A novel regulatory gene promotes novel cell fate by suppressing ancestral fate in the sea anemone Nematostella vectensis

Abstract

Cnidocytes (i.e., stinging cells) are an unequivocally novel cell type used by cnidarians (i.e., corals, jellyfish, and their kin) to immobilize prey. Although they are known to share a common evolutionary origin with neurons, the developmental program that promoted the emergence of cnidocyte fate is not known. Using functional genomics in the sea anemone, Nematostella vectensis, we show that cnidocytes develop by suppression of neural fate in a subset of neurons expressing RFamide. We further show that a single regulatory gene, a C2H2-type zinc finger transcription factor (ZNF845), coordinates both the gain of novel (cnidocyte-specific) traits and the inhibition of ancestral (neural) traits during cnidocyte development and that this gene arose by domain shuffling in the stem cnidarian. Thus, we report a mechanism by which a truly novel regulatory gene (ZNF845) promotes the development of a truly novel cell type (cnidocyte) through duplication of an ancestral cell lineage (neuron) and inhibition of its ancestral identity (RFamide).

Keywords: cell identity; cnidarians; evolution; novelty.

Fig. 1.

ZNF845 specifies cnidocyte identity in N. vectensis. (A–C) ZNF845 is partially coexpressed with: (A) EdU (proliferating cells), (B) SoxB2 mRNA (neural progenitor cells), and (C) PaxA mRNA (cnidocytes). Insets: white arrowheads show coexpression; nuclei are white (DAPI stain). (D) ZNF845 expression after knockdown of SoxB2 and PaxA (by MO) assayed by in situ hybridization and qPCR. *P < 1E-02 for SoxB2 MO or PaxA MO versus Ctrl MO. (E) Cnidocyte differentiation (α-Mcol4 antibody) after ZNF845 knockdown (shRNA). (F) qPCR of target gene expression after ZNF845 knockdown; fold-change relative to housekeeping gene EF1B. (G) Response of RFamide- and PaxC-expressing cells to ZNF845 knockdown. (H) RFamide and PaxC are not coexpressed. (I) Expression of RFamide and PaxC after knockdown of PaxC (MO). (J) Model for ZNF845-mediated specification of cnidocyte identity from an RFamide-expressing ancestral cell lineage. *P < 1E-02 (SI Appendix, Table S2).

Fig. 2.

RFamide identity is inhibited by NR12 in developing cnidocytes. (A) NR11, NR12, and NR13 are down-regulated in ZNF845 knockdowns (shRNA) as assayed by in situ hybridization and qPCR. (B–D) NR11, NR12, and NR13 are coexpressed with Mcol4 (cnidocytes; cyan), but not with each other (C and D). (E and F) NR12 is partially coexpressed with (E) ZNF845 and (F) PaxA. (G) Response of RFamide- and PaxC-expressing cells after NR12 knockdown. (H and I) There is no effect of NR12 knockdown on (H) cnidocyte differentiation (Mcol4) or (I) expression of NR11 or NR13. (J) Modular regulation of neural traits (e.g., axons, vesicles plus neuropeptides) explains how some traits can be lost (e.g., axons) and others retained but inhibited (RFamide) in developing cnidocytes. *P < 1E-02. See SI Appendix, Table S2 for detailed information. TF, transcription factor.

Fig. 3.

Evolutionary history of two six-domain C2H2 ZNF proteins. (A and B) Relationships among ZF-C₂H₂ domains from (A) ZNF845 (N. vectensis; JGI Protein Identification [ID]: 81344) and (B) GFI1B (N. vectensis JGI Protein ID: 112378). Broken gray lines represent the locations of the other ∼11,000 branch tips of the complete tree. (C) Proposed model for the emergence of ZNF845: 1. In the common ancestor of cnidarians and bilaterians, ZF-C₂H₂ domains were in distinct proteins. 2. In the stem cnidarian, domain shuffling brought domains 1/5, 2, 3, 4 and 6 together in a single protein, 3. Domain 1/5 then duplicated and diverged to become two distinct domains (1 and 5) before the diversification of extant cnidarians (SI Appendix, Figs. S5–S8).

Fig. 4.

Origin of novelty through lineage duplication and suppression of ancestral fate. A stepwise model for the origin of cnidocytes by duplication and divergence of an RFamide neural lineage and a proposal for the evolutionary timing of these events are presented.