which is expressed more medially, surrounding the otic vesicle. Expression in both the caudal and pectoral fin folds persists throughout the extent of our analysis up to 72 hpf (Fig. 2O–Q). We observed no significant expression in other tissue components including the pronephros, musculature or otic vesicle. As with frem1a, we were not able to detect early embryonic expression of frem2b despite repeated attempts using two different riboprobes.

Expression of fras1

fras1 has a more diverse and dynamic pattern of expression than frem1a or frem2a. We first detect fras1 message in the axial midline shortly before the initiation of somitogenesis (Fig. 3A). fras1 expression is initiated in the developing somites as they form and shortly after the emergence of the tail bud at the midbrain/hindbrain boundary (Fig. 3C). Expression at the MHB is particularly prominent from 20 to 30 hpf (Fig. 3D) after which it gradually declines, and is strongest in the medial aspect of this structure (Fig. 3E). Similarly, expression in the somites peaks around 24 hpf and then gradually declines as the musculature differentiates (compare Fig. 3D,G). We were unable to detect prominent expression of the gene in the musculature of the jaws and head at later developmental stages. From 24 hpf, we observe expression of fras1 in the developing branchial arches. This expression is initially detected at low levels throughout the arches (Fig. 3E) but becomes stronger in the arch endoderm (Fig. 3F). By 48 hpf, expression has declined in the most anterior arches but remains strongest in the two posterior-most endodermal pouches (4 and 5), specifically in their most ventral aspects (Fig. 3I, inset). Like frem1a, fras1 is also expressed in the PEM of the hyoid arch, and we also observed low levels of gene expression in the oral ectoderm from 48 hpf (Fig. 3G,H,J). At later stages, we detect expression in the pectoral fin folds and operculum (Fig. 3K,L). Like frem1a, fras1 is strongly expressed in the caudal fin fold from the early tail bud stage and we detail the temporal and spatial expression in the fin fold in a later section of this study. Our expression analysis correlates with the results of two similar analyses available on the ZFIN community database (Kudon et al.,2001; Thisse and Thisse,2004).

thumbnail image

Figure 3. A: Expression of fras1 during development. fras1 expression is first noted in the axial midline 8 hr postfertilization (A, arrowhead). B–D: Transcripts are then detected in the developing somites (B, dorsal; C and D, white arrowheads). D,E: Expression is noted in the midbrain–hindbrain boundary (D, black arrowhead) toward the medial aspect of the future cerebellum (E, arrowhead). F: Transcripts are detected in the branchial arches specifically in the differentiating endodermal pouches (arrowheads). C,D,G: Strong expression is noted in the caudal fin fold shortly after its differentiation at the early tail bud stage (red arrowheads). F–I,K: Expression is detected in the pectoral fin fold (F) and this persists throughout the period investigated (G–I,K). G,H: By 48 hpf expression is noted in the oral ectoderm (black arrowheads) and in the posterior margin of the hyoid arch. I: By this stage, expression in the branchial arches is strongest in the pouches 4 and 5, in the most ventral aspects of these structures (inset arrowhead). J–L: Expression in the oral ectoderm persists at 56 hpf (J, red arrowhead) as does expression in the posterior ectodermal margin (PEM) at 56 hpf (J and K, black arrowheads) and 72 hpf (L, section, arrowhead). op, oropharynx; ope, operculum.

Download figure to PowerPoint

Somitic Expression of frem3

frem3 has the most dynamic and diverse expression pattern of the extracellular blebs genes. frem3 transcripts are first detected shortly after the commencement of somitogenesis in the developing rostral adaxial mesoderm cells (Fig. 4A–C) and in the flanking ectoderm of the developing tail (Fig. 4B). By the tail bud stage expression in developing adaxial cells is consolidated and as development progresses, expression in the somitic muscle persists (Fig. 4D,E). At 24 hpf, expression in more differentiated rostral somites is detected throughout the musculature (Fig. 3F,H), whereas in less mature somites expression is highest in the newly specified adaxial cells (Fig. 4G,I) To determine whether activation of frem3 expression in the somites was dependant on migration of slow muscle cells from the midline, we examined frem3 expression in you too (yot) embryos in which these cells fail to form (Brand et al.,1996). However, we did not note any obvious differences in either the timing or level of expression of frem3 (data not shown). Expression levels in the majority of the somites decreases progressively from 30 hpf, by which time tail extension and somitogenesis is complete. At this stage, however, frem3 expression persists in the most anterior somites adjacent to the pectoral fin as well as in the differentiating fin muscles themselves (Fig. 4J).

thumbnail image

Figure 4. Expression of frem3 during development. frem3 is broadly expressed during embryonic development in zebrafish. A–C: Expression is first detected during early somitogenesis and, at the bud stage is detected in the lateral head mesoderm (A, anterior), somites (B, posterior; C, dorsal), and differentiating tail ectoderm (B, arrowhead). E,I: In newly differentiating caudal somites, expression is highest in the adaxial cells adjacent to the neural tube (black arrowheads). F,H: By 24 hpf expression is detected throughout the somitic musculature (F, lateral; H, in section, arrowhead). J: Expression is noted in the developing pectoral fin musculature (asterisk) which persists in the adjacent somites 1 and 2 (arrowhead). F–I: As with the other frem-related genes, expression is detected in the caudal fin folds. D,E,G,I: In addition to somitic expression, transcripts are observed in the hypochord (red arrowheads) as a single medial line of cells. K–R: During branchial arch development frem3 is expressed in the endodermal pouches (K–O; in section P,Q,R, red arrowheads). Expression is also noted in the otic placode (F, arrow) and by later developmental stages this expression is highest in the dorsolateral aspect of the otic vesicle (N,Q). S–V: Expression is initiated in the musculature of the developing head and in the hyoid posterior ectodermal margin and extending into the medial ectoderm (S–U, and in section, V). W: Expression in the operculum persists during later embryogenesis. nt, neural tube; nc, notochord; hb, hindbrain; bae, branchial arch endoderm; ov, otic vesicle; pff, pectoral fin fold; som, somite; pfm, pectoral fin muscle; op, oropharynx; ia, intermandibularis posterior muscle; ope, operculum.

Download figure to PowerPoint

Hypochord Expression of frem3

Shortly before the tail rudiment begins to extend from the yolk at 16 hpf, frem3 is expressed in the hypochord, a single stripe of midline cells ventral to the notochord (Fig. 4E–G,I). The hypochord arises from the lateral edges of the organizer during development (Latimer et al.,2002) and its secretion of VEGF has been implicated in the establishment of the dorsal aorta (Cleaver and Krieg,1998). We did not detect significant expression of frem3 before the 10-somite stage nor did we observe an expression pattern similar to that of the hypochord precursor markers notch5 or her4 (Latimer et al.,2002). This suggests that frem3 expression is activated only when cells of the hypochord become significantly differentiated from a midline precursor population; an observation suggesting characteristic changes in ECM complement when cells of the hypochord differentiate. Similar expression of other ECM components at this stage have previously been noted in zebrafish (Yan et al.,1995; Higashijima et al.,1997) and morphological studies of the hypochord have shown that it develops ultrastructural characteristics of an epithelia, including a well-defined basal lamina (Eriksson and Lofberg,2000). Mammalian studies suggest that many Fras/Frem interactions also take place in the context of a basement membrane (reviewed in Short et al.,2007). We noted persistent expression of frem3 in this structure until approximately 27 hpf, the point at which expression in the myotome is also beginning to decline.

Other Expression of frem3

Cells of the otic vesicle also express frem3 shortly after its formation at the 20-somite stage (19 hpf), which persists until at least 60 hpf (Fig. 4F,J–L,O,S). In the vesicle, staining is restricted to the dorsal and ventral aspects of lateral surface ectoderm (Fig. 4N,Q). We also observed frem3 expression in the developing pharyngeal arches from around 22 hpf. Sections of embryos at this and later stages detail robust frem3 staining in the endoderm of all of the pharyngeal arches with strongest staining in the second (hyoid) arch (Fig. 4L–N,P,Q). After 50 hpf, expression decreases in all but the posterior ectodermal margin (PEM) of the hyoid arch. Unlike frem1a and fras1, expression of frem3 in the PEM is quite broad, extending up much of the medial side of the arch and several cell diameters across the lateral margin (Fig. 4T,V). PEM expression was evident in the later stages we examined (Fig. 4W). As is the case during somitogenesis, frem3 expression is also activated in the developing musculature of the jaw and face as it develops around 56 hpf (Fig. 4T–V).

Expression of the Fras and Frem Genes During Fin Morphogenesis

The caudal fin fold of the zebrafish develops from a sheet of cuboidal epidermal cells approximately six to nine cells wide which undergo an orchestrated remodeling to form a folded epidermal structure (Dane and Tucker,1985). This morphological study has indicated that the formation of the fin fold is mediated largely by alterations in the shape of these cells rather than by proliferation, and specifically by changes in their extracellular matrix. During this remodeling process the basomedial edges of the cuboidal cells come together at the midline resulting in the formation of a “fan” of wedge shaped cells which subsequently separate from the underlying mesoderm to form a distinct ridge. The juxtaposed cells on either side of this ridge establish a transient basal association which is subsequently interrupted by the formation of a basal lamina surrounding an ECM rich subepidermal space in which the actinotrichia will eventually form. The apical cell in this ridge, known as the “cleft” cell, is morphologically distinct from the other “ridge” cells which flank it, and it has been suggested that this specialized cell type might play a distinct functional role in the subsequent development of the fin (Dane and Tucker,1985).

We noted that both fras1 and frem1a are coexpressed in medial cells of the developing fin fold shortly after the outgrowth of the tail bud from the yolk (20 hpf; Fig. 5A,D), an observation confirmed by two-color in situ hybridization (Fig. 5T,U). Initially, the genes are expressed in a band ∼2–3 cell diameters wide (Fig. 5A,D); however, as the fin fold matures this expression consolidates to a line of cells in the midline of the fin fold (Fig. 5F,I,K,N). Sectioning of stained embryos indicated that expression at this stage is restricted to the apical cleft cell (Fig. 5P,S). frem3, on the other hand, is expressed in two distinct bands of cells on either side of the midline around the same time that fras1 and frem1a is initiated (Fig. 5C). At the time when expression of these genes is initiated in the caudal fin fold, there has been little or no morphological differentiation, indicating that differential gene expression is established before the remodeling of these cells. As the fin fold differentiates, frem3 expression is maintained in these lateral cell populations (Fig. 5H,M) and as the fold separates from the mesoderm it is expressed in a position consistent with the ridge cells (Fig. 5R). Two-color in situ hybridization confirmed that frem3 expression is complimentary to that of fras1 and frem1a (Fig. 5E,J,O). The expression of frem2a is more complex. At early stages, highest expression is detected in frem3 expressing ridge cells (Fig. 5B) but within several hours expression spreads to include cleft and ridge cells (Fig. 5G,L,Q). Expression of all four genes in the caudal fin fold persists up to 60 hpf. Our investigations also highlighted a similar complementary pattern of gene expression in the developing pectoral fin (Figs. 2–4 and data not shown), suggesting that mechanisms of differentiation are shared between different types of growing fin.

thumbnail image

Figure 5. Expression of the frem and fras genes during median fin morphogenesis. Upper panel: Expression of the frem/fras genes in the developing fin fold. At 20 hours postfertilization (hpf), expression of frem1a and fras1 are detected in a midline stripe of cells (A,D), whereas frem3, and to a lesser extend frem2a, are expressed in two complementary cell populations cells (B,C). At 27 hpf, this pattern of complementary expression is continued, although frem2a is now expressed in both medial and lateral cell populations (F–I, dorsal/ventral; K–N, posterior. Dual in situ hybridization confirms complementary expression of frem3 and frem1a (E, lateral; J, section; O, lateral) and co-expression of frem1a and fras1 (T,U). Sections of embryos at 27 hpf confirm the observation made in whole embryos (P–S, caudal fold). Lower panel: We propose a model of complementary and overlapping gene expression, which corresponds to ridge and cleft cell populations first identified morphologically by Dane and Tucker,1985. nc, notochord.

Download figure to PowerPoint

Our studies also indicate that the fin fold, and the expression of the fras and frem genes within it, varies significantly in both an anterior–posterior and dorsal–ventral axes. This variation appears to be driven both by the stage of differentiation of the fold and by its position on the body. These differences invariably relate to the relative thickness of the fold itself, rather than in the complementary patterns of gene expression which we observe as the fold differentiates. We also note a significant and extensive degree of intercalation between cells of the fold, especially of those which correspond to the cleft position. To the best of our knowledge, the fras and frem genes represent the first well characterized markers of the cleft and ridge cells of the developing fish fin fold. Furthermore, more precise, characterization of differential expression of other genes might therefore provide a useful model system by which to study protein interactions in differentiating epithelia.

CONCLUSIONS

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

We have investigated the complement and expression of the frem/fras genes in zebrafish and have identified gene duplications and structural alterations in this gene family. The most notable of these is the difference in gene structure encoding orthologous Frem3 proteins, in which a “complete” C-terminal domain comprising 5 Calxβ, a transmembrane and a PDZ interaction domain are present in fish but are “lost” in higher eutherians. This difference is significant, because in mouse Fras1 and Frem2, these domains mediate their interaction and subsequent traffic with the intracellular Grip1 protein, as evidenced by the mislocalization of these proteins in its absence (Takamiya et al.,2004; Kiyozumi et al.,2006). Consequently, the absence of these domains in the mouse essentially renders Frem3 independent of Grip1, notionally allowing the protein to assume functions independent of the Frem2-Frem1-Fras1 complex. Indeed both the semi-independent pattern of Frem3 expression in the mouse, and the observation that its normal localization is refractory to perturbations of the ternary complex (Kiyozumi et al.,2007) suggest that this is the case. It is notable that, in the fish, expression of frem3 most closely mirrors that of Frem2 in mice, with strong but transient expression in the developing musculature and branchial arch endoderm (Jadeja et al.,2005, and our unpublished data), whereas in the mouse, Frem3 expression is restricted relative to Frem1, Frem2, and Fras1 (Kiyozumi et al.,2007).

In assessing the similarities between the developmental expression patterns of the fish and mouse Frem/Fras1 genes, it is evident that there is an excellent correlation with respect to regions in which basal lamina are being formed/remodeled and in which epithelia are undergoing structural rearrangement (e.g., hypochord, fin fold, PEM). The blistering observed in the blebs mice and FS patients is thought to be a consequence of fragility of these basement membranes and studies of mutants or morpholino induced knockdowns of these genes in the fish will be required to determine whether this essential function is conserved in fish. We have, however, detected only low levels of frem/fras1 expression in the zebrafish ectoderm (other than the fin fold) and no obvious expression in the differentiating nephrons of the pronephros. The kidney is an organ severely affected in mammalian mutants of these proteins (Smyth and Scambler,2005), and the genes are expressed at very early stages of kidney differentiation in the mouse. However, on the basis of the embryonic stages surveyed as part of these studies, we cannot exclude a role for the proteins in the later forming zebrafish mesonephros.

Our results demonstrate that exquisite gene regulation characterizes the initial stages of specification and morphogenesis of the fin fold, identifying markers of cell types previously only studied at the morphological level. Our previous studies of mouse organogenesis suggested that Frem1 is expressed in a complementary pattern to Fras1 and Frem2 in regions of epithelial–mesenchymal interaction (Smyth et al.,2004), although recent studies indicate that Frem1 protein is detected in epithelial and distinct cell types in certain contexts (Petrou et al.,2007a). In the fin fold, a region of extensive matrix/basement membrane synthesis, this pattern of divergent and coexpression is also apparent and we propose the teleost fin fold as a model system in which to study these interactions. Whether these expression differences reflect changes in modularity and reshuffling of gene expression over evolutionary time is unclear, however further studies of fras and frem genes in the fish will shed light on their specific roles in altering cell shape and adhesion and provide an insight into the etiology of Fraser syndrome.

EXPERIMENTAL PROCEDURES

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

Bioinformatic Analysis

Sequence searches and comparisons were made using Blast and Blast2seq and gene prediction using Genewise using previously defined vertebrate gene and protein sequences as templates (Smyth et al.,2004) and predictions were supported by RT-PCR generated and expressed sequence tag (EST) sequences. Coding sequences for all six genes are available as Genbank Accessions BK006468-BK00647, and these submissions include identifiers for EST and RT-PCR sequences used in the assembly of cDNAs. Multiple alignments of the genes/proteins were made using MUSCLE and CLUSTALW. All sequences used were from Ensembl v.46 (Aug 2007) and analysis was undertaken using Zebrafish (Zv.7 Apr 2007), Medaka (HdrR Oct 2005), Fugu (FUGU4.0, Jun 2005), Tetraodon (TETRAODON 8, Mar 2007), and Stickleback (BROAD S1, Feb 2006) genome assemblies. Assembly of all phylogenetic trees were constructed by the neighbor-joining method, based on the proportion of amino acid sites at which sequences compared were different and omitting alignment gaps as necessary using the Pairwise-deletion option. The reliability of each branch was assessed using 1,000 bootstrap replications. All phylogenetic trees were carried out using MEGA software.

In Situ Hybridization and Histology

Embryos were collected from matings of golden or St Kilda Aquarium stock zebrafish and staged according to the criteria of (Kimmel et al.,1995). Embryos were fixed in fresh 4% paraformaldehyde, dehydrated in methanol and stored at −20°C until use. In situ probes to fras1 (674-bp probe, nucleotides 9683-10357, GenBank accession #BK006468), frem1a (630-bp probe, nucleotides 936-1566, GenBank accession #BK006469), frem1b (653-bp probe#1, nucleotides 3326-3979; 344-bp probe#2, nucleotides 2347-2691, GenBank accession #BK006470), frem2a (523-bp probe, nucleotides 8649-9172, GenBank accession #BK006471), and frem2b (343-bp probe#1, nucleotides 4746-5089; 505-bp probe#2, nucleotides 8601-9106, GenBank accession #BK006472), and frem3 (795-bp probe, nucleotides 246-1041, GenBank accession #BK006473) were amplified from total RNA derived from 24 hpf embryos and subcloned into pGEM-TEasy (Promega). Control sense probes were included for all genes. In situ probe synthesis was performed using the digoxigenin RNA Labeling Kit (SP6/T7; Roche Diagnostics). Single- and two-color stained in situ hybridization was performed as described by (Jowett,1999). Stained embryos were paraffin embedded and 5- to 7-μm sections were de-waxed and in some cases counterstained with Nuclear Fast Red (Vector Laboratories) before dehydration and mounting under DEPEX (Sigma Aldrich). Whole-mount in situ stained embryos were cleared and mounted in 75% glycerol for imaging on a Leica SZ16 stereo microscope or Olympus DX70 upright microscope.

Acknowledgements

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

We thank Dave Keenan, Andrew Trotter, Kelly Turner, and the staff of the Ludwig Institute aquarium for their assistance and Dave Daggett and Pete Currie for performing initial in situ hybridizations. We also thank Joan Heath for providing embryos and Joan Heath, Robert Bryson-Richardson, and Heather Verkade for critical discussions and review of the manuscript.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. CONCLUSIONS
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  • Brand M, Heisenberg CP, Warga RM, Pelegri F, Karlstrom RO, Beuchle D, Picker A, Jiang YJ, Furutani-Seiki M, van Eeden FJ, Granato M, Haffter P, Hammerschmidt M, Kane DA, Kelsh RN, Mullins MC, Odenthal J, Nusslein-Volhard C. 1996. Mutations affecting development of the midline and general body shape during zebrafish embryogenesis. Development 123: 129142.
  • Chiotaki R, Petrou P, Giakoumaki E, Pavlakis E, Sitaru C, Chalepakis G. 2007. Spatiotemporal distribution of Fras1/Frem proteins during mouse embryonic development. Gene Expr Patterns 7: 381388.
  • Cleaver O, Krieg PA. 1998. VEGF mediates angioblast migration during development of the dorsal aorta in Xenopus. Development 125: 39053914.
  • Dane PJ, Tucker JB. 1985. Modulation of epidermal cell shaping and extracellular matrix during caudal fin morphogenesis in the zebra fish Brachydanio rerio. J Embryol Exp Morphol 87: 145161.
  • Eriksson J, Lofberg J. 2000. Development of the hypochord and dorsal aorta in the zebrafish embryo (Danio rerio). J Morphol 244: 167176.
  • Higashijima S, Nose A, Eguchi G, Hotta Y, Okamoto H. 1997. Mindin/F-spondin family: novel ECM proteins expressed in the zebrafish embryonic axis. Dev Biol 192: 211227.
  • Jadeja S, Smyth I, Pitera JE, Taylor MS, van Haelst M, Bentley E, McGregor L, Hopkins J, Chalepakis G, Philip N, Perez Aytes A, Watt FM, Darling SM, Jackson I, Woolf AS, Scambler PJ. 2005. Identification of a new gene mutated in Fraser syndrome and mouse myelencephalic blebs. Nat Genet 37: 520525.
  • Jowett T. 1999. Analysis of protein and gene expression. San Diego: Academic Press. p 6385.
  • Kimmel CB, Ballard WW, Kimmel SR, Ullmann B, Schilling TF. 1995. Stages of embryonic development of the zebrafish. Dev Dyn 203: 253310.
  • Kiyozumi D, Sugimoto N, Sekiguchi K. 2006. Breakdown of the reciprocal stabilization of QBRICK/Frem1, Fras1, and Frem2 at the basement membrane provokes Fraser syndrome-like defects. Proc Natl Acad Sci U S A 103: 1198111986.
  • Kiyozumi D, Sugimoto N, Nakano I, Sekiguchi K. 2007. Frem3, a member of the 12 CSPG repeats-containing extracellular matrix protein family, is a basement membrane protein with tissue distribution patterns distinct from those of Fras1, Frem2, and QBRICK/Frem1. Matrix Biol 26: 456462.
  • Kudon T, Tsang M, Hukriede NA, Chen X, Dedekian M, Clarke CJ, Kiang A, Schultz S, Epstein JA, Toyama R, Dawid IB. 2001. A gene expression screen in zebrafish embryogenesis. In: ZFIN direct data submission.
  • Latimer AJ, Dong X, Markov Y, Appel B. 2002. Delta-Notch signaling induces hypochord development in zebrafish. Development 129: 25552563.
  • McGregor L, Makela V, Darling SM, Vrontou S, Chalepakis G, Roberts C, Smart N, Rutland P, Prescott N, Hopkins J, Bentley E, Shaw A, Roberts E, Mueller R, Jadeja S, Philip N, Nelson J, Francannet C, Perez-Aytes A, Megarbane A, Kerr B, Wainwright B, Woolf AS, Winter RM, Scambler PJ. 2003. Fraser syndrome and mouse blebbed phenotype caused by mutations in FRAS1/Fras1 encoding a putative extracellular matrix protein. Nat Genet 34: 203208.
  • Petrou P, Chiotaki R, Dalezios Y, Chalepakis G. 2007a. Overlapping and divergent localization of Frem1 and Fras1 and its functional implications during mouse embryonic development. Exp Cell Res 313: 910920.
  • Petrou P, Pavlakis E, Dalezios Y, Chalepakis G. 2007b. Basement membrane localization of Frem3 is independent of the Fras1/Frem1/Frem2 protein complex within the sublamina densa. Matrix Biol 26: 652658.
  • Shafeghati Y, Kniepert A, Vakili G, Zenker M. 2008. Fraser syndrome due to homozygosity for a splice site mutation of FREM2. Am J Med Genet A 146: 529531.
  • Short K, Wiradjaja F, Smyth I. 2007. Let's stick together: the role of the Fras1 and Frem proteins in epidermal adhesion. IUBMB Life 59: 427435.
  • Slavotinek A, Li C, Sherr EH, Chudley AE. 2006. Mutation analysis of the FRAS1 gene demonstrates new mutations in a propositus with Fraser syndrome. Am J Med Genet A 140: 19091914.
  • Smyth I, Scambler P. 2005. The genetics of Fraser syndrome and the blebs mouse mutants. Hum Mol Genet 14 Spec No. 2: R269R274.
  • Smyth I, Du X, Taylor MS, Justice MJ, Beutler B, Jackson IJ. 2004. The extracellular matrix gene Frem1 is essential for the normal adhesion of the embryonic epidermis. Proc Natl Acad Sci U S A 101: 1356013565.
  • Staub E, Hinzmann B, Rosenthal A. 2002. A novel repeat in the melanoma-associated chondroitin sulfate proteoglycan defines a new protein family. FEBS Lett 527: 114118.
  • Takamiya K, Kostourou V, Adams S, Jadeja S, Chalepakis G, Scambler PJ, Huganir RL, Adams RH. 2004. A direct functional link between the multi-PDZ domain protein GRIP1 and the Fraser syndrome protein Fras1. Nat Genet 36: 172177.
  • Thisse B, Thisse C. 2004. Fast release clones: a high throughput expression analysis. In: ZFIN direct data submission.
  • Vrontou S, Petrou P, Meyer BI, Galanopoulos VK, Imai K, Yanagi M, Chowdhury K, Scambler PJ, Chalepakis G. 2003. Fras1 deficiency results in cryptophthalmos, renal agenesis and blebbed phenotype in mice. Nat Genet 34: 209214.
  • Winter RM. 1990. Fraser syndrome and mouse 'bleb' mutants. Clin Genet 37: 494495.
  • Yan YL, Hatta K, Riggleman B, Postlethwait JH. 1995. Expression of a type II collagen gene in the zebrafish embryonic axis. Dev Dyn 203: 363376.