Functional studies on the role of Notch signaling in Hydractinia development

Abstract

The function of Notch signaling was previously studied in two cnidarians, Hydra and Nematostella, representing the lineages Hydrozoa and Anthozoa, respectively. Using pharmacological inhibition in Hydra and a combination of pharmacological and genetic approaches in Nematostella, it was shown in both animals that Notch is required for tentacle morphogenesis and for late stages of stinging cell maturation. Surprisingly, a role for Notch in neural development, which is well documented in bilaterians, was evident in embryonic Nematostella but not in adult Hydra. Adult neurogenesis in the latter seemed to be unaffected by DAPT, a drug that inhibits Notch signaling. To address this apparent discrepancy we studied the role of Notch in Hydractinia echinata, an additional hydrozoan, in all life stages. Using CRISPR-Cas9 mediated mutagenesis, transgenesis, and pharmacological interference we show that Notch is dispensable for Hydractinia normal neurogenesis in all life stages but is required for the maturation of stinging cells and for tentacle morphogenesis. Our results are consistent with a conserved role for Notch in morphogenesis and nematogenesis across Cnidaria, and a lineage specific loss of Notch dependence in neurogenesis in hydrozoans.

The colonial cnidarian Hydractinia

“…Current research on Hydractinia focuses on a number of topics, including embryonic development [14], neurogenesis [15,16], stem cells, germ cells, and regeneration [17][18][19][20], allorecognition [21], metabolism [22], immunity [23], and natural product chemistry [24]. Allorecognition refers to the ability to discriminate 'self ' from 'nonself ' within the same species, a phenomenon observed in most colonial cnidarians, but not in Hydra or Nematostella, the two most commonly used cnidarian model systems for molecular work.…”

“…Interestingly, data published to date suggest that both stem cell behavior and the mode of regeneration differ substantially between cnidarian species [18,28,30]. For example, hydrozoan neuronal cells derive from migratory i-cells, whereas in anthozoans, neural progenitor cells are epithelial [16]. As to regeneration modes, Hydra can reform the main head structures following decapitation in the absence of cell proliferation whereas in Hydractinia and Nematostella cell proliferation is essential for regeneration [18].…”

“…A more targeted way to genetically manipulate the animals is provided by CRISPR-Cas9 technology. In Hydractinia, this is performed by microinjecting site-specific short guide [34] and licensed under CC BY 4.0 (link: https ://creat iveco mmons .org/licen ses/by/4.0/)) RNAs (sgRNA) together with recombinant Cas9 to generate loss-of-function mutations [16,20]. Adding to the injecting cocktail a plasmid including a fragment of DNA, flanked by two homology arms, can be used for targeted knock-in of fragments [34].…”

CRISPR/Cas9-mediated genome editing in a reef-building coral

“…Recently, the range and power of such methods has been dramatically expanded by the emergence of the CRISPR/Cas9 genome-editing technology, which can be applied to diverse organisms and has facilitated not only the generation of loss-of-function mutations but also the introduction of more subtly modified genes, the tagging of proteins, and large-scale genomic restructuring (17)(18)(19). In cnidarians, this tool has been used in the sea anemone Nematostella vectensis and the hydrozoan Hydractinia echinata by microinjecting single-guide RNA (sgRNA)/Cas9 ribonucleoprotein complexes into one-cell zygotes (20)(21)(22), a powerful method for producing genetic changes at an early developmental stage (19,23,24). An obstacle to applying this technique to corals is the limited availability of gametes to generate zygotes.…”

CRISPR/Cas9-mediated gene knockin in the hydroid Hydractinia symbiolongicarpus

“…With the advent of CRISPR/Cas9 genome editing, this goal is now within reach. To date, CRISPR/Cas9 has been used to disrupt genes in several cnidarians, including Nematostella [37][38][39][40] , Clytia [41] , Acropora [42] , and Hydractinia [43] . These studies have each employed embryonic injection of one or more guide RNAs (sgRNAs) to disrupt a gene of interest, then evaluated phenotypes in developing embryos or adult animals derived directly from them.…”