Octopod Hox genes and cephalopod plesiomorphies.

Few other invertebrates captivate our attention as cephalopods do. Octopods, cuttlefish, and squids amaze with their behavior and sophisticated body plans that belong to the most intriguing among mollusks. Little is, however, known about their body plan formation and the role of Hox genes. The latter homeobox genes pattern the anterior-posterior body axis and have only been studied in a single decapod species so far. Here, we study developmental Hox and ParaHox gene expression in Octopus vulgaris. Hox genes are expressed in a near-to-staggered fashion, among others in homologous organs of cephalopods such as the stellate ganglia, the arms, or funnel. As in other mollusks Hox1 is expressed in the nascent octopod shell rudiment. While ParaHox genes are expressed in an evolutionarily conserved fashion, Hox genes are also expressed in some body regions that are considered homologous among mollusks such as the cephalopod arms and funnel with the molluscan foot. We argue that cephalopod Hox genes are recruited to a lesser extent into the formation of non-related organ systems than previously thought and emphasize that despite all morphological innovations molecular data still reveal the ancestral molluscan heritage of cephalopods.

Staggered Hox expression is more widespread among molluscs than previously appreciated.

Hox genes are expressed along the anterior-posterior body axis in a colinear fashion in the majority of bilaterians. Contrary to polyplacophorans, a group of aculiferan molluscs with conserved ancestral molluscan features, gastropods and cephalopods deviate from this pattern by expressing Hox genes in distinct morphological structures and not in a staggered fashion. Among conchiferans, scaphopods exhibit many similarities with gastropods, cephalopods and bivalves, however, the molecular developmental underpinnings of these similar traits remain unknown. We investigated Hox gene expression in developmental stages of the scaphopod Antalis entalis to elucidate whether these genes are involved in patterning morphological traits shared by their kin conchiferans. Scaphopod Hox genes are predominantly expressed in the foot and mantle but also in the central nervous system. Surprisingly, the scaphopod mid-stage trochophore exhibits a near-to staggered expression of all nine Hox genes identified. Temporal colinearity was not found and early-stage and late-stage trochophores, as well as postmetamorphic individuals, do not show any apparent traces of staggered expression. In these stages, Hox genes are expressed in distinct morphological structures such as the cerebral and pedal ganglia and in the shell field of early-stage trochophores. Interestingly, a re-evaluation of previously published data on early-stage cephalopod embryos and of the gastropod pre-torsional veliger shows that these developmental stages exhibit traces of staggered Hox expression. Considering our results and all gene expression and genomic data available for molluscs as well as other bilaterians, we suggest a last common molluscan ancestor with colinear Hox expression in predominantly ectodermal tissues along the anterior-posterior axis. Subsequently, certain Hox genes have been co-opted into the patterning process of distinct structures (apical organ or prototroch) in conchiferans.

Brain regionalization genes are co-opted into shell field patterning in Mollusca.

The 'brain regionalization genes' Six3/6, Otx, Pax2/5/8, Gbx, and Hox1 are expressed in a similar fashion in the deuterostome, ecdysozoan, and the cephalopod brain, questioning whether this holds also true for the remaining Mollusca. We investigated developmental Gbx-expression in representatives of both molluscan sister groups, the Aculifera and Conchifera. Gbx is expressed in the posterior central nervous system of an aculiferan polyplacophoran and solenogaster but not in a conchiferan bivalve suggesting that Gbx, together with Six3/6, Otx, Pax2/5/8, and Hox1, is involved in central nervous system regionalization as reported for other bilaterians. Gbx is, however, also expressed in the anterior central nervous system, i.e. the anlagen of the cerebral ganglia, in the solenogaster, a condition not reported for any other bilaterian so far. Strikingly, all Gbx-orthologs and the other 'posterior brain regionalization genes' such as Pax2/5/8 and Hox1 are expressed in the mantle that secretes shell(s) and spicules of mollusks (except cephalopods). In bivalves, the ancestral condition has even been lost, with Gbx and Pax2/5/8 not being expressed in the developing central nervous system anymore. This suggests an additional role in the formation of the molluscan shell field(s) and spicule-bearing cells, key features of mollusks.

The ParaHox gene Gsx patterns the apical organ and central nervous system but not the foregut in scaphopod and cephalopod mollusks.

BACKGROUND: It has been hypothesized that the ParaHox gene Gsx patterned the foregut of the last common bilaterian ancestor. This notion was corroborated by Gsx expression in three out of four lophotrochozoan species, several ecdysozoans, and some deuterostomes. Remarkably, Gsx is also expressed in the bilaterian anterior-most central nervous system (CNS) and the gastropod and annelid apical organ. To infer whether these findings are consistent with other mollusks or even lophotrochozoans, we investigated Gsx expression in developmental stages of representatives of two other molluscan classes, the scaphopod Antalis entalis and the cephalopod Idiosepius notoides. RESULTS: Gsx is not expressed in the developing digestive tract of Antalis entalis and Idiosepius notoides. Instead, it is expressed in cells of the apical organ in the scaphopod trochophore and in two cells adjacent to this organ. Late-stage trochophores express Aen-Gsx in cells of the developing cerebral and pedal ganglia and in cells close to the pavilion, mantle, and foot. In postmetamorphic specimens, Aen-Gsx is expressed in the cerebral and pedal ganglia, the foot, and the nascent captacula. In early squid embryos, Ino-Gsx is expressed in the cerebral, palliovisceral, and optic ganglia. In late-stage embryos, Ino-Gsx is additionally expressed close to the eyes and in the supraesophageal and posterior subesophageal masses and optic lobes. Developmental stages close to hatching express Ino-Gsx only close to the eyes. CONCLUSIONS: Our results suggest that Gsx expression in the foregut might not be a plesiomorphic trait of the Lophotrochozoa as insinuated previously. Since neither ecdysozoans nor deuterostomes express Gsx in their gut, a role in gut formation in the last common bilaterian ancestor appears unlikely. Gsx is consistently expressed in the bilaterian anterior-most CNS and the apical organ of lophotrochozoan larvae, suggesting a recruitment of Gsx into the formation of this organ in the Lophotrochozoa. The cephalopod posterior subesophageal mass and optic ganglia and the scaphopod pedal ganglia also express Gsx. In summary, Gsx expression only appears to be conserved in the anterior-most brain region during evolution. Accordingly, Gsx appears to have been recruited into the formation of other expression domains, e.g., the apical organ or the foregut, in some lophotrochozoans.

Ancestral role of Pax2/5/8 in molluscan brain and multimodal sensory system development.

BACKGROUND: Mollusks represent the largest lophotrochozoan phylum and exhibit highly diverse body plans. Previous studies have demonstrated that transcription factors such as Pax genes play important roles during their development. Accordingly, in ecdysozoan and vertebrate model organisms, orthologs of Pax2/5/8 are among others involved in the formation of the midbrain/hindbrain boundary, the auditory/geosensory organ systems, and the excretory system. METHODS: Pax2/5/8 expression was investigated by in situ hybridization during the development of representatives of the two major molluscan subclades, Aculifera and Conchifera. RESULTS: Compared to the investigated polyplacophoran and bivalve species that lack larval statocysts as geosensory organs and elaborate central nervous systems (CNS), cephalopods possess highly centralized brains and statocysts. Pax2/5/8 is expressed in regions where sensory cells develop subsequently during ontogenesis. Expression domains include esthetes and the ampullary system in polyplacophorans as well as the eyes of cephalopods. No Pax2/5/8 expression was observed in the less centralized CNS of bivalve, polyplacophoran, and gastropod embryos, thus arguing for a loss of Pax2/5/8 involvement in CNS development in these lineages. In contrast, Pax2/5/8 is expressed among others in brain lobes along the trajectory of the esophagus that divides the cephalopod brain. CONCLUSIONS: Our results, along with those on Otx- and Hox-gene expression, demonstrate that the cephalopod condition is similar to that in mouse and fruit fly, with Otx being expressed in the anterior-most brain region (except for the vertical lobe) and a Pax2/5/8 expression domain separating the Otx-domain from a Hox-gene expressing posterior brain region. Thus, Pax2/5/8 appears to have been recruited independently into regionalization of non-homologous complex brains of organisms as different as squid, fruit fly, and mouse. In addition, Pax2/5/8 is expressed in multimodal sensory systems in mollusks such as the esthetes and the ampullary system of polyplacophorans as well as the eyes of cephalopods. Pax2/5/8-expressing cells are present in regions where the future sensory cells such as the polyplacophoran esthetes are situated and hence Pax2/5/8 expression probably predates sensory cell development during ontogeny. In mollusks, Pax2/5/8 is only expressed in derivatives of the ectoderm and hence an ancestral role in molluscan ectoderm differentiation is inferred.