The Hippo pathway controls a switch between retinal progenitor cell proliferation and photoreceptor cell differentiation in zebrafish

Abstract

The precise regulation of numbers and types of neurons through control of cell cycle exit and terminal differentiation is an essential aspect of neurogenesis. The Hippo signaling pathway has recently been identified as playing a crucial role in promoting cell cycle exit and terminal differentiation in multiple types of stem cells, including in retinal progenitor cells. When Hippo signaling is activated, the core Mst1/2 kinases activate the Lats1/2 kinases, which in turn phosphorylate and inhibit the transcriptional cofactor Yap. During mouse retinogenesis, overexpression of Yap prolongs progenitor cell proliferation, whereas inhibition of Yap decreases this proliferation and promotes retinal cell differentiation. However, to date, it remains unknown how the Hippo pathway affects the differentiation of distinct neuronal cell types such as photoreceptor cells. In this study, we investigated whether Hippo signaling regulates retinogenesis during early zebrafish development. Knockdown of zebrafish mst2 induced early embryonic defects, including altered retinal pigmentation and morphogenesis. Similar abnormal retinal phenotypes were observed in zebrafish embryos injected with a constitutively active form of yap [(yap (5SA)]. Loss of Yap's TEAD-binding domain, two WW domains, or transcription activation domain attenuated the retinal abnormalities induced by yap (5SA), indicating that all of these domains contribute to normal retinal development. Remarkably, yap (5SA)-expressing zebrafish embryos displayed decreased expression of transcription factors such as otx5 and crx, which orchestrate photoreceptor cell differentiation by activating the expression of rhodopsin and other photoreceptor cell genes. Co-immunoprecipitation experiments revealed that Rx1 is a novel interacting partner of Yap that regulates photoreceptor cell differentiation. Our results suggest that Yap suppresses the differentiation of photoreceptor cells from retinal progenitor cells by repressing Rx1-mediated transactivation of photoreceptor cell genes during zebrafish retinogenesis.

Figure 1. Mst2 is essential for early zebrafish embryogenesis.

(A) Early developmental abnormalities of mst2 morphants. Control or mst2 morpholino (MO) at the indicated dose was injected into zebrafish embryos and phenotypes were analyzed at 52 hpf. Embryos were classified into five color categories on the basis of their phenotypes: blue, normal embryos; green, short body length (SL); orange, abnormal eye pigmentation (AP) accompanied by SL; red, abnormal eye morphology (AM) plus AP plus SL; and brown, dead or malformed embryos. Results are presented as the percentage of the total number of embryos examined (N). (B) Representative control and mst2 morphants at 52 hpf. Embryos were injected with control MO (13.3 ng) or mst2 MO (13.3 ng). Top panels, lateral views of whole embryos. Middle panels, higher magnification images of the head regions of the embryos in the top panels. Bottom panels, dorsal views of the head regions of the embryos in the top panels. (The head is at the top of each panel.) White arrowhead, representative area of AM.

Figure 2. Forced expression of mRNA encoding constitutively active yap alters early zebrafish embryogenesis.

(A) Representative images of EGFP mRNA-injected (control) or yap (WT) mRNA-injected zebrafish embryos at 52–54 hpf. Top panels, lateral views of whole embryos. Bottom panels, higher magnification images of the head regions of the embryos in the top panels. N, total number of embryos examined. Embryos injected with either yap (WT) mRNA or EGFP mRNA had normal phenotypes. (B) Representative images of EGFP mRNA-injected (control) or yap (5SA) mRNA-injected zebrafish embryos at 48 hpf. Embryos injected with Yap (5SA) mRNA (10 pg) showed the same spectrum of abnormal phenotypes as mst2 morphants. Data are presented as for Fig. 1B.

Figure 3. The TEAD-binding, WW and transcription activation domains of Yap contribute to early zebrafish development.

Left panel, schematic illustration of constructs of Yap (WT), Yap (5SA), and the indicated variants with deletion (Δ) or mutation (*) of the indicated domains. Specific amino acid alterations are indicated. A, Lats phosphorylation site replaced by an alanine. In vitro-synthesized mRNAs (10 pg) derived from these constructs were injected into zebrafish embryos and phenotypes were quantified as shown in the right panel. Color classification is as for Fig. 1A. Results are presented as the percentage of the total number of embryos examined (N).

Figure 4. Yap is directly involved in zebrafish retinogenesis.

(A) Schematic illustration of the base hsp70-EGFP-yap construct (left panel) and the procedure for the heat shock experiment (right panel). Zebrafish embryos at the one-cell stage were injected with plasmid DNA containing the heat shock promoter constructs indicated in (B). At 21 hpf, injected embryos were immersed in a 37°C water bath for 1 h to apply heat shock and thus induce expression of EGFP-fused Yap. At 54 hpf, EGFP-expressing embryos were isolated and classified on the basis of their phenotypic features. (B) Representative images of the embryos in (A) that were injected with heat shock promoter constructs as indicated on the left side of panels. For each column, top right panels show lateral views of whole embryos, top left panels show higher magnification images of the head regions of the embryos, and bottom panels are fluorescent images of the corresponding top panels. White arrowheads, areas of AP. (C) Quantification of phenotypes of the embryos injected with heat shock promoter constructs in (A, B) as analyzed at 54 hpf. Color classification is as for Fig. 1A except that the phenotype of AP alone is indicated by striped orange shading. Results are presented as the percentage of the total number of embryos examined (N).

Figure 5. Retina-specific expression of yap (5SA) induces retinogenesis defects without affecting body axis formation.

(A) Schematic illustration of the base rx-EGFP-yap construct (left panel) and the procedure for the experiment (right panel). Zebrafish embryos at the one-cell stage were injected with plasmid DNA containing the rx promoter constructs indicated in (B). (B) Representative dorsal and lateral views of the embryos in (A) that were injected with rx promoter constructs as indicated on the left side of panels. Data are presented as for Fig. 4B. White arrowheads, areas of AM plus AP. (C) Quantification of phenotypes of the embryos injected with rx promoter constructs in (A, B) as analyzed at 32 hpf. Color classification is as for Fig. 4C except that the phenotype of AM plus AP is indicated by striped red shading. Results are presented as the percentage of the total number of embryos examined (N). Note that expression of yap (5SA) variants mutated in both the WW1 and WW2 domains prevented the appearance of abnormal eye phenotypes.

Figure 6. Yap (5SA) mRNA-injected embryos exhibit dramatic downregulation of retinal photoreceptor genes.

(A) The top five GO categories for genes downregulated by over 4.0-fold in yap (5SA)-expressing embryos at 48 hpf as determined by microarray analysis. (B) A summary of microarray results for the top 50 downregulated genes in the yap (5SA)-expressing embryos in (A) compared with yap (WT)-expressing embryos at 42, 48 and 54 hpf. The expression levels of genes in the yap (5SA)-injected embryos are shown as Log₂ (fold change) values relative to yap (WT)-injected embryos. The order of the genes is based on expression levels detected at 48 hpf. Red lettering indicates retinal photoreceptor genes whose expression was severely decreased in yap (5SA)-injected embryos. (C) RT-PCR analysis of mRNA expression of the indicated retinal genes in zebrafish embryos injected with yap (WT) or yap (5SA) mRNA and examined at 48 hpf. β-actin, loading control. Yap (5SA)-expressing embryos are grouped by abnormal phenotype, as indicated. Results are representative of two independent experiments.

Figure 7. The WW domains of Yap interact with the PPXY motif of Rx1.

(A) Schematic illustration of the zebrafish Rx1 and Rx1 (ΔPPXY) constructs. A partial amino acid sequence including the PPXY motif of zebrafish Rx1 was aligned with the sequences of Rx from the indicated species. The PPXY motif (red lettering) is highly conserved among vertebrates. A detailed alignment of Rx family proteins can be found in Fig. S5A. (B) Co-immunoprecipitation analysis of HEK293T cells transiently expressing Myc-Rx1 that were co-transfected with empty vector (–), or vector expressing Yap (5SA), Yap (5SA/WW1^*, 2^*) or Yap (5SA/TEAD^*). Lysates were immunoprecipitated (IP) with anti-FLAG Ab to isolate Yap, followed by Western blotting (WB) with anti-Myc Ab to detect Myc-Rx1 (top), or with anti-FLAG Ab to detect FLAG-Yap (middle). (C) Co-immunoprecipitation analysis of HEK293T cells transiently expressing FLAG-Yap (5SA) that were co-transfected with empty vector (–), or vector expressing Rx1 or Rx1 (ΔPPXY). Lysates were IP’d using anti-FLAG Ab and subjected to WB with anti-Myc Ab to detect Rx1 (top), and with anti-FLAG Ab to detect Yap (middle). Bottom, WB analysis of total cell lysate using anti-Myc Ab to detect Rx1.