The basic helix-loop-helix (bHLH) transcription factor Twist is highly conserved from Drosophila to vertebrates and plays a major role in mesoderm specification of triploblasts. The presence of a Twist homologue in diploblasts such as the cnidarian Podocoryne carnea raises questions on the evolution of mesoderm, the third cell layer characteristic for triploblasts. Podocoryne Twist is expressed in the early embryo until the myoepithelial cells of the larva differentiate and then again during medusa development. There, the gene is detected first when the myoepithelial cells of the polyp dedifferentiate to form the medusa bud and later Twist is found transiently in the entocodon, a mesoderm-like cell layer which differentiates into the smooth muscle and striated muscle of the bell. On the other hand, in later bud stages and the medusa, expression is seen where non-muscle tissues differentiate. Experimental analysis of in vitro transdifferentiation and regeneration demonstrates that Twist activity is not needed when isolated striated muscle regenerate medusa organs. Developmental roles of Twist are discussed with respect to early animal evolution from a common ancestor of cnidarians and bilaterians.

The development of visual organs is regulated in Bilateria by a network of genes where members of the Six and Pax gene families play a central role. To investigate the molecular aspects of eye evolution, we analyzed the structure and expression patterns of cognate members of the Six family genes in jellyfish (Cnidaria, Hydrozoa), representatives of a basal, non-bilaterian phylum where complex lens eyes with spherical lens, an epidermal cornea, and a retina appear for the first time in evolution. In the jellyfish Cladonema radiatum, a species with well-developed lens eyes in the tentacle bulbs, Six1/2-Cr and Six3/6-Cr, are expressed in the eye cup. Six4/5-Cr is mainly expressed in the manubrium, the feeding, and sex organ. All three Six genes are expressed in different subsets of epidermal nerve cells, possibly of the RFamide type which are part of a net connecting the different eyes with each other and the effector organs. Furthermore, expression is found in other tissues, notably in the striated muscle. During eye regeneration, expression of Six1/2-Cr and Six3/6-Cr is upregulated, but not of Six4/5-Cr. In Podocoryne carnea, a jellyfish without eyes, Six1/2-Pc and Six3/6-Pc are also expressed in the tentacle bulbs, Six1/2-Pc additionally in the manubrium and striated muscle, and Six3/6-Pc in the mechanosensory nematocytes of the tentacle. The conserved gene structure and expression patterns of all Cladonema Six genes suggest broad conservation of upstream regulatory mechanisms in eye development.

The function of basic helix-loop-helix (bHLH) proteins in cell differentiation was shown to be conserved from Drosophila to vertebrates, exemplified by the function of MyoD in striated muscle differentiation. In phylogeny striated muscle tissue appears first in jellyfish and the question of its evolutionary position is controversially discussed. For this reason we have studied the developmental role of myogenic bHLH genes in medusa development. Based on their dimerization ability, four genes of the bHLH family of transcription factors were isolated from the hydrozoan jellyfish Podocoryne carnea. While the proteins Id and Ash group with cognate family members from bilaterians, Net-like and JellyD1 could not be unequivocally classified. Id is expressed during the medusa budding process and in the adult medusa, Ash and Net-like are expressed in all life cycle stages from egg to adult medusa and JellyD1 is expressed in the blastula and gastrula stages, the planula larva, and in late medusa bud stages. The dimerization specificity, the expression pattern, and the conservation of two residues specific for a MyoD bHLH domain suggest that JellyD1 is related to an ancestral MyoD gene. Id, Net-like, and JellyD1 are either expressed in the entocodon or its derived tissues, the striated and smooth muscle of the bell. These findings strengthen the hypothesis that the entocodon is a mesoderm-like structure and that the common ancestor of Cnidaria and Bilateria was more complex in cell-type architecture and body organization than commonly thought.

Pax transcription factors are involved in a variety of developmental processes in bilaterians, including eye development, a role typically assigned to Pax-6. Although no true Pax-6 gene has been found in nonbilateral animals, some jellyfish have eyes with complex structures. In the cubozoan jellyfish Tripedalia , Pax-B , an ortholog of vertebrate Pax-2/5 / 8 , had been proposed as a regulator of eye development. Here we have isolated three Pax genes ( Pax-A , Pax-B , and Pax-E ) from Cladonema radiatum , a hydrozoan jellyfish with elaborate eyes. Cladonema Pax-A is strongly expressed in the retina, whereas Pax-B and Pax-E are highly expressed in the manubrium, the feeding and reproductive organ. Misexpression of Cladonema Pax-A induces ectopic eyes in Drosophila imaginal discs, whereas Pax-B and Pax-E do not. Furthermore, Cladonema Pax-A paired domain protein directly binds to the 5′ upstream region of eye-specific Cladonema opsin genes, whereas Pax-B does not. Our data suggest that Pax-A , but not Pax-B or Pax-E , is involved in eye development and/or maintenance in Cladonema . Phylogenetic analysis indicates that Pax-6 , Pax-B , and Pax-A belong to different Pax subfamilies, which diverged at the latest before the Cnidaria–Bilateria separation. We argue that our data, showing the involvement of Pax genes in hydrozoan eye development as in bilaterians, supports the monophyletic evolutionary origin of all animal eyes. We then propose that during the early evolution of animals, distinct classes of Pax genes, which may have played redundant roles at that time, were flexibly deployed for eye development in different animal lineages.

Bone morphogenetic proteins (BMPs) have key roles in gastrulation, mesoderm induction and axial patterning. The multitude of bilaterian BMPs employed in these morphogenetic processes contrasts starkly with the scarcity of BMPs in Cnidaria, the most basal eumetazoan phylum. In coral, sea anemone and hydra species, BMPs have been found to be associated with larval and polyp axial patterning. In the hydrozoan jellyfish Podocoryne (Hydractinia) carnea the BMP2/4 and BMP5-8 genes are expressed unilaterally in the larva, corroborating a possible role in larval axial development. With the focal area of BMP expression in the anterior region, however, the jellyfish larva may have a developmental reversal of spatial polarity compared to the anthozoan larva. In medusa development, BMP genes are expressed in divergent expression territories within the presumptive radial canals and in various parts of the endoderm, indicative of an involvement in mesoderm patterning and gastrovascular system formation reminiscent of bilaterian BMP functions. In addition, the BMP2/4 and BMP5-8 genes may play roles in wound response and dedifferentiation or S-phase re-entry, respectively, as the former is expressed in striated muscle cells immediately after excision from the bell and the latter in the initial phase of muscle cell transdifferentiation.