1. Cell Biology
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The EMT transcription factor Snai1 maintains myocardial wall integrity by repressing intermediate filament gene expression

  1. Alessandra Gentile
  2. Anabela Bensimon-Brito
  3. Rashmi Priya
  4. Hans-Martin Maischein
  5. Janett Piesker
  6. Stefan Guenther
  7. Felix Gunawan  Is a corresponding author
  8. Didier YR Stainier  Is a corresponding author
  1. Max Planck Institute for Heart and Lung Research, Department of Developmental Genetics, Germany
  2. DZHK German Centre for Cardiovascular Research, Partner Site Rhine-Main, Germany
  3. Max Planck Institute for Heart and Lung Research, Microscopy Service Group, Germany
  4. Max Planck Institute for Heart and Lung Research, Bioinformatics and Deep Sequencing Platform, Germany
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Cite this article as: eLife 2021;10:e66143 doi: 10.7554/eLife.66143

Abstract

The transcription factor Snai1, a well-known regulator of epithelial-to-mesenchymal transition, has been implicated in early cardiac morphogenesis as well as in cardiac valve formation. However, a role for Snai1 in regulating other aspects of cardiac morphogenesis has not been reported. Using genetic, transcriptomic, and chimeric analyses in zebrafish, we find that Snai1b is required in cardiomyocytes for myocardial wall integrity. Loss of snai1b increases the frequency of cardiomyocyte extrusion away from the cardiac lumen. Extruding cardiomyocytes exhibit increased actomyosin contractility basally as revealed by enrichment of p-myosin and α-catenin epitope α-18, as well as disrupted intercellular junctions. Transcriptomic analysis of wild-type and snai1b mutant hearts revealed the dysregulation of intermediate filament genes, including desmin b (desmb) upregulation. Cardiomyocyte-specific desmb overexpression caused increased cardiomyocyte extrusion, recapitulating the snai1b mutant phenotype. Altogether, these results indicate that Snai1 maintains the integrity of the myocardial epithelium, at least in part by repressing desmb expression.

Introduction

As the contractile units of the heart, cardiomyocytes (CMs) need to maintain a cohesive tissue-level cytoskeleton to beat synchronously and withstand the high mechanical forces (Sequeira et al., 2014; Gautel and Djinović-Carugo, 2016). Using zebrafish as a model to analyse CM cytoskeletal organization at single-cell resolution, we searched for candidate transcription factors that regulate CM cytoskeletal and tissue integrity. Amongst the transcription factors involved in cardiac development, we focused on Snai1 (Nieto, 2002; Nieto et al., 2016), whose orthologues regulate cytoskeletal remodelling and epithelial tissue integrity in Drosophila embryos (Martin et al., 2010; Weng and Wieschaus, 2016) and in mammalian cells in culture (Wee et al., 2020). During vertebrate heart formation, Snai1 has been implicated in myocardial precursor migration towards the midline (Qiao et al., 2014) and in valve formation (Tao et al., 2011), but a role in myocardial wall development, during which an epithelial-to-mesenchymal (EMT)-like process occurs (Staudt et al., 2014; Jiménez-Amilburu et al., 2016; Priya et al., 2020), has not been reported.

Results

The transcription factor Snai1b maintains myocardial wall integrity

We focused our attention on one of the zebrafish snai1 paralogues (Blanco et al., 2007), snai1b, the knockdown of which has been reported to cause embryonic cardiac defects (Qiao et al., 2014). To analyse snai1b function, we generated a promoter-less snai1b allele (Figure 1—figure supplement 1A), which displays almost undetectable levels of snai1b mRNA and no transcriptional upregulation of its paralogue (El-Brolosy et al., 2019; Figure 1—figure supplement 1B). Approximately half of the mutant embryos exhibit cardiac looping defects (Figure 1—figure supplement 1C–D’), as reported for snai1b morphants (Qiao et al., 2014). Upon close examination of the snai1b mutant hearts, we observed a new and surprising phenotype leading to a disruption in myocardial wall integrity: CMs extrude away from the cardiac lumen (Figure 1A–D’). We found that both heterozygous and homozygous snai1b mutant embryos, including ones that display cardiac looping defects, exhibit a significant increase in the number of extruding CMs compared with their wild-type siblings (Figure 1A–E, Figure 1—figure supplement 1E). The frequency of this CM extrusion is higher in the atrioventricular canal (AVC) (Figure 1—figure supplement 1F), where the cells are exposed to stronger mechanical forces from the blood flow and from looping morphogenesis (Auman et al., 2007; Dietrich et al., 2014; Bornhorst et al., 2019). CM extrusion can be observed as early as 48 hours post fertilization (hpf), as well as during larval stages including at 78 (Figure 1—figure supplement 2A–C) and 100 (Figure 1—figure supplement 2D–F) hpf. By imaging beating hearts over a >18 hours period starting at 52 hpf, we observed that a few extruding CMs in snai1b mutants appear to detach from the myocardium and remain in the pericardial cavity for several hours (Figure 1—figure supplement 1I–K, Figure 1—video 1B); in contrast, we did not observe CMs in the pericardial cavity in wild types (Figure 1—video 1A). These results uncover a previously uncharacterized role for Snai1b in maintaining myocardial wall integrity.

Figure 1 with 6 supplements see all
Loss of snai1b leads to cardiomyocyte (CM) extrusion, disrupting myocardial wall integrity.

(A–B”) Single-plane images of Tg(myl7:BFP-CAAX) snai1b+/+ (A) and snai1b-/- (B) hearts at 50 hpf. Close-up of boxed areas (A’, B’) and schematic (A”, B”). (C–D’) 3D surface rendering of the myocardium of Tg(myl7:BFP-CAAX) snai1b+/+ (C, C’) and snai1b-/- (D, D’) embryos at 50 hpf. CM extrusions are clearly observed in snai1b-/- embryos (magenta arrowheads in B, B’, B”, D, D’). (E) More CMs are extruding in Tg(myl7:BFP-CAAX) snai1b-/- embryos compared with snai1b+/+ and snai1b+/- siblings at 50 hpf (snai1b+/+, n = 20; snai1b+/-, n = 23; snai1b-/-, n = 24). (F–H) Blocking cardiac contractions with tnnt2a MO leads to a reduced number of extruding CMs in snai1b-/- embryos, comparable with uninjected snai1b+/+ embryos. (F–G’) 3D surface rendering of the myocardium of snai1b+/+ (F) and snai1b-/- (G) uninjected embryos and snai1b-/- embryos injected with tnnt2a MO (G’). (H) Fewer CMs are extruding (magenta arrowheads in G) in snai1b-/- embryos injected with tnnt2a MO (n = 14) compared with uninjected snai1b-/- (n = 6) and snai1b+/+ (n = 9) embryos at 50 hpf. (I–L) 3D surface rendering of the myocardium showing snai1b+/+ donor-derived CMs in a snai1b+/+ (I) or snai1b-/- (J) heart, and snai1b-/- donor-derived CMs in a snai1b+/+ heart (K). (L) The percentage of donor-derived CMs that extrude is higher when snai1b-/- donor-derived CMs are in snai1b+/+ hearts (n = 8) than when snai1b+/+ donor-derived CMs are in snai1b+/+ (n = 5) or snai1b-/- (n = 14) hearts. (M–P) Overexpression of snai1b specifically in CMs partially rescues the CM extrusion phenotype in snai1b-/- embryos. 3D surface rendering of the myocardium of a snai1b-/- embryo (M), and snai1b-/- embryo overexpressing snai1b under a myl7 (N) or a fli1a (O) promoter. (P) Fewer CMs are extruding (magenta arrowheads) in snai1b-/- embryos (n = 19) overexpressing snai1b in CMs (N, P) compared with snai1b-/- embryos (M, P, n = 38), and this number is comparable to that in snai1+/+ embryos (n = 24). The number of extruding CMs does not change in snai1b-/- embryos (n = 16) when snai1b is overexpressed in endothelial cells (fli1a) (O, P). Plot values represent means ± S.D.; p-values determined by one-way ANOVA followed by multiple comparisons with Dunn test (E, H, L, P). Scale bars: 20 µm. V: ventricle; A: atrium; ap: apical; ba: basal; n: number of embryos.

For all further analyses, we decided to focus on the snai1b mutants displaying apparently unaffected cardiac looping. We first investigated whether the extruding CMs in snai1b mutants were apoptotic as dying epithelial cells are frequently removed by extrusion (Rosenblatt et al., 2001). However, we did not observe a significant difference in the rate of dying cells, as assessed by terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL), between snai1b+/+ (Figure 1—figure supplement 3A) and snai1b-/- (Figure 1—figure supplement 3A’, A”) hearts, indicating that CM extrusion in snai1b mutants is not due to cell death.

We next asked whether the defects in myocardial integrity have an impact on cardiac morphology and function. We observed only a small reduction (five cells on average) in CM numbers at 50 hpf (Figure 1—figure supplement 3B–C). However, we observed a significant decrease in the number of delaminating CMs in snai1b-/- larvae at 78 hpf (Figure 1—figure supplement 4B), resulting in fewer trabecular CMs at 100 hpf (Figure 1—figure supplement 4A, C, D) compared with wild-type siblings. Furthermore, snai1b-/- embryos exhibited an increased CM aspect ratio, as well as reduced apical cell surface and ventricular volume compared with wild-type embryos at 52 (Figure 1—figure supplement 5A–E) and 74 (Figure 1—figure supplement 5F–J) hpf, indicating a requirement for Snai1b in maintaining CM morphology at both cellular and tissue levels. Although snai1b-/- embryos did not exhibit differences in heart rate, ejection fraction, or fractional shortening compared with wild types at 52 hpf (Figure 1—figure supplement 5K–M), we observed a significant reduction in all these parameters at 74 hpf (Figure 1—figure supplement 5N–P). Taken together, these data suggest that the loss of snai1b disrupts cardiac wall morphology, and subsequently cardiac function.

A role for contractility-induced mechanical forces on myocardial wall integrity has recently been reported (Fukuda et al., 2017; Rasouli et al., 2018; Fukuda et al., 2019). Hence, we sought to test whether the loss of cardiac contractility would eliminate the CM extrusion phenotype in snai1b mutants, as previously shown for klf2 mutants (Rasouli et al., 2018). We observed that after injecting a tnnt2a morpholino (Sehnert et al., 2002) to prevent cardiac contraction, the number of extruding CMs in snai1b mutants at 50 hpf was significantly reduced (Figure 1G', H), and in fact became comparable with that in uninjected snai1b+/+ embryos (Figure 1F, H). These data indicate that mechanical forces due to cardiac contraction are required for the increased frequency of CM extrusion observed in snai1b mutants.

To test whether Snai1b plays a cell-autonomous role in promoting myocardial wall integrity, we generated mosaic hearts by cell transplantation (Figure 1I–L). We observed that donor-derived snai1b+/+ CMs remained integrated in the snai1b-/- myocardial wall (Figure 1J), whereas donor-derived snai1b-/- CMs in a snai1b+/+ heart were significantly more prone to extrude than their wild-type neighbours (Figure 1K, L). Together, these data indicate that snai1b is required in a CM-autonomous manner to maintain myocardial wall integrity. Furthermore, we found that CM-specific, but not endothelial-specific, snai1b overexpression rescued the snai1b-/- CM extrusion phenotype (Figure 1M–P, Figure 1—figure supplement 1G, H), further indicating that Snai1b is required in CMs to suppress their extrusion away from the lumen.

Snai1b limits cardiomyocyte extrusion by regulating the actomyosin machinery

During the process of cardiac trabeculation, some CMs undergo an EMT-like process, lose their apicobasal polarity, and delaminate towards the cardiac lumen (Staudt et al., 2014; Jiménez-Amilburu et al., 2016). We wanted to determine whether the extruding CMs in snai1b mutants also lose their apicobasal polarity. Notably, we observed that the polarity marker Podocalyxin remained on the apical side of the extruding CMs in snai1b mutants (Figure 2—figure supplement 1A–B’’), suggesting that apicobasal polarity is maintained.

Studies in Drosophila embryos and in mammalian cells in culture have revealed the importance of cell extrusion in limiting tissue overcrowding and eliminating dying cells to maintain tissue homeostasis and/or determine cell fate (Kocgozlu et al., 2016; Wee et al., 2020). Other experiments have shown that a contractile actomyosin ring around the cell cortex is necessary for their extrusion (Rosenblatt et al., 2001; Eisenhoffer et al., 2012; Kocgozlu et al., 2016). Using a monoclonal antibody against the α-catenin epitope α-18 (Yonemura et al., 2010), which recognizes the activated conformation of α-catenin, a mechanosensitive protein, and polyclonal antibodies against phosphorylated/activated myosin light chain (p-myosin), we assessed cellular contractility in extruding CMs in snai1b+/+ and snai1b-/- embryos (Figure 2A–B”, E–F”). Increased α-catenin epitope α-18 and p-myosin immunofluorescence intensity was observed in the basal side of extruding CMs in snai1b-/- (Figure 2B–C’, F–G’) and snai1b+/+ (Figure 2C–C’, G–G’) embryos. As cellular extrusions also involve the rearrangement of cell-cell junctions (Grieve and Rabouille, 2014; Lubkov and Bar-Sagi, 2014; Teng et al., 2017), we assessed the localization of the major CM adhesion molecule, N-cadherin (Bagatto et al., 2006; Cherian et al., 2016). We observed an overall reduction in N-cadherin levels in the junctions between CMs in snai1b mutants compared with those in wild-type siblings (Figure 2A–B”, D–D’), suggesting that Snai1 regulates N-cadherin localization to stabilize actomyosin tension at the junctions, thereby sustaining adhesion between CMs.

Figure 2 with 1 supplement see all
Extruding cardiomyocytes (CMs) exhibit changes in actomyosin components.

(A–B”) Orthogonal projections in the YZ plane of a 52 hpf snai1b+/+ heart (A) immunostained for α-catenin epitope α-18, N-cadherin, and GFP compared with a snai1b-/- sibling heart (B). Close-up of boxed areas of snai1b+/+ (A’) and snai1b-/- (B’) CMs. Schematics illustrate the localization of activated α-catenin (magenta) in the basal domain of extruding CMs in snai1b-/- embryos and defects in N-cadherin (yellow) localization in the junctional domain of snai1b-/- CMs (A”–B”). (C–D’) Fluorescence intensity profile (FIP) (C–D) and mean fluorescence intensity (mFI) (C’–D’) of α-catenin epitope α-18 and N-cadherin immunostaining in 52 hpf snai1b+/+ and snai1b-/- CMs, and in snai1b+/+ and snai1b-/- extruding CMs. The α-catenin epitope α-18 is observed in the basal domain (white arrowhead in B’) of extruding CMs (white asterisks in B’) in snai1b-/- embryos, and a reduction in junctional N-cadherin (red arrowhead in B’) is observed in snai1b-/- CMs. (FIP α-catenin epitope α-18: snai1b+/+ CMs, N = 179; snai1b+/+ extruding CMs, N = 60; snai1b-/- CMs, N = 140; snai1b-/- extruding CMs, N = 54; mFI α-catenin epitope α-18: snai1b+/+ CMs, N = 90; snai1b+/+ extruding CMs, N = 24; snai1b-/- CMs, N = 88; snai1b-/- extruding CMs, N = 44. FIP N-cadherin: snai1b+/+ CMs, N = 90; snai1b+/+ extruding CMs, N = 12; snai1b-/- CMs, N = 98; snai1b-/- extruding CMs, N = 49; mFI N-cadherin: snai1b+/+ CMs, N = 90; snai1b+/+ extruding CMs, N = 25; snai1b-/- CMs, N = 92; snai1b-/- extruding CMs, N = 70.) (E–F”) Representative images of a 52 hpf snai1b-/- heart (F) immunostained for p-myosin and GFP compared with a snai1b+/+ sibling heart (E). Schematics illustrate the basal enrichment of p-myosin (magenta) in extruding CMs in snai1b-/- embryos (E”–F”). (G–G’) FIP (G) and mFI (G’) of p-myosin immunostaining in snai1b+/+ and snai1b-/- CMs, and in snai1b+/+ and snai1b-/- extruding CMs. p-myosin is enriched basally (orange arrowheads in F’) in snai1b-/- extruding CMs in (white asterisks in F’). (FIP p-myosin: snai1b+/+ CMs, N = 204; snai1b+/+ extruding CMs, N = 60; snai1b-/- CMs, N = 140; snai1b-/- extruding CMs, N = 49; mFI p-myosin: snai1b+/+ CMs, N = 100; snai1b+/+ extruding CMs, N = 29; snai1b-/- CMs, N = 153; snai1b-/- extruding CMs, N = 48). Plot values represent means ± S.E.M. (C, D, G). In the violin plots (C’, D’, G’), solid black lines indicate median. p-values determined by Kruskal–Wallis test (C’, D’, G’). Scale bars: 20 µm (A, B, E, F); 2 µm (A’, B’, E’, F’). ap: apical; ba: basal; N: number of CMs. See also Figure 2—figure supplement 1.

Intermediate filament gene expression is dysregulated in snai1b-/- hearts

To further understand how the transcription factor Snai1b is required to maintain myocardial wall integrity, we compared the snai1b+/+ and snai1b-/- cardiac transcriptomes at 48 hpf, a time when CM extrusion is starting to be observed (Figure 3A). Since Snai1 primarily acts as a transcriptional repressor (Baulida et al., 2019), we focused on the genes upregulated in snai1b-/- hearts compared with wild type. In the 339 upregulated genes, gene ontology analysis revealed an enrichment of genes related to the cytoskeleton (Figure 3—figure supplement 1A), particularly an upregulation of intermediate filament (IF) genes (Figure 3B). Mutations that modify posttranslational modification sites in IF proteins have been associated with cardiomyopathy (Rainer et al., 2018), but how IF genes are regulated at the transcriptional level remains poorly understood. Interestingly, the muscle-specific IF gene desmin b (desmb) was upregulated in snai1b-/- hearts (Figure 3C), further suggesting that Snai1 modulates CM development cell-autonomously. Desmin is localized to Z-discs and desmosomes within intercalated discs in muscle cells, and an imbalance in Desmin levels is a major cause of cardiomyopathies (Capetanaki et al., 2015).

Figure 3 with 1 supplement see all
Transcriptomic analysis reveals upregulation of intermediate filament genes in snai1b-/- hearts.

(A) RNA extracted from 48 hpf snai1b+/+ and snai1b-/- hearts was used for transcriptomic analysis. (B) GO analysis of cellular processes shows enrichment of intermediate filament components in snai1b-/- hearts. (C) Heatmap of a list of upregulated cytoskeletal genes, including desmb. (D) Relative mRNA levels of desmb are significantly increased in snai1b-/- hearts at 48 hpf; n = 4 biological replicates, 30 hearts each. (E) Schematic of snai1b overexpression under a myl7 promoter; snai1b and desmb mRNA levels analysed at 48 hpf. (F) Relative mRNA levels of desmb are significantly reduced in snai1b cardiomyocyte (CM)-specific overexpressing hearts at 48 hpf; n = 4 biological replicates, 30 hearts each. (G–H”) Orthogonal projections in the YZ plane of a 52 hpf snai1b-/- heart (H) immunostained for Desmin and membrane GFP compared with a snai1b+/+ heart (G). Close-up of boxed areas of snai1b+/+ (G’) and snai1b-/- (H’) CMs. Schematics (Desmin in magenta) illustrate the basal enrichment of Desmin in extruding CMs in snai1b-/- embryos (G”–H”). (I–I’) Fluorescence intensity profile (FIP) (I) and mean fluorescence intensity (mFI) (I’) of Desmin in snai1b+/+ and snai1b-/- CMs, and in snai1b+/+ and snai1b-/- extruding CMs. Desmin immunostaining is observed throughout the snai1b-/- myocardium, with an enrichment in the basal domain (white arrowheads in H’–G’) in extruding CMs (white asterisks in H’). (FIP: snai1b+/+ CMs, N = 49; snai1b+/+ extruding CMs, N = 41; snai1b-/- CMs, N = 45; snai1b-/- extruding CMs, N = 41; mFI: snai1b+/+ CMs, N = 56; snai1b+/+ extruding CMs, N = 30; snai1b-/- CMs, N = 65; snai1b-/- extruding CMs, N = 46). Plot values represent means ± S.D. (D, F) or mean ± S.E.M. (I). In the violin plot (I’), solid black lines indicate median. p-Values determined by Student’s t-test (D, F) or Kruskal–Wallis test (I’). Scale bars: 20 µm (G, H); 2 µm (G’, H’). ap: apical; ba: basal; n: number of embryos; N: number of CMs; FC: fold change. All Ct values are listed in Supplementary file 2. See also Figure 3—figure supplement 1.

Using quantitative PCR and immunostaining to analyse desmin at the mRNA and protein levels, respectively, we first examined the upregulation of desmb/Desmin in snai1b-/- hearts compared with wild type (Figure 3D, G–I’). Notably, extruding snai1b-/- CMs exhibit an enrichment of Desmin in their basal domain and a correlative loss of Desmin at intercellular junctions (Figure 3I–I’), indicating abnormal Desmin localization. As IFs are known to regulate actomyosin contractility in keratinocytes and astrocytes (van Bodegraven and Etienne-Manneville, 2020), these data suggest that basal enrichment of Desmin promotes CM extrusion in snai1b-/- hearts.

To further test whether Snai1 represses desmb expression, we analysed desmb transcript levels upon snai1b overexpression. qPCR analysis 4.5 hours after mRNA injection confirmed downregulation of desmb transcript levels when snai1b was overexpressed (Figure 3—figure supplement 1B, C), compared with gfp mRNA injected controls. Similarly, qPCR analysis of hearts overexpressing snai1b specifically in their CMs showed a reduction of desmb transcript levels by 40% (Figure 3E, F). ChIP-seq experiments using mouse skeletal myoblasts have shown that murine Snai1 binds to the proximal promoter of Desmin (Soleimani et al., 2012). Additionally, in silico analysis of zebrafish desmin has uncovered potential Snai1b binding sites in the promoter of desmb, but not desma (Kayman Kürekçi et al., 2021). To test whether zebrafish Snai1b can repress the promoter activity of desmb, we performed luciferase assays in HEK293T cells. We cloned 800 bp of the proximal promoter of desmb upstream of the Firefly luciferase gene, and the open reading frame of snai1b under a constitutively active promoter. The desmb promoter region alone induced transcriptional activation of Luciferase compared with control. However, co-expression of Snai1b led to a significant reduction of the Luciferase signal (Figure 3—figure supplement 1D), suggesting that Snai1b can repress the promoter activity of desmb. Taken together, these data suggest that Snai1b regulates desmb transcription.

desmb overexpression in cardiomyocytes promotes their extrusion

Both loss (Taylor et al., 2007; Ramspacher et al., 2015) and gain (Chen et al., 2018) of Desmin expression have been associated with cardiac defects. Thus, we asked whether an imbalance in desmb expression could lead to CM extrusion by overexpressing desmb mosaically in CMs. We observed that desmb overexpressing CMs were more prone to extrude compared with gfp overexpressing CMs (Figure 4A–C), suggesting that IFs are needed at their endogenous levels to maintain myocardial wall integrity. We hypothesized that increased Desmin levels induce CM extrusion by disrupting desmosome organization leading to compromised cell-cell adhesion and/or by increasing cell contractility basally. We first used electron microscopy to analyse desmosomes at the ultrastructural level, but observed no obvious defects in snai1b-/- CMs compared with wild type (Figure 4—figure supplement 1A–D). This result is consistent with a previous study that shows intact desmosomes in extruding epithelial cells (Thomas et al., 2020). To test whether overexpression of Desmin in CMs was associated with increased cell contractility, we performed immunostaining on desmb overexpressing hearts using α-catenin epitope α-18, p-myosin, and Desmin antibodies. desmb overexpressing CMs exhibited a basal enrichment of Desmin (Figure 4H–I’), as well as of the activated actomyosin factors α-catenin epitope α-18 and p-myosin (Figure 4D–G’). As we observed in snai1b-/- CMs, desmb overexpressing CMs also exhibited reduction of N-cadherin at the junctions compared with control (Figure 4—figure supplement 2A–B’). Taken together, these data show that increasing desmb expression in CMs compromises their adhesion (reduced N-cadherin) and increases their basal actomyosin contractility (increased α-catenin epitope α-18 and p-myosin), recapitulating snai1b mutant phenotypes.

Figure 4 with 3 supplements see all
desmb overexpression in cardiomyocytes (CMs) induces their extrusion.

(A, B) Single-plane images of snai1b+/+ embryos injected with myl7:GFP (A) or with myl7:desmb-p2a-GFP (B) at 50 hpf. (C) A higher percentage of CMs extrude when overexpressing desmb (n = 23) compared with control (n = 15) (magenta arrowheads in B, B’). (D–D’, F–F’, H–H’) Orthogonal projections in the YZ plane of hearts from 52 hpf embryos injected with myl7:desmb-p2a-GFP and immunostained for α-catenin epitope α-18, GFP, and BFP (D–D’), p-myosin, GFP, and Alcama (F–F’), or Desmin, GFP, and Alcama (H–H’). Close-up of boxed areas of desmb-overexpressing and adjacent wild-type CMs (D’, F’, H’). (E–E’, G–G’, I–I’) Fluorescence intensity profile (FIP) (E, G, I) and mean fluorescence intensity (mFI) (E’, G’, I’) of α-catenin epitope α-18 (E–E’), p-myosin (G–G’), and Desmin (I–I’) in CMs that overexpress desmb (magenta arrowheads) and CMs that do not overexpress desmb (white arrowheads). α-catenin epitope α-18, p-myosin, and Desmin immunostaining signals are enriched in the basal domain in desmb overexpressing CMs. FIP: desmb OE CMs, (E) N = 132, (G) N = 120, (I) N = 88; desmb OE extruding CMs, (E) N = 47, (G) N = 54, (I) N = 49; control CMs, (E) N = 153, (G) N = 133, (I) N = 86. mFI: desmb OE CMs, (E’) N = 49, (G’) N = 65, (I’) N = 63; desmb OE extruding CMs, (E’) N = 33, (G’) N = 60, (I’) N = 59; control CMs, (E’) N = 55, (G’) N = 62, (I’) N = 64. Plot values represent means ± S.D. (C) or means ± S.E.M. (E, G, I). In the violin plots (E’, G’, I’), solid black lines indicate median. p-values determined by Mann–Whitney U (C) or Kruskal–Wallis (E’, G’, I’) test. Scale bars: 20 µm (A–B’, D, F, H); 2 µm (D’, F’, H’). V: ventricle; A: atrium; ap: apical; ba: basal; n: number of embryos; N: number of CMs. See also Figure 4—figure supplements 13.

Discussion

A role for Snai1 in cell extrusion has been reported in Drosophila embryos as well as in mammalian cells in culture. During Drosophila gastrulation, Snai1 promotes the medio-apical pulsations of contractile Myo-II that drive apical constriction (Martin et al., 2009; Martin et al., 2010; Mitrossilis et al., 2017). However, the transcriptional targets of Snai1 that promote cellular contractility in this system remain unknown. Recent in vitro studies have reported Snai1-mediated upregulation of active RhoA, leading to increased cortical actomyosin activity and apical extrusion (Wee et al., 2020). Here, our work uncovers a previously unsuspected role for the EMT-inducing factor Snai1 in limiting CM extrusions by regulating IF gene expression. We show that the CM extrusions in snai1b mutants are associated with increased accumulation of actomyosin basally, providing more evidence for a role of Snai1 in regulating cell contractility through actin networks. This function appears to be partly independent of Snai1’s role in EMT as no obvious changes in Podocalyxin localization were observed. Our data show the requirement of Snai1 in maintaining epithelial tissue integrity in a vertebrate organ and add to the growing evidence that Snai1 has EMT-independent roles in epithelial tissues.

Furthermore, we report a previously uncharacterized function of Snai1 in regulating desmin expression and find that an increase in Desmin levels perturbs tissue integrity. Although IFs including vimentin (Kajita et al., 2014) and keratin (Kadeer et al., 2017; Thomas et al., 2020) have been reported to accumulate at the interface between extruding cells and their neighbours, our study provides evidence that increased Desmin levels are correlated with mislocalization of the actomyosin machinery in the basal domain of extruding cells. These data are consistent with previous findings that IFs can regulate the actomyosin network, with factors such as vimentin binding to actin and modulating RhoA activity (Jiu et al., 2017), and keratin binding to myosin (Kwan et al., 2015). In addition to a role for Desmin in maintaining nuclear membrane architecture in CMs (Heffler et al., 2020), our results shed light on the function of Desmin in maintaining myocardial wall integrity.

Our results also uncover the requirement of Snai1 and the correct levels of Desmin in maintaining myocardial wall integrity under contraction-induced mechanical pressure. Cardiac contraction is essential in patterning the cardiac tissue: without a heartbeat, cardiac valves and the trabecular network fail to form (Granados-Riveron and Brook, 2012; Collins and Stainier, 2016). Our results further indicate that without a strong intracellular cytoskeletal network regulated by Snai1, the heartbeat-induced mechanical forces can lead to an increase in CM extrusion and loss of myocardial wall integrity. While it has been shown that an increase in cell contractility due to changes in morphology, adhesion, or cell density drives cell extrusion (Eisenhoffer et al., 2012; Levayer et al., 2016; Kocgozlu et al., 2016; Saw et al., 2017; Miroshnikova et al., 2018; Campinho et al., 2020; Priya et al., 2020), we present evidence that external mechanical forces contribute to non-apoptotic CM extrusion during cardiac development, and that actomyosin and IF cytoskeletal regulation prevent CM extrusion.

In conclusion, our findings uncover molecular mechanisms that suppress cell extrusion in a tissue under constant mechanical pressure and show a multifaceted, context-dependent role for Snai1 in promoting EMT (Nieto et al., 2016), and also in maintaining tissue integrity during vertebrate organ development (Figure 4—figure supplement 3).

Materials and methods

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
AntibodyAnti-tRFP (rabbit polyclonal)EvrogenRRID:AB_2571743IF(1:200)
AntibodyAnti-GFP (chicken polyclonal)AvesLabRRID:AB_10000240IF(1:800)
AntibodyAnti-N-cadherin (rabbit polyclonal)AbcamRRID:AB_444317IF(1:250)
AntibodyAnti-p-myosin (rabbit polyclonal)AbcamRRID:AB_303094IF(1:200)
AntibodyAnti-Desmin (rabbit polyclonal)SigmaRRID:AB_476910IF(1:100)
AntibodyAnti-α-catenin epitope α-18 (rat monoclonal)Gift from Prof. Akira NagafuchiIF(1:300)
AntibodyAnti-Alcama (mouse monoclonal)DSHBRRID:AB_531904IF(1:50)
AntibodyAlexa Fluor 488 Goat anti Chicken IgG (H + L)Thermo Fisher ScientificRRID:AB_142924IF(1:500)
AntibodyAlexa Fluor 647 Goat anti Rabbit IgG (H + L)Thermo Fisher ScientificRRID:AB_141663IF(1:500)
AntibodyAlexa Fluor 647 Goat anti Rat IgG (H + L)Thermo Fisher ScientificRRID:AB_141778IF(1:500)
AntibodyAlexa Fluor 568 Goat anti Rabbit IgG (H + L)Thermo Fisher ScientificRRID:AB_2534123IF(1:500)
AntibodyAlexa Fluor 568 Goat anti Rat IgG (H + L)Thermo Fisher ScientificRRID:AB_2534121IF(1:500)
Chemical compound, drugAgarose, low gelling temperatureSigmaCat# A9414-25g
Chemical compound, drugBovine serum albuminSigmaCat# A-9418
Chemical compound, drugChloroformMerckCat# 102445
OtherDAPISigmaCat# D954(1 µg/mL)
Chemical compound, drugDimethyl sulfoxide (DMSO)SigmaCat# D8418
Chemical compound, drugDMEM(1X)+GlutamaxThermo Fisher ScientificCat# 31966-021
Chemical compound, drugDyNAmo ColorFlash SYBR Green qPCR MixThermo Fisher ScientificCat# F416S
Chemical compound, drugEthanol, undenatured, absoluteServaCat# 11093.01
Chemical compound, drugFBS superiorBiochromCat# S0615
Chemical compound, drugGlycineSigmaCat# 50046
Chemical compound, drug2-PropanolRothCat# 6752.4
Chemical compound, drugLipofectamine 3000 Transfection ReagentThermo Fisher ScientificL3000001
Chemical compound, drugMethanolRothCat# 4627.5
Chemical compound, drugNormal Goat SerumThermo Fisher ScientificCat# 16210072
Chemical compound, drugParaformaldehydeSigmaCat# P6148
Chemical compound, drugPhosphate buffered saline (PBS)SigmaCat# P4417
Recombinant DNA reagentpT3TS-nCas9n (plasmid)AddgeneCat# 46757
Recombinant DNA reagentpCS2z vector (plasmid)AddgeneCat# 62214
Recombinant DNA reagentpCMV-Tol2 (plasmid)AddgeneCat# 31823
Recombinant DNA reagentpGl4.14-luc; SV40:hRLuc (plasmid)Bensimon-Brito et al., 2020
Chemical compound, drugSodium citrate monobasicSigmaCat# 71497-1KG
Chemical compound, drugTriton X-100SigmaCat# X-100
Chemical compound, drugTRIzol ReagentThermo Fisher ScientificCat# 15596026
Chemical compound, drugTween 20SigmaCat# P1379
Commercial assay or kitDual-Luciferase Reporter Assay SystemPromegaCat# E1910
Commercial assay or kitIn Situ Cell Death Detection Kit, FluoresceinRoche11684795910
Commercial assay or kitMaxima First Strand cDNA kitThermo Fisher ScientificCat# K1641
Commercial assay or kitMegaShortScript T7 Transcription KitThermo Fisher ScientificCat# AM1354
Commercial assay or kitMegaScript T3 Transcription KitThermo Fisher ScientificCat# AM1348
Commercial assay or kitmMESSAGE mMACHINE T7 Transcription KitThermo Fisher ScientificCat# AM1344
Commercial assay or kitmMESSAGE mMACHINE T3 Transcription KitThermo Fisher ScientificCat# AM1348
Commercial assay or kitRNA Clean and Concentrator KitZymo ResearchCat# R1013
Cell line (Homo sapiens)HEK-293TATCCCat# CRL-3216RRID:CVCL_0063
Strain, strain background (Danio rerio)Tg(myl7:BFP- CAAX)bns193Guerra et al., 2018ZFIN:bns193
Strain, strain background (Danio rerio)Tg(myl7:H2B-EGFP)zf521TgMickoleit et al., 2014ZFIN:zf521Tg
Strain, strain background (Danio rerio)Tg(myl7:mVenus-gmnn)ncv43TgJiménez-Amilburu et al., 2016ZFIN:ncv43Tg
Strain, strain background (Danio rerio)Tg(−0.2myl7:snai1b-p2a-GFP) bns555This paperZFIN:bns555
Strain, strain background (Danio rerio)Tg(−0.2myl7:EGFP-podocalyxin) bns10Jiménez-Amilburu et al., 2016ZFIN:bns10
Strain, strain background (Danio rerio)Tg(fli1a:Gal4)ubs4Zygmunt et al., 2011ZFIN:ubs4
Strain, strain background (Danio rerio)Tg(UAS:snai1b-p2a-GFP) bns442This paperZFIN:bns442
Strain, strain background (Danio rerio)Tg(myl7:EGFP-Hsa.HRAS)s883TgD'Amico et al., 2007ZFIN:s883Tg
Strain, strain background (Danio rerio)snai1bbn351 mutantThis paperZFIN:bns351
Sequence-based reagentqPCRThis paperTable S1
Sequence-based reagentGenotypingThis paperTable S1
Sequence-based reagentPCRThis paperTable S1
Software, algorithmFiJi ImageJ 1.53 cSchindelin et al., 2012RRID:SCR_002285
Software, algorithmGraphPad Prism 6GraphPadRRID:SCR_002798
Software, algorithmImaris, version 8.4.0BitplaneRRID:SCR_007370
Software, algorithmZen Digital ImagingCarl Zeiss MicroscopyRRID:SCR_013672

Zebrafish husbandry

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Zebrafish husbandry was performed in accordance with institutional (MPG) and national (German) ethical and animal welfare regulation. Larvae were raised under standard conditions. Adult zebrafish were maintained in 3.5 L tanks at a stock density of 10 zebrafish/L with the following parameters: water temperature: 27–27.5°C; light:dark cycle: 14:10; pH: 7.0–7.5; conductivity: 750–800 µS/cm. Zebrafish were fed 3–5 times a day, depending on age, with granular and live food (Artemia salina). Health monitoring was performed at least once a year. All embryos used in this study were raised at 28°C and staged at 75% epiboly for synchronization.

All procedures performed on animals conform to the guidelines from Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes and were approved by the Animal Protection Committee (Tierschutzkommission) of the Regierungspräsidium Darmstadt (reference: B2/1218).

Zebrafish lines

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The following lines were used in this study: Tg(myl7:BFP-CAAX)bns193 (Guerra et al., 2018); Tg(myl7:H2B-EGFP)zf521 (Mickoleit et al., 2014); Tg(myl7:mVenus-gmnn)ncv43 (Jiménez-Amilburu et al., 2016); Tg(−0.2myl7:EGFP-podocalyxin)bns10 (Jiménez-Amilburu et al., 2016); Tg(fli1a:Gal4)ubs4 (Zygmunt et al., 2011); Tg(myl7:EGFP-Hsa.HRAS)s883 (D'Amico et al., 2007); Tg(UAS:snai1b-p2a-GFP)bns442 (this study); Tg(−0.2myl7:snai1b-p2a-GFP)bns555 (this study); and snai1bbns351 (this study).

Generation of transgenic lines

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To generate the snai1b overexpression lines, the full coding sequence was amplified by PCR using the following primers: forward – 5′-ATGCCACGCTCATTTCTTGT-3′ and reverse – 5′-GAGCGCCGGACAGCAGCC-3′. The 765 bp amplicon was cloned into pT2-UAS and into an iSce-I plasmid downstream of a −0.2myl7 promoter and upstream of a P2A linker and GFP. All cloning experiments were performed using ColdFusion Cloning (System Biosciences). The plasmids were then injected into AB embryos at the one-cell stage (25 pg/embryo) together with Tol2 mRNA (25 pg/embryo) to generate Tg(UAS:snai1b-p2a-GFP) and Tg(−0.2myl7:snai1b-p2a-GFP), respectively.

Generation of the snai1bbns351 allele

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The snai1b mutant allele was generated using the CRISPR/Cas9 technology. Guide RNA (gRNA) sequences were designed using the CRISPOR program (http://crispor.tefor.net/). To generate a promoter-less allele, two gRNAs were designed: one targeting the proximal promoter (5′-GTCTATAAGTGGCGCAG-3′) and another targeting exon 1, immediately after the sequence encoding the SNAG domain (5′-GTAGTTTGGCTTCTTGT-3′), resulting in a deletion of 1300 bp. The gRNAs were transcribed using a MegaShortScript T7 Transcription Kit (Thermo Fisher Scientific). cas9 mRNA was transcribed using an mMESSAGE mMACHINE T3 Transcription Kit (Thermo Fisher Scientific) using pT3TS-nCas9n as a template. The RNAs were purified with an RNA Clean and Concentrator Kit (Zymo Research). gRNAs (~12.5 pg/embryo for each gRNA) and cas9 mRNA (~300 pg/embryo) were co-injected at the one-cell stage. High-resolution melt analysis (HRMA) was used to determine the efficiency of the gRNAs. For genotyping, a reverse primer (5′-AATTTCACTCTCACCAGTCTGA-3′) was combined with a forward primer in the promoter region (5′-ACCTTCTTGTTGTGAGGCGA-3′) to detect the mutant allele, and with a forward primer in exon 1 (5′-ATGCCACGCTCATTTCTTGTCAA-3′) to detect the wild-type allele.

Overexpression of snai1b

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A full-length snai1b cDNA was cloned from 48 hpf cDNA into the pCS2+ vector (Addgene). In vitro transcription using a mMESSAGE mMACHINE T7 Transcription Kit (Thermo Fisher Scientific) generated snai1b mRNA. Wild-type embryos were injected at the one-cell stage with 25 pg of snai1b or gfp mRNA. RNA from 40 4.5 hpf embryos was extracted using a standard phenol/chloroform protocol.

Overexpression of desmb

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To generate the desmb overexpression plasmid, the full coding sequence was amplified by PCR using the following primers: forward – 5′-ATGAGCCACTCTTATGCCAC-3′ and reverse – 5′-CATGAGGTCCTGCTGGTG-3′. The 1419 bp amplicon was cloned into a iSce-I plasmid downstream of a −0.2myl7 promoter and upstream of a P2A linker and GFP. All cloning experiments were performed using ColdFusion Cloning (System Biosciences). The plasmid was then injected into Tg(myl7:BFP-CAAX) embryos at the one-cell stage (25 pg/embryo) together with Tol2 mRNA (25 pg/embryo) to obtain mosaic expression.

Immunohistochemistry

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Embryos were collected, treated with 1-phenyl-2-thiourea (PTU) at 24 hpf to prevent pigmentation, and fixed in 4% PFA for 2 hours at room temperature, after stopping the heart with 0.4% Tricaine to prevent it from collapsing during fixation. After exchanging the fixative with PBS/0.1% Tween washes, yolks were removed using forceps, incubated in 0.1 M glycine for 10 min, and then washed with PBS/1% BSA/1% DMSO/0.5% Triton-X (PBDT), and blocked with PBDT/10% goat serum before incubating in primary antibody at 4°C overnight. The embryos were washed in PBDT and incubated in secondary antibody for 2 hours at room temperature, then incubated with DAPI (2 µg/mL) for 10 min and washed with PBS/0.1% Tween.

Primary antibodies used were GFP (Abcam, 1:800 dilution); N-cadherin (Abcam, 1:250 dilution); p-myosin (Abcam, 1:200); tRFP (Evrogen, 1:200 dilution); Desmin (Sigma, 1:100); and Alcama (DSHB ZN-8, 1:50). α-catenin epitope α-18 (1:300) antibody was a generous gift from Prof. Akira Nagafuchi. Secondary antibodies (1:500 dilution) used were Alexa Fluor 568, Alexa Fluor 488, and Alexa Fluor 647 (Thermo Fisher Scientific).

Imaging

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Confocal microscopes were used to image stopped hearts. Embryos were mounted in 1% low-melting agarose with 0.2% Tricaine, and the stopped hearts were imaged using a Zeiss LSM700 or LSM880 confocal microscope with a 20× or 40× dipping lens. Fixed embryos were mounted in 1% low-melting agarose and were imaged using a Zeiss LSM700 or LSM880 confocal microscope with a 20× or 40× dipping lens, and genotyped afterwards.

Heart rate, ventricular ejection fraction, and ventricular fractional shortening quantification

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Live imaging of beating hearts was performed using a Zeiss Spinning Disk confocal microscope. Zebrafish at 48, 78, and 100 hpf were mounted in 2% low-melting agarose without Tricaine. 20–30 s movies were recorded with 5 ms exposure. Light intensity and duration were kept to a minimum to avoid light-induced twitching. Kymographs were generated using ImageJ, and ventricular ejection fraction and ventricular fractional shortening were quantified with ImageJ.

TUNEL assay

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Embryos at 50 hpf were fixed in 4% PFA for 2 hours at room temperature, washed in PBS/0.1% Tween, and manually deyolked with insulin needles. Samples were dehydrated and stored in 100% MeOH at −20°C overnight. After rehydration, embryos were processed for antibody staining (Evrogen, tRFP 1:200) . Subsequently, samples were permeabilized with 0.1% sodium citrate in PBS for 2 min on ice. After washes in PBS/0.3% Triton-X, embryos for the positive control were incubated for 15 min at 37°C with DNAaseI. All the embryos were incubated for 1 hour at 37°C in the TUNEL solution (In Situ Cell Death Detection Kit Fluorescein, Roche). After washes, embryos were mounted for imaging.

Quantitative PCR analysis

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Dissected hearts were homogenized in TRIzol (Thermo Fisher Scientific) using a NextAdvance Bullet Blender Homogenizer, followed by standard phenol/chloroform extraction. At least 500 ng of total RNA was used for reverse transcription using a Maxima First Strand cDNA synthesis kit (Thermo Fisher Scientific). For all experiments, DyNAmo ColorFlash SYBR Green qPCR Mix (Thermo Fisher Scientific) was used on a CFX connect Real-time System (Bio-Rad) with the following program: pre-amplification 95°C for 7 min, amplification 95°C for 10 s and 60°C for 30 s (39 cycles), melting curve 60–92°C with increment of 1°C each 5 s. Each point in the dot plots represents a biological replicate from three technical replicates. Gene expression values were normalized using the housekeeping gene rpl13a and fold changes were calculated using the 2−ΔΔCt method; all Ct values are listed in Supplementary file 2. Primer sequences can be found in Table S1.

Blastomere transplantations and morpholino injections

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Cells obtained from mid-blastula stage donor embryos were transplanted along the blastoderm margin of age-matched host embryos. A tnnt2a ATG-MO was injected into the yolk at the one-cell stage at 0.3 ng per embryo. The embryos were then imaged at 52 hpf.

Image analysis

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All immunostainings were analysed in the YZ orthogonal plane to better visualize CM extrusion. The line scan function of Fiji was used to quantify fluorescence intensity at the junctional and basal domains. To visualize the fluorescence profile of N-cadherin immunostaining, a line of uniform thickness was drawn from junction to junction in adjacent CMs. To analyse the localization of α-catenin epitope α-18, p-myosin, and Desmin, a line of uniform thickness was drawn from the basal to the apical domain of CMs. Asymmetry in fluorescence intensity appears due to variable CM length. To assess fluorescence intensity, the mean grey values were used, drawing a line of uniform thickness at the junctional (N-cadherin) or basal (α-catenin epitope α-18, p-myosin, and Desmin) domain of CMs. Images were processed and analysed with Fiji. A background subtraction of rolling ball radius 20 was applied, followed by a mean filter of radius 1. Brightness and contrast were adjusted to remove any background fluorescence. Apical cell surface and aspect ratio were quantified using the line function of ImageJ.

The total number of CMs was counted using the Spots function, and 3D cardiac surface rendering and ventricular volume quantification were obtained with the Surfaces function of the Imaris Bitplane Software.

Luciferase assay and plasmids

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To generate the plasmid with a zebrafish desmb promoter driving Firefly Luciferase expression (pGl4.14-luc; SV40:hRLuc) (Bensimon-Brito et al., 2020), we cloned 800 bp of the promoter region of desmb using the following primers: forward – 5′-GAAAGCATAGTCTGCTTTCTCG-3′ and reverse – 5′-GAGCGCCGGACAGCAGCC-3′.

The zebrafish snai1b coding sequence was inserted downstream of the CMV promoter in the pCMV-Tol2 plasmid (Addgene). The full-length snai1b coding sequence was amplified using the following primers: forward – 5′-ATGCCACGCTCATTTCTTGT-3′ and reverse – 5′-GAGCGCCGGACAGCAGCCGGAC3′. Per well in a 24-well plate, HEK-293T cells were transfected with 200 ng of the luciferase plasmid and 200 ng of pCMV-snai1b or the empty plasmid as control, as well as 1.5 μL Lipofectamine 3000 Transfection Reagent (Thermo Fisher Scientific). The cells were incubated with the transfection mix for 5–6 hours in DMEM + Glutamax (Thermo Fisher Scientific)/10% FBS Superior (Biochrom) without antibiotics. The cells were then incubated in DMEM + Glutamax/10% FBS/1% penicillin-streptomycin (PenStrep, Sigma) overnight. After 24 hours, they were rinsed in PBS and lysed with PLB buffer for Luciferase Assay (Promega). The supernatants were used to perform the luciferase assay, using the Dual-Luciferase Reporter Assay System (Promega), following the manufacturer’s instructions. Each experiment was carried out in triplicates (three wells per condition) in four independent experiments.

Cell line

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We used human Embryonic Kidney cells (HEK293T, ATCC Cat# CRL-3216), which were certified by STR profiling by ATCC, and tested negative for mycoplasma contamination.

RNA-seq

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48 hpf Tg(myl7:BFP-CAAX) snai1b+/+ and snai1b-/- hearts were manually dissected using forceps. Approximately 20 hearts per replicate were pooled, and total RNA was isolated using the miRNeasy micro kit, combined with on-column DNase digestion. Total RNA and library integrity were verified with LabChip Gx Touch 24 (Perkin Elmer). Approximately 10 ng of total RNA was used as input for SMART-Seq v4 Ultra Low Input RNA Kit (Takara Clontech) for cDNA pre-amplification. Obtained full-length cDNA was checked on LabChip GX Touch 24 and fragmented by Ultrasonication by E220 machine (Covaris). Final Library Preparation was performed by Low Input Library Prep Kit v2 (Takara Clontech). Sequencing was performed on a NextSeq500 instrument (Illumina) using v2 chemistry, resulting in an average of 30M reads per library with 1 × 75 bp single-end setup. The resulting raw reads were assessed for quality, adapter content, and duplication rates with FastQC (available online at http://www.bioinformatics.babraham.ac.uk/projects/fastqc). Trimmomatic version 0.39 was used to trim reads with a quality drop below a mean of Q20 in a window of 10 nucleotides (Bolger et al., Trimmomatic: a flexible trimmer for Illumina sequence data). Only reads between 30 and 150 nucleotides were used in subsequent analyses. Trimmed and filtered reads were aligned versus the Ensembl Zebrafish genome version DanRer11 (GRCz11.92) using STAR 2.6.1d with the parameter ‘outFilterMismatchNoverLmax 0.1’ to increase the maximum ratio of mismatches to mapped length to 10% (Dobin et al., 2013). The number of reads aligning to genes was counted with feature Counts 1.6.5 tool from the Subread package (Liao et al., 2014). Only reads mapping at least partially inside exons were admitted and aggregated per gene, while reads overlapping multiple genes or aligning to multiple regions were excluded from further analyses. Differentially expressed genes were identified using DESeq2 version 1.18.1 (Love et al., 2014). The Ensembl annotation was enriched with UniProt data (release 06.06.2014) based on Ensembl gene identifiers (Activities at the Universal Protein Resource (UniProt)).

For the gene ontology analysis, all genes with a p-value≤0.05 were used as a query list. Genes with >5 normalized reads in at least one sample were used as a background list. The analysis was performed with the Gitools 2.3.1 (http://www.gitools.org) software. Z-scores were calculated using the default settings, and multiple test correction with Benjamini–Hochberg FDR was performed.

Transmission electron microscopy (TEM)

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Larvae were collected at 60 hpf from a wild-type or mutant incross. The embryos were immediately fixed in ice-cold 1% PFA, 2% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) for 30 min on ice, and then stored at 4°C overnight. Samples were washed in 0.1 M sodium cacodylate buffer and postfixed in 2% (w/v) OsO4, followed by en bloc staining with 2% uranyl acetate. Samples were dehydrated with a graded series of washes in acetone, transferred to acetone/Epon solutions, and eventually embedded in Epon. Ultra-thin sections (approximately 70 nm thick) obtained with a Reichert-Jung Ultracut E microtome were collected on copper slot grids. Sections were post-stained with 2% uranyl acetate for 20 min and 1% lead citrate for 2 min. Sections were examined with a Jeol JEM-1400 Plus transmission electron microscope (Jeol, Japan), operated at an accelerating voltage of 120 kV. Digital images were recorded with an EM-14800 Ruby Digital CCD camera unit (3296px × 2472px).

Randomization and blinding procedures

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All experiments using snai1b mutants were randomized as follows: animals from heterozygous crosses were collected, imaged, and analysed, and subsequently genotyped. For all immunostainings, the genotyping was performed after the analysis. The only exceptions were for the RNAseq, TEM, and tnnt2a MO experiments, for which the mutants were obtained from maternal zygotic incrosses using snai1b zygotic mutants (approximately 70% of them reach adulthood). All experiments shown in Figure 1—figure supplement 4 were performed with first generation cousin animals. Transgenic animals were selected by fluorescence before imaging, and therefore could not be randomized. The investigators were blinded to allocation during experiments and outcome assessment whenever possible.

Statistical analysis

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All statistical analyses were performed in GraphPad Prism (version 6.07). A Gaussian distribution was tested for every sample group using the D’Agostino–Pearson omnibus normality test. For the experiments that passed the normality test, all samples were further analysed using the following parametric tests: the Student’s t-test for comparison of two samples or the one-way ANOVA test followed by correction for multiple comparisons with Dunn's test for three or more samples. For all the experiments that did not pass the normality test, all samples were further analysed using non-parametric tests: p-values were determined using the Mann–Whitney test for comparison of two samples or the Kruskal–Wallis test followed by correction for multiple comparisons with Dunn's test for three or more samples.

Data availability

Sequencing data have been deposited in GEO under accession code GSE162604.

The following data sets were generated

References

  1. Book
    1. Collins MM
    2. Stainier DYR
    (2016) Chapter Twenty-Five - Organ Function as a Modulator of Organ Formation: Lessons from Zebrafish
    In: Wassarman P. M, editors. Essays on Developmental Biology, Part B. Academic Press. pp. 417–433.
    https://doi.org/10.1016/bs.ctdb.2015.10.017

Decision letter

  1. Edward E Morrisey
    Senior and Reviewing Editor; University of Pennsylvania, United States
  2. Chinmay M Trivedi
    Reviewer; University of Massachusetts Medical School, United States
  3. Benoit Bruneau
    Reviewer; University of California, San Francisco, United States

Our editorial process produces two outputs: i) public reviews designed to be posted alongside the preprint for the benefit of readers; ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.

Acceptance summary:

The examination into how EMT related transcriptional pathways regulate cardiomyocyte gene expression provides critical insight into how heart wall integrity is developed and maintained. These current findings will also likely have an important impact on how these pathways regulate the injury response to the heart.

Decision letter after peer review:

Thank you for submitting your article "The EMT transcription factor Snai1 maintains myocardial wall integrity by repressing intermediate filament genes" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by Edward Morrisey as the Senior and Reviewing Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Chinmay M Trivedi (Reviewer #1); Benoit Bruneau (Reviewer #3).

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Essential revisions:

1. Additional immunohistochemistry to assess the shape changes in Snai1 deficient cardiomyocytes.

2. Further examination of the effects of Desmin over expression on cardiomyocyte behavior as noted by reviewer 2 and 3.

3. All noted text changes from the reviewers should be addressed through careful editing.

Reviewer #1 (Recommendations for the authors):

Gentile A et al. show a novel role of Snai1b in growth regulation of zebrafish myocardial wall. Specifically, authors show that zebrafish lacking Snai1b exhibit cardiac looping defects (~50% penetrance), consistent with previously described morpholino mediated Snai1b knockdown phenotype. Extruding cardiomyocytes away from cardiac lumen, mostly in the atrioventricular canal region were observed in remaining 50% of Snai1b knockout zebrafish. Using RNA-seq, authors identified several dysregulated genes, including enrichment of intermediate filament genes in Snai1b knockout zebrafish. Among these dysregulated genes, authors suggest that increased Desmin expression and its aberrant localization promote cardiomyocyte extrusion in Snai1b knockout zebrafish hearts. Overall, present manuscript describes a novel phenomenon during cardiac development, hence, it is of interest to developmental biologists. Major concerns are:

1. Snai1 is known to affect cushion formation in atrioventricular canal region. It would be helpful to establish cause and effect relationship for Snai1b in this region. Zebrafish lack global Snai1b expression – so it would be helpful to show if defective cushion promotes cardiomyocyte extrusion in atrioventricular canal region. Tnnt2 morpholino experiments provides some insights, however, it does not rule out role of defective atrioventricular cushion (defective EMT).

2. For Figure 2 – additional histology / immunohistology to show extrusion, cohesion, and orientation of cardiomyocytes at a section level (2D) in Snai1b knockout hearts could help to characterize phenotype at a cellular level. It is assumed that all cardiomyocytes lack Snai1b protein (immunostaining would help), however, only few cardiomyocyte show extrusion. Minor point – Cartoon images in figure 2 are somewhat disconnected from immunostaining images.

3. Do Snai1b knockout hearts exhibit defective contractile phenotype? Is there a cardiac phenotype in surviving adult zebrafish? Do RNA-seq and SEM from adult zebrafish heart represent embryonic extrusion and intermediate filament defects?

4. It is unclear why only few cardiomyocytes show extrusion when most of cardiomyocytes, if not all, overexpressing Desmin gene.

5. Molecular link connecting Snai1b and cardiac filaments genes is not determined.

Reviewer #2 (Recommendations for the authors):

An intact myocardium is essential for cardiac function, yet much remains unknown regarding the cell biological mechanisms maintaining this specialized epithelium during embryogenesis. In this manuscript, Gentile and colleagues discover a novel role for the repressive transcription factor Snai1b in supporting myocardial integrity. In the absence of Snai1b, cardiomyocytes exhibit an enrichment of intermediate filament genes, including desmin b. In addition, the authors detect mislocalization of Desmin, along with adherens junction and actomyosin components, to the basal membrane in snai1b mutant cardiomyocytes, and these mutant cells exhibit an increased likelihood of extrusion from the myocardium. Ultimately, the authors put forward a model wherein Snai1b protects cardiomyocytes from extrusion at least in part by regulating the amount and organization of Desmin in the cell, thereby supporting myocardial integrity.

Overall, the authors highlight an important aspect of epithelial maintenance in an environment that experiences significant biomechanical stress due to cardiac function. By generating a promoter-less allele of snai1b, the authors have created a clean genetic model in which to work. Coupled with beautiful microscopy and transcriptomics, this story has the potential to enlighten both cell biologists and cardiovascular biologists on the underpinnings of myocardial integrity. However, clarifications regarding the overall model would be particularly beneficial for the reader.

1. A clearer discussion of the proposed molecular mechanism for Snai1b function would aid a reader's overall contextualization of this work. At one point, the authors suggest that Snai1b regulates N-cadherin localization to adherens junctions, thereby stabilizing actomyosin tension at cell junctions. Later, it is suggested that Desmin activates the actomyosin contractile network at the basal membrane. It is unclear whether the authors believe that these are separate events or whether they may be coupled, perhaps through Desmin disruption at the lateral membranes, leading to modifications in nearby adherens junctions. A more thorough investigation of the phenotype resulting from desmin b overexpression may clarify this relationship.

2. It appears that extruded cells do not bud off from the myocardium, but rather remain on the apical surface of the existing myocardium. However, it is unclear whether this change in tissue architecture affects cardiac function or the overall morphology of the chamber. A brief discussion of these possibilities would have helped to contextualize the significance of this phenotype.

3. The authors show that cardiomyocyte extrusion is most prevalent near the atrioventricular canal, and they suggest that this regionalized effect is due to the different types of extrinsic factors, like biomechanical forces, that this region experiences. However, it is also possible that regional differences in certain intrinsic factors are involved, such as junctional plasticity, actomyosin activity at the basal membrane, etc. To distinguish between these possibilities, it would have been informative to know whether the extent of N-cadherin/α-18/p-Myosin/Desmin mislocalization varies depending on the regional location of cardiomyocytes within the snai1b mutant heart. For example, do cardiomyocytes near the atrioventricular canal exhibit more extreme effects on N-cadherin/α-18/p-Myosin/Desmin localization than cardiomyocytes in further away portions of the ventricle? Or, do these cells exhibit similar degrees of protein mislocalization, but cells near the atrioventricular canal have a lower threshold for extrusion?

The following adjustments and/or clarifications would strengthen the manuscript.

1. Regarding the precise mechanism by which extrusion occurs, it would be helpful to know which aspects are a direct cause of Desmin overexpression/mislocalization. The authors begin to investigate this question by staining for α-18. However, these data seem incomplete, as they are not subdivided into extruding vs. non-extruding cells (as in other experiments). Additionally, if the authors wish to assemble further support for the model that Desmin regulates actomyosin activity at the basal surface, then assaying p-Myosin would bolster this idea, rather than relying on α-18 as an indicator of contractility. Finally, assaying N-cadherin localization in Desmin overexpressing cells would enable the authors to distinguish whether the N-cadherin mislocalization observed in snai1b mutants is dependent on Desmin overexpression or a parallel effect of Snai1b deficiency – both of these would be interesting outcomes that would help to clarify their overall model.

2. Given the reduction of N-cadherin on lateral membranes and the proposal that junctional actomyosin is destabilized in snai1b mutant cardiomyocytes, one might expect that adherens junctions are not intact in mutants. The authors did not compare the integrity and/or abundance of adherens junctions in their EM experiments, which seems like a missed opportunity. Are adherens junctions visible in any of the existing EM data?

3. In the N-cadherin fluorescence intensity profiles, why is the signal routinely higher on one side the of the cell? Shouldn't the signal be equivalent, independent of the side where the profile begins?

4. Is cardiac function affected in snai1b mutants? If they are functionally deficient (e.g. reduced contractility), this would somewhat lessen the impact of the mutant-into-wild-type transplant experiments, as it has been observed that cardiomyocytes with impaired sarcomeric contraction are extruded when they are mosaically present within a functional myocardium. If mutants are functionally normal, however, adding this information would strengthen the meaningfulness of the cell autonomy experiments.

5. Do the authors believe that the cardiomyocytes that are not extruded in snai1b mutants are affected in any meaningful way? For example, is the observed trabeculation defect believed to be related to the extrusion phenotype?

Reviewer #3 (Recommendations for the authors):

Gentile, A. et al. generated snai1b mutant zebrafish embryos and showed that loss of Snai1b led to two mutant phenotypes in the heart: i) hearts with clear looping defects, ii) hearts without looping defects that displayed abnormal cardiomyocyte (CM) extrusion. The authors focused on the second class of mutants and found that loss of Snai1b led to reduction of N-cadherin at cell junctions and basal accumulation of phosphorylated myosin light chain and the α-18 epitope of α-catenin, indicative of mechanical activation. Bulk RNA-sequencing of isolated hearts revealed an upregulation of intermediate filament (IF) genes in Snai1b mutants, and of particular interest, the authors identified upregulation of the muscle-specific IF gene desmin b. Immunofluorescent imaging revealed that Desmin was not only upregulated in Snai1b mutants, but mis-localized away from cell junctions and accumulated at the basal side of extruding cells along with actomyosin machinery. Accordingly, CM-specific overexpression of Desmin was sufficient to promote cell extrusion.

The presented work is particularly interesting because it identifies a new role for the Snai1b transcription factor in maintaining proper tissue structure, independent of its typical function in regulating epithelial to mesenchymal transition (EMT). Overall, the experiments were well designed and controlled, and the data is clearly and logically presented. However, some of the findings could be explained by alternative hypotheses and other interesting aspects of the data were left unexplored.

One hypothesis that was not sufficiently discussed is that loss of Snai1b may prevent cardiomyocytes from undergoing the EMT that is necessary for normal delamination and trabeculation, and thus cells are instead extruded away from the lumen to prevent overcrowding in the developing myocardium. In fact, the authors present evidence that EMT is blocked and acknowledge that extrusion is a known mechanism for preventing overcrowding. It would be interesting to see whether extrusion away from the lumen also occurs if EMT is blocked through other means.

The authors show that extruding cells do not seem to be dead or dying, and that a small number of CMs do extrude in wild type embryos. This raises the intriguing possibility that some amount of CM extrusion is necessary for normal development and that these cells may give rise to epicardial or other cell types. Live-imaging and lineage-tracing studies would inform whether the extrusion observed in mutant embryos is an enhancement of a normal morphogenetic process or an additional abnormal response to loss of Snai1 function.

One particularly interesting observation that was left unexplored was the identification of a second class of Snai1b mutants with defective heart looping. It isn't clear whether these embryos also display enhanced CM extrusion, or if there are other clearly aberrant cell behaviors. Furthermore, it would be very interesting to know whether there is any evidence that the defective looping is due to the same changes in cytoskeletal gene expression and protein organization observed in the class of Snai1b mutants that were detailed throughout the manuscript.

The authors suggest that Snai1b regulates Desmin in two ways: 1) overall expression levels, and 2) post-translationally to control its localization at cell junctions. Although the first claim is sufficiently supported, the second claim lacks experimental evidence. An alternative explanation is that overexpression of Desmin in response to loss of Snai1b leads to mislocalization independent of an interaction with Snai1b. This point could be clarified by examining Desmin localization in the desmb overexpression system. In addition, assaying for co-IP of Snai1b and Desmin could demonstrate a direct interaction between the two and better support a role for Snai1 in regulating post-translational localization of Desmin.

Although the authors convincingly show that Desmin accumulates with other contractile machinery at the basal side of extruding CMs in Snai1b muntants, additional evidence is needed to support a causal link between basal Desmin accumulation and extrusion. For instance, if knockdown or inhibition of Desmin prevents extrusion in the Snai1b mutants, the causal relationship would be much clearer.

https://doi.org/10.7554/eLife.66143.sa1

Author response

Essential revisions:

1. Additional immunohistochemistry to assess the shape changes in Snai1 deficient cardiomyocytes.

We have now included additional in-depth characterization of the shape changes in snai1b mutant CMs. We quantified CM surface areas and aspect ratios, as well as ventricular volumes, at 52 and 74 hpf. These quantifications were carried out in live fish carrying the transgenic CM membrane marker Tg(myl7:HRAS-EGFP) in order to preserve CM morphology and avoid potential damage from the immunostaining procedure. We found that the snai1b mutant CMs are significantly smaller and more rounded at both time points. This result suggests that the reduction in intercellular adhesion and changes in cytoskeletal architecture caused by the loss of Snai1b lead to morphological changes and abnormal rounding of the CMs. We also found significantly smaller ventricular volumes at 52 and 74 hpf in snai1b mutant hearts, suggesting that the cellular defects impact tissue architecture. These new results are shown in Figure 1 – supplement 5.

2. Further examination of the effects of Desmin over expression on cardiomyocyte behavior as noted by reviewer 2 and 3.

We showed in the previous version of the manuscript that desmb overexpression in CMs leads to basal localization of the α-catenin epitope α-18 and cell extrusion. We have now included additional data using anti-Desmin, anti-p-myosin, and anti-N-cadherin immunostaining to further characterize the effects of desmb overexpression in CMs. First, we observed the enrichment of Desmin at the basal domain of desmb overexpressing CMs. We also observed in desmb overexpressing CMs an increased localization of p-myosin in their basal domain (similar to the basal enrichment of the α-catenin epitope α-18 shown in figure 4D-D’) and a reduction of junctional N-cadherin. These results further show that CM-specific desmb overexpression leads to dysregulation of the actomyosin cytoskeleton and junctional adhesion, phenocopying snai1b mutant CMs. We have also included more detailed quantitative analysis to differentiate the fluorescence signal between extruding and non-extruding desmb overexpressing CMs. These new results are shown in Figure 4 and Figure 4 – supplement 1.

3. All noted text changes from the reviewers should be addressed through careful editing.

We have carefully edited the text as suggested by the Reviewers.

Reviewer #1 (Recommendations for the authors):

[…] Major concerns are:

1. Snai1 is known to affect cushion formation in atrioventricular canal region. It would be helpful to establish cause and effect relationship for Snai1b in this region. Zebrafish lack global Snai1b expression – so it would be helpful to show if defective cushion promotes cardiomyocyte extrusion in atrioventricular canal region. Tnnt2 morpholino experiments provides some insights, however, it does not rule out role of defective atrioventricular cushion (defective EMT).

We thank the reviewer for these suggestions. While we agree that this point is interesting, it must be noted that atrioventricular (AV) valve formation in zebrafish starts at ⁓56 hpf, with the collective migration of the valve endothelial cells (Gunawan et al., 2019; Gunawan et al., 2020). Zebrafish heart valves are functional starting at approximately 72 hpf, when they can efficiently close the lumen (Gunawan et al., 2019; Gunawan et al., 2020). Thus, the endothelial-to-mesenchymal transition process in the AV canal takes place after CMs start extruding in snai1b mutants (48 hpf).

Nevertheless, we examined valve formation in snai1b mutants and observed that the early stages of valve development seem unaffected, and that wild-type like valve leaflets appear by 72 hpf (Author response image 1). Together, these data indicate that the CM extrusion defects in snai1b mutants are not a secondary effect of valve dysfunction.

Author response image 1
Single-plane images of Tg(kdrl:nls-mCherry)snai1b+/+ (A) and snai1b-/- (B) valve leaflets at 72 hpf.

(snai1b+/+, n=9; snai1b-/-, n=11). Scale bars: 20 µm. n, number of embryos.

2. For Figure 2 – additional histology / immunohistology to show extrusion, cohesion, and orientation of cardiomyocytes at a section level (2D) in Snai1b knockout hearts could help to characterize phenotype at a cellular level. It is assumed that all cardiomyocytes lack Snai1b protein (immunostaining would help), however, only few cardiomyocyte show extrusion. Minor point – Cartoon images in figure 2 are somewhat disconnected from immunostaining images.

We thank the reviewer for these suggestions. While we agree with the point regarding the Snai1b immunostaining, there are no commercially available or published antibodies that detect zebrafish Snai1b, particularly one that differentiates between Snai1a and Snai1b. To better characterize the snai1b mutant phenotype at the cellular level, we have now included quantification of CM apical surface areas and aspect ratios at 52 and 74 hpf. We found that the CMs in snai1b mutants appear smaller and more rounded compared with those in wild-type embryos. We also found a smaller ventricular volume in snai1b mutant hearts, potentially due to the changes in CM shape. These new results are shown in Figure 1 – supplement 5. We also changed the cartoons in Figure 2, as suggested.

3. Do Snai1b knockout hearts exhibit defective contractile phenotype? Is there a cardiac phenotype in surviving adult zebrafish? Do RNA-seq and SEM from adult zebrafish heart represent embryonic extrusion and intermediate filament defects?

We thank the reviewer for these comments. However, characterizing the snai1b mutant adult phenotypes is beyond the scope of this manuscript. It is important to clarify that the RNA-seq and SEM experiments were performed in embryonic hearts.

4. It is unclear why only few cardiomyocytes show extrusion when most of cardiomyocytes, if not all, overexpressing Desmin gene.

We agree with the reviewer. As we showed by immunostaining in wild-type hearts (Figure 3I), a subset of CMs – the few extruding wild-type CMs – exhibit Desmin enrichment in their basal domain. We speculate that only this subset of CMs exhibits basal Desmin enrichment because their position within the myocardium exposes them to higher mechanical forces due to increased blood flow and looping morphogenesis, which in turn raises their propensity to extrude. Indeed, as we show in Figure 1 – supplement 1F, most of the CM extrusions in snai1b mutants are observed at the AV canal, where CMs experience the highest level of mechanical forces (Lombardo et al., 2019; Campinho et al., 2020).

5. Molecular link connecting Snai1b and cardiac filaments genes is not determined.

We have now used a luciferase assay in HEK293T cells to test the regulation of desmb expression by Snai1b. It was previously shown by ChIP-seq in mouse skeletal myoblasts that Snai1 can bind to the proximal promoter of Desmin (Soleimani et al., 2012). Our in silico analysis uncovered an 800 base pair region upstream of the start codon of zebrafish desmb that exhibits a high degree of similarity (>45%) with the mammalian sequence and is thus a promising proximal promoter for desmb. Furthermore, Kürekçi et al. recently reported that the zebrafish desmb promoter contains putative Snai1b-binding sites (Kayman Kürekçi et al., 2021). We cloned this 800 bp region upstream of a luciferase reporter and co-transfected the resulting plasmid with a plasmid expressing zebrafish Snai1b, which led to a significant decrease of luciferase activity compared with the proximal promoter alone. These data suggest that Snai1b binds to the proximal promoter of desmb and represses its transcription, potentially implicating Snai1b as a direct regulator of desmb expression. This new result is shown in Figure 3 – supplement 1D.

Reviewer #2 (Recommendations for the authors):

An intact myocardium is essential for cardiac function, yet much remains unknown regarding the cell biological mechanisms maintaining this specialized epithelium during embryogenesis. In this manuscript, Gentile and colleagues discover a novel role for the repressive transcription factor Snai1b in supporting myocardial integrity. In the absence of Snai1b, cardiomyocytes exhibit an enrichment of intermediate filament genes, including desmin b. In addition, the authors detect mislocalization of Desmin, along with adherens junction and actomyosin components, to the basal membrane in snai1b mutant cardiomyocytes, and these mutant cells exhibit an increased likelihood of extrusion from the myocardium. Ultimately, the authors put forward a model wherein Snai1b protects cardiomyocytes from extrusion at least in part by regulating the amount and organization of Desmin in the cell, thereby supporting myocardial integrity.

Overall, the authors highlight an important aspect of epithelial maintenance in an environment that experiences significant biomechanical stress due to cardiac function. By generating a promoter-less allele of snai1b, the authors have created a clean genetic model in which to work. Coupled with beautiful microscopy and transcriptomics, this story has the potential to enlighten both cell biologists and cardiovascular biologists on the underpinnings of myocardial integrity. However, clarifications regarding the overall model would be particularly beneficial for the reader.

1) A clearer discussion of the proposed molecular mechanism for Snai1b function would aid a reader's overall contextualization of this work. At one point, the authors suggest that Snai1b regulates N-cadherin localization to adherens junctions, thereby stabilizing actomyosin tension at cell junctions. Later, it is suggested that Desmin activates the actomyosin contractile network at the basal membrane. It is unclear whether the authors believe that these are separate events or whether they may be coupled, perhaps through Desmin disruption at the lateral membranes, leading to modifications in nearby adherens junctions. A more thorough investigation of the phenotype resulting from desmin b overexpression may clarify this relationship.

We thank the reviewer for these comments. We have now included Desmin and p-myosin immunostaining of desmb overexpressing CMs. We observed an increased level of Desmin protein in the desmb overexpressing CMs, as well as its basal localization. We also observed increased p-myosin localization basally, as we did in snai1b mutant CMs. N-cadherin immunostaining at the junctions was reduced in desmb overexpressing CMs, as in snai1b mutant CMs. Altogether, these results indicate that myocardial-specific desmb overexpression phenocopies snai1b mutants. We have also included deeper quantitative analysis of the immunostaining, now distinguishing the results between extruding and non-extruding desmb overexpressing CMs. These new results are shown in Figure 4F-I’ and Figure 4 – supplement 2.

Although both reduced junctional N-cadherin and abnormal basal localization of actomyosin factors are consistently observed in snai1b mutants and in desmb overexpressing embryos, additional tools will need to be developed and used to determine whether they are separate or coupled events.

2) It appears that extruded cells do not bud off from the myocardium, but rather remain on the apical surface of the existing myocardium. However, it is unclear whether this change in tissue architecture affects cardiac function or the overall morphology of the chamber. A brief discussion of these possibilities would have helped to contextualize the significance of this phenotype.

We thank the reviewer for this interesting point. We have now included a time-lapse spinning disk movie of wild-type and snai1b mutant hearts from 52 to 70 hpf. At the starting timepoint (t0) in snai1b mutant hearts, we observed extruding CMs that were still embedded within the myocardium. Within 6 hours, we did not observe extruding CMs in the same location as we had at t0, but instead found CMs outside of the myocardial wall and they remained in the pericardial cavity for several hours. These new results suggest that CMs do indeed extrude out of the myocardium in snai1b mutant hearts, and they are shown in Figure 1 – supplement 1I-K and video 1.

Additionally, we quantified the heart rate, ejection fraction, and fractional shortening at 52 and 74 hpf. At 52 hpf, we did not find significant differences between wild-type and snai1b mutants, but at 74 hpf, the heart rate, ejection fraction, and fractional shortening were significantly lower in snai1b mutants compared to wild types. Furthermore, snai1b mutants exhibited reduced ventricular volume at 52 and 74 hpf. As the reduction in cardiac function occurs after CMs start to extrude, these data indicate that CM extrusion has an impact on the overall morphology of the ventricle and cardiac function. These new results are shown in Figure 1 – supplement 5.

3) The authors show that cardiomyocyte extrusion is most prevalent near the atrioventricular canal, and they suggest that this regionalized effect is due to the different types of extrinsic factors, like biomechanical forces, that this region experiences. However, it is also possible that regional differences in certain intrinsic factors are involved, such as junctional plasticity, actomyosin activity at the basal membrane, etc. To distinguish between these possibilities, it would have been informative to know whether the extent of N-cadherin/α-18/p-Myosin/Desmin mislocalization varies depending on the regional location of cardiomyocytes within the snai1b mutant heart. For example, do cardiomyocytes near the atrioventricular canal exhibit more extreme effects on N-cadherin/α-18/p-Myosin/Desmin localization than cardiomyocytes in further away portions of the ventricle? Or, do these cells exhibit similar degrees of protein mislocalization, but cells near the atrioventricular canal have a lower threshold for extrusion?

We thank the reviewer for this interesting point. We hypothesize that CMs closer to the AV canal exhibit more severe effects on N-cadherin/α-catenin epitope α-18/p-myosin/Desmin localization, due to the higher mechanical forces they experience (Lombardo et al., 2019; Campinho et al., 2020). However, our immunostaining procedure for zebrafish embryos requires deyolking to allow access of the tissue to the antibody, which unfortunately leads to the loss of the atrium and the part of the AV canal closest to the atrium; thus, this procedure renders it difficult to perform quantitative analysis of the immunostaining signal throughout the heart.

The following adjustments and/or clarifications would strengthen the manuscript.

1. Regarding the precise mechanism by which extrusion occurs, it would be helpful to know which aspects are a direct cause of Desmin overexpression/mislocalization. The authors begin to investigate this question by staining for α-18. However, these data seem incomplete, as they are not subdivided into extruding vs. non-extruding cells (as in other experiments). Additionally, if the authors wish to assemble further support for the model that Desmin regulates actomyosin activity at the basal surface, then assaying p-Myosin would bolster this idea, rather than relying on α-18 as an indicator of contractility. Finally, assaying N-cadherin localization in Desmin overexpressing cells would enable the authors to distinguish whether the N-cadherin mislocalization observed in snai1b mutants is dependent on Desmin overexpression or a parallel effect of Snai1b deficiency – both of these would be interesting outcomes that would help to clarify their overall model.

We thank the reviewer for these comments. We have now included Desmin and p-myosin immunostaining of desmb overexpressing CMs. We observed an increased level of Desmin protein, as well as its basal localization. We also observed increased p-myosin localization basally, as we did in snai1b mutant CMs. N-cadherin immunostaining at the junctions were reduced in desmb overexpressing CMs, as in snai1b mutant CMs. Altogether, these results indicate that myocardial-specific desmb overexpression phenocopies snai1b mutants. We have also included deeper quantitative analyses of the immunostaining, now distinguishing the results between extruding and non-extruding desmb overexpressing CMs. These new results are shown in Figure 4F-I’ and Figure 4 – supplement 2.

2. Given the reduction of N-cadherin on lateral membranes and the proposal that junctional actomyosin is destabilized in snai1b mutant cardiomyocytes, one might expect that adherens junctions are not intact in mutants. The authors did not compare the integrity and/or abundance of adherens junctions in their EM experiments, which seems like a missed opportunity. Are adherens junctions visible in any of the existing EM data?

We thank the reviewer for this interesting comment. Unfortunately, adherens junctions were not visible in any of the existing EM data.

3. In the N-cadherin fluorescence intensity profiles, why is the signal routinely higher on one side the of the cell? Shouldn't the signal be equivalent, independent of the side where the profile begins?

We have now fixed these profiles and thank the reviewer for pointing out this issue.

4. Is cardiac function affected in snai1b mutants? If they are functionally deficient (e.g. reduced contractility), this would somewhat lessen the impact of the mutant-into-wild-type transplant experiments, as it has been observed that cardiomyocytes with impaired sarcomeric contraction are extruded when they are mosaically present within a functional myocardium. If mutants are functionally normal, however, adding this information would strengthen the meaningfulness of the cell autonomy experiments.

We thank the reviewer for this important point. We compared cardiac function in snai1b+/+ and snai1b-/- embryos by quantifying their heart rate, ejection fraction, and fractional shortening at 52 hpf, a timepoint at which CM extrusions are already observed in snai1b mutants. Notably, we found that at this stage loss of snai1b does not significantly affect cardiac function (Figure 1—figure supplement 4).

5. Do the authors believe that the cardiomyocytes that are not extruded in snai1b mutants are affected in any meaningful way? For example, is the observed trabeculation defect believed to be related to the extrusion phenotype?

We thank the reviewer for these questions. We hypothesize that the transcriptome of all CMs in snai1b mutants are affected in a similar manner. However, the extruding CMs are potentially exposed to higher mechanical forces from being in, or close to, the AV canal. The trabeculation defects in snai1b mutants could indeed be related to the CM extrusion phenotype as trabeculation requires the actomyosin machinery to be enriched in the apical domain to induce apical constriction and basal delamination (Priya et al., 2020). Thus, the ectopic enrichment of actomyosin factors in the basal domain of CMs in snai1b mutants might interfere with trabeculation.

Reviewer #3 (Recommendations for the authors):

Gentile, A. et al. generated snai1b mutant zebrafish embryos and showed that loss of Snai1b led to two mutant phenotypes in the heart: i) hearts with clear looping defects, ii) hearts without looping defects that displayed abnormal cardiomyocyte (CM) extrusion. The authors focused on the second class of mutants and found that loss of Snai1b led to reduction of N-cadherin at cell junctions and basal accumulation of phosphorylated myosin light chain and the α-18 epitope of α-catenin, indicative of mechanical activation. Bulk RNA-sequencing of isolated hearts revealed an upregulation of intermediate filament (IF) genes in Snai1b mutants, and of particular interest, the authors identified upregulation of the muscle-specific IF gene desmin b. Immunofluorescent imaging revealed that Desmin was not only upregulated in Snai1b mutants, but mis-localized away from cell junctions and accumulated at the basal side of extruding cells along with actomyosin machinery. Accordingly, CM-specific overexpression of Desmin was sufficient to promote cell extrusion.

The presented work is particularly interesting because it identifies a new role for the Snai1b transcription factor in maintaining proper tissue structure, independent of its typical function in regulating epithelial to mesenchymal transition (EMT). Overall, the experiments were well designed and controlled, and the data is clearly and logically presented. However, some of the findings could be explained by alternative hypotheses and other interesting aspects of the data were left unexplored.

One hypothesis that was not sufficiently discussed is that loss of Snai1b may prevent cardiomyocytes from undergoing the EMT that is necessary for normal delamination and trabeculation, and thus cells are instead extruded away from the lumen to prevent overcrowding in the developing myocardium. In fact, the authors present evidence that EMT is blocked and acknowledge that extrusion is a known mechanism for preventing overcrowding. It would be interesting to see whether extrusion away from the lumen also occurs if EMT is blocked through other means.

We thank the reviewer for these interesting questions regarding CM overcrowding, EMT, and CM extrusion. To test the hypothesis that CMs in snai1b mutants are extruding to prevent overcrowding in the developing myocardium, we treated embryos with an ErbB2 inhibitor to reduce CM proliferation (however, ErbB2 also regulates EMT). We did not observe a significant difference in the number of extruding CMs in treated snai1b mutants compared with control (Author response image 2), while when we treated wild-type embryos with the ErbB2 inhibitor, we observed an increase in CM extrusion (data not shown). In addition, previous cell transplantation studies (Liu et al., 2010) analysed wild-type hearts with a few erbb2 mutant CMs and did not report CM extrusion. Altogether, these data suggest that increased CM extrusion in snai1b mutants is not caused by increased CM proliferation or defective EMT. However, additional analysis using tools that specifically block CM proliferation versus EMT will be needed to further investigate these interesting questions.

Author response image 2
The number of extruding CMs is not significantly different between DMSO and ErbB2 inhibitor treated snai1b-/- embryos at 52 hpf.

(snai1b-/- DMSO, n=23; snai1b-/- ErbB2 inhibitor, n=35). Plot values represent means ± S.D.; p-values determined by Mann-Whitney U test. n, number of embryos.

The authors show that extruding cells do not seem to be dead or dying, and that a small number of CMs do extrude in wild type embryos. This raises the intriguing possibility that some amount of CM extrusion is necessary for normal development and that these cells may give rise to epicardial or other cell types. Live-imaging and lineage-tracing studies would inform whether the extrusion observed in mutant embryos is an enhancement of a normal morphogenetic process or an additional abnormal response to loss of Snai1 function.

We thank the reviewer for these interesting comments. We have now included a time-lapse spinning disk movie of the snai1b mutant hearts from 52 to 70 hpf, in which we observed CMs outside of the myocardial wall and they remained in the pericardial cavity for several hours (see our rebuttal to Major Point #2 in reviewer 2’s comment).

To test the hypothesis that CM extrusion is a normal process that gives rise to other cell types outside the myocardial wall, we performed a lineage-tracing experiment in wild-type embryos. We treated Tg(myl7:creERT2);(-3.5ubb:loxP-EGFP-loxP-mCherry) embryos with tamoxifen from 24 to 72 hpf, and imaged the larvae at 96 hpf. We found no switched (EGFP to mCherry) CM-derived cells in the pericardial cavity (Author response image 3). From our lineage tracing analysis, we believe that the extruded CMs do not contribute to other cardiac cells. Our time-lapse movies also show that in snai1b mutants, extruded CMs are not attached to and are positionally distant from the heart (Figure 1 —figure supplement 1I-K), thereby indicating that it is unlikely the extruded CMs give rise to epicardial or other cardiovascular cells.

Author response image 3
Single-plane images of Tg(myl7:creERT2);(-3.5ubb:loxP-EGFP-loxP-mCherry);(myl7:BFP-CAAX) larvae at 96 hpf.

No switched cells are found in the pericardial cavity. n=23. Scale bar: 20 µm. n, number of embryos.

One particularly interesting observation that was left unexplored was the identification of a second class of Snai1b mutants with defective heart looping. It isn't clear whether these embryos also display enhanced CM extrusion, or if there are other clearly aberrant cell behaviors. Furthermore, it would be very interesting to know whether there is any evidence that the defective looping is due to the same changes in cytoskeletal gene expression and protein organization observed in the class of Snai1b mutants that were detailed throughout the manuscript.

We thank the reviewer for these comments. We have now examined in more detail CM morphology in unlooped snai1b mutant hearts and have included some quantification in the revised manuscript. We found that the number of extruding CMs is similar in all snai1b mutants regardless of the looping phenotype. These new data are included in Figure 1 —figure supplement 1C-E. It will indeed be interesting to investigate whether changes in cytoskeletal gene and protein expression are also an underlying cause of the cardiac looping phenotype in snai1b mutants.

The authors suggest that Snai1b regulates Desmin in two ways: 1) overall expression levels, and 2) post-translationally to control its localization at cell junctions. Although the first claim is sufficiently supported, the second claim lacks experimental evidence. An alternative explanation is that overexpression of Desmin in response to loss of Snai1b leads to mislocalization independent of an interaction with Snai1b. This point could be clarified by examining Desmin localization in the desmb overexpression system. In addition, assaying for co-IP of Snai1b and Desmin could demonstrate a direct interaction between the two and better support a role for Snai1 in regulating post-translational localization of Desmin.

We thank the reviewer for these comments. We have now performed anti-Desmin immunostaining in desmb overexpressing CMs, and found that Desmin is enriched basally. This result suggests that an overabundance of Desmin can lead to its basal enrichment. However, whether Snai1b and Desmin interact at the protein level will need additional tools and analyses, and thus we have removed the corresponding sentence from the manuscript.

Although the authors convincingly show that Desmin accumulates with other contractile machinery at the basal side of extruding CMs in Snai1b muntants, additional evidence is needed to support a causal link between basal Desmin accumulation and extrusion. For instance, if knockdown or inhibition of Desmin prevents extrusion in the Snai1b mutants, the causal relationship would be much clearer.

We thank the reviewer for this suggestion. We used a desmb ATG morpholino to knock down desmb in snai1b mutants. Although we used a low concentration of the morpholino (0.5 ng), we surprisingly observed an increased number of extruding CMs in both wild types and snai1b mutants compared with standard control morpholino injections (Author response image 4). We hypothesize that the right balance of Desmin expression is needed to preserve myocardial wall integrity; too much or too little of Desmin increases CM extrusions. However, we cannot exclude the possibility that the effects observed are due to off-target effects of the morpholino. Due to these uncertainties, we did not include the desmb morpholino data in the manuscript.

Author response image 4
The number of extruding CMs increases in snai1b+/+ and snai1b-/-desmb morphants compared with the respective control morphants at 52 hpf.

(snai1b+/+ control MO, n=5; snai1b+/+ desmb MO, n=8; snai1b-/- Control MO, n=5; snai1b-/- desmb MO, n=9). Plot values represent means ± S.D.; p-values determined by Mann-Whitney U test. n, number of embryos..

https://doi.org/10.7554/eLife.66143.sa2

Article and author information

Author details

  1. Alessandra Gentile

    Max Planck Institute for Heart and Lung Research, Department of Developmental Genetics, Bad Nauheim, Germany
    Contribution
    Conceptualization, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - original draft, Writing - review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5423-7295
  2. Anabela Bensimon-Brito

    1. Max Planck Institute for Heart and Lung Research, Department of Developmental Genetics, Bad Nauheim, Germany
    2. DZHK German Centre for Cardiovascular Research, Partner Site Rhine-Main, Bad Nauheim, Germany
    Present address
    Aix Marseille Univ, INSERM, Marseille Medical Genetics, Marseille, France
    Contribution
    Conceptualization, Supervision, Methodology, Writing - review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1663-2232
  3. Rashmi Priya

    1. Max Planck Institute for Heart and Lung Research, Department of Developmental Genetics, Bad Nauheim, Germany
    2. DZHK German Centre for Cardiovascular Research, Partner Site Rhine-Main, Bad Nauheim, Germany
    Present address
    The Francis Crick Institute, London, United Kingdom
    Contribution
    Conceptualization, Writing - review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0510-7515
  4. Hans-Martin Maischein

    Max Planck Institute for Heart and Lung Research, Department of Developmental Genetics, Bad Nauheim, Germany
    Contribution
    Methodology
    Competing interests
    No competing interests declared
  5. Janett Piesker

    Max Planck Institute for Heart and Lung Research, Microscopy Service Group, Bad Nauheim, Germany
    Contribution
    Investigation, Methodology, Writing - review and editing
    Competing interests
    No competing interests declared
  6. Stefan Guenther

    1. DZHK German Centre for Cardiovascular Research, Partner Site Rhine-Main, Bad Nauheim, Germany
    2. Max Planck Institute for Heart and Lung Research, Bioinformatics and Deep Sequencing Platform, Bad Nauheim, Germany
    Contribution
    Formal analysis, Investigation, Methodology, Writing - review and editing
    Competing interests
    No competing interests declared
  7. Felix Gunawan

    1. Max Planck Institute for Heart and Lung Research, Department of Developmental Genetics, Bad Nauheim, Germany
    2. DZHK German Centre for Cardiovascular Research, Partner Site Rhine-Main, Bad Nauheim, Germany
    Contribution
    Conceptualization, Supervision, Methodology, Writing - original draft, Writing - review and editing
    For correspondence
    Felix.Gunawan@mpi-bn.mpg.de
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5592-9680
  8. Didier YR Stainier

    1. Max Planck Institute for Heart and Lung Research, Department of Developmental Genetics, Bad Nauheim, Germany
    2. DZHK German Centre for Cardiovascular Research, Partner Site Rhine-Main, Bad Nauheim, Germany
    Contribution
    Conceptualization, Supervision, Funding acquisition, Writing - original draft, Project administration, Writing - review and editing
    For correspondence
    Didier.Stainier@mpi-bn.mpg.de
    Competing interests
    Senior editor, eLife
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0382-0026

Funding

Max Planck Society

  • Alessandra Gentile
  • Anabela Bensimon-Brito
  • Rashmi Priya
  • Hans-Martin Maischein
  • Janett Piesker
  • Stefan Guenther
  • Felix Gunawan
  • Didier YR Stainier

Deutsches Zentrum für Herz-Kreislaufforschung

  • Rashmi Priya
  • Stefan Guenther
  • Felix Gunawan
  • Didier YR Stainier

European Molecular Biology Organization (ALTF 642–2018)

  • Felix Gunawan

Cardio Pulmonary Institute Grant (EXC 2026 390649896)

  • Rashmi Priya

CIHR (293898)

  • Felix Gunawan

European Molecular Biology Organization (LTF 1569-2016)

  • Rashmi Priya

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

This work was supported by funds from the Max Planck Society to DYRS, a European Molecular Biology Organization (EMBO) Advanced Fellowship (ALTF 642-2018) and a Canadian Institute for Health Research Fellowship (293898) to FG, and an EMBO fellowship (LTF 1569-2016), a Humboldt fellowship and a Cardio-Pulmonary Institute Grant (EXC 2026, project ID 390649896) to RP. We would like to thank Michelle Collins, Paolo Panza, Chi-Chung Wu, Mridula Balakrishnan, Srinivas Allanki, Giulia Boezio, Simon Perathoner, and Honorine Destain for comments on the manuscript, Prof. Akira Nagafuchi for the α-catenin epitope α-18 antibody, and Gabrielius Jakutis for help with the HEK293T cells.

Ethics

Animal experimentation: Zebrafish husbandry was performed in accordance with institutional (MPG) and national (German) ethical and animal welfare regulation. All procedures performed on animals conform to the guidelines from Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes and were approved by the Animal Protection Committee (Tierschutzkommission) of the Regierungspräsidium Darmstadt (reference: B2/1218).

Senior and Reviewing Editor

  1. Edward E Morrisey, University of Pennsylvania, United States

Reviewers

  1. Chinmay M Trivedi, University of Massachusetts Medical School, United States
  2. Benoit Bruneau, University of California, San Francisco, United States

Publication history

  1. Received: December 29, 2020
  2. Accepted: June 7, 2021
  3. Version of Record published: June 21, 2021 (version 1)

Copyright

© 2021, Gentile et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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