We have shown earlier that irradiation with UV induces duplication of foot in regenerating middle pieces of hydra. The present study was undertaken to elucidate the underlying mechanism(s) leading to this curious phenomenon. UV irradiation induced duplicated foot in about 30% of regenerating middle pieces. Metalloproteinases are important in foot formation, while Wnt pathway genes are important in head formation in hydra. The effect of UV irradiation on expression of these genes was studied by in situ hybridization and q-PCR. In whole polyps and middle pieces, UV irradiation led to up-regulation of HMP2 and HMMP, the two metalloproteinases involved in foot formation in hydra. HMP2 expression was significantly increased starting from 30 min post exposure to UV at 254 nm (500 J/m 2 ), while HMMP showed significant up-regulation 6 h post UV exposure onwards. In middle pieces, increased expression of both metalloproteinases was observed only at 48 h. In whole polyps as well as in middle pieces, expression of Wnt3 and b-catenin was detected within 30 min of UV exposure and was accompanied by up-regulation of GSK3b, DKK3 and DKK1/2/4, inhibitors of the Wnt pathway. These conditions likely lead to inactivation of Wnt signaling. We therefore conclude that duplication of foot due to UV irradiation in regenerating middle pieces of hydra is a combined effect of up-regulation of metalloproteinases and inactivation of the Wnt pathway. Our results suggest that UV irradiation can be employed as a tool to understand patterning mechanisms during foot formation in hydra.

Mannose 6-phosphate (M6P)-dependent lysosomal enzyme targeting to endosome/lysosome complex is poorly understood among lower invertebrates. So far, only a M6P-independent lysosomal enzyme sorting protein, named LERP, has been described in Drosophila. Here, we have identified mannose 6-phosphate receptor (MPR) homologues in Hydra vulgaris, a basal Cnidarian, at genome level and further purified a cation-dependent MPR-like protein from hydra using affinity chromatography. Structural comparisons of hydra MPRs with mammalian MPRs confirm that the residues important for interacting with the M6P ligand are conserved. Based on our results, we report for the first time the occurrence of MPR-related proteins and M6P-dependent lysosomal enzyme targeting in H. vulgaris.

Background: Mechanisms regulating BMP and Wnt pathways and their interactions are not well studied in Hydra. Results: We report identification of BMP inhibitor gremlin, comparison of its expression with that of noggin and possible antagonism between Wnt and BMP signaling in Hydra. Gremlin is expressed in body column with high levels in budding region and in early buds. Noggin, on the other hand, is expressed in the hypostome, base of tentacles, lower body column, and basal disc. During budding, noggin is expressed at the sites of tentacle emergence. This was confirmed in ectopic tentacles in polyps treated with alsterpaullone (ALP), a GSK-3β inhibitor that leads to upregulation of Wnt pathway. RT-PCR data show that upregulation of Wnt is accompanied by downregulation of bmp 5-8b though noggin and gremlin remain unaltered till 24 hours. Conclusions: Different expression patterns of gremlin and noggin suggest their roles in budding and patterning of tentacles, respectively. Further, bmp 5-8b inhibition by activated Wnt signaling does not directly involve noggin and gremlin in Hydra. Our data suggest that Wnt/BMP antagonism may have evolved early for defining the oral-aboral axis, while the involvement of BMP antagonists during axial patterning is a recent evolutionary acquisition within the Bilateria lineage.

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Hydra, a fresh water Cnidarian, has been used as a model system to study regeneration and pattern formation. Here we report a newly identified hydra from Naukuchiatal, India. Comparison of Hydra vulgaris Naukuchiatal with Hydra magnipapillata, Hydra vulgaris AEP and Hydra vulgaris Ind-Pune showed variations in morphology. Nema arrangement in holotrichous isorhiza nematocytes showed transverse coiling pattern, a characteristic feature of the 'vulgaris' group. Phylogenetic analysis using conserved mitochondrial genes confirmed that Naukuchiatal hydra belongs to the 'vulgaris' group though it is a different strain particular to the type locality. Its morphological peculiarities could be the result of pristine environment.

13Mechanisms regulating BMP and Wnt signaling pathways have been widely studied in many 14 organisms. One of the mechanisms by which these pathways are regulated is by binding of 15 extracellular ligands. In the present study, we report studies with two BMP antagonists, gremlin 16 and noggin from Hydra vulgaris Ind-Pune and demonstrate antagonistic relationship between 17 BMP and Wnt pathways. Gremlin was ubiquitously expressed from the body column to head 18 region except in the basal disc and hypostome. During budding, gremlin was expressed 19 predominantly in the budding region suggesting a possible role in budding; this was confirmed in 20 polyps with different stages of buds. Noggin, on the other hand, was predominantly expressed in 21 the endoderm of hypostome, base of the tentacles, lower body column and at the basal disc in 22 whole polyps. During budding, noggin was expressed at the sites of emergence of tentacles 23 suggesting a role in tentacle formation. This was confirmed in alsterpaullone-treated polyps, 24 which showed noggin expression as distinct spots where ectopic organizers and ectopic tentacles 25 eventually formed. Using RT-PCR, we found that up-regulation of Wnt is accompanied with 26 down-regulation of BMP5-8b demonstrating antagonism between the two pathways. Down-27 regulation of noggin and gremlin, however, occurred only after 24 h recovery. The data suggest 28 that inhibition of BMP pathway by Wnt signaling in hydra does not directly involve noggin and 29 gremlin. Our findings indicate that the BMP/Noggin antagonism evolved early for setting up 30 2 and/or maintaining the head organizer while involvement of these BMP antagonists during 31 vertebrate axial patterning are recent evolutionary acquisitions. 32 33 Summary statement 36 We show that setting up of the Organizer by BMP/Noggin antagonism and role of BMP 37 inhibitors in tissue patterning are evolutionarily ancient, probably arising for the first time in 38 hydra 39 40 Introduction 41 Bone morphogenetic protein family, a subfamily of transforming growth factor-β (TGF-β) 42 superfamily was originally identified for role of some of its members in formation and 43 regeneration of bone in vivo. Subsequently, their roles in crucial developmental processes, such 44 as, proliferation, migration, differentiation and fate specification of embryonic cells, 45 morphogenesis and dorso-ventral patterning were discovered (Massagué, 1998; Komiya and 46 Habas, 2008). Non-optimal BMP signaling leads to a variety of developmental abnormalities and 47 disease conditions including cardiovascular diseases, symphalangism, diabetic nephropathy and 48 several types of cancers (Kattamuri et al., 2017). BMPs initiate downstream signaling by two 49 mechanisms. The first is a canonical smad-dependent pathway which involves phosphorylation 50 of Serine/Threonine kinase receptors, Type I and Type II receptor complexes (BMPRI and 51 BMPRII), and activation of smad-1/5/8. The second is a smad-independent pathway, which 52 involves TGF-β Activated Tyrosine Ki...