Lhx2 is a progenitor-intrinsic modulator of Sonic Hedgehog signaling during early retinal neurogenesis

  1. Xiaodong Li
  2. Patrick J Gordon
  3. John A Gaynes
  4. Alexandra W Fuller
  5. Randy Ringuette
  6. Clayton P Santiago
  7. Valerie Wallace
  8. Seth Blackshaw
  9. Pulin Li
  10. Edward M Levine

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Abstract

An important question in organogenesis is how tissue-specific transcription factors interact with signaling pathways. In some cases, transcription factors define the context for how signaling pathways elicit tissue- or cell-specific responses, and in others, they influence signaling through transcriptional regulation of signaling components or accessory factors. We previously showed that during optic vesicle patterning, the Lim-homeodomain transcription factor Lhx2 has a contextual role by linking the Sonic Hedgehog (Shh) pathway to downstream targets without regulating the pathway itself. Here, we show that during early retinal neurogenesis in mice, Lhx2 is a multilevel regulator of Shh signaling. Specifically, Lhx2 acts cell autonomously to control the expression of pathway genes required for efficient activation and maintenance of signaling in retinal progenitor cells. The Shh co-receptors Cdon and Gas1 are candidate direct targets of Lhx2 that mediate pathway activation, whereas Lhx2 directly or indirectly promotes the expression of other pathway components important for activation and sustained signaling. We also provide genetic evidence suggesting that Lhx2 has a contextual role by linking the Shh pathway to downstream targets. Through these interactions, Lhx2 establishes the competence for Shh signaling in retinal progenitors and the context for the pathway to promote early retinal neurogenesis. The temporally distinct interactions between Lhx2 and the Shh pathway in retinal development illustrate how transcription factors and signaling pathways adapt to meet stage-dependent requirements of tissue formation.

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  1. Version published to 10.7554/elife.78342 on eLife
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    Referee #2

    Evidence, reproducibility and clarity

    Summary:

    The manuscript entitled "Lhx2 is a progenitor-intrinsic modulator of Sonic Hedgehog signaling during early retinal neurogenesis" by Li et al is a very interesting study in which the importance of Lhx2 is studied in conditional knock-out background to decipher the importance during retinal neurogenesis of developing embryo. The study reveal importance of co-receptors essential to Shh signalling. Data presented are clean and would add depth of knowledge to the literature. The study/manuscript do have some lacunae which are listed below which would be good to address before it is published

    Major comments

    1. Authors do not seem to have performed a rescue experiment with Lhx2 CKO. If this is absolutely not possible, a conditional overexpression could have given confirmatory clues on the Lhx CKO phenotype discussed. In any case some sort of rescue experiments are essential with respect to Lhx2 as done with Cdon and Gas1
    2. Also, overexpression studies with the following genes, Cdon, and Gas1 is interesting in the CKO background. What about their over expression phenotype in Wild-type, Ptch1-CKO, and purmorphamine/Shh-N treated conditions. Alternatively, an ex-vivo or in vitro approach using cultured cells may also prove worthy to prove this point.
    3. A logical explanation of Tamoxifen administration at a window E11.5 - E15.5 is good to have in the results section.
    4. Authors say ".........that Lhx2-deficient RPCs can respond to recombinant Shh-N at more physiologically relevant concentrations, but their response is still attenuated compared to Lhx2-expressing RPCs." If the Shh-N dose is increased above physiological concentrations in Lhx-CKO conditions does the Gli1 read-out will restore to normalcy ? This would give insight on to the roles of co-receptors such as Cdon, Gas1.

    Minor comments

    1. In the 'Introduction' the authors write "Pathway activation is not achieved, however, by simple ligand binding to Patched, but also requires one of three co-receptors: Cell Adhesion, Oncogene Regulated (Cdon), Brother of Cdon (Boc), or Growth Arrest Specific 1 (Gas1)." Please give a citation of appropriate literature.
    2. In the introduction authors write "In this case, an interaction with Notch signaling is partly responsible, through Lhx2-dependent expression of ligand (Notch1), receptors (Dll1, Dll3), and downstream transcriptional effectors (Hes1, Hes5)" I feel Dll1 and Dll3 are ligand and Notch is receptor. Please check.
    3. Figure 1c: Western blots tubulin is less in CKO alongside Gli1. It would be better to do quantification for showing any significant changes.
    4. Figure 2A and Result 2: Why harvesting at E14.5 specifically for qPCR/ChIP sequencing, while for RNA sequencing and in situ , it was done E15.5.
    5. Figure 5D, E: It is not clear why there were more mCitrine+ cells in CKO explants at 96 hours.
    6. Figure 6 C,D,E,F: Does Lhx2 CKO, cause cell death as the levels of vsx2 are low in vehicle in CKO (D,F) as compared to ctrl (C,E).
    7. The levels of Gli1 are higher in CKO vehicle (D,F) than the control panel (C,E). This difference in Gli1 expression is more evident D versus C. Does it mean that CKO increases Gli1 expression in explants, which seems to be opposite of what shown in the first results (Figure 1D, E).
    8. It is not clear why the increase in Gli1 expression with Shh-N (E,F) is not that much evident than with purmorphamine (C,D) in both control and CKO explants.
    9. Figure 7 7-F: The changes in the levels of Cyclin D1 and Hes1 in Ctrl/Lhx2 CKO/ dCKO are not very clear from the data in present form. It would be better to show the changes in their mRNA levels by qPCR analysis. Further, it is not clear how the changes in CyclidD1 and Hes1 levels are proving that Lhx2 acts downstream of Shh signaling.
    10. Supplementary table 4: In the page 3 have typo error "centrations at 72 hr"
    11. Supplementary Figure 8 is labeled as Supplementary Figure 7, so there are two figures labeled as Supplementary Figure 7.

    Significance

    Study is significant, and adds more depth to existing knowledge in this science field. Developmental and cell biologists would benefit from this study.

    Retina regeneration, Cellular signalling, Epigenetics, One knock outs/knockdowns, transgenics, RNAseq, Microarray, ChIPseq, Cell sorting

  4. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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    Referee #1

    Evidence, reproducibility and clarity

    The manuscript by Li et al., entitled: "Lhx2 is a progenitor-intrinsic modulator of Sonic Hedgehog signaling during early retinal neurogenesis," focuses on an important topic in developmental biology-the regulatory interaction between transcription factors (TFs) and signaling pathways, namely how the TF confers cells' competence to respond to extrinsic cues. The study focuses specifically on Lhx2 regulation of Sonic Hedgehog (HH)-pathway genes in retinal development. The authors approach this complex topic through global transcriptomic analyses combined with elegant functional in-vivo studies, including a systematic examination of the pathway genes through the use of inhibitors and ligand on cKO retinal explants. The results reveal complex regulation by Lhx2 of several HH-pathway genes in the developing mouse retina, including Ptch, Gli1 and the co receptors Cdon and Gas1. The finding that a single TF controls several components of the signaling pathway is interesting. Nevertheless, probably due to the complex activity of Lhx2, which functions on additional targets, it remains unclear how regulation of HH-pathway genes by Lhx2 impacts the eventual phenotype of the Lhx2 cKO retinal progenitor cells (RPCs). In the following, I list the main findings and several comments that need to be addressed:

    Fig. 1 presents the experimental system using inducible Hes1-CreERT to mutate Lhx2 on E11.5 and examine the expression of Gli1 and Sonic Hedgehog in the control and mutant.

    • The authors should present the distribution of the Lhx2 protein in the control vs. mutant. Considering that the deletion is of only part of the gene (as shown in Fig. 2), it is important to present the loss of the protein as well as the efficiency of Cre activity. • On the figure, add a characterization of the cellular phenotype of the cKO retinas on E15.5 by presenting the expression of markers for ganglion cells and RPCs. Figs. 2 & 3 present the bulk RNA-seq analysis of the Lhx2 cKO retinas, including experimental design, validation and results. The integration of previously published ATAC-seq and ChIP-seq data for Lhx2 point to the direct targets and bound regulatory regions.
    • "4 biological repeats per genotype" - Specify if four eyes were sampled from two embryos or from four different embryos. Were the embryos from different litters? • Add GSEA analysis for the HH-pathway genes. Fig. 4 presents a published approach to quantifying the response to HH using a cellular reporter assay (Li et al., 2018), whereas in Fig. 5, availability of HH ligand is evaluated by elegantly implementing the cellular reporter assay. The results suggest that Lhx2 does not regulate ligand availability. • Fig. 4 presents a published approach and thus can be included in Fig. 5.
    Fig. 6 presents evidence that in the Lhx2 cKO, the Shh pathway is functional downstream of Smo, because the expression of Gli1 increases in cKO cells following Smo activation (with purmorphamine). Furthermore, the response to Shh-N is shown to be partly attenuated in the Lhx2 cKO retina.
    Figure 7 examines whether Ptch deletion can rescue aspects of the Lhx2 phenotype. This was done by comparing the phenotypes of cKOs of Lhx2, Ptch, or both Ptch and Lhx2. The results revealed partial rescue, in the Ptch and Lhx2 cKO, of the expression of Ptch1 and Gli1, but not of the proliferation and premature differentiation phenotypes based on expression of Cyclin D1, EDU, PCNA and Hes1. • Add images of the control to Fig. 7B,C. • Explain how the deletion of Ptch1 was examined. They next investigated regulation of the Ptch co - receptors Cdon and Gas1 by Lhx2 (Figs. 8, 9). Fig. 8 presents the developmental expression pattern of Cdon and Gas1 in the control, and their downregulation in the Lhx2 cKO (although Cdon is maintained in the dorsal optic cup). The results show that Cdon is the co-receptor that is normally expressed in RPCs. GAS1 seems to play a role in the peripheral progenitors destined to ciliary body and iris.
    Electroporation of both receptors into the Lhx2 cKO retinas resulted in increased pathway activity (based on Gli1 reporter). • Both Cdon and Gas1 were electroporated into the Lhx2 cKO retina, although Gas1 is not expressed in control RPCs (based on the analysis in previous panels). Explain why both were co-electroporated and the outcome of electroporating only Cdon. • The outcome of electroporation of the co-receptors into control retina should be presented. • It is important to include staining for Lhx2; it is possible that the cells that respond to the co-receptors are those that were not mutated (escapers). Presenting the loss of Lhx2 (or Cre activity through the use of a reporter) and comparing it to the outcome of electroporation into the control retina are therefore required.

    Finally, the authors present evidence that Lhx2 cKO, on E13.5 when Cdon is no longer expressed in the RPCs, continues to compromise the HH - pathway genes. This further supports continued regulation of several HH-pathway genes in early and late RPCs.

    • The finding that a Lhx2 controls several components of HH pathway could be relevant to Lhx2 activity in patterning of the cortex - I suggest to discuss the possible relevance of the findings to other organs.

    Additional comments:

    • Fig. 4E: Add explanation of the quantitative analysis. • Fig. 5: Explain how results were normalized based on retinal size (which is significantly smaller in cKO retinas). How many independent experiments were run here? How many different retinas were tested? Were retinas taken from the same mouse considered 'independent'?
    • Fig. 8B: Indicate the genotype of the presented tissue. • Fig. 8 A,B should be presented in one panel, in the same orientation. • Fig. 8D: Present the different channels, in addition to the merge image.

    Significance

    The study focuses on an important topic in developmental biology-the regulatory interaction between transcription factors (TFs) and signaling pathways, namely how the TF confers cells' competence to respond to extrinsic cues. The study focuses specifically on Lhx2 regulation of Sonic Hedgehog (HH)-pathway genes in retinal development. The results reveal complex regulation by Lhx2 of several HH-pathway genes in the developing mouse retina, including Ptch, Gli1 and the co receptors Cdon and Gas1. The finding that a single TF controls several components of the signaling pathway is interesting. Nevertheless, probably due to the complex activity of Lhx2, which functions on additional targets, it remains unclear how regulation of HH-pathway genes by Lhx2 impacts the eventual phenotype of the Lhx2 cKO retinal progenitor cells (RPCs).

  5. Version published to 10.1101/2021.04.24.441277 on bioRxiv