Npr3 regulates neural crest and cranial placode progenitors formation through its dual function as clearance and signaling receptor

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Natriuretic peptide signaling has been implicated in a broad range of physiological processes, regulating blood volume and pressure, ventricular hypertrophy, fat metabolism, and long bone growth. Here we describe a completely novel role for natriuretic peptide signaling in the control of neural crest (NC) and cranial placode (CP) progenitors formation. Among the components of this signaling pathway, we show that natriuretic peptide receptor 3 (Npr3) plays a pivotal role by differentially regulating two developmental programs through its dual function as clearance and signaling receptor. Using a combination of MO-based knockdowns, pharmacological inhibitors and rescue assays we demonstrate that Npr3 cooperate with guanylate cyclase natriuretic peptide receptor 1 (Npr1) and natriuretic peptides (Nppa/Nppc) to regulate NC and CP formation, pointing at a broad requirement of this signaling pathway in early embryogenesis. We propose that Npr3 acts as a clearance receptor to regulate local concentrations of natriuretic peptides for optimal cGMP production through Npr1 activation, and as a signaling receptor to control cAMP levels through inhibition of adenylyl cyclase. The intracellular modulation of these second messengers therefore participates in the segregation of NC and CP cell populations.

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  1. eLife assessment

    This fundamental work reports the unique finding that specific ligands and receptors in the natriuretic peptide signaling pathway act during early embryogenesis to discriminate between neural crest and cranial placode fates using two distinct mechanisms. This work will be of broad interest to both developmental and cell biologists.

  2. Reviewer #1 (Public Review):

    This manuscript reports the unique finding that specific ligands and receptors in the natriuretic peptide signaling pathway act during early embryogenesis to discriminate between neural crest (NC) and cranial placode (CP) fates. This is a significant finding for two reasons: 1) the developmental role of this pathway has not been studied in any detail; and 2) how cells located in the border between the neural ectoderm and non-neural ectoderm decide on NC versus CP fates is of broad interest and being actively pursued by a number of laboratories. The authors present logical and experimentally convincing support for their conclusions. They report the expression patterns by in situ hybridization and qPCR of the various ligands and receptors of the natriuretic peptide signaling pathway, clearly demonstrating that several of these molecules are expressed in the right place at the right time to influence NC and/or CP formation. They establish that Npr3 is a target of Pax3 and Zic1, two transcription factors previously shown to be required for NC and CP formation, further illustrating that it is part of the appropriate regulatory network. Next, the authors use morpholino knock down of Npr3 to show that the resulting embryos have deficient expression of two NC genes and two CP genes. The controls used for the knock-down are the correct ones and were confirmed by treatment with a high-affinity and selective Npr3 antagonist. The function of Npr3 was further explored by discriminating between its known two functions - clearance of natriuretic peptides and inhibition of adenylyl cyclase - by expressing either WT or mutant versions of human NPR3 in Npr3 morphant embryos. That WT rescued both NC and CP genes but the mutant version only rescued NC genes leads to the appropriate conclusion that Npr3 regulates NC and CP fates via different mechanisms. This conclusion was confirmed by treating Npr3 morphants with a specific adenylyl cyclase inhibitor, which restored CP gene expression, and treating CP promoting explants with an adenylyl cyclase activator, which repressed CP gene expression. Using similar knock-down approaches the authors convincingly demonstrate that Npr2 does not participate in NC/CP formation, but Npr1 does; again, the knock-down results were confirmed by treating embryos with a specific Npr1 antagonist. Finally, the authors complete the story by determining by equally well-controlled knock-down experiments which of the three natriuretic peptides participate in this process. In short, the many different experiments strongly support the conclusions, and the experiments are well controlled and include large numbers of embryos to provide exceptional rigor.

  3. Reviewer #2 (Public Review):

    This study reports a novel role of the natriuretic receptors Npr3 and Npr1 in the formation of neural crest (NC) and cranial placode (CP) progenitor populations in frog embryos. The authors discovered this receptor family in a screen for genes activated during NC development. They show the relevant expression of these receptors and the corresponding ligands in the NC and CP populations. Knockdown and rescue experiments combined with pharmacological drug treatment demonstrated that Npr3 clearance activity is required for NC progenitor formation. Surprisingly, adenylyl cyclase inhibition was required for cGMP production and the effect on CP development. The authors conclude that the two second messengers downstream participate in the segregation of the NC and CP progenitors in embryonic development.

    The significance of this study is in the demonstration of two distinct developmental programs that are separately controlled by different activities of the same receptor. The study is well designed and executed with proper controls. Nevertheless, the data suggesting that Npr3 regulates NC and CP fates via different mechanisms are limited and need further support, such as the analysis of additional markers for CP progenitors, to be unambiguously interpreted. The work is likely to impact two different areas: early embryonic development and natriuretic peptide signaling.

  4. Reviewer #3 (Public Review):

    The current manuscript describes the expression of multiple Natriuretic peptides and their receptor during the early embryo development in the amphibian Xenopus laevis. This signaling pathway is well known to control a broad range of physiological processes but its role in embryogenesis has not been studied before. Thus, the study presents some important novel findings. After defining the combination of ligands and receptors expressed during embryogenesis, they used loss of function experiment to test the requirement of this signaling pathway to the development of neural crest cells.

    The loss of function experiments are well controlled as they use both chemical inhibitors and Morpholino that block the translation of the protein. They also rescue the phenotypes by using either mRNA from the human protein (receptor), or purified peptides (Ligands).

    The results clearly show that loss of either Npr1 or Npr3 affects the development of both neural crest and placodes, while Npr2 had no visible effect. Similarly, they found that the loss of the ligands Nppa and Nppc affected neural crest and placode development while Nppb had no effect. Again, the loss of function was achieved with both Morpholino KD and inhibitors. In general, the loss of neural crest and placodal marker are associated with an expansion of neural marker (Sox2) and a corresponding decrease of epidermal marker (Keratin).

    For Npr3 the author show that the loss of the protein is associated with an increase in cell death and decrease in cell proliferation which match some previous work on the role of the receptor in other cell type. It is unclear how much this can account for the striking difference in patterning observed and experiment to test this have not been performed.
    Overall, this work is important for the field as it shows novel genes that are critical for craniofacial and sensory development. It is likely that mutations in any genes involved in this pathway could result in birth defect which could be corrected pharmacologically.