Expression of Hox genes during regeneration of nereid polychaete Alitta (Nereis) virens (Annelida, Lophotrochozoa)
- Elena L Novikova†1Email author,
- Nadezhda I Bakalenko†1,
- Alexander Y Nesterenko1 and
- Milana A Kulakova1
© Novikova et al.; licensee BioMed Central Ltd. 2013
Received: 29 November 2012
Accepted: 5 February 2013
Published: 2 May 2013
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Hox genes are the key determinants of different morphogenetic events in all bilaterian animals. These genes are probably responsible for the maintenance of regenerative capacities by providing positional information in the regenerating animal body. Polychaetes are well known for their ability to regenerate the posterior as well as the anterior part of the body. We have recently described the expression of 10 out of 11 Hox genes during postlarval growth of Alitta (Nereis) virens. Hox genes form gradient overlapping expression patterns, which probably do not contribute to the morphological diversity of segments along the anterior-posterior axis of the homonomously segmented worm. We suggest that this gradient expression of Hox genes establishes positional information along the body that can be used to maintain coordinated growth and regeneration.
We showed that most of the Hox gene expression patterns are reorganized in the central nervous system, segmental ectoderm and mesoderm. The reorganization takes place long before regeneration becomes apparent. The most rapid reorganization was observed for the genes with the largest differences in expression levels in the amputation site and the terminal structures (pygidium and growth zone). Moreover, we revealed the expression of two antisense Hox RNAs (Nvi-antiHox5 and Nvi-antiHox7) demonstrating unique expression patterns during regeneration.
Hox genes probably participate in the maintenance and restoration of the positional information in A. virens. During postlarval growth and regeneration, Hox genes do not alter the diversity of segments but provide the positional information along the anterior-posterior axis. The reorganization of at least some Hox gene patterns during regeneration may be regulated by their anti-sense transcripts, providing a rapid response of Hox gene transcripts to positional failure. The capacity of Hox genes to maintain the positional information in the adult body is present in different bilaterian animals (planarias, polychaetes and mammals) and might be an ancestral function inherited from the common evolutionary remote ancestor.
KeywordsRegeneration ncRNA Hox genes Positional information Polychaetes Lophotrochozoa
Regeneration capacity stands for the ability of the adult organism to restore lost parts of the body by de novo growth due to cell proliferation and/or reorganization of somatic tissues . Regeneration processes are studied in many animal models, including anamniotes (Anura and Urodella, fishes), insects (cricket), planaria and hydrozoa (Hydra), and mammals [2–6]. Intriguingly, regenerative capacities are dramatically different in different animals. On the one hand, planarians can grow any part of the body and even regenerate completely from a tiny body fragment, consisting of 1.5–4 thousands of cells, which makes up about 6–10% of the total cell count . On the other hand, mammals are capable of physiological regeneration [8–10] and can restore parts of internal organs after injury, e.g., the heart after myocardial infarction or the bone after fracture [11, 12]. Newborn mice and children can regenerate fingers after injury or amputation of the distal phalanx .
It was noticed years ago that molecular programs of reparative and embryological morphogenesis have several features in common. For instance, embryonic patterns of key transcriptional factors are restored during regenerative processes, e.g., in the case of Sonic hedgehog (Shh), FGF, Wnt and Hox genes [3, 13–16]. The question arises what differences in molecular factor functioning during regeneration of different animals define their varying capacities for reparative morphogenesis.
Hox genes encode transcriptional factors, which are involved in multiple morphogenetic processes . Their main and conservative function is considered to be the regionalization of the anterior-posterior (AP) axis in both protostome and deuterostome animals during embryonic development. Hox genes pattern the bilateral body according to the rules of spatial and temporal colinearity . At the same time, Hox genes were also shown to participate in processes in the adult body. Hox genes are necessary for reparative morphogenesis in different model organisms, e.g. planaria, zebrafish, axolotl and Xenopus laevis[4, 19–22]. In higher vertebrates, mostly mammals, Hox genes are expressed in the adult tissues capable of remodeling and/or constant renewal. Thus, in vertebrates the genes of the Hox cluster are involved in physiological regeneration, e.g., cyclic renewal of hair follicles  or hematopoiesis [9, 10]. Genes HOXA7, HOXB3, HOXA3 and HOXB13 were shown to regulate differentiation of mesenchymal stem cells (MSCs), which play an active role in reparative morphogenesis in vertebrates .
Expression analysis of genes in human dermal fibroblasts revealed differential Hox gene expression in fibroblasts with different localization. This “Hox code” is maintained during the whole cell life and is probably needed for the correct establishment of regenerative processes . It is becoming obvious that one of the main Hox gene functions in the adult organism is the maintenance of the positional information, which is provided by Hox proteins .
Previously we described the expression of 11 Hox genes in the ontogenesis of errant marine polychaetes A. virens (Nereididae, Annelida, Lophotrochozoa) and Platynereis dumerilii. The ontogenesis of nereididae polychaetes comprises the stages of spherical trochophore larva, segmented nechtochaete larva and postlarval growth . Hox genes demonstrated canonical collinear expression in segment larval ectoderm. However, during the formation of the definitive multisegmental worm’s body, they pattern the morphogenetic territory in a different, and unique, way. Hox genes are expressed as a gradient, their expression domains overlapping and not retaining the anterior boundaries in the postlarval segments (Bakalenko, Novikova and Kulakova, unpublished data). We suggest that in this case, Hox genes are involved in the establishment and maintenance of positional coordinates in the growing homonomously segmented body rather than in specification of segments with similar morphology. If it is indeed the case, the expression pattern of Hox genes should be reorganized after positional failure, for example, due to a loss of a body part.
Here we studied the expression dynamics of Hox genes during different regeneration stages of the polychaete A. virens. Nereid polychaetes, capable of rapid unipolar regeneration, are an excellent model for studying reparative morphogenesis. We believe that characteristic features of Hox gene behavior during regeneration of A. virens will support our hypothesis concerning the role of these genes in creating the postlarval worm’s body and partially explain the capability of these polychaetes for rapid axial regeneration.
Adult A. virens were collected near “Kartesh” Marine Biological Station of the Zoological Institute (Russian Academy of Sciences) in the Chupa Inlet of the White Sea. Mature worms were caught with a hand net near the water surface during their spawning period (June and July). Artificial fertilization and cultivation of the embryos were carried out at 10.5°C . The culture of postlarval animals was kept in the laboratory of experimental embryology (Petergof, Russia) under the following conditions: temperature, 18°C; salinity, 230/00; artificial sea water (Red Sea salt).
Cloning of A. virens Hox genes
A. virens Hox genes were cloned as described previously . Gene fragments, except Nvi-Hox3, were inserted into pGEM®-T Easy Vector (Promega). Nvi-Hox3 was inserted into pBluescript II SK+ (Fermentas). The vector sequence allows one to obtain sense and antisense probes from different promoters (T7 and Sp6). Antisense probes were used for the detection of sense transcripts’ expression. Sense probes were used for antisense transcripts’ detection.
Juvenile worms consisting of 20–30 segments were relaxed in clove oil (Sigma) with a low concentration for 5 min and then cut into two pieces approximately in the middle of the body. The anterior parts and the “tails” were incubated separately, each part in a separate petri dish (3 cm in diameter). Regenerating worms were fixed with 4% PFA in 1.75× PBS at the following time points: 0 h, 4 h, 10 h, 18 h, 1, 2, 3 and 7 days. Eight to ten worms were used for each time point in in situ hybridization.
Whole mount in situ hybridization (WMISH)
WMISH was performed for A. virens as described in Irvine et al. (1999)  with some modifications. A detailed protocol is available upon request. Digoxigenin-labeled RNA probes were prepared according to the manufacturer’s protocol (Roche). Collagenase treatment [collagenase (Sigma) 100 γ/ml, 2.5 mM DTT; 1mM CaCl2] was used for 5 min, and incubation in SDS/Tween buffer was performed for 30 min to improve probe penetration. Proteinase K (Sigma) treatment was performed for 8–10 min (10 γ/ml). Prehybridization and hybridization steps in Hybridization (Hybe) buffer were carried out overnight at 65°C. Washings from the probe were performed as follows: 100% Hybe 2 × 60 min, 80% prehybe/20% PTw 2 × 20 min, 50% prehybe/50% PTw 4 × 30 min, 20% prehybe/80% PTw 2 × 20 min and 100% PTw 2 × 20 min at 67°C. Incubation in the blocking buffer [1% B-M Blocking Rgt. (Roche)/5% normal sheep serum/PBS/0.1% Tween] took 60 min. Incubation in Anti-Digoxigenin-AP, Fab fragments from sheep (Roche) (1:4,000) was performed overnight at +4°C. Washings from antibodies were carried out for 10 × 20 min in PTw on the shaker. Incubation in AP buffer was performed before colored reaction for 3 × 5 min. BM-purple (Roche) was used as a chromogenic substrate to localize the hybridized probe. The results were imaged on a DMRXA microscope (Leica) with a Leica DC500 digital camera with Nomarski optics. The worms were mounted in clove oil before the microscopic analysis. Optical sections were assembled with the use of Helicon Focus software. Brightness, contrast and color values were corrected in all the images using image processing software Adobe Photoshop CS5. SDS/Tween buffer: 150 mM NaCl, 50 mM Tris, pH 7.5, 1 mM EDTA, 1% SDS, 0.5% Tween 20. Hybridization buffer: 50% formamide, 5× SSC pH 4.5, 50 μg/ml yeast tRNA, 50 μg/ml heparin, 0.1% Tween 20, 1% SDS, 100 μg/ml salmon ssDNA. Prehybe buffer: 50% formamide, 5× SSC pH 4.5, 1% SDS, 0.1% Tween 20. PBS: 1.5 M NaCl, 70 mM Na2HPO4, 30 mM NaH2PO4. PTw: PBS + 0.2% Tween 20. AP buffer: 100 mM NaCl, 50 mM MgCl2, 100 mM Tris, pH 9.5, 0.01% Tween 20. In situ hybridization for each time point was performed for at least 3 times.
Regeneration time course
Though many polychaetes are capable of extensive regeneration, members of the Nereidae can restore only the posterior body end. During posterior regeneration the pygidial structures and prepygidial growth zone (GZ) are formed first, and the segments form after that sequentially, as during normal growth. By 0–4 h post-amputation (hpa), the edges of the wound are tightened as a result of ring muscle contraction, and the gut lumen is closed. The gut comes in tight contact with the ectoderm to prevent the efflux of fluid from the coelomic cavity. The blastema is formed around 1 day post amputation (dpa). The cells of the superficial epithelium around the wound proliferate and migrate toward the injury. By 2 dpa, two rudiments, the primordia of the pygidial lobes, appear on the ventral side laterally in respect to the anus. The pygidium of the regenerating worm, with two well-developed lobes and anal cirri, is fully formed after 3 days. Neither parapodial rudiments nor any signs of segmentation can be seen on the surface of the regenerating region. However, the beginning of the segmentation process can be visualized on histological sections (Starunov and Lavrova, personal communication). After the pygidium and the GZ have been formed, the segmentation process proceeds as during normal growth.
Hox gene expression during the regeneration of A. virens
We divided Hox genes into four groups by their expression dynamics during the posterior regeneration of A. virens.
Early response genes
The first group comprises Nvi-Lox5, Nvi-Lox2 and Nvi-Post2. The expression patterns of these genes are reorganized very early in the neural system, within 4 h after amputation.
Middle response genes, expressed in neural system
Nvi-Hox5 and Nvi-Hox7 display pattern reorganization by 10 and 18 hpa, respectively, before the active proliferation starts.
Middle response genes, expressed in the growth zone
This group comprises Nvi-Hox2 and Nvi-Hox3 genes. Both of them do not form gradients in the postlarval body and are activated de novo in 10 hpa.
Late response genes
The fourth group comprises the genes whose expression patterns change only with the emergence of new structures, i.e., after the start of proliferation and organogenesis.
Expression of antisense transcripts
Hox genes in posterior regeneration of A. virens
Here we described the expression dynamics of 10 Hox genes during the posterior regeneration of polychaete A. virens. The expression was recorded at different stages of regeneration: 0, 4, 10, 18 hpa, 1, 2, 3 and 7 dpa. The 0-hpa point was taken as a control for the RNA probe penetration into the adult tissues: at this point we expected to get the same transcript distribution as in normal worms.
The second regeneration phase, overlapping with the first one, starts at 24 hpa. At this stage, the blastema is formed because of proliferation of dedifferentiated cells under the wound epithelium. Most of the Hox genes are expressed intensively in the blastema from the beginning of its formation and continue to be active in the rudiment of the terminal structures, which are morphologically distinguished by 3 dpa. During this period, all the Hox genes involved in the patterning of terminal structures and nascent segments are expressed in the regenerating worm. At the onset of organogenesis, the transcription of some Hox genes is downregulated in the nervous system (Figure 2h,p; Figure 3g-i, o-q).
Recently, we have suggested a hypothesis that during postlarval growth of A. virens, Hox genes play an unusual role different from their function in the formation of the nectochaete body . Indeed, Hox expression patterns in the larval and the postlarval body differ considerably. During nectochaete development, most of the Hox genes participate in the formation of the body of the segmented larva, functioning in accordance with the rule of spatial colinearity, as in embryogenesis of other bilaterian animals. On the contrary, during postlarval development, Hox genes are expressed in a gradient manner in the worm’s body with morphologically similar segments. We suggested that the gradient expression of Hox genes in postlarval ontogenesis does not lead to the morphological diversity of segments, but creates the positional information, which is needed to determine the position of body parts in a homonomously segmented body (Bakalenko, Novikova and Kulakova, unpublished data).
Our data from this study support the hypothesis of a coordinating function of Hox genes. Indeed, for some of the genes we observed a rapid reorganization of expression patterns in differentiated cells long before the morphological signs of regeneration were visible. The patterns of most of the genes from the early and the middle response group are reorganized in such a way that the last segments of the operated worm acquire the Hox expression pattern characteristic of the posterior body end.
Normal Hox gene activity in A. virens lacks temporal colinearity, but the anterior expression boundaries are colinear. The anterior boundaries of the genes that pattern the larval body are stabilized in larval segments. The middle genes (Nvi-Hox7, Nvi-Lox2 and Nvi-Lox4) are turned on after metamorphosis and have diffuse anterior boundaries, which are established late in the postlarval body (Bakalenko, Novikova and Kulakova, unpublished data). Interestingly, during pattern reorganization, the anterior boundaries of the genes that pattern the body of nectochaete larva, except for Nvi-Post2, remain stable. Oppositely, the expression boundaries of the genes that are turned on in the postlarval body can shift widely, but never spread to the larval segments. This interesting feature obviously reflects the difference in the epigenetic regulation of Hox transcription in larval and postlarval segments. In the segments where middle genes have been expressed at some moment (postlarval segments), their expression is easily induced, but in larval segments these genes have never been active and so cannot be turned on. It is interesting to note that the order of activation/repression of different Hox genes in the amputation site is not consistent with their presumed genomic order (Figure 13a). Here we observe that the greater the difference between the gene expression level at the prospective amputation site and that in terminal structures (pygidium and GZ) during normal development, the faster up- or downregulation of the gene expression proceeds during regeneration. Indeed, all the early response genes (Nvi-Post2, Nvi-Lox2 and Nvi-Lox5) display high expression levels in terminal structures and low expression levels at the potential amputation site during normal development. Oppositely, the late response genes (Nvi-Hox1, Nvi-Hox4, Nvi-Lox4) display low expression levels both at the potential amputation site and in the GZ.
Hox genes in regeneration of bilaterian animals
Hox gene expression during regeneration was studied in many representatives of Bilateria [4, 19, 20, 22]. Nevertheless, only one work describes the expression pattern of almost the whole set of Hox genes (with the exception of Lox4 and Hox7) during the polychaete regeneration . The authors studied the expression of Hox genes in regeneration of a homonomously segmented polychaete P. dumerilii. Unfortunately, it is difficult to compare our results with those from that study. Pfeifer and colleagues analyze the gene expression at a stage when the blastema has already formed (1 dpa), active formation of terminal structures is in progress, and differentiation of tissues and organogenesis has started. During these stages of regeneration, the expression pattern of many Pdu-Hox genes is similar to the expression pattern of Nvi-Hox genes. The only difference is the expression of Nvi-Lox2 and Pdu-Lox2. Nvi-Lox2 is the expressed in the segmental ectoderm and the neural system, whereas the domain of Pdu-Lox2 expression is located in coelothelia. However, the authors miss the early expression phase of some genes in differentiated tissues. This very phase is the reorganization of the expression pattern according to the new body proportions. Pfeifer and colleagues consider the expression of the Hox gene cluster in regenerating structures of P. dumerilii to pattern the neural system of nascent segments .
The expression of some Hox genes during regeneration was shown for another representative of Lophotrochozoa, planaria Dugesia japonica[20, 21]. Notably, DjAbd-Ba, Plox4-Dj and Plox5-Dj genes, which display a gradient expression pattern in normal development, show reorganization of expression patterns in definitive tissues during regeneration similar to A. virens.
Among vertebrates (Deuterostomia), the best capacities for axial regeneration are characteristics of Urodela [3, 16]. Surprisingly, these animals do display the persisting expression of Hox genes in adult tissues, which is upregulated during the regeneration processes [30, 31]. Upregulation of 5′HoxC genes after tail excision was found in the neural system of adult newt Pleurodeles waltl. The authors consider that such a persisting expression of Hox genes in the neural system of an adult animal provides the positional information necessary for regeneration .
In earlier investigations, the expression of some Hox genes was found in the limbs and the tail of adult Urodela [31, 32]. The most intriguing fact is that their orthologs are not expressed in the limb of X. laevis, whose adults cannot regenerate limbs . The ability of tailed amphibians to regenerate is probably associated with the maintenance of Hox gene expression in definitive tissues [30 – 32].
In higher vertebrates, Hox gene transcripts were found in populations of stem cells, such as fibroblasts and mesenchymal stem cells, and in tissues where constant renewal is possible [9, 10, 23, 24]. Mammals are incapable of epimorphic regeneration, and their wound healing is followed by scar formation, after which regeneration is impossible . Nevertheless, mammals can regenerate internal organs because of activity of stem cells present in certain types of tissue [11, 12]. Fibroblasts and mesenchymal stem cells were shown to carry the Hox code, which they get during embryogenesis [34, 35]. Once established, this Hox code in stem cells is retained throughout the lifetime and cannot be changed in case of positional failure.
Thus, the mechanism of maintaining and restoring positional values seems to have a similar basis among representatives of different bilaterian lineages. The important element of this complex system is the genes of the Hox cluster, which are thought to be capable of maintaining positional information in adult tissues and are consistently expressed in response to injuries to restore the positional coordinates in a regenerating animal. We suggest that this capacity of Hox genes is expressed in different ways in different taxa. In mammals, the rigidity of positional memory, necessary for the correct differentiation of fibroblasts and mesenchymal stem cells in the context of their background, provides a well-adjusted physiological and organ regeneration at the expense of epimorphic regeneration. A large variety of tissues and the necessity to maintain tissue homeostasis and integrity in mammals call for the early commitment of the Hox code. The other strategy is used by animals capable of epimorphic regeneration: tailed amphibians, planarians and polychaetes. These animals maintain the positional information in the adult body because of Hox gene expression in differentiated tissues and can rapidly reorganize these expression patterns when the body proportions change.
Regeneration of nereid polychaetes: regulation in more detail
When considering the early stages of regeneration of A. virens in more detail, we can see that immediately after the operation, the edges of the entodermal gut contact with the edges of the covering epithelium. During normal growth, the boundary between the ectodermal and the endodermal structures is mediated by the terminal structure, the pygidium. The narrow GZ, consisting of cells capable of rare synchronic divisions, lies between the ectoderm and the pygidium . Based on well-known experimental works on regeneration of nereid polychaetes (reviewed in ) and other model organisms [16, 38, 39], we propose that the contact of the tissues on the border between the gut and the segmental ectoderm, which never come in contact during normal development, leads to the formation of an organizer, the source of morphogenic signals. These signals, addressed to the adjacent tissues, can initiate the reorganization of positional information, dedifferentiation, proliferation and formation of a new pygidium and GZ. We observe the first expression answer to these signals from the wound surface by 3–4 hpa in case of Nvi-Hox genes, which are expressed in the GZ and the pygidium during normal development (Figure 1c,k; Figure 2c,k, Figure 3c,k), as well as for Nvi-Cad. Nvi-Cad belongs to the ParaHox gene group, which is known to be upstream regulators for Hox genes [41, 42]. This may suggest a role for Nvi-Cad as a positional marker, whose well-timed changes in the expression boundaries lead to reorganization of spatial coordinates in the regenerating worm (Figure 13b).
All Hox genes of early response are activated in the nervous system. As it happens before the blastema formation, one can suggest the existence of a link between these two processes. Indeed, the blastema starts to form after the expression domains of the Hox genes have been reorganized according to the new body proportions. However, in a series of studies on nereid polychaete Nereis diversicolor performed in the 1970s (reviewed in ), it was shown that the restoration of the pygidium, the GZ and new segments happened in the area of contact between the gut and body wall independently of the neural system. According to the authors’ description, without the nervous system, the pygidium did not produce the anal cirri, and the nascent segments lacked the “cephalo-caudal differentiation” . The early reorganization of Hox patterns probably does not define the blastema formation. Nevertheless, Hox genes may prepattern the blastema itself and its sources so that the new structures (pygidium, GZ and new segments) are integrated into the whole body correctly. Experiments on functional knockout of one or several genes of early response might provide interesting results, helping to reveal the regulatory connections between the Hox genes and to show the influence of Hox expression failure on segmentation and segmental differentiation.
Expression of antisense transcripts
Rapid reorganization of Hox expression patterns during regeneration of A. virens demands well-coordinated regulation of this process. While studying Hox gene expression in the postlarval development of A. virens, expression of long non-coding RNAs, complementary to sense RNA probes of Hox genes, was shown for the first time for a lophotrochozoan animal (Bakalenko, Novikova and Kulakova, unpublished data). Long non-coding RNA transcripts are the transcripts that lack long open reading frames and do not code the proteins . Discovered transcripts may belong to natural antisense transcripts (NAT) as they are at least partially complementary to known sequences of Hox genes. We found that antisense ncRNAs display unique expression patterns during normal growth of the worm (Bakalenko, Novikova and Kulakova, unpublished data). In this work we analyzed the expression dynamics of ncRNA for Nvi-Нох5 and Nvi-Нох7 during worm regeneration.
Expression of Nvi-antiНох5 is upregulated in the ganglion of the last body segment by 4 hpa (Figure 11c, k). At this point, expression of the sense transcript hasn’t been reorganized yet. But in several hours, Nvi-Hox5 expression moves towards the head, and Nvi-antiНох5 expression intensifies on this territory. By 18–24 hpa, transcript patterns overlap in 1–2 ganglia in the middle of the body, and after 48 hpa transcripts are expressed in complementary domains. Thus, we can assume we observe the negative regulation of the sense transcript by the antisense one.
In the case of Nvi-Нох7, the different pattern of sense and antisense transcript overlapping is observed. These transcripts are expressed in the same ganglia of the nerve cord nearly at all regeneration stages, though the expression of Nvi-antiНох7 is much less intensive (see “Results”).
Based on the complementary expression of Nvi-Hox5 and Nvi-antiHox5 transcripts, we can suggest that preceding activation of the antisense transcript provides very rapid negative regulation of the coding transcript by the non-coding one. The similar expression pattern and dynamics were shown for Ubx and antiUbx transcripts during the development of several myriapoda species: the activation of antisense Ubx occurred a bit earlier than Ubx expression, and transcripts displayed complementary expression patterns [44, 45]. The authors proposed some models of negative regulation of the sense Ubx transcript by the antisense one [46–48], but all of these mechanisms of regulation are implemented in the cell nucleus. However, in our case and in the study on myriapoda, long ncRNA transcripts were observed in the cytoplasm, where they were detected by in situ hybridization. It is unclear why ncRNAs leave the nucleus and go to the cytoplasm and what mechanism may be activated to perform potential regulation of sense transcripts by antisense ones. Moreover, overlapping of sense and antisense expression domains was shown for both Nvi-Hox5 and Nvi-Hox7 genes on some regeneration stages. We had no opportunity to describe the distribution of Hox proteins at that time point. The suppression of gene expression may occur here on the translational level because of RNAi or another, still unknown mechanism.
We showed that gradient Hox gene expression patterns undergo a rapid and consistent reorganization during the earliest stages of regeneration. We consider these changes to be orchestrated to compensate for the positional failure as they restore the native expression pattern in the rest of the body long before the first morphological signs of regeneration. The expression dynamics of Hox genes in a regenerating polychaete A. virens shows that the establishment, maintenance and restoration of positional memory in a multisegmental worm may be mediated by this group of genes.
Antisense transcripts of Nvi-Hox genes are expressed in spatial domains partially complementary to protein-encoding RNA. Their expression dynamics during regeneration indicate repression rather than activation of complementary mRNA. Regulatory transcripts are localized in cytoplasm, which implies their participation in translational silencing, probably with the use of the mechanism of RNA interference.
Comparing the definitive expression of Hox genes in representatives of different evolutionary branches, we can suppose that the common ancestor of Bilateria already possessed the system of establishing and maintaining the positional information in the adult body using the Hox genes. This Hox genes’ ability was utilized in different ways in various animal taxa. Thus, mammals used the system of positional markers for establishing different types of differentiation of multipotent stem cells along the body axis and in this way acquired the ability to maintain tissue homeostasis. Tailed amphibians, planarians and polychaetes use the same principle of positional marking of cell territories by means of Hox genes, but they have become capable of (or retained the ability to) rapid reorganization of this information in the case of positional failure, thus providing themselves with outstanding regeneration capacities.
Hour post amputation
Day post amputation
Ventral neural cord.
The authors are grateful to Tanya Andreeva, who laid the foundation for this research, and to Michael Akam and Charles Cook, who helped to clone Nvi-Hox gene fragments and supported us. The authors thank Olga B. Lavrova for valuable comments and maintaining the A. virens culture in the laboratory of experimental embryology. We are also grateful to the staff of the White Sea Biological Station “Kartesh” (Zoological Institute, Russian Academy of Science) for help in collecting and maintaining A. virens. We thank the “Chromas” center for providing the opportunity to use Leica microscope. The research was supported by RFBR grants 06-04-49654-a and 09-04-01322-a.
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