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  1. Author Response

    Reviewer #2 (Public Review):

    In this study, the authors identify a role for mindbomb (mib) in the trafficking of the Ryk receptor, a Receptor Like Tyrosine Kinase with roles in planar cell polarity (PCP). The authors use a combination of tools and mutants in the zebrafish to propose that the role in gastrulation is notch-independent. A strength is the generation of new genetic alleles. The authors show a genetic interaction between Mib and Ryk. An intriguing finding from the double mutant (mib;MZryk), is that the impact of Mib is mediated through Ryk. This study highlights the need to consider complex signaling networks when evaluating phenotypes, especially convergent extension.

    A concern is that most of the analyses are with morpholino knockdown. It is understood that the use of the MO allows for higher sample sizes, but the authors would need to also investigate the functional rescue in the genetic mutants.

    In the revised version of our manuscript we have included a new series of experiments in which Ryk-GFP overexpression was used to rescue the CE defects of mib1tfi91 mutants (Fig.3L). In accordance with the data already obtained in mib1 morphants, this additional experiment confirms our previous observation that Ryk overexpression allows to rescue the morphogenetic defects of Mib1 depleted animals.

    Moreover, the CE defects shown in Figures 2-4 were analyzed by morphological characteristics. The conclusions can be strengthened by measurement of axial structures with molecular markers.

    To further strengthen our analysis of the impact of Mib1 / Ryk loss of function on the development of axial structures we performed in situ hybridizations with shhb (labeling the notochord) and foxa3 (labeling notochord and prechordal plate) on mib1 morphant, mib1ΔRF123- or mib1ΔRF3-injected embryos as well as MZ ryknce4g mutant embryos. The corresponding pictures and quantifications have been included in Fig.4H,I and the newly added Figure 1-figure supplement1. Importantly these new experiments confirm our observations that Mib1 or Ryk loss of function both result in a decrease of axial elongation and an increase of notochord width that are diagnostic for embryos that display an impairment of PCP-dependent CE movements.

    Ryk has been proposed to be a substrate for Mib and in the manuscript, there is a clear demonstration that Mib overexpression is sufficient to stimulate Ryk-GFP internalization. It is noteworthy that Mib over-expression does not stimulate Vangl internalization. However, the difference of the number of Ryk-GFP endosomes/cell in the mib mutant compared to would-type is less clear. Also, the MO alone is not shown.

    The impact of the Mib MO alone on the number of Ryk-GFP labeled endosomes is now displayed in Figure 3-figure supplement 4A,B in addition to the quantifications already displayed documented in Fig3E.

    Using an early endosomal marker with the Ryk-GFP would be a more effective way to evaluate endocytosis.

    The data displayed in Figure 3-figure supplement 1A show that a majority of RYK-GFP labeled intracellular vesicles are also positive for the early endosomal marker Rab5, providing thereby evidence that the intracellular structures quantified in Fig.3E and J correspond essentially to endosomal compartments.

    In the manuscript, the authors generate a zebrafish ryk mutant. In reference to the functional analysis of the ryknce4g product, the authors would need to perform a Western blot of the injected embryos to determine how efficiently the RNA is made into protein and how stable the protein product is, compared to levels of the expressed wt RNA. This will be important when making conclusions about a lack of rescue and lack of over-expression defects.

    We do not claim that the loss of Ryk function in ryknce4g mutants is solely due to a loss of Ryk protein. Our analysis of ryk gene expression in ryk mutants (through in situ hybridization in Figure 4-figure supplement1D and newly included qPCR analysis in Figure 4-figure supplement1G) indeed suggests that ryknce4g transcripts are subject to nonsense mediated degradation. It is therefore not possible to estimate the stability of the Ryk mutant protein through Western Blot, as the resultant protein levels would be affected not only by premature termination of Ryk protein translation, but also by the degradation of the mutant transcript. Independent of the relative importance of these two processes, our antibody staining data presented in Fig.4C provide evidence that the ryknce4g mutation results in a complete absence of full length protein. Taken together, our data provide collective evidence that our ryk mutants present a complete loss of Ryk activity, as further documented by the observation that ryknce4g loss of function phenotypes cannot be enhanced through ryk morpholino injection (Figure 4-figure supplement1I).

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  2. Evaluation Summary:

    This manuscript describes a novel role of the E3 ubiquitin ligase Mindbomb1 (Mib1), a known key regulator of Notch signaling, in regulating convergent extension movements of the zebrafish gastrula, which are dependent on planar cell polarity (PCP) signaling. The authors show that the ability of Mib1 to modulate PCP is totally dependent on the receptor tyrosine kinase Ryk via endocytosis. This paper will be of interest to scientists studying cell signaling and cell movement.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

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  3. Reviewer #1 (Public Review):

    The paper by Saraswathy et al follows up on an astute observation in a previous study from the same group, which showed that mib1 morphants are associated with an early phenotype reflective of impaired convergence extension. This study allows them to link this observation to a role for Mib1 in internalization of Ryk, which previous studies have identified as a target of Mib1 dependent ubiquitination and for its role in Planar Cell Polarity. The study suggests that RING domains required for ubiquitination of Mib1 target genes are required for this function as ectopic expression of mutant forms of Mib1 that lack either all three RING domains or just the last RING domain are capable of exaggerating CE deficits in morphants or specific mib1 mutants, which retain some maternal mib1 function.

    In this context, it remains unclear why the ta52 allele, which has a point mutation that interferes with mib1 function in the context of its other interactions, fails to have this CE phenotype or why ectopic expression of this mutant form of mib1 does not seem to cause similar exaggeration of the early CE phenotype.

    The authors go on to suggest the CE phenotype is not related to a role for mib1 in Notch signaling as ectopic expression of an activated form of Notch (ICD) does reduce the CE phenotype. On the other hand, ectopic expression of RhoA, which functions downstream of Ryk in PCP, is able to reduce the CE phenotype.

    The authors demonstrate that impaired mib1 function is associated with increased accumulation of Ryk-GFP on the cell surface and reduced intracellular Ryk-GFP, which the authors describe are part of an endocytic compartment based on its colocalization with Rab5. To determine if the CE phenotype produced by reduced Mib1-dependent endocytosis, arises as a result of too much Ryk on the cell surface or of as a result of reduction in the endosomal compartment, they determine the effects of ectopically expressing Ryk-GFP. Ectopic Ryk-GFP reduces CE associated defects and results in both increased surface expression and an increase in the amount of internalized Ryk-GFP. This leads the authors to conclude that it is not the increase of Ryk at the cell but rather, the reduction of internalized Ryk that is responsible for CE defect in embryos with impaired Mib1 function.

    The authors go on to demonstrate that effects of Mib1 on internalization of Ryk are specific and not seen with other PCP components like Vangl2, Fz2 or Fz7.
    Finally, as Mib1 function is hypothesized to be relevant only in the context of Ryk endocytosis for its CE defects, the authors show that loss of Mib1 function does not increase the severityof these defects in Ryk null mutants.

    The paper describes a straightforward set of logical experiments to explore the link between Mib1, Ryk and its role in CE. The primary conclusions of the paper are supported by data presented. Nevertheless, many questions remain unanswered. It remains unclear to me why the ta52b allele has such a minimal effect, the authors could elaborate on this. It also remains unclear how Ryk endocytosis contributes to effective PCP or how reduced Ryk endocytosis alters the organization or function of other PCP components.

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  4. Reviewer #2 (Public Review):

    In this study, the authors identify a role for mindbomb (mib) in the trafficking of the Ryk receptor, a Receptor Like Tyrosine Kinase with roles in planar cell polarity (PCP). The authors use a combination of tools and mutants in the zebrafish to propose that the role in gastrulation is notch-independent. A strength is the generation of new genetic alleles. The authors show a genetic interaction between Mib and Ryk. An intriguing finding from the double mutant (mib;MZryk), is that the impact of Mib is mediated through Ryk. This study highlights the need to consider complex signaling networks when evaluating phenotypes, especially convergent extension.

    A concern is that most of the analyses are with morpholino knockdown. It is understood that the use of the MO allows for higher sample sizes, but the authors would need to also investigate the functional rescue in the genetic mutants. Moreover, the CE defects shown in Figures 2-4 were analyzed by morphological characteristics. The conclusions can be strengthened by measurement of axial structures with molecular markers.

    Ryk has been proposed to be a substrate for Mib and in the manuscript, there is a clear demonstration that Mib overexpression is sufficient to stimulate Ryk-GFP internalization. It is noteworthy that Mib over-expression does not stimulate Vangl internalization. However, the difference of the number of Ryk-GFP endosomes/cell in the mib mutant compared to would-type is less clear. Also, the MO alone is not shown. Using an early endosomal marker with the Ryk-GFP would be a more effective way to evaluate endocytosis.

    In the manuscript, the authors generate a zebrafish ryk mutant. In reference to the functional analysis of the ryknce4g product, the authors would need to perform a Western blot of the injected embryos to determine how efficiently the RNA is made into protein and how stable the protein product is, compared to levels of the expressed wt RNA. This will be important when making conclusions about a lack of rescue and lack of over-expression defects.

    Was this evaluation helpful?
  5. Reviewer #3 (Public Review):

    The manuscript by Muraleedharan Saraswathy et al. describes a novel role of the E3 ubiquitin ligase Mindbomb1 (Mib1), a known key regulator of Notch signaling, in regulating convergent extension (CE) movements of the zebrafish gastrula, which is dependent on planar cell polarity (PCP) signaling. The authors demonstrate that mib1 null mutant embryos exhibit CE defects and modulate PCP, independently of Notch signaling. The authors also show that the ability of Mib1 to modulate PCP is totally dependent on the receptor tyrosine kinase Ryk via endocytosis.

    The strength of the manuscript is that Mib1 regulates PCP-dependent CE in a Notch-independent manner. Also, the genetic epistasis analysis demonstrates that Mib1 loss of function has no effect on CE in maternal zygotic ryk null mutants, suggesting that Mib1 is an essential regulator of Ryk endocytosis during zebrafish CE. These demonstrations are convincing. However, the weakness is the conclusion that Mib1 is an 'essential' regulator of PCP is not fully supported by the data. Rather, Mib1 could be a context-dependent modulator of PCP. To fulfill the scope of eLife requires the clarification of the weakness. It would be worth considering publication if this is clarified.

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