The expression patterns of the Hox genes

 

4.1. Chaetopterus variopedatus

 

The first study of Polychaete’s Hox-genes transcription patterns was performed on Chaetopterus variopedatus (Irvine and Martindale, 2000; Peterson et al., 2000). This polychaete is world-wide. It builds U-shaped burrow and, while chasing water through the burrow, gathers food particles by specialized organs. Despite its belonging to basal Annelids, Chaetopterus has a very complex heteronomous organization. The body of adult worm has three functionally and morphologically different parts – the tagmas (A, B and C). The tagma A consists of a presegmental prostomium and a peristomium that are fused together. Peristomium includes ten chaete-carrying segments – setigers (A1-A9). There are some morphological differencies among the setigers (a bundle of dark spines on A4 and neuropodial uncini on A9) (Irvine et al., 1999). The tagma B consists of five specialized segments (B1-B5) that are necessary for the food particle filtration and for the water pumping through the animal's tube. Each of these segments has an unique morphology (Fig. 2a). The tagma C consists of a variable number of uniform segments, in which gametes are formed.

The function of five cloned genes in Chaetopterus (CH-Hox1, CH-Hox2, CH-Hox3, CH-Hox4 и CH-Hox5) intrigued researchers very much. First, it was important to understand, how polychaete’s Hox-gene transcription relates with the time of segment setting up along the body axis and with tagma’s morphological boundaries. If Chaetopterus Hox-genes obey to spatial colinearity rule (that is common for vertebrates and for arthropods), than probably this rule worked in their common ancestor. And, of course, Chaetopterus pelagian larva arouse especial interest. Is Hox-gene cluster used for it’s building? If yes, then what is happening during definitive structure formation?

The place, the time and the intensity of transcription were determined by the WMISH (Whole Mount In Situ Hybridization) method (Irvine and Martindale, 2000) and by the quantitative assessment of CH-Hox-genes transcript amount in an organism (Peterson et al., 2000). It appears that all polychaete’s Hox-genes begin to work in larva’s posterior zone long before the visible segmentation. Topologically this zone relates to a growth zone. Later there will appear all larva’s and definitive segments. The CH-Hox1 and CH-Hox3 begin to transcribe after 24 hpf (L1), with lower initial CH-Hox3 transcription level than for CH-Hox1. The CH-Hox4 and CH-Hox5 are activated after 48 hpf (L2), and CH-Hox5 transcription level at this stage is lesser than for CH-Hox4. At the later stages the signal intensity for all these genes become equal. It may point at temporal gradient (Irvine and Martindale, 2000). The quantitative analysis confirmed this assumption (Peterson et al., 2000). Thus, 3’-associated genes of Chaetopterus are activated according the rule of temporal colinearity with only one exception: CH-Hox2 has maternal matrix and is present in embryo from the start (Peterson et al., 2000). Authors aptly note that other animals with teloblastic growth (leeches, crustaceans, insects with short germ band) haven’t such early persisted Hox-expression in growth zone. This is probably a feature of polychaetes or ancestral condition, that was lost in studied representatives of other groups (Irvine and Martindale, 2001).

During the larva’s growth to the stage L5 areas of Hox-genes expression aren’t limited by the growth zone anymore and they spread at the setigers. As a result, CH-Hox1, CH-Hox2, CH-Hox3, CH-Hox4, and CH-Hox5 have anterior boundaries in segments 2, 1, 3, 4, and 5 respectively. That is obvious that spatial colinearity principle is spreading at the CH-Hox-genes, and only gene that violates both rules (temporal and spatial colinearity) is CH-Hox2.

During the larva growth and development, CH-Hox-genes begin to work in forming nervous system, in parapodia and in superficial ectoderma of segments. Besides, CH-Hox1 and CH-Hox2 are expressed at the foregut/midgut boundary. This domain persists for CH-Hox1 until larva’s metamorphosis and is localized in the caudal end of the pharynx (Irvine and Martindale, 2000).

The transcription boundaries of CH-Hox-genes are related to tagma’s morphological boundaries. It is especially true for posterior borders of transcription. So the posterior boundary of CH-Hox1 and CH-Hox2 is located at the ninth segment, where tagma A ends. The posterior boundary of CH-Hox5 is placed at B2 segment. This is coincides with anterior morphological boundary of subsequent palette-bearing segments B3-B5, which serve to water flow generating. Anterior boundaries of all five genes pass through relatively homogeneous tagma A. But there also is present certain correlation. A4 is only one setiger, where CH-Hox3 domain isn’t covered by CH-Hox4. And the bundle of dark spines is forming only at A4.

Unfortunately, there still isn’t a data about central and posterior genes of Hox-cluster in Chaetopterus. Let’s hope, that the Chaetopterus’ s belonging to basal Annelids, which was recently revealed by molecular phylogeny methods (Weigert and Bleidorn, 2016), will renew an researcher interest to this object.

 

 

4.2. Capitellateleta

The polychaete Capitella teleta (formerly known as Capitella sp. I) belongs to Sedentaria branch. Unlike Chaetopterus, it’s body tagmization is feebly marked. It has thoracic and abdominal parts, their segments are similar. However chaetes at thoracal and at abdominal segments have different morphology, ganglions of the thorax are placed more closely, and FMRF-amid immunoreactive neurons lie only within fifth segment (de Jong and Seaver, 2016). The embryogenesis and larval development in Capitella passes inside the brood tube. Each female produces 100-250 offsprings, which are developing synchronously. Nonfeeding free-swimming larvae come out from the tube at 8^th-9^th day after the fertilization (Stage 9). After short pelagian stage larvae begin a metamorphosis: they lengthen, loss ciliary bands (prototroch, telotroch, neurotroch), settle and begin to feed. The subsequent growth proceeds by prepygidial (subterminal) growth zone. A juvenile has 13-14 segments. It grows to maturation for 8-10 weeks, reaching the size of 55-65 segments. The embryonic and larval development of Capitella had been described in details and well-staged, the fate of single blastomeres was traced and cell lines were mapped (Seaver et al., 2005; Meyer et al., 2010). Many of Capitella ’s developmental molecular markers were cloned. It makes the work with this object much easier (Seaver and Kaneshige, 2006; Frobius and Seaver, 2006; Shimeld et al., 2010; Meyer et al., 2015). Besides, Capitella able to regenerate posterior part of the body.

The larva’s morphogenesis in Capitella is noteworthy. This is sophisticated 13-segment larva (Seaver et al., 2005). According to the BrdU-labeling data, initial larval segments are forming from bilaterally-symmetric growth zone, which is placed ventro-laterally (Seaver et al., 2005). This growth zone covers almost all length of embryo’s anterior-posterior axis and produces first ten larval segments within short time period (~24 h). The remaining larval segments (A2-A4) are originated from posterior growth zone. These segments are formed slowly – one segment per day. The late pelagian larva come out from the brood tube and don’t segment until the metamorphosis ends (Frobius et al., 2008).

How are related the Hox-genes expression pattern and the position segment identity in polychaete with poorly defined tagmosis? Is there difference between larval and postlarval expression? Are there common traits between Hox-genes transcription pattern in Annelids from different evolutionary branches? In 2008 these questions were answered.

The expression of all Capitella ’s Hox-cluster genes was analyzed by WMISH, from the early larval stage to the third day after the metamorphosis (Frobius et al., 2008). It appears that all larval CapI-Hox genes transcripts form the wide gradients with different intensity in the nervous system and in the setiger’s ectoderm. Their anterior expression boundaries are located along anterior-posterior body axis, according to spatial colinearity principle (Fig.2b). The only gene which violates this rule is CapI-pb (PG2). It begins to work in subesophageal ganglion at the T1 level, while CapI-lab (PG1) has a boundary at T2 level. Posterior boundaries of most genes are localized at the level of last larval segments and ectodermal growth zone. This rule also has exceptions. So CapI-Hox3 is probably only one gene that works in mesodermal part of growth zone, and CapI-Hox5 is only one gene with evident larval posterior boundary at the level of thoracic segments T7-T8. In late larva occurs down regulation of all Hox-genes with persisting of weak transcription in the nervous system and within the growth zone.

The CapI-lab, CapI-pb, CapI-Hox3 begin to transcribe almost simultaneously, before the segmentation. After this consecutively CapI-Dfd и CapI-Scr become activated, then CapI-lox5, CapI-Antp, and CapI-lox4 and at the finish of larval stage - CapI-lox2 and CapI-Post2 (Frobius et al., 2008). Generally the rule of temporal colinearity is held. However in some cases the transcription signal of 5’-genes looks more brightly and more complex, that in 3’-neighbours at the same stage. For example CapI-lox5 and CapI-Post2 patterns are brighter and more detailed than CapI-Scr and CapI-lox4 / CapI-lox2 respectively.

Besides an axial transcription in nervous system and ectoderm, addition domains have been found for some Capitella Hox-genes. So CapI-lab, CapI-pb, CapI-Hox3, and CapI-Antp are expressed in the presumptive foregut (Frobius et al., 2008). The CapI-lab expression begins in dorsal part of the stomodaeum and later is localized in esophagus; CapI-pb is transiently expressed in dorsal part of the pharynx; CapI-Hox3 is weakly detected in the pharynx and in the esophagus; CapI-Antp is weakly and transiently expressed in the pharynx, more ventrally than CapI-pb.

Besides that, CapI-lab is transiently expressed in two cell clusters in medium larva’s cranial epidermis, and CapI-Hox3 and CapI-Antp are weakly expressed in brain. The CapI-Post1, fallen out from Hox-cluster, contributes to the specification of chaete-bearing cells – the chaeteblasts (Frobius et al., 2008).

Such sophisticated and dynamic pattern of expression reduces dramatically to the start of the worm’s postlarval life. Spatial expression domains of all Hox-genes are weakened and become restricted by CNS (Central Nervous System) ganglions. Moreover, transcription disappears in growth zone for all genes except CapI-Hox3, CapI-Dfd and CapI-lox5 (Frobius et al., 2008; de Jong and Seaver, 2016). Anterior boundaries of expression remain stable (but CapI-Antp shifts in juvenile worm by half of segment to the anterior). It is interesting then exactly at this stage are established posterior boundaries of Hox-genes expression between thorax and abdomen. CapI-lb, CapI-Hox3, CapI-Dfd, CapI-Scr, and CapI-lox5 all have posterior expression boundaries at the anterior edge of T9 in juveniles, CapI-Antp has it at the posterior edge of T9. CapI-lox2 and CapI-Post2 have anterior boundaries at the stage A1 (Frobius et al., 2008).

 

 

4.3. Alitta virens and Platynereis dumerilii

 

Nereid polychaetes Platynereis dumerilii (Pdu) and Alitta virens (Avi) (formerly known as Nereis virens) belong to Errantia branch. Unlike Chaetopterus and Capitella, they are homonomously segmented worms, i.e. all their segments are similar. In adult Platynereis the number of segments is nearly 70, while in Alitta the number of metameres slightly more than 200. These worms grow by new segments forming in prepygidial growth zone. They breed once in the life and produce a great quantity of offsprings (~2000 in Platynereis and about one million in Alitta). Both species are able to caudal regeneration.

The Pdu and Avi ontogenesis includes two consecutive larval stages – a spherical lecithotrophic trochophore and a segmented nectochaete (Fig.2c). Nectochaete sinks to the bottom and after the long pause (5 days of development for Pdu; 16–17 days of development for Avi) begins to form postlarval segments from subterminal (prepygidial) growth zone. The temperature optimum for normal larva’s development is different for these two species (18⁰С Pdu; 10,5⁰С Avi). At an average Pdu develops three times faster than Avi. The presence of two larvae in their ontogenesis with cardinally different organization (segmented and non-segmented) attracts worthy attention of classic embryologists, zoologists and development biologists (Wilson, 1892; Fischer et al., 2010; Starunov et al., 2017).

After the finding that Nereid’s genome includes eleven Hox-genes (deRosaetal., 1999), suspicions had arisen: may be, these genes work by different way than in other segmented animals? There was unclear, whether Hox-genes take part in larval morphogenesis. If they are, then big set of regionalizing genes looked obviously overabundant for four-segment nectochaete and even more so for spherical trochophore. Also was intriguing, what are Hox-genes doing at postlarval stage? Indeed why such big Hox-cluster is needed, if all body segments are equal?

It appears that main part of Hox-genes in A. virens and P. dumerilii are expressing during metamorphosis of spherical trochophore into the nectochaete (Kulakova et al., 2007). The expression is colinear to anterior-posterior axis of larva’s body and involves segmental ectoderm, neuromeres of neural cord and pygidium, i.e. all derivates of primary somatoblast (2d-micromere). Generally, orienting by larval expression pattern in whole, these genes may be classified to the four groups.

The first group includes Hox1, Hox4, Hox5, Lox5 and Post2. The early expression of these are genes isn’t fading during development and looks colinear. There is reason to think, that they are used in larval body regionalization. Anterior boundaries of expression of Hox1, Hox4, Hox5, and Lox5 are consecutively bound to the boundaries of three chaete-bearing segments and their neuromers (Fig.2c). The Post2 expression marks pygidium and pygidial cirri (Kulakova et al., 2007).

Genes Hox2 and Hox3 belong to the second group. They become activated in 2d derivates much earlier than other Hox-genes. Their dynamic transcription accompanies the complex process of definitive axis establishment within the larval body. Later these Hox-genes acquire expression pattern that is similar to well-ordered axial expression pattern of the first group genes. While doing so, the anterior boundary of Hox2 expression lies at the cephalic segment level, i.e. more arnterior than for the Hox1. The anterior Hox3 expression boundary passes at the level of first chaete-bearing segment and coincides with Hox1 boundary. At the end of metamorphosis to nectochaete an expression of Hox2 and Hox3 fades gradually and becomes limited by subterminal zone – the subsequent juvenile worm’s growth zone.

The third group is formed by central Hox-genes (Hox7, Lox4 and Lox2), which not become activated until nectochaete stage. They work in thin subterminal zone. At the beginning of postlarval growth they become involved to the new segment patterning process.

The last group is represented by only one gene – Post1, which lost its Hox-function and specifies chaetal sacks cells (Kulakova et al., 2002).

Like the Capitella and the Chaetopterus, some nereid’s Hox-genes have additional expression domains. For example, Hox1 and Hox2 genes are expressed in the pharynx and at the esophagus-midgut boundary. Besides, Pdu-Hox1 (but not Avi-Hox1) is expressed in apical tuft (Kulakova et al., 2007; Bakalenko et al., 2013).

Postlarval Hox-genes expression is studied in details for Avi (Bakalenko et al., 2013). According to our workgroup data, Pdu Hox-genes are involved in same processes, and their patterns don’t differ much (Novikova, Bakalenko – unpublished data). All nereid’s Hox-genes work in postlarval ontogenesis. Their expression (except Hox3) looks like wide differently directed gene-specific gradients in segment superficial tissues, neural ganglions andparapodiums.

Hox3 orthologs are expressed in juvenile nereid only in ectodermal growth zone. The intermediate pattern is described for Avi-Hox2. This gene has strong transcription in mesodermal growth zone and the weak one in some ectodermal clusters, which are placed at the median line of each chaete-bearing segment (Bakalenko et al., 2013).

The postlarval expression of nereid’s Hox-genes differs from larval one so much, that there is possible to suppose the presence of two different morphogenetic programs of body construction for the larva and for the worm. The first program forms larva’s oligomerous body according the rule of spatian colinearity. Each of four segments has its own stable Hox-code. Anterior boundaries of the transcription become established in larval segments and remain unchanged during all life of worm.

The second program forms polymer and constantly growing body which hasn’t tagmas. The only one difference within postlarval segments is their position in relation to body ends. Hox-gene’s transcription gradients change in proportion with worm’s growth, i.e. are zoomed. It suggests that same segment is placed in different Hox-context according the growth zone (Fig.3). Each postlarval segment expresses full set of Hox-genes, but at different time and expression’s boundaries aren’t stable.

Such complex system must be for some purpose. There is possible that juvenile polychaete uses Hox-genes for assignment and maintenance of position value for each segment of the many. The polychaete “numbers” segments with the use of Hox-protein set, for the control of their growth and number. If it is true, then position failure would affect Hox-genes functioning. Indeed, most of Avi Hox-genes quickly reorganize pattern during the regeneration (Novikova et al., 2013). Boundaries of these genes expression rapidly shift in new spatial coordinates of regenerate to restore normal proportional relations between domains of their activity. It was revealed that position failure become smooth within less than 8 hours. A segment which is nearest to cut line acquires a “Hox-code” of most posterior segment. After that a new structure begins to form from regeneration blastema cell. This is pygidium and new growth zone (Novikova et al., 2013). How often polychaetes use Hox-cluster for maintenance and for restoration of position values in regenerating body? It is still unknown. But thereis a reason to think that this isn’t unique phenomenon for nereids. For example in Capitella during regeneration occurs a shift of anterior expression boundaries for three genes: CapI-lox4, CapI-lox2 and CapI-Post2 (de Jong and Seaver, 2016). There is crucial that in this case CapI-lox4 and CapI-Post2 shift to the thoracic area which is never occurred in the normal condition. The shift happens at the different time for different genes. The stronger a failure in normal Hox-gene expression position the earlier transcription begins. It all points at common mechanism of position value restore for polychaetes, even if they are divided by 500 million years of evolution.