Expression of 'segmentation' genes during larval and juvenile development in the polychaetes Capitella sp. I and H. elegans

Polychaete annelids and arthropods are both segmented protostome invertebrates. To investigate whether the segmented body plan of these two phyla share a common molecular ground pattern, we report the developmental expression of orthologues of the arthropod segment polarity genes engrailed (en), hedgehog (hh), and wingless (wg/Wnt1) in larval and juvenile stages of the polychaete annelid Capitella sp. I and en in a second polychaete, Hydroides elegans. Temporally, neither Wnt1 nor hh are detected in the segmented region of the larval body until after morphological segmentation is apparent. Expression of CapI-Wnt1 is limited to a ring of ectoderm marking the future anus during larval segmentation. CapI-hh is expressed in a ring of the hindgut internal to that of CapI-Wnt1, as well as in a subset of ventral nerve cord neurons, anterior gut tissue, and mesoderm. In both H. elegans and Capitella sp. I, en is expressed in a spatially and temporally dynamic manner in segmentally iterated structures as well as a population of cells that migrate internally from ectoderm to mesoderm, possibly representing a population of ecto-mesodermal precursors. Significantly, the expression patterns we report for wg, en, and hh orthologues in Capitella sp. I and for en in larval development of H. elegans are not comparable to the highly conserved ectodermal segment polarity pattern observed in arthropods at any life history stage, consistent with distinct origins of segmentation between annelids and arthropods.     

The leech hunchback protein is expressed in the epithelium and CNS but not in the segmental precursor lineages

We are interested in identifying the regulatory genes involved in segmental pattern formation in annelids. The Drosophila segmentation gene hunchback (hb) is critical for the proper anteroposterior development of the fly embryo, but its function outside the diptera is currently unknown. Here, the protein expression pattern of Leech Zinc Finger II (LZF2), a leech orthologue of hb is characterized. In early embryogenesis, LZF2 protein is expressed in a subset of micromeres and is later expressed in the micromere-derived epithelium of the provisional epithelium and prostomium. LZF2 protein is detected in the ventral nerve cord during organogenesis, first in interganglionic muscle cells and later in subsets of neurons in each neuromere of the CNS. The location of immunoreactive cells during development and the similarity of the expression pattern of LZF2 to the expression of the Caenhorhabditis elegans hb homologue hbl-1 suggests that LZF2 plays a role in the morphogenetic movements of leech gastrulation and later in CNS specification but not in anteroposterior pattern formation. Received: 28 May 1999 / Accepted: 21 December 1999     

Capitella sp. I homeobrain-like, the first lophotrochozoan member of a novel paired-like homeobox gene family

The paired-like class of homeobox genes contains numerous distinct families, many of which have been implicated in a variety of developmental functions. We report the isolation and expression of a gene with high similarity to Drosophila melanogaster homeobrain from the polychaete annelid Capitella sp. I. The homeobrain-like (hbnl) gene is a paired-like gene that contains a conserved homeodomain, octapeptide region, alanine stretches, and an OAR domain. Gene orthology analyses of the homeodomain from CapI-hbnl places this gene in a new family of paired-like homeodomain genes that includes D. melanogaster homeobrain (hbn) and representatives from all major bilaterian clades as well as a cnidarian gene. CapI-hbnl expression is largely restricted to subsets of cells in the brain and eyes during larval development in Capitella sp. I. The earliest expression of CapI-hbnl is in small discrete cell clusters in the cerebral ganglia. This expression persists through late larval developmental stages whereas expression is absent in postmetamorphic juveniles. Outside the brain, expression is present on the ventral side of the larva in two small cell clusters, at the brain/pharyngeal border and in the anterior-most segment. CapI-hbnl shares features of brain expression with hbn, although in contrast to hbn, which is expressed along the length of the ventral nerve cord, CapI-hbnl has a restricted anterior expression pattern. CapI-hbnl represents an important neural marker for characterization of the annelid nervous system.     

Non-canonical functions of hunchback in segment patterning of the intermediate germ cricket Gryllus bimaculatus

In short and intermediate germ insects, only the anterior segments are specified during the blastoderm stage, leaving the posterior segments to be specified later, during embryogenesis, which differs from the segmentation process in Drosophila, a long germ insect. To elucidate the segmentation mechanisms of short and intermediate germ insects, we have investigated the orthologs of the Drosophila segmentation genes in a phylogenetically basal, intermediate germ insect, Gryllus bimaculatus (Gb). Here, we have focused on its hunchback ortholog (Gb'hb), because Drosophila hb functions as a gap gene during anterior segmentation, referred as a canonical function. Gb'hb is expressed in a gap pattern during the early stages of embryogenesis, and later in the posterior growth zone. By means of embryonic and parental RNA interference for Gb'hb, we found the following: (1) Gb'hb regulates Hox gene expression to specify regional identity in the anterior region, as observed in Drosophila and Oncopeltus; (2) Gb'hb controls germband morphogenesis and segmentation of the anterior region, probably through the pair-rule gene, even-skipped at least; (3) Gb'hb may act as a gap gene in a limited region between the posterior of the prothoracic segment and the anterior of the mesothoracic segment; and (4) Gb'hb is involved in the formation of at least seven abdominal segments, probably through its expression in the posterior growth zone, which is not conserved in Drosophila. These findings suggest that Gb'hb functions in a non-canonical manner in segment patterning. A comparison of our results with the results for other derived species revealed that the canonical hb function may have evolved from the non-canonical hb functions during evolution.     

The Annelid Ancestry of the Chordates and the Origin of the Chordate Central Nervous System and the Notochord

It is maintained that the segmentation of the chordates is not a re-emergent condition and that their ancestors must not be sought among oligomerous coelomates but among the kolygomerous forms. It is proposed that the chordates developed from tube-dwelling Polychaeta resembling the present members of the Sabellidae , by reverting to a free-living existance and a reversal of the dorsoventrality. The chordate neural tube is regarded as a new structure developed from the closed-over faecal groove and the surrounding gland cells of the glandular mass of the ventral gland-shields of the polychaete ancestor.
The disappearance of the annelid cerebral ganglia and their gradual replacement by a new co-ordinating centre in the form of a dilation of the anterior end of the neural tube is postulated. The author accepts D elsman's (1922) suggestion that the paired segmental ganglia of the annelid ancestor persist as the spinal ganglia of the vertebrates.
The chordate mouth is regarded as the same as that of its annelid ancestor.
A strip of dense connective tissue wedged in between the ventral nerve chords and in which are embedded the fibres of the longitudinal muscle overlying the nerve chords of the presumed polychaete ancestor is regarded as the precursor of the notochord, while the longitudinal musculature of the chordate trunk developed from the adjacent longitudinal muscle bands.
A cartilage-like internal skeleton supports the branchial crown of the tubicolous polychaetes examined.     

Ecdysone receptor homologs from mollusks, leeches and a polychaete worm

The genomes of the mollusk Lottia gigantea, the leech Helobdella robusta and the polychaete worm Capitella teleta each have a gene encoding an ecdysone receptor homolog. Publicly available genomic and EST sequences also contain evidence for ecdysone receptors in the seahare Aplysia californica, the bobtail squid Euprymna scolopes and the medicinal leech Hirudo medicinalis. Three-dimensional models of the ligand binding domains of these predicted ecdysone receptor homologs suggest that each of them could potentially bind an ecdysone-related steroid. Thus, ecdysone receptors are not limited to arthropods and nematodes.     

A gap gene, hunchback, regulates the spatial expression of Ultrabithorax

We have examined the distribution of Ultrabithorax (Ubx) proteins in embryos mutant for the zygotic gap class of segmentation genes. Members of this class include hunchback (hb), knirps (kni), and Krüppel (Kr). All three mutations disrupt segmentation in specific regions of the embryo. Mutations in kni and Kr produce complex alterations in the Ubx expression pattern. In hb mutants Ubx is ectopically expressed both anterior and posterior to its wild-type boundaries. Thus, the hb gene may play an important role in the specification of the boundaries of Ubx expression. Using the Ubx protein distribution as a marker for metameric organization and using Hoechst dye to monitor cell death, we could follow early events that lead to the final gap-segmentation phenotype in the larval cuticle.     

Spatial regulation of the gap gene giant during Drosophila development

We describe the regulated expression of the segmentation gene giant (gt) during early embryogenesis. The gt protein is expressed in two broad gradients in precellular embryos, one in anterior regions and the other in posterior regions. Double immunolocalization studies show that the gt patterns overlap with protein gradients specified by the gap genes hunchback (hb) and knirps (kni). Analysis of all known gap mutants, as well as mutations that disrupt each of the maternal organizing centers, indicate that maternal factors are responsible for initiating gt expression, while gap genes participate in the subsequent refinement of the pattern. The maternal morphogen bicoid (bcd) initiates the anterior gt pattern, while nanos (nos) plays a role in the posterior pattern. Gene dosage studies indicate that different thresholds of the bcd gradient might trigger hb and gt expression, resulting in overlapping but noncoincident patterns of expression. We also present evidence that different concentrations of hb protein are instructive in defining the limits of kni and gt expression within the presumptive abdomen. These results suggest that gt is a bona fide gap gene, which acts with hb, Krüppel and kni to initiate striped patterns of gene expression in the early embryo.     

Expression of <Emphasis Type="Italic">hunchback</Emphasis> protein in a subset of ectodermal teloblasts of the oligochaete annelid <Emphasis Type="Italic">Tubifex</Emphasis>

The early expression patterns of hunchback protein (T-hb protein) were examined in the oligochaete Tubifex, using an antibody raised to the LZF2 protein in leech. This antibody recognizes a 60-kDa polypeptide in the Tubifex embryo. Before teloblastogenesis, T-hb protein is expressed in every cleavage-stage blastomere. At the completion of teloblastogenesis, the only cells expressing T-hb are a fraction of the micromere-derived epithelial cells. During gastrulation, nuclear T-hb is seen in spreading micromere-derived epithelial cells and also in a subset of ectodermal teloblasts. Comparisons of these results with those from other annelids suggest that hb expression in the early cleavage blastomeres and the micromere-derived epithelium are features highly conserved among annelids. In contrast, hb expression in teloblasts appears to be an innovation evolved in the oligochaetes.     

The bx region enhancer, a distant cis-control element of the Drosophila Ubx gene and its regulation by hunchback and other segmentation genes.

The Drosophila homeotic gene Ultrabithorax (Ubx) is regulated by complex mechanisms that specify the spatial domain, the timing and the activity of the gene in individual tissues and in individual cells. In early embryonic development, Ubx expression is controlled by segmentation genes turned on earlier in the developmental hierarchy. Correct Ubx expression depends on multiple regulatory sequences located outside the basal promoter. Here we report that a 500 bp DNA fragment from the bx region of the Ubx unit, approximately 30 kb away from the promoter, contains one of the distant regulatory elements (bx region enhancer, BRE). During early embryogenesis, this enhancer element activates the Ubx promoter in parasegments (PS) 6, 8, 10, and 12 and represses it in the anterior half of the embryo. The repressor of the anterior Ubx expression is the gap gene hunchback (hb). We show that the hb protein binds to the BRE element and that such binding is essential for hb repression in vivo, hb protein also binds to DNA fragments from abx and bxd, two other regulatory regions of the Ubx gene. We conclude that hb represses Ubx expression directly by binding to BRE and probably other Ubx regulatory elements. In addition, the BRE pattern requires input from other segmentation genes, among them tailless and fushi tarazu but not Krüppel and knirps.     

Role of hunchback in segment patterning of Locusta migratoria manilensis revealed by parental RNAi

In long germ embryos, all body segments are specified simultaneously during the blastoderm stage. In contrast, in short germ embryos, only the anterior segments are specified during the blastoderm stage, leaving the rest of the body plan to be specified later. The striking embryological differences between short and long germ segmentation imply fundamental differences in patterning at the molecular level. To gain insights into the segmentation mechanisms of short germ insects, we have investigated the role of the homologue of the Drosophila gap gene hunchback (hb) in a short germ insect Locusta migratoria manilensi by paternal RNA interference (RNAi). Phenotypes resulting from hb knockdown were categorized into three classes based on severity. In the most extreme case, embryos developed the most anterior structures only, including the labrum, antennae and eyes. The following conclusions were drawn: (i) L. migratoria manilensis hb (Lmm'hb) controls germ band morphogenesis and segmentation in the anterior region; (ii) Lmm'hb may function as a gap gene in a wide domain including the entire gnathum and thorax; and (iii) Lmm'hb is required for proper growth of the posterior germ band. These findings suggest a more extensive role for L. migratoria manilensis hunchback in anterior patterning than those described in Drosophila.