Hedgehog signaling is required for endomesodermal patterning and germ cell development in Nematostella vectensis

Cheng-Yi Chen, Sean A. McKinney, Lacey R. Ellington, Matthew C. Gibson

Preprint posted on January 15, 2020

On the (h)edge: the germline precursors of a basal metazoa are induced at the interface between Hedgehog signalling domains

Selected by Paul Gerald L. Sanchez and Stefano Vianello



Organisms that reproduce sexually do so by fusion of two specialised cells: sperm and egg (gametes). By carrying genetic information from one generation to the other these cells ensure the maintenance of the species over time. While in some organisms (e.g. plants) gametes are derived from cells that also give rise to body types, in (most) animals this is not the case. Sperm and eggs are instead produced by a dedicated line of cells put aside very early as to spare them from the accumulation of mutations over time. Throughout reproductive life gametes will be produced by dedicated, germline, stem cells. In turn, these cells have their origin in the embryo and ultimately descend from so-called Primordial Germ Cells (PGCs). 

As the early setting aside of PGCs will ultimately originate the germline, PGC specification is a milestone of early development, and understanding the mechanisms leading to their specification is an area of great interest. Comprehensive surveys of PGC specification across the evolutionary tree report two main strategies through which embryos specify their PGCs:

  • preformation, whereby maternal factors are quickly deposited within a subset of cells in the embryo to lock them into PGC fate
  • induction, whereby PGCs are specified later by signalling cues from the zygote/embryo itself (e.g.  BMP signalling in mouse)

Most model organisms such as C.elegans (roundworm), Drosophila (fruitfly), zebrafish, and Xenopus (frog) all specify their PGCs by preformation, while induction seems to be an exclusive of e.g. chicken, mouse, and human. While preformation might thus intuitively appear to be “ancestral”, most species actually specify PGCs by inductive mechanisms.  Even more interestingly, research on cnidarians (sister group of all bilaterians) has found that here too PGCs are induced rather than locked away by early maternal determinants. The common ancestor of all animals would thus induce their PGCs, but how this actually takes place has remained a mystery.


Key Findings

The authors identify the mechanisms of PGC specification in the sea anemone Nematostella vectensis (bilaterian sister group), and confirm that here too specification occurs by induction rather than preformation. Specifically, PGCs are induced out of a field of endomesodermal cells (which surround the pharynx), where these cells abut the ectoderm of the pharynx itself. As can be seen in Figure A, this interface and site of PGC specification crucially coincides with the interface between Hedgehog-signalling domains (Hh and its receptor, Patched).


Hedgehog-signaling expression domains, showing juxtaposed expression of the ligand (hh1) and its receptor (ptc). Putative PGC clusters, marked by the Vasa2 protein, form at the interface of the two expression domains. [Modified from Figure 5 of the preprint. Red was changed to magenta using the “Replace Red with Magenta” plug-in in Fiji.]

While maternally inherited germline-determinants (e.g. Vasa2) are indeed detected, these do not differentially accumulate in any cell, and germline transcripts are expressed homogeneously throughout the entire endomesodermal field. Instead, one observes clear segregation between cells expressing the signalling molecule Hedgehog (the pharynx ectoderm), and cells expressing its receptor Patched (the surrounding endomesoderm). At the interface between these domains, where Hedgehog signalling is thus productive, PGCs are specified.

The authors then track these cells as they delaminate out of the endomesoderm by epithelial to mesenchymal transition (EMT)-like mechanisms, and reach the developing gonad rudiments, in which they will mature to germline stem cells.

Results are particularly interesting in the use of Hox mutants, altered in their body plan, where PGCs specify in altered locations (Figure B), indicative of an inductive mode of specification. That is, even though these mutants miss the primary structures where PGCs would be expected to emerge (primary mesenteries), PGCs emerge nonetheless,  if in an altered location. Conversely, even in the wild-type body plan, PGCs will not get specified if Hedgehog signalling is absent (CRISPR experiments).


Localization of putative PGC clusters in Hox mutants with altered body plans. [Figure S7 of the preprint. Red was changed to magenta using the “Replace Red with Magenta” plug-in in Fiji.]



The current study builds on the beautiful body of previous work that has investigated the formation of precursor germ cells in a basal metazoa, Nematostella vectensis. The authors provide here several pieces of evidence supporting inductive PGC specification (versus preformation) as an ancestral strategy in eumetazoans, and they capitalize on experimental advances that were not previously available (e.g. immunostaining with antibody against Vasa2 and generation of mutants using CRISPR/Cas9).

The identification of the role of Hedgehog signaling in specification of PGCs is remarkable and sparks interesting developmental questions. Most noteworthy is the description of the reciprocal expression of the Hedgehog ligand and its receptor, and how putative PGCs (Vasa2+ cells) form at the interface of these juxtaposed expression domains. 

The preprint provides thought-provoking mechanistic insight into the possible earliest forms of animal PGC specification.


Questions to the authors:

  • What is known about the role of Hedgehog signalling for PGC induction in other organisms and model systems? Are there traces (or conservation) of such ancestral evolutionary strategy?
  • Would it be possible to implant a bead with Hh ligand (hh1) in the endomesoderm of the planula, adjacent to the domain expressing the Hh receptor (ptc)? Would this result in specification of an ectopic putative PGC cluster?
  • BMP signalling is described here as having a purely attractive/repulsive role, almost like a chemokine. This is in contrast with its key PGC-inductive role usually associated to it based on studies in e.g. mouse (and even cricket, cfr. Extavour and Nakamura 2016). Could you elaborate on this?
  • It is particularly remarkable that PGCs form near the mouth in Nematostella, while their site of induction is posterior in mouse (and human?). Could a model be made whereby the site of PGC specification correlates with the site of Gastrulation?
  • If zygotic specification is ancestral, what would be the model explaining loss of this strategy in favour of determinant deposition (preformation) in some later species?


Further Reading

  • Extavour, Cassandra G., and Michael Akam. “Mechanisms of germ cell specification across the metazoans: epigenesis and preformation.” Development 130.24 (2003): 5869-5884.
  • Extavour, Cassandra G., et al. “Vasa and nanos expression patterns in a sea anemone and the evolution of bilaterian germ cell specification mechanisms.” Evolution & development 7.3 (2005): 201-215.
  • Johnson, Andrew D., and Ramiro Alberio. “Primordial germ cells: the first cell lineage or the last cells standing?.” Development 142.16 (2015): 2730-2739.
  • He, Shuonan, et al. “An axial Hox code controls tissue segmentation and body patterning in Nematostella vectensis.” Science 361.6409 (2018): 1377-1380.
  • Matus, David Q., et al. “The Hedgehog gene family of the cnidarian, Nematostella vectensis, and implications for understanding metazoan Hedgehog pathway evolution.” Developmental biology 313.2 (2008): 501-518.

Tags: hh, nematostella, pgc, primordial germ cells

Posted on: 25th February 2020


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