ParaHox Genes Revisited: From Gut Patterning to Integrated Axial and Neural Organization in Rotifera

The ParaHox homeobox genes Gsx, Xlox, and Cdx are evolutionarily related to Hox genes and form part of the ANTP-class homeobox gene repertoire. Comparative genomic data indicate that ParaHox genes were already present before the cnidarian-bilaterian split. Across metazoans, ParaHox genes often show conserved associations with the anteroposterior body axis and have been implicated in both gut patterning and neural development, although gene complements and genomic organization vary substantially among lineages. To investigate ParaHox gene deployment in Gnathifera, we identified orthologs of Gsx and Cdx across gnathiferan lineages, including Chaetognatha, Monogononta, Bdelloidea, Seisonidea, and Acanthocephala, but found no evidence of Xlox, indicating a reduced ParaHox gene complement. We analyzed the genomic organization and embryonic expression of Gsx and Cdx in the monogonont rotifer Brachionus manjavacas (Bm). Genomic mapping revealed a dispersed ParaHox configuration, with Bm-Gsx and Bm-Cdx separated by 4.4 Mb. Using whole-mount in situ hybridization, we detected Bm-Gsx expression in neurons of the foot region, as well as in a small number of cells with neuronal characteristics and probable involvement in stomatogastric system development. Bm-Cdx was expressed in FMRFamide-positive cells associated with the bladder, consistent with a neuroepithelial identity. Together, these data indicate that in rotifers, ParaHox gene expression is predominantly associated with neural structures. We propose that this pattern represents a derived condition reflecting the compact body plan and reduced gut organization characteristic of rotifers, highlighting the evolutionary flexibility of ParaHox gene deployment under lineage-specific developmental constraints.