Altered neuroepithelial morphogenesis and migration defects in iPSC-derived cerebral organoids and 2D neural stem cells in familial bipolar disorder.

Bipolar disorder (BD) is a severe mental illness that can result from neurodevelopmental aberrations, particularly in familial BD, which may include causative genetic variants. In the present study, we derived cortical organoids from BD patients and healthy (control) individuals from a clinically dense family in the Indian population. Our data reveal that the patient organoids show neurodevelopmental anomalies, including organisational, proliferation and migration defects. The BD organoids show a reduction in both the number of neuroepithelial buds/cortical rosettes and the ventricular zone size. Additionally, patient organoids show a lower number of SOX2-positive and EdU-positive cycling progenitors, suggesting a progenitor proliferation defect. Further, the patient neurons show abnormal positioning in the ventricular/intermediate zone of the neuroepithelial bud. Transcriptomic analysis of control and patient organoids supports our cellular topology data and reveals dysregulation of genes crucial for progenitor proliferation and neuronal migration. Lastly, time-lapse imaging of neural stem cells in 2D in vitro cultures reveals abnormal cellular migration in BD samples. Overall, our study pinpoints a cellular and molecular deficit in BD patient-derived organoids and neural stem cell cultures.

LSD1 Regulates Neurogenesis in Human Neural Stem Cells Through the Repression of Human-Enriched Extracellular Matrix and Cell Adhesion Genes.

Neurogenesis begins with neural stem cells undergoing symmetric proliferative divisions to expand and then switching to asymmetric differentiative divisions to generate neurons in the developing brain. Chromatin regulation plays a critical role in this switch. Histone lysine-specific demethylase LSD1 demethylates H3K4me1/2 and H3K9me1/2 but the mechanisms of its global regulatory functions in human neuronal development remain unclear. We performed genome-wide ChIP-seq of LSD1 occupancy, RNA-seq, and Histone ChIP-seq upon LSD1 inhibition to identify its repressive role in human neural stem cells. Novel downstream effectors of LSD1 were identified, including the Notch signaling pathway genes and human-neural progenitor-enriched extracellular matrix (ECM) pathway/cell adhesion genes, which were upregulated upon LSD1 inhibition. LSD1 inhibition led to decreased neurogenesis, and overexpression of downstream effectors mimicked this effect. Histone ChIP-seq analysis revealed that active and enhancer markers H3K4me2, H3K4me1, and H3K9me1 were upregulated upon LSD1 inhibition, while the repressive H3K9me2 mark remained mostly unchanged. Our work identifies the human-neural progenitor-enriched ECM pathway/cell adhesion genes and Notch signaling pathway genes as novel downstream effectors of LSD1, regulating neuronal differentiation in human neural stem cells.

Regulation of chromatin accessibility and gene expression in the developing hippocampal primordium by LIM-HD transcription factor LHX2.

In the mammalian cerebral cortex, the hippocampal primordium (Hcp) occupies a discrete position in the dorsal telencephalic neuroepithelium adjacent to the neocortical primordium (Ncp). We examined transcriptomic and chromatin-level features that distinguish the Hcp from the Ncp in the mouse during the early neurogenic period, embryonic day (E)12.5. ATAC-seq revealed that the Hcp was more accessible than the Ncp at this stage. Motif analysis of the differentially accessible loci in these tissues revealed LHX2 as a candidate transcription factor for modulating gene regulatory networks (GRNs). We analyzed LHX2 occupancy profiles and compared these with transcriptomic data from control and Lhx2 mutant Hcp and Ncp at E12.5. Our results revealed that LHX2 directly regulates distinct genes in the Hcp and Ncp within a set of common pathways that control fundamental aspects of development namely pluripotency, axon pathfinding, Wnt, and Hippo signaling. Loss of Lhx2 caused a decrease in accessibility, specifically in hippocampal chromatin, suggesting that this factor may play a unique role in hippocampal development. We identified 14 genes that were preferentially enriched in the Hcp, for which LHX2 regulates both chromatin accessibility and mRNA expression, which have not thus far been examined in hippocampal development. Together, these results provide mechanistic insight into how LHX2 function in the Hcp may contribute to the process by which the hippocampus acquires features distinct from the neocortex.

Combinatorial delivery of CPI444 and vatalanib loaded on PEGylated graphene oxide as an effective nanoformulation to target glioblastoma multiforme: In vitro evaluation.

Glioblastoma multiforme (GBM) is known as the primary malignant and most devastating form of tumor found in the central nervous system of the adult population. The active pharmaceutical component in current chemotherapy regimens is mostly hydrophobic and poorly water-soluble, which hampers clinical implications. Nanodrug formulations using nanocarriers loaded with such drugs assisted in water dispersibility, improved cellular permeability, and drug efficacy at a low dose, thus adding to the overall practical value. Here, we successfully developed a water-dispersible and biocompatible nanocargo (GO-PEG) based on covalently modified graphene oxide (GO) with a 6-armed poly(ethylene glycol) amine dendrimer for effective loading of the two hydrophobic anticancer drug molecules, CPI444 and vatalanib. These drug molecules target adenosine receptor (A2AR), vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR), and type III stem cell receptor tyrosine kinase (c-KIT), which plays a crucial role in cancers. The effective cellular delivery of the drugs when loaded on GO-PEG is attributed to the increased permeability of the drug-nanoconjugate formulation. We observed that this combinatorial drug treatment with nanocargo resulted in a significant reduction in the overall cell survival as supported by reduced calcium levels and stem cell markers such as Oct4 and Nanog, which are two of the prime factors for GBM stem cell proliferation. Furthermore, reduced expression of CD24 upon treatment with nanoformulation impeded cellular migration. Cellular assays confirmed inhibition of cell proliferation, migration, and angiogenic potential of GBM treated with GO-PEG-Drug conjugates. Ultimately, GBM U87 cells assumed programmed cell death at a very low concentration due to nanocarrier-mediated drug delivery along with the chosen combination of drugs. Together, this study demonstrated the advantage of GO-PEG mediated combined delivery of CPI444 and vatalanib drugs with increased permeability, a three-pronged combinatorial strategy toward effective GBM treatment.

Dendrimer-Functionalized Nanodiamonds as Safe and Efficient Drug Carriers for Cancer Therapy: Nucleus Penetrating Nanoparticles.

Nanodiamonds (NDs) are increasingly being assessed as potential candidates for drug delivery in cancer cells and they hold great promise in overcoming the side effects of traditional chemotherapeutics. In the current work, carboxylic acid functionalized nanodiamonds (ND-COOH) were covalently modified with poly(amidoamine) dendrimer (PAMAM) to form amine-terminated nanodiamonds (NP). Unlike ND-COOH, the chemically modified nanodiamond platform NP revealed a pH-independent aqueous dispersion stability, enhancing its potential as an effective carrier. Physical encapsulation of poorly water soluble cabazitaxel (CTX) drug on NP formed ND-PAMAM-CTX (NPC) nanoconjugates and substantially reduced the size of CTX from micrometer to nanometer. CTX was localized within the pores of nanoparticle aggregates and the cavities of the PAMAM dendrimer, thus facilitating the loaded drug's controlled and sustained release. NPC's cumulative CTX release efficiency was determined to be ∼95% at pH 4 after 96 h. A high cellular uptake of NPC both within the cytoplasm and nucleus of U87 cells is confirmed, accounting for a reduced IC50 value (1 nM). Both the cell cycle and Western blot analyses confirmed enhanced cell death and suppressed tubulin protein expression in NPC-treated cells. A significantly high inhibition to cell division with early apoptosis and reduced metastasis demonstrates the effective loading of CTX dosages on the nanocarrier. The present work highlights the potential of a newly designed nanocarrier NP as an efficient nanocargo for cellular delivery applications and may provide future insights to treat one of the most aggressive tumors in neuro-oncological research, glioblastoma multiforme (GBM).

Identification of Components of the Hippo Pathway in Hydra and Potential Role of YAP in Cell Division and Differentiation.

The Hippo signaling pathway has been shown to be involved in regulating cellular identity, cell/tissue size maintenance and mechanotransduction. The Hippo pathway consists of a kinase cascade which determines the nucleo-cytoplasmic localization of YAP in the cell. YAP is the effector protein in the Hippo pathway, which acts as a transcriptional cofactor for TEAD. Phosphorylation of YAP upon activation of the Hippo pathway prevents it from entering the nucleus and abrogates its function in the transcription of the target genes. In Cnidaria, the information on the regulatory roles of the Hippo pathway is virtually lacking. Here, we report the existence of a complete set of Hippo pathway core components in Hydra for the first time. By studying their phylogeny and domain organization, we report evolutionary conservation of the components of the Hippo pathway. Protein modelling suggested the conservation of YAP-TEAD interaction in Hydra. Further, we characterized the expression pattern of the homologs of yap, hippo, mob and sav in Hydra using whole-mount RNA in situ hybridization and report their possible role in stem cell maintenance. Immunofluorescence assay revealed that Hvul_YAP expressing cells occur in clusters in the body column and are excluded in the terminally differentiated regions. Actively proliferating cells marked by Ki67 exhibit YAP colocalization in their nuclei. Strikingly, a subset of these colocalized cells is actively recruited to the newly developing bud. Disruption of the YAP-TEAD interaction increased the budding rate indicating a critical role of YAP in regulating cell proliferation in Hydra. Collectively, we posit that the Hippo pathway is an essential signaling system in Hydra; its components are ubiquitously expressed in the Hydra body column and play a crucial role in Hydra tissue homeostasis.

Satb2 acts as a gatekeeper for major developmental transitions during early vertebrate embryogenesis.

Zygotic genome activation (ZGA) initiates regionalized transcription underlying distinct cellular identities. ZGA is dependent upon dynamic chromatin architecture sculpted by conserved DNA-binding proteins. However, the direct mechanistic link between the onset of ZGA and the tissue-specific transcription remains unclear. Here, we have addressed the involvement of chromatin organizer Satb2 in orchestrating both processes during zebrafish embryogenesis. Integrative analysis of transcriptome, genome-wide occupancy and chromatin accessibility reveals contrasting molecular activities of maternally deposited and zygotically synthesized Satb2. Maternal Satb2 prevents premature transcription of zygotic genes by influencing the interplay between the pluripotency factors. By contrast, zygotic Satb2 activates transcription of the same group of genes during neural crest development and organogenesis. Thus, our comparative analysis of maternal versus zygotic function of Satb2 underscores how these antithetical activities are temporally coordinated and functionally implemented highlighting the evolutionary implications of the biphasic and bimodal regulation of landmark developmental transitions by a single determinant.

Epigenetic Regulation in Hydra: Conserved and Divergent Roles.

Transitions in gene regulatory processes responsible for the emergence of specialized cell types and spatiotemporal regulation of developmental signaling prior to the divergence of Cnidaria and Bilateria are poorly understood. As a sister group of Bilateria, the phylum Cnidaria can provide significant insights into these processes. Among the cnidarians, hydrae have been studied for >250 years to comprehend the mechanisms underlying their unique immortality and robust regenerative capacity. Studies on Hydra spp. and other pre-bilaterians alike have advanced our understanding of the evolutionary underpinnings governing eumetazoan tissue development, homeostasis, and regeneration. In addition to its regenerative potential, Hydra exhibits continuously active axial patterning due to its peculiar tissue dynamics. These distinctive physiological processes necessitate large scale gene expression changes that are governed by the multitude of epigenetic mechanisms operating in cells. This review highlights the contemporary knowledge of epigenetic regulation in Hydra with contemporary studies from other members of Cnidaria, as well as the interplay between regulatory mechanisms wherever demonstrated. The studies covered in the scope of this review reveal both ancestral and divergent roles played by conserved epigenetic mechanisms with emphasis on transcriptional regulation. Additionally, single-cell transcriptomics data was mined to predict the physiological relevance of putative gene regulatory components, which is in agreement with published findings and yielded insights into the possible functions of the gene regulatory mechanisms that are yet to be deciphered in Hydra, such as DNA methylation. Finally, we delineate potentially rewarding epigenetics research avenues that can further leverage the unique biology of Hydra.

Epigenomic landscape of enhancer elements during Hydra head organizer formation.

BACKGROUND: Axis patterning during development is accompanied by large-scale gene expression changes. These are brought about by changes in the histone modifications leading to dynamic alterations in chromatin architecture. The cis regulatory DNA elements also play an important role towards modulating gene expression in a context-dependent manner. Hydra belongs to the phylum Cnidaria where the first asymmetry in the body plan was observed and the oral-aboral axis originated. Wnt signaling has been shown to determine the head organizer function in the basal metazoan Hydra. RESULTS: To gain insights into the evolution of cis regulatory elements and associated chromatin signatures, we ectopically activated the Wnt signaling pathway in Hydra and monitored the genome-wide alterations in key histone modifications. Motif analysis of putative intergenic enhancer elements from Hydra revealed the conservation of bilaterian cis regulatory elements that play critical roles in development. Differentially regulated enhancer elements were identified upon ectopic activation of Wnt signaling and found to regulate many head organizer specific genes. Enhancer activity of many of the identified cis regulatory elements was confirmed by luciferase reporter assay. Quantitative chromatin immunoprecipitation analysis upon activation of Wnt signaling further confirmed the enrichment of H3K27ac on the enhancer elements of Hv_Wnt5a, Hv_Wnt11 and head organizer genes Hv_Bra1, CnGsc and Hv_Pitx1. Additionally, perturbation of the putative H3K27me3 eraser activity using a specific inhibitor affected the ectopic activation of Wnt signaling indicating the importance of the dynamic changes in the H3K27 modifications towards regulation of the genes involved in the head organizer activity. CONCLUSIONS: The activation-associated histone marks H3K4me3, H3K27ac and H3K9ac mark chromatin in a similar manner as seen in bilaterians. We identified intergenic cis regulatory elements which harbor sites for key transcription factors involved in developmental processes. Differentially regulated enhancers exhibited motifs for many zinc-finger, T-box and ETS related TFs whose homologs have a head specific expression in Hydra and could be a part of the pioneer TF network in the patterning of the head. The ability to differentially modify the H3K27 residue is critical for the patterning of Hydra axis revealing a dynamic acetylation/methylation switch to regulate gene expression and chromatin architecture.

Differential tissue stiffness of body column facilitates locomotion of Hydra on solid substrates.

The bell-shaped members of the Cnidaria typically move around by swimming, whereas the Hydra polyp can perform locomotion on solid substrates in an aquatic environment. To address the biomechanics of locomotion on rigid substrates, we studied the 'somersaulting' locomotion in Hydra We applied atomic force microscopy to measure the local mechanical properties of Hydra's body column and identified the existence of differential Young's modulus between the shoulder region versus rest of the body column at 3:1 ratio. We show that somersaulting primarily depends on differential tissue stiffness of the body column and is explained by computational models that accurately recapitulate the mechanics involved in this process. We demonstrate that perturbation of the observed stiffness variation in the body column by modulating the extracellular matrix polymerization impairs the 'somersault' movement. These results provide a mechanistic basis for the evolutionary significance of differential extracellular matrix properties and tissue stiffness.

Origin of RNA Polymerase II pause in eumetazoans: Insights from Hydra.

Multicellular organisms have evolved sophisticated mechanisms for responding to various developmental, environmental and physical stimuli by regulating transcription. The correlation of distribution of RNA Polymerase II (RNA Pol II) with transcription is well established in higher metazoans, however genome-wide information about its distribution in early metazoans, such as Hydra, is virtually absent. To gain insights into RNA Pol II-mediated transcription and chromatin organization in Hydra, we performed chromatin immunoprecipitation (ChIP)-coupled high-throughput sequencing (ChIP-seq) for RNA Pol II and Histone H3. Strikingly, we found that Hydra RNA Pol II is uniformly distributed across the entire gene body, as opposed to its counterparts in bilaterians such as human and mouse. Furthermore, correlation with transcriptome data revealed that the levels of RNA Pol II correlate with the magnitude of gene expression. Strikingly, the characteristic peak of RNA Pol II pause typically observed in bilaterians at the transcription start sites (TSSs) was not observed in Hydra. The RNA Pol II traversing ratio in Hydra was found to be intermediate to yeast and bilaterians. The search for factors involved in RNA Pol II pause revealed that RNA Pol II pausing machinery was most likely acquired first in Cnidaria. However, only a small subset of genes exhibited the promoter proximal RNP Pol II pause. Interestingly, the nucleosome occupancy is highest over the subset of paused genes as compared to total Hydra genes, which is another indication of paused RNA Pol II at these genes. Thus, this study provides evidence for the molecular basis of RNA Pol II pause early during the evolution of multicellular organisms.

Molecular signature of an ancient organizer regulated by Wnt/ß-catenin signalling during primary body axis patterning in Hydra.

Wnt/ß-catenin signalling has been shown to play a critical role during head organizer formation in Hydra. Here, we characterized the Wnt signalling regulatory network involved in formation of the head organizer. We found that Wnt signalling regulates genes that are important in tissue morphogenesis. We identified that majority of transcription factors (TFs) regulated by Wnt/ß-catenin signalling belong to the homeodomain and forkhead families. Silencing of Margin, one of the Wnt regulated homeodomain TFs, results in loss of the ectopic tentacle phenotype typically seen upon activation of Wnt signalling. Furthermore, we show that the Margin promoter is directly bound and regulated by ß-catenin. Ectopic expression of Margin in zebrafish embryos results in body axis abnormalities suggesting that Margin plays a role in axis patterning. Our findings suggest that homeobox TFs came under the regulatory umbrella of Wnt/ß-catenin signalling presumably resulting in the evolution of primary body axis in animal phyla.

Cellular and Molecular Mechanisms of Hydra Regeneration.

Regeneration of lost body parts is essential to regain the fitness of the organism for successful living. In the animal kingdom, organisms from different clades exhibit varied regeneration abilities. Hydra is one of the few organisms that possess tremendous regeneration potential, capable of regenerating complete organism from small tissue fragments or even from dissociated cells. This peculiar property has made this genus one of the most invaluable model organisms for understanding the process of regeneration. Multiple studies in Hydra led to the current understanding of gross morphological changes, basic cellular dynamics, and the role of molecular signalling such as the Wnt signalling pathway. However, cell-to-cell communication by cell adhesion, role of extracellular components such as extracellular matrix (ECM), and nature of cell types that contribute to the regeneration process need to be explored in depth. Additionally, roles of developmental signalling pathways need to be elucidated to enable more comprehensive understanding of regeneration in Hydra. Further research on cross communication among extracellular, cellular, and molecular signalling in Hydra will advance the field of regeneration biology. Here, we present a review of the existing literature on Hydra regeneration biology and outline the future perspectives.

NF-κB Signaling and IL-4 Signaling Regulate SATB1 Expression via Alternative Promoter Usage During Th2 Differentiation.

SATB1 is a genome organizer protein that is expressed in a lineage specific manner in CD4+ T-cells. SATB1 plays a crucial role in expression of multiple genes throughout the thymic development and peripheral differentiation of T cells. Although SATB1 function has been subjected to intense investigation, regulation of SATB1 gene expression remains poorly understood. Analysis of RNA-seq data revealed multiple transcription start sites at the upstream regulatory region of SATB1. We further demonstrated that SATB1 gene is expressed via alternative promoters during T-helper (Th) cell differentiation. The proximal promoter "P1" is used more by the naïve and activated CD4+ T-cells whereas the middle "P2" and the distal "P3" promoters are used at a significantly higher level by polarized T-helper cells. Cytokine and TCR signaling play crucial roles toward SATB1 alternative promoter usage. Under Th2 polarization conditions, transcription factor STAT6, which operates downstream of the cytokine signaling binds to the P2 and P3 promoters. Genetic perturbation by knockout and chemical inhibition of STAT6 activation resulted in the loss of P2 and P3 promoter activity. Moreover, chemical inhibition of activation of NF-κB, a transcription factor that operates downstream of the TCR signaling, also resulted in reduced P2 and P3 promoter usage. Furthermore, usage of the P1 promoter correlated with lower SATB1 protein expression whereas P2 and P3 promoter usage correlated with higher SATB1 protein expression. Thus, the promoter switch might play a crucial role in fine-tuning of SATB1 protein expression in a cell type specific manner.

Rapid divergence of histones in Hydrozoa (Cnidaria) and evolution of a novel histone involved in DNA damage response in hydra.

Histones are fundamental components of chromatin in all eukaryotes. Hydra, an emerging model system belonging to the basal metazoan phylum Cnidaria, provides an ideal platform to understand the evolution of core histone components at the base of eumetazoan phyla. Hydra exhibits peculiar properties such as tremendous regenerative capacity, lack of organismal senescence and rarity of malignancy. In light of the role of histone modifications and histone variants in these processes it is important to understand the nature of histones themselves and their variants in hydra. Here, we report identification of the complete repertoire of histone-coding genes in the Hydra magnipapillata genome. Hydra histones were classified based on their copy numbers, gene structure and other characteristic features. Genomic organization of canonical histone genes revealed the presence of H2A-H2B and H3-H4 paired clusters in high frequency and also a cluster with all core histones along with H1. Phylogenetic analysis of identified members of H2A and H2B histones suggested rapid expansion of these groups in Hydrozoa resulting in the appearance of unique subtypes. Amino acid sequence level comparisons of H2A and H2B forms with bilaterian counterparts suggest the possibility of a highly mobile nature of nucleosomes in hydra. Absolute quantitation of transcripts confirmed the high copy number of histones and supported the canonical nature of H2A. Furthermore, functional characterization of H2A.X.1 and a unique variant H2A.X.2 in the gastric region suggest their role in the maintenance of genome integrity and differentiation processes. These findings provide insights into the evolution of histones and their variants in hydra.

Comparative sequence analyses of genome and transcriptome reveal novel transcripts and variants in the Asian elephant Elephas maximus.

The Asian elephant Elephas maximus and the African elephant Loxodonta africana that diverged 5-7 million years ago exhibit differences in their physiology, behaviour and morphology. A comparative genomics approach would be useful and necessary for evolutionary and functional genetic studies of elephants. We performed sequencing of E. maximus and map to L. africana at ~15X coverage. Through comparative sequence analyses, we have identified Asian elephant specific homozygous, non-synonymous single nucleotide variants (SNVs) that map to 1514 protein coding genes, many of which are involved in olfaction. We also present the first report of a high-coverage transcriptome sequence in E. maximus from peripheral blood lymphocytes. We have identified 103 novel protein coding transcripts and 66-long non-coding (lnc)RNAs. We also report the presence of 181 protein domains unique to elephants when compared to other Afrotheria species. Each of these findings can be further investigated to gain a better understanding of functional differences unique to elephant species, as well as those unique to elephantids in comparison with other mammals. This work therefore provides a valuable resource to explore the immense research potential of comparative analyses of transcriptome and genome sequences in the Asian elephant.

A comprehensive epigenome map of Plasmodium falciparum reveals unique mechanisms of transcriptional regulation and identifies H3K36me2 as a global mark of gene suppression.

BACKGROUND: Role of epigenetic mechanisms towards regulation of the complex life cycle/pathogenesis of Plasmodium falciparum, the causative agent of malaria, has been poorly understood. To elucidate stage-specific epigenetic regulation, we performed genome-wide mapping of multiple histone modifications of P. falciparum. Further to understand the differences in transcription regulation in P. falciparum and its host, human, we compared their histone modification profiles. RESULTS: Our comprehensive comparative analysis suggests distinct mode of transcriptional regulation in malaria parasite by virtue of poised genes and differential histone modifications. Furthermore, analysis of histone modification profiles predicted 562 genes producing anti-sense RNAs and 335 genes having bidirectional promoter activity, which raises the intriguing possibility of RNA-mediated regulation of transcription in P. falciparum. Interestingly, we found that H3K36me2 acts as a global repressive mark and gene regulation is fine tuned by the ratio of activation marks to H3K36me2 in P. falciparum. This novel mechanism of gene regulation is supported by the fact that knockout of SET genes (responsible for H3K36 methylation) leads to up-regulation of genes with highest occupancy of H3K36me2 in wild-type P. falciparum. Moreover, virulence (var) genes are mostly poised and marked by a unique set of activation (H4ac) and repression (H3K9me3) marks, which are mutually exclusive to other Plasmodium housekeeping genes. CONCLUSIONS: Our study reveals unique plasticity in the epigenetic regulation in P. falciparum which can influence parasite virulence and pathogenicity. The observed differences in the histone code and transcriptional regulation in P. falciparum and its host will open new avenues for epigenetic drug development against malaria parasite.

Evolution of Hox-like genes in Cnidaria: Study of Hydra Hox repertoire reveals tailor-made Hox-code for Cnidarians.

Hox and ParaHox genes play decisive roles in patterning the anterior-posterior body axis in Bilateria. Evolutionary origin of Hox genes and primary body axis predate the divergence of Bilateria and Cnidaria. However, function of Cnidarian Hox-like genes and their regulation in axis determination is obscure due to studies limited to a few representative model systems. Present investigation is conducted using Hydra, a Hydrozoan member of phylum Cnidaria, to gain insights into the roles of Cnidarian Hox-like genes in primary axis formation. Here, we report identification of six Hox-like genes from our in-house transcriptome data. Phylogenetic analysis of these genes shows bilaterian counterparts of Hox1, Gsx and Mox. Additionally, we report CnoxB_HVUL, CnoxC2_HVUL and CnoxC3_HVUL belonging to two Cnidarian specific groups. In situ hybridization analysis of Hydra homologues provided important clues about their possible roles in pattern formation of polyps and bud development. Specifically, Hox1_HVUL is regulated by Wnt signaling and plays critical role in head formation. Collating information about expression patterns of different Hox-like genes from previous reports and this study reveals no conformity within Cnidaria. Indicating that unlike in Bilateria, there is no consolidated Hox-code determining primary body axis in Cnidaria.