Toddler – an embryonic signal hidden in non-coding RNA

It has been assumed that most if not all signals that control early development are known. However, using genomics studies we identified several new candidate signals. We find that one of these - Toddler/Apela - is essential for embryogenesis by promoting the movement of cells during zebrafish gastrulation. Toddler/Apela signals through G-protein coupled receptors and might be the first in a series of previously uncharacterized developmental signals.

HFSP Long-Term Fellows Andrea Pauli and Eivind Valen and colleagues
authored on Tue, 28 January 2014

Secreted proteins provide essential instructive cues for vertebrate development. By regulating the activities of specific signalling pathways, they control a wide range of processes, including early embryonic patterning, axis determination, germ layer specification, cell differentiation, cell migration, morphogenesis and organ formation. In light of the sheer diversity of regulated processes, it is striking how few signalling pathways (e.g. FGF-, Wnt-, TGF-beta-, Hedgehog- and Notch-signalling) have been implicated in the control of embryogenesis. While the importance of each of these pathways remains undisputed, many processes are still poorly understood, raising the possibility that some important regulators of embryogenesis are yet to be identified.

To identify putative new regulators of early development, we used genomics approaches to search for previously uncharacterized coding and non-coding sequences in zebrafish. Using evolutionary conservation and ribosome profiling to identify translated RNA sequences (Pauli et al., 2012; Chew et al., 2013), we uncovered more than 300 novel zebrafish proteins, including more than 20 candidate signaling proteins.     

Figure: Toddler promotes gastrulation movements via Apelin receptor signaling. Toddler is a previously unrecognized, essential, short, conserved embryonic signal that promotes cell migration during zebrafish gastrulation. The internalization movement highlighted by the colored cell tracks requires Toddler signaling. Toddler signals via the G-protein-coupled APJ/Apelin receptor and may be the first of several uncharacterized embryonic signals.  

To test whether any of these novel signals might have a function, we focused on Toddler (official gene name: Apela), a gene that had been incorrectly annotated as a long non-coding RNA in zebrafish, mouse and human, but that we predicted to encode a highly conserved 58 amino acid peptide. We generated mutants for Toddler in zebrafish and discovered that Toddler is essential for embryogenesis by promoting normal gastrulation movements. Without Toddler, cells are slow to internalize and migrate sluggishly, resulting in embryos with no, or small, hearts.

How does Toddler regulate cell movements? Small peptides often signal through G-protein coupled receptors (GPCRs). Indeed, Toddler was found to activate the Apelin GPCR, and lack of this GPCR leads to very similar phenotypes as seen in toddler mutants. Most motility cues are expressed in specific regions of the embryo and attract or repel cells. This is not the case for Toddler: it is expressed broadly and appears to function as a motogen, a signal that promotes the movement but not the directionality of Apelin receptor-expressing cells.

The discovery of Toddler does not only identify a GPCR agonist and promoter of cell movement, it also raises the possibility that several other signaling proteins remain to be characterized, and that Toddler might just be the first in a series of novel developmental signals.


1) Toddler: An Embryonic Signal That Promotes Cell Movement via Apelin Receptors. Pauli A, Norris ML, Valen E, Chew GL, Gagnon JA, Zimmerman S, Mitchell A, Ma J, Dubrulle J, Reyon D, Tsai SQ, Joung JK, Saghatelian A, Schier AF. Science. 2014 Jan 9. [Epub ahead of print] PMID: 24407481

Other references

2) Ribosome profiling reveals resemblance between long non-coding RNAs and 5' leaders of coding RNAs. Chew GL, Pauli A, Rinn JL, Regev A, Schier AF, Valen E. Development. 2013 Jul;140(13):2828-34. doi: 10.1242/dev.098343. Epub 2013 May 22.

3) Systematic identification of long noncoding RNAs expressed during zebrafish embryogenesis. Pauli A, Valen E, Lin MF, Garber M, Vastenhouw NL, Levin JZ, Fan L, Sandelin A, Rinn JL, Regev A, Schier AF. Genome Res. 2012 Mar;22(3):577-91. doi: 10.1101/gr.133009.111. Epub 2011 Nov 22.

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