Lophotrochozoan Zic Genes
5.2 Comparisons of the Expression Patterns of Zic Genes in Polychaete and Oligochaete Annelids
5.2.1 Annelid Zic Genes
We will first look at Zic genes in the annelids, also known as ringed worms or seg- mented worms. Phylum Annelida includes two main classes: Polychaeta and Clitellata (Ruppert et al. 2004). The latter includes Oligochaeta and Hirudinea as subclasses. Polychaeta, Oligochaeta, and Hirudinea are represented by the rag worm, earthworm, and leech, respectively. Their body is composed of three regions:
the prostomium, trunk, and pygidium (Fig. 5.1). The elongate trunk consists of a longitudinal series of similar body units: the segments. At the anterior end of the trunk is the prostomium, which contains the brain and sense organs. The pygidium includes the anus at the posterior end of the body. In each segment, there are chaetae (chitinous bristles) that project outward from the epidermis to provide traction and perform other tasks. Typically, they appear in bilaterally paired bundles, with one dorsolateral and one ventrolateral pair per segment (Fig. 5.1). Each chaeta arises from a pit-like epidermal follicle composed of follicle cells and a single chaetoblast at the base of the follicle. The nervous system consists of the anterior dorsal brain in the prostomium and a ventral pair of longitudinal nerve cords. The nerve consists of the suprapharyngeal ganglia and subpharyngeal ganglia, which are connected cir- cumferentially around the pharynx (Fig. 5.1). In each segment, the ventral nerve cords have a pair of segmental ganglions from which segmental nerves enter the body wall and innervate the body wall musculature (Fig. 5.1). The overall structure of the annelid nervous system is known as a segmental nervous system.
Zic homologues have been isolated from polychaete Capitella teleta and oligo- chaete Tubifex tubifex, and their expression profiles during development have been described (Layden et al. 2010; Takahashi et al. 2008). Although their embryonic development, in particular the segmentation process, largely differs between these two species, similarities in their developmental program have been pointed out in recent studies. This includes the colinearity of Hox gene expression (Endo et al.
2016) and axis organizers (Seaver 2014). Furthermore, the development of both animals starts with spiral egg cleavage in a similar fashion. However, the segmenta- tion processes are largely different. In polychaete Platynereis, the segments are acquired after the trochophore (swimming plankton) stage (Fig. 5.1). The segmenta- tion process is regulated by the hedgehog signaling pathway (Dray et al. 2010), similar to that in Drosophila. The hedgehog signaling components (hh, ptc, and Gli) are expressed in segment polarity-like patterns, and the chemicals that antagonize the hedgehog signaling disrupt segment formation (Dray et al. 2010). On the other hand, segmentation in oligochaete Tubifex derives from cell bandlet-produced prog-
5 Lophotrochozoan Zic Genes
enies of the large posterior teloblast (Figs. 5.1 and 5.2). There are five pairs of telo- blasts arranged bilaterally (M, N, O, P, and Q). The teloblasts generate daughter cells one by one through asymmetrical cell division, and the older daughter cells are located on the anterior side of each bandlet. Among the teloblasts, the M blast lin- eage contributes solely to the mesodermal structures (Goto et al. 1999).
Fig. 5.1 Annelid body plan. Polychaete annelids acquire segments after a trochophore-like stage, whereas oligochaete annelids directly develop an adult-like segmented body without metamorpho- sis. Gross CNS organization is similar. All cartoons except Capitella embryo (top middle, ventral view) are lateral view. Left side is the anterior in all cartoons
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Fig. 5.2 Tubifex Zic expression. (a) Summary of Tubifex development (for details, see Shimizu 1982). Ar, anterior; Dl, dorsal; E, endodermal cells; GB, germ band; Pr, posterior; S, setae; Sg, segment; Vl, ventral; double arrowheads, prostomium. M, N, O, P, and Q indicate teloblasts, and NOPQ and OPQ indicate their precursors. (b) Antero-dorsal view of a stage 12 embryo shortly after division of NOPQ into N and OPQ. Arrows indicate a pair of anteriorly located surface cells.
Solid arrowheads indicate stained cells located internally. (c) Stage 15 embryos viewed from the left side (anterior to left, cleared specimen). Mesodermal germ bands are demarcated by broken lines. Circles of broken lines indicate M teloblasts. Arrow indicates CNS progenitor-like signal;
open arrowhead indicates a primary m-blast cell. (d) Stage 16 embryo side view (anterior to left).
X, Y, and Z indicate the three domains; open arrowhead indicates a primary m-blast cell. (e) Schematic illustration of Zic expression in the mesodermal germ bands derived from M teloblasts.
Each square represents a mesodermal segment that coincides with the body segment. Anterior is to the left; dorsal is at the top. The three domains X, Y, and Z correspond to those indicated in (d).
Roman numerals represent the tentative triphasic expression pattern of Zic (Reprinted from Takahashi et al. 2008)
5 Lophotrochozoan Zic Genes
5.2.2 Comparison of Mesodermal Expression in Oligochaetes and Polychaetes
In oligochaete mesodermal development, Zic expression was first detected in the newest M daughter cells (called m-blasts) and, later, in differentiating mesodermal cells (Takahashi et al. 2008). The earliest expression is transient and ceases upon the first cell division (Fig. 5.2c–e). The later phase of expression was observed initially as a dumbbell-shaped structure and subsequently as intensely stained paired dots (Fig. 5.2d, e). The dots are thought to be mesodermal tissues surrounding chaetal sacs (Takahashi et al. 2008).
In polychaetes, mesodermal Zic expression has been detected in one or a few cells in an anterior portion of the bilateral mesodermal bands, in the visceral meso- derm surrounding the foregut, in the head mesoderm at the anterior tip of the ani- mal, and in mesoderm associated with forming chaetae. The earliest mesodermal expression was found in the anterior portion of the bilateral mesodermal domain (Fig. 5.3a, b) (Layden et al. 2010). However, its expression in the bilateral mesoder- mal band seems to cease by the following stage, and segmentally reiterated Zic- expressing cell cluster expression can be seen in the lateral bands (Fig. 5.3c, d). The number of segmental clusters increases along the anterior-posterior axis as new seg- ments form. Later, the expression of bilateral mesoderm derivatives can be seen in cells wrapping the chaetal sacs (Layden et al. 2010).
Thus, the expression of polychaete Zic in the segmented mesoderm is somewhat similar to that of oligochaete Zic. It is remarkable that the expression profiles are conserved despite the largely different segmentation processes in polychaetes and oligochaetes. The expression of oligochaete Zic in visceral mesoderm surrounding the foregut and in the head mesoderm has not been described but awaits further expression analysis.
5.2.3 Comparisons of Zic Expression in Oligochaete and Polychaete Neural Development
In oligochaete Tubifex, the brain (cerebral ganglions) is known to derive from micromeres. In developing oligochaete embryos, one of the earliest Zic expressions is in a pair of anterior surface micromere clusters (Fig. 5.2b). It is possible that these cells include primitive cerebral ganglions. However, no lineage-tracing data exists for brain-forming cells in oligochaetes today, and thus their cell identity remains uncertain. Zic-expressing anterior end cell clusters can be detected during the fol- lowing stages and appear to be internalized (Fig. 5.2c). The Zic-expressing cell clusters in the anterior tip (prostomium) partly overlap with Emx expression (Takahashi et al. 2008). Considering that Emx homologues are expressed in early embryonic brains in both Drosophila and mouse (Lichtneckert and Reichert 2005), oligochaete Zic is likely to be expressed in the cerebral ganglions, at least in part.
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Fig. 5.3 Capitella Zic expression. Expression of Zic at cleavage (M), stage 3 (A and B), stage 4 (C, D, and N), stage 5 (E, F, and O), stage 6 (G and H), stage 7 (I and, J), and stage 8 (K, L, P and Q). Views are as indicated except that H is a ventral-lateral view; M is an animal view; N and O are anterior views; P is a lateral view; and Q is a ventral view. In all lateral and ventral views, the anterior is to the left, and ventral is down in all lateral views. An asterisk (*) indicates the relative mouth position. Zic is detectable in micromeres on an animal pole (M). Zic mesodermal expres- sion is detected in the anterior mesodermal band on the ventral side of stage 3 animals (A and B) and in two bilateral mesoderm domains adjacent to the foregut at stages 4 and 5 (C and E, arrow- heads). The mesodermal expression expands around the foregut during stages 6 (G) and 7 (I, notched arrowhead). At stage 5, four lateral mesodermal domains (F, arrowheads) are detected. At stage 7, lateral mesoderm domains form two longitudinal rows along the anterior-posterior (A-P) axis (J, arrowheads). At stage 8, the longitudinal domains are clearly mesoderm associated with chaetae (L, arrowheads, and P), but not the chaetal sac (P, arrows). Zic is expressed in the pre- sumptive brain ectoderm at all stages (B, arrow, C-L, and N). Ventrolateral expression at the lateral edge of ventral neural ectoderm is detected at stage 4 (C, arrow) and stage 5 (F and O, arrows).
The lateral neural ectoderm domain is downregulated in an A-P wave apparent at stage 6 (G, white line demarcates anterior border of expression) but remains expressed in the growth zone (I and J, arrows). Zic is also detectable in neurons in the brain (H, arrow) and, beginning at stage 6, in the ventral nerve cord (VNC; G, white arrowhead). VNC expression persists through stage 8 (K, arrowhead, and Q). Medial and lateral ganglions are detected in each segment (Q, which is a higher magnification of L). In Q, the white lines demarcate segmental boundaries (Reprinted from Layden et al. 2010)
5 Lophotrochozoan Zic Genes
In polychaete Capitella, neuroectodermal expression of Zic is detected at the bilaterally symmetrical anterior neuroectoderm and in a subregion of the developing brain. Expression in the anterior neuroectoderm region is maintained throughout development. Initially, Zic-expressing cells are thought to form brain neurons, based on previous fate-mapping studies (Meyer and Seaver 2009; Meyer et al. 2010). The expression of the Capitella bHLH-type neurogenic gene ash seems to partly overlap with the Zic-expressing region (Layden et al. 2010; Meyer and Seaver 2009). In addition to the anterior neuroectoderm, Zic-expressing cells exist in the ventrolat- eral neuroectoderm to form the ventral nerve chord. The Zic-expressing ventrolat- eral neuroectoderm partly overlaps with that of ash expression and is proposed to be the lateral edge of the presumptive ventral neuroectoderm (Layden et al. 2010). At a later stage, Zic expression is observed at the medial and lateral ganglions in each segment of the ventral nerve cord.
Expression in the brain may be a common feature of the oligochaete and poly- chaete Zic genes. Although properties of the early neuroectoderm in the oligochaete have not been described, expression profiles prior to brain establishment look simi- lar in oligochaete and polychaete animals. In the polychaete Capitella, neuroecto- dermal development has been described. The neuronal precursor cells are generated by ingression as single cells from the anterior surface cell layer (ectoderm), and prior to this process, ash is expressed (Meyer and Seaver 2009). Capitella Zic is likely to be co-expressed in the neural ectoderm at the gastrulation stage (Meyer and Seaver 2009; Layden et al. 2010). This is reminiscent of the vertebrate neural induc- tion process (Nakata et al. 1997; Kuo et al. 1998). Together with the sequential expression of Zic and ash in cnidarians (Chap. 2), studies on annelid Zic implicate the eumetazoan-wide usage of Zic family genes in the initial stage of neurogenesis.