1 Box 351800, Zoology Department and Center for Developmental Biology, University of Washington, Seattle, WA 98195-1800, USA
2 Friday Harbor Laboratories, 620 University Road, University of Washington, Friday Harbor, WA 98250-9299, USA
*Author for correspondence (e-mail: bjswalla{at}u.washington.edu)
Accepted 13 June 2002
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SUMMARY |
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Key words: Ascidian, Metamorphosis, Innate immunity, Subtractive hybridization
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INTRODUCTION |
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Initially, hatched larvae are unable to respond to either natural or artificial settlement cues. Within a discrete, species-specific number of hours after hatching, larvae become competent to respond to settlement cues and commence a series of rapid morphological changes (Degnan et al., 1997). Within the first 20 minutes of settlement, the papillae facilitate initial attachment, the tail is rapidly resorbed and the trunk mesenchyme undergoes extensive migration both within the body and across the epidermis into the tunic (Fig. 1C-G). During the next few hours, the newly settled juvenile molts the outer larval tunic, extends epidermal ampullae, rotates the viscera to the adult feeding position, and begins to resorb the cerebral vesicle (Cloney, 1978
) (summarized in Fig. 1H,I). The rudiments of adult structures including the heart, blood cells, musculature, gut and chordate pharyngeal slits then differentiate over a period ranging from days to weeks within the settled juvenile (Fig. 1B) (Hinman et al., 2000
; Jeffery and Swalla, 1997
).
Competent ascidian larvae can be induced to undergo settlement by a wide variety of both natural and artificial settlement cues (Cloney, 1990; Degnan et al., 1997
). These include a variety of stressful stimuli such as trauma, crowding and osmotic changes. We have recently demonstrated that Boltenia larvae also settle in response to specific bacterial cues (B. D., B. J. S. and A. Aderem, unpublished). In addition, research has indicated that nitric oxide and Hsp-90 signaling may be involved in the initial settlement response (Bishop et al., 2001
; Jackson and Swalla, 2001
). Work in the Degnan lab on Herdmania curvata has established that settlement is triggered by a signal secreted at the anterior papillary region of the larvae (Degnan et al., 1997
). They have further demonstrated a crucial role for EGF signaling in triggering settlement (Eri et al., 1999
). The EGF-like molecule Hemps is secreted at the initiation of settlement by a group of anterior cells termed the papillae associated tissue (PAT) cells (Eri et al., 1999
). They demonstrated that application of an antibody against Hemps specifically blocks settlement, whereas exposure of competent larvae to Hemps protein induces settlement (Eri et al., 1999
). We have shown that a Boltenia cornichon homolog expressed in this anterior region during competency may potentiate the Hemps EGF signaling pathway (Davidson and Swalla, 2001
). In this report, we demonstrate that these PAT cells are actually connected to the external environment by a tunnel through the anterior tunic and that they migrate out through this tunnel during metamorphosis (Davidson et al., 2001
). Thus the PAT cells are positioned to respond to external signals and trigger settlement through the secretion of Hemps.
We employed suppressive subtractive hybridization in order to examine gene expression during metamorphosis in the solitary ascidian, Boltenia villosa. We conducted three screens focused on differential gene expression during: (1) the acquisition of larval competence, (2) the first hour of settlement and (3) the first two days of juvenile differentiation. These screens have led to the isolation of several transcripts whose putative proteins have been identified through matches in GenBank. These identified transcripts include genes with potentially interesting roles in the post-larval differentiation of adult rudiments (Nakayama et al., 2001) (B. D., S. Smith and B. J. S., unpublished). Intriguingly, a significant proportion of the identifiable transcripts putatively code for proteins involved in invertebrate innate immunity, including Mannose-specific lectin (MBL), MBL-associated serine protease (MASP), Hemocytin, four complement factors, two selectins, two von Willibrand factors, and Pentraxin.
Although it is established that ascidians are capable of innate immune-related reactions such as inflammation and cytotoxicity, it is only recently that the molecules involved in the ascidian innate immune system have been identified. It is now clear that ascidians possess a mature complement system, including both the lectin and alternative pathways (Nonaka, 2000). Research also indicates that ascidian immune responses include the use of an IL1-like cytokine that can activate and guide blood cells (Raftos et al., 1998
). In addition, a variety of lectins have been identified in ascidians with putative roles in pathogen recognition (Abe et al., 1999
; Kenjo et al., 2001
; Matsumoto et al., 2001
; Nair et al., 2001
).
Our isolation and characterization of several innate-immunity-related transcripts expressed during Boltenia metamorphosis includes several novel ascidian immune-related genes but, more importantly, it is the first description of the developmental expression of innate immune-related genes in an ascidian. Our results detail the temporally discrete upregulation of innate immune-related genes during B. villosa post-larval development and metamorphosis. Such discrete upregulation suggests that innate immunity may have an unforeseen developmental role during ascidian metamorphosis. Innate immune-related genes have described roles in vertebrate developmental processes, particularly related to restructuring of differentiated tissues. For example, it has been shown that the complement component C3 is expressed during regeneration in both the axolotol limb (Del Rio-Tsonis et al., 1998) and during liver regeneration (Mastellos et al., 2001
).
Here, we survey the initial results of our three subtractive hybridization screens. We then characterize the expression of several of the immune-related transcripts and discuss our hypotheses about the possible roles for innate immunity in ascidian metamorphosis. Characterization and discussion of other isolated transcripts with putative developmental roles will be published separately (B. D., S. Smith and B. J. S., unpublished). Research on the specific functions of these genes during ascidian metamorphosis is ongoing in the Swalla laboratory.
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MATERIALS AND METHODS |
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Subtractive hybridization
Total RNA was isolated from 50-100 mg samples of pre-competent larvae, competent larvae and 1 hour juveniles using RNAzol B (Tel-Test, Friendswood, TX, USA), a guanidinium thiocyanate-phenol-chloroform method. PolyA+ RNA was collected from 50 mg-100 mg samples of 1 hour and two day juveniles using magnetized oligo dT beads (Dynal, Oslo, Norway). cDNA was then prepared by oligo-dT reverse transcription and amplified following the protocols of the Clontech Smart-PCR cDNA synthesis kit (Clontech Laboratories, Palo Alto, CA, USA).
Differentially expressed transcripts were isolated using a series of three suppressive subtractive hybridization screens including: (1) Competent larval cDNA for the tester and pre-competent larval cDNA as the driver; (2) 1 hour juvenile cDNA for the tester and competent larval cDNA as the driver; and (3) two day juvenile cDNA for the tester and 1 hour juvenile cDNA as the driver following the protocols of the Clontech PCR-select cDNA subtraction kit. The resulting differentially expressed transcripts were ligated into the pT-Adv Vector (Clontech TA cloning; Clontech) and colonies were randomly selected, prepped and then sent off for sequencing by the UW Health Sciences Sequencing Facility. Additional sequencing was also conducted at the Institute for Systems Biology (Seattle, WA, USA). Protein homologies were detected through NCBI Blast (Altschul et al., 1997).
RT-PCR analysis
PolyA+ RNA was collected from 50-100 mg samples of 6 hour (gastrula), 11 hour (tailbud) and 16-hour-old embryos, larvae 2 and 11 hours after hatching (pre-competent and competent), and juveniles 1 hour, two days, four days and 10 days after induction of settlement using magnetized oligo dT beads (Dynal). These samples were then reverse transcribed using random primers. RT-PCR was performed on equalized amounts of these cDNA samples using the following protocol: 2 minutes at 95°C then 20-25 cycles: 95°C for 45 seconds, 50°C for 45 seconds and 72°C for 80 seconds, using primers specific for each gene of interest. Primers specific to B. villosa 16S ribosomal RNA were used as a control.
Isolation of full-length transcripts
Rapid amplification of cDNA ends was used to generate 5' and 3' PCR products. A pool of double stranded cDNA from larval and juvenile stages was initially isolated for subtractive hybridizations, amplified following the protocols of the Clontech Smart-PCR cDNA synthesis kit (Clontech Laboratories) (see above), and was then ligated to double stranded adaptors (Marathon cDNA Amplification Kit, Clontech). PCR was used to amplify 3' and 5' ends of specific transcripts using gene-specific primers and primers that anneal to the ligated adaptors as described in the Clontech protocol. Gel purified PCR products were cloned into the pT-Adv Vector (Clontech TA cloning) and sequenced at the Institute for Systems Biology.
Propidium iodide staining
Larvae and juveniles were fixed at 4°C in 4% (w/v) paraformaldehyde in 100 mM HEPES pH 6.9, 2 mM MgSO4, 1 mM EGTA for 24-48 hours. They were then washed three times for 10 minutes in phosphate-buffered saline (PBS) and stored at 4°C in PBS. Animals were then treated with 20 µg/ml RNase A for 2 hours at 37°C, followed by 2 µg/ml PI for 1 hour. Samples were washed four times in PBS and mounted in PBS:glycerol (1:1). Fluorescent images were obtained on a BioRad 600 laser scanning confocal microscope.
Whole-mount in situ hybridization
Larvae and juveniles were fixed at 4°C in 4% (w/v) paraformaldehyde in 100 mM HEPES pH 6.9, 2 mM MgSO4, 1 mM EGTA for 24-48 hours. They were then dehydrated in 50% ethanol, then 80% ethanol (30 minutes each) and stored at 20°C in 80% ethanol. Digoxigenin-labeled antisense probes were synthesized from linearized plasmids according to the protocols supplied with the DIG RNA Labeling kit (Roche Molecular Biochemicals, Indianapolis, IN, USA). Whole mount in situ hybridizations were performed by a similar protocol to those of Swalla et al. (Swalla et al., 1994). Samples were washed with phosphate buffered saline with 0.1% Tween 20 (PBT) then treated with 10 µg/ml Proteinase K in PBT at 37°C for 10 minutes. The reaction was stopped in 2 mg/ml glycine in PBT, then washed with PBT. Samples were post-fixed in 4% paraformaldehyde in PBS, washed with PBT and treated with 0.25% anhydrous acetic acid in 0.1 M triethanolamine (pH 8.0) prepared just before use. Samples were hybridized overnight at 45°C, washed with 2XSSC at 45°C and treated with 20 µg/ml RNase at 37°C. Samples were blocked in 0.1% blocking reagent in PBT, then incubated in 1/2000 anti-DIG-AP in PBT, both from the DIG Nucleic Acid Detection Kit (Roche Molecular Biochemicals). AP detection buffer contained levamisole and NBT/BCIP. After the desired staining was reached, samples were rinsed in PBS. Samples were then mounted in benzyl alcohol:benzyl benzoate after being dehydrated through a series of ethanol washes: 30%, 50%, 80%, 90%, 100% along with two washes in benzyl alcohol:benzyl benzoate 1:1.
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RESULTS |
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An overview of the results for each subtraction is displayed in Table 1. Of the 195 transcripts sequenced, 132 are potential protein-coding genes. Only 37 transcripts were duplicates (representing 16 unique sequences), indicating that the isolated transcripts comprise only a fraction of the diverse genes transcribed during the targeted time periods. Only matches with less than a 1.0 e5 probability of a chance occurrence were classified as significant. Of the 132 potential proteins, 65 showed a significant match to known proteins or protein domains. In addition there were 26 sequences matching mitochondrial 16S rRNA.
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A complete list of identified transcripts is displayed in Table 2. Transcripts are sub-divided into the three screens by which they were identified. In addition each transcript has been assigned a provisional functional identity based on its match to known proteins. These seven functional categories include housekeeping, extracellular matrix, cytoskeletal, muscle-related, stress proteins, developmental (signaling and transcription) and immune-related proteins (as described in the Table legend). Some proteins are assigned to more than one potential category, as they have overlapping functions.
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Isolation of full-length transcripts
The majority of the transcripts isolated in our screens represented fragments of the full-length cDNAs ranging in size from 200 bp to 2 kb. Many of these fragments contained the 3' end of the transcript. We employed RACE to isolate the 5' and 3' ends of these incomplete fragments, focusing specifically on some of the immune-related transcripts, whose identities were initially unclear. A summary of our results is displayed in Table 3, which describes the length and match of the fuller length fragments that we obtained.
Temporal expression of isolated transcripts
In order to determine when the immune-related transcripts were first expressed and how their expression changed over time, we conducted a series of RT-PCR reactions. This series included all the stages used in our screens (pre-competent larvae, competent larvae, 1 hour post-settlement, and two-day old juveniles). We also extended the series to include stages representing embryogenesis and juvenile differentiation: fertilized eggs, gastrulae, tailbud and 16 hours post-fertilization (midway between tailbud and hatching), four-day old juveniles (when differentiation of organ rudiments is just beginning), and 10-day old juveniles (which have completed differentiation of the organs necessary to begin feeding). Therefore, our series represents a complete range of developmental stages from fertilized egg to feeding juvenile.
We have conducted RT-PCR reactions for seven immune-related transcripts (shown in Fig. 2) along with 10 transcripts with putative developmental roles (Bv-Crn is included in Fig. 2 and the rest will be published separately). 16S mitochondrial ribosomal transcript levels were used as controls.
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The majority of the immune-related transcripts display dynamic patterns of temporal expression (Fig. 2). Bv-VWa1, Bv-Ptx and Bv-Ccp3 show distinct peaks of expression followed by declining levels of expression. Bv-LRR and Bv-Ccp2 had the most dynamic expression patterns, both displaying two peaks of expression during larval or post-larval development. Only Bv-Masp and possibly Bv-Sccp2 showed more linear patterns of temporal expression, both of them increasing during larval competence and then remaining relatively stable.
Of the 17 transcripts analyzed, 13 are distinctly upregulated at the time points at which they were isolated by subtractive hybridization (developmental transcript data not shown). This pattern is demonstrated in Fig. 2, in which arrowheads indicate the time points of the subtractive screens. Bv-Lrr, Bv-Vwa2, Bv-Ccp2, Bv-Ptx and Bv-Ccp3 all show dramatic upregulation at the expected time points. Bv-Crn displays a more gradual rise in expression. Bv-Sccp2 and Bv-Masp both show the greatest increase in expression at the acquisition of competence, prior to the post-settlement time point at which they were isolated, indicating that they were probably isolated because of a gradual increase in expression rather than a dramatic upregulation.
Expression patterns of immune-related transcripts
We have conducted whole mount in situ hybridizations for several of the immune-related transcripts, including Bv-MASP, Bv-Sccp2 (Selectin), Bv-Ptx (Pentraxin), Bv-Ccp3 (Complement factor 3) and Bv-Ccp2 (Complement factor 2). An overview of the expression patterns of these five transcripts is displayed in Fig. 3. We have also included the expression patterns from a non-immune-related transcript, Bv-HspBP2 (Hsp-70 binding protein), as a control (Fig. 3U-W). We have examined a range of different stages but show only the four stages included in our screens; namely, pre-competent larvae (Fig. 3A-E), competent larvae (Fig. 3F-U), 1 hour after settlement (Fig. 3K-V) and two days after settlement (Fig. 3P-W). In addition, Fig. 4 displays some more detailed photos of particular expression patterns. In the pre-competent larvae (Fig. 3A-E), none of the immune-related transcripts displayed detectable expression. In contrast, developmental transcripts are often expressed in the mesenchyme of pre-competent larvae, as shown for Bv-Crn (Fig. 4A). In competent larvae (Fig. 3F-U), Bv-Ptx is expressed at low levels in the anterior papillary region (Fig. 3H), whereas Bv-Sccp2, Bv-MASP and Bv-Ccp2 all display stronger expression in this same region (Fig. 3F,G,J). Bv-Ccp3 is expressed in the anterior trunk epidermis as well as in a subset of cells along the mid-trunk region where they appear to have migrated into the tunic (Fig. 3I). In contrast, non-immune transcripts show either no significant expression at this time (Bv-HspBP2; Fig. 3U) or show distinct patterns of expression (Bv-Crn; Fig. 4B). One hour after settlement, Bv-Ccp2 shows strong expression throughout the trunk epidermis (Fig. 3O). The other four immune genes are expressed in the anterior papillary region (Fig. 3K-N). Bv-Ptx is also expressed in the area of the resorbing cerebral vesicle (Fig. 3M; Fig. 4C). The control (Bv-HspBP2) shows no significant expression (Fig. 3V). We have conducted in situ hybridizations for Tenascin (Bv-Tenc) expression and have included a photo of Bv-Tenc expression 1 hour after settlement to show that this putative ECM remodeling gene is expressed in the resorbing cerebral vesicle, tail and muscle granules as well as in the papillary region (Fig. 4D). In juveniles, two days after settlement, Bv-MASP expression is limited to faint staining of the body wall epidermis (Fig. 3P). The other four immune-related transcripts are expressed strongly in the epidermis of the ampullae as well as body wall epidermis. (Fig. 3Q-T). At higher magnification of these two-day juveniles, expression of Bv-Sccp2 (Fig. 4E) and Bv-Ccp3 (Fig. 4F) can be discerned in both the epidermis and in nearby blood cells. In contrast, expression of Bv-HspBP2 is not observed in the ampullae, and displays a distinctive pattern concentrated around the bases of the ampullae (Fig. 3W).
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DISCUSSION |
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Innate immune signaling may coordinate the resorption of larval tissues
The comprehensive resorption of larval tissues may require a highly coordinated immune response. The differential transcription of genes involved in coagulation and targeting of tissues for phagocytosis, including complement factors, von Willebrand factors, Hemocytin and Pentraxin support this hypothesis. In addition, the differential transcription of putative extracellular matrix-modifying genes such as Tenascin c, Thrombospondin, Arylsulfatase and Tenascin-x indicate the possible involvement of immune-related cells, such as macrophages, in the restructuring and repairing of transforming tissues, in parallel with the function of these genes in similar vertebrate processes (Jones and Jones, 2000a; Jones and Jones, 2000b
; Murphy-Ullrich, 2001
). The complement system has also been implicated as playing roles in liver regeneration (Mastellos et al., 2001
) and in the regeneration of urodele limbs (Del Rio-Tsonis et al., 1998
). Our results demonstrating expression of Bv-Ptx (Pentraxin) and Bv-Tenc (Tenascin) in and around resorbing Boltenia larval tissues lends further support to these putative functions (Fig. 5C,D). A related hypothesis is that innate immunity is upregulated during metamorphosis not only to coordinate larval tissue resorption, but also as a response to the extraordinary levels of stress entailed in the death and reorganization of larval tissues. Such a hypothesis emerges from Matzingers danger model of immune activation (Gallucci and Matzinger, 2001
; Matzinger, 2002
). According to the danger model, immune activation is more responsive to damaged self-tissue than to non-self antigens. Matzinger describes how signals produced by stressed or damaged cells, including heat shock proteins and cell surface fragments, are integral activators of the immune system. Thus immune activation during Boltenia metamorphosis would not require the detection of a foreign antigen, as in classic immune responses, but only the detection of endogenous signals indicative of tissue damage.
Maturation of immune competence during metamorphosis
The upregulation of immune-related transcripts during metamorphosis may represent the programmed maturation of the adult immune system. The timing of this immune maturation may simply be tied to an overall maturation of adult tissues. Alternatively, settling ascidians may initiate immune competence to deal with the new set of threats posed by the benthic habitat they are about to occupy. Clearly, there is a much higher concentration of bacteria, fungus and other pathogens on the substrate than in the open ocean, and therefore ascidians may not need a mature immune system until settlement. The migration of blood cells into the tunic before and during settlement may be considered part of this immune maturation, converting the newly synthesized tunic into an immune-ready tissue containing immune-competent blood cells. Temporal expression patterns of some of the isolated immune transcripts (Bv-MASP and Bv-Sccp2) support this hypothesis as they are upregulated in larvae and then maintained at steady levels throughout juvenile differentiation (Fig. 2). However, the more dynamic temporal expression patterns of the other immune transcripts, particularly Bv-LRR and Bv-Ccp2, suggest a more specific developmental role.
Pan-epithelial migration of trunk mesenchyme during metamorphosis as an inflammatory process
During metamorphosis in both colonial and solitary ascidians, blood cells and mesenchymal stem cells undergo a variety of targeted migrations (Cloney, 1982; Cloney and Grimm, 1970
). Some migrate within the body, eventually forming the adult mesoderm, others are observed to migrate across the epidermis into the tunic. The discovery that inflammation-related genes are expressed during metamorphosis leads us to the novel hypothesis that this blood cell migration represents an inflammatory reaction akin to the extravasation of leukocytes across endothelia. The differential transcription of putative complement factors and selectins support this hypothesis (Fig. 7). Complement signaling is involved in initiating an inflammatory response (Goldsby et al., 2000
), whereas selectins are specifically involved in the initial adherence of migrating blood cells to the endothelia during extravasation (Vestweber and Blanks, 1999
). Furthermore, the expression of these same transcripts in the migrating blood cells and the areas of the epidermis (the anterior papillary region and ampullae) across which they migrate reinforce this hypothesis (Figs 3, 4). A similar pattern of cross-epithelia blood cell migration also occurs during inflammatory responses in colonial ascidians (Magor et al., 1999
; Rinkevich and Weissman, 1992
). A well-described inflammatory response occurs during rejection reactions between two unrelated ascidian colonies which contact each other. In this case, inflammation is mediated by blood cells that migrate across the ampullae epidermis bordering the colony, mirroring the similar migration of blood cells across the ampullae epidermis during metamorphosis in Boltenia.
Innate immune-related transcripts may have purely developmental functions
The upregulation of innate-immune-related genes during Boltenia metamorphosis does not necessarily involve an immune response per se. It may be that in B. villosa these immune-related proteins function in the developmental regulation of cell adhesion and migration. Some innate immunity genes, such as selectins and tolls, have overlapping immune and non-immune functions. Functional characterizations of upregulated genes will help to clarify if they actually represent an immune response or are part of more general developmental processes. Presently, our research into the response of B. villosa larvae to specific bacterial cues, discussed below, strongly supports our hypothesis that the upregulation of immune-related genes during metamorphosis is indicative of an immune response.
Innate immune signaling may underlie ascidian larval competence
Another novel hypothesis is that ascidians may use their innate immune system to initiate metamorphosis in response to specific bacterial cues. There is a growing body of research indicating the prevalent use of specific bacterial cues and/or host-produced sugar molecules by settling marine invertebrates to detect appropriate microhabitats and initiate settlement (Beckmann et al., 1999; Chia and Bickell, 1978
; Johnson and Sutton, 1994
; Maki and Mitchell, 1985
; Orlov, 1996
; Strathmann, 1978
; Unabia and Hadfield, 1999
). However, such responses have generally been assumed to involve detection by sensory neurons. Some researchers have previously hypothesized that marine invertebrate larvae may employ lectins to detect bacterial settlement cues, however, this was proposed in a non-immune context (Maki and Mitchell, 1985
; Orlov, 1996
).
Propidium iodide staining of Boltenia villosa larvae demonstrates that cell migration across the epidermis is occurring before metamorphosis (Fig. 5). This migration leads to a set of regularly spaced cells along the outside of the epidermis in competent larvae. These cells were previously undescribed and their function is still unknown. Thus, there are at least two waves of trans-epidermal cell migration, one, described here for the first time, occurs during the acquisition of competence and the other occurs soon after settlement (Cloney and Grimm, 1970). In addition, we have detected a group of cells, presumed to be the PAT cells (Eri et al., 1999
), that migrate through an anterior tunnel in the tunic into the external environment (Davidson et al., 2001
). This tunnel forms during the acquisition of larval competence (B. D., unpublished). Our Bv-Crn in situ hybridizations (Fig. 4A,B) along with the cell lineage work of Hirano and Nishida (Hirano and Nishida, 1997
) indicate that all of these migrating cells originate from the trunk mesenchyme. Together, these observations indicate that correct positioning of trunk mesenchyme cells through targeted migrations may have an important role in establishing competence to undergo metamorphosis.
Our in situ hybridization results indicate that migrating mesenchyme cells in the papillae and trunk region express immune-related transcripts including a putative Selectin (Bv-Sccp2) and two complement factors (Bv-Ccp2 and Bv-Ccp3). This may indicate that these immune-related genes mediate migration across an epithelial layer as they do in vertebrate inflammation (Fig. 7) (see above). Alternatively, expression of these genes may be involved in regulating adhesion and migration of mesenchyme cells in a manner unrelated to immunity (see above). A third possibility is that immune responsiveness of cells within the tunic, and those with access to the external environment through an anterior tunnel, may mediate the detection of and response to bacterial settlement cues.
We have recently completed a set of experiments demonstrating that Boltenia larvae will undergo metamorphosis in response to the presence of specific types of marine bacteria (B. D., B. J. S. and A. Aderem, unpublished). Our results indicate that this settlement response is mediated through the detection of peptidoglycans from the bacterial cell wall and can be inhibited through the application of immunosuppressant drugs (in preparation). Thus, our results strongly support our hypothesis that detection of bacterial settlement cues in Boltenia is mediated by the innate immune system. Furthermore, our data suggests that this immune reception of bacterial cues probably occurs in the PAT or tunic cells. The exposure of the PAT cells to the external environment through an anterior opening in the tunic, the central role of these cells in initiating metamorphic signaling (Eri et al., 1999), and the expression of putative innate immune mediators such as complement factors in the PATs and tunic cells together lend strong support to the idea that the PAT or tunic cells are employed to detect and respond to specific bacterial cues. This hypothesis has important implications for settlement of marine invertebrate larvae. The use of bacterial settlement cues is widespread among marine invertebrates, although the nature of the receptors and signaling systems mediating this response has remained largely unexplored. Conserved components of the innate immune system are ideally suited to coordinate the rapid physiological response to bacterial and/or host-specific cues involved in larval settlement.
Perspectives
We have begun to characterize some of the molecular signals involved in coordinating the complex developmental events of ascidian metamorphosis. Our results indicate that ascidian metamorphosis represents a valuable resource for exploring the origins of innate immune function in chordates. It is conjectured that the lack of an adaptive immune system in ancestral chordates and jawless vertebrates may be compensated for by a highly developed innate immune system (Nonaka, 2000; Smith et al., 2001
). The differential expression of a wide variety of innate immune-related factors during ascidian metamorphosis represents an excellent model for investigating innate immune function in ascidians, and raises intriguing possibilities of an overlap between developmental and immune signaling during invertebrate metamorphosis. In order to discover the function of this metamorphic immune response, we are attempting to specifically disrupt single genes or components of the complement pathway and examine potential effects on metamorphosis.
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ACKNOWLEDGMENTS |
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