Elsevier

Differentiation

Volume 113, May–June 2020, Pages 19-25
Differentiation

Spatial and temporal deletion reveals a latent effect of Megf8 on the left-right patterning and heart development

https://doi.org/10.1016/j.diff.2020.03.002Get rights and content

Abstract

Laterality disease is frequently associated with congenital heart disease (CHD). However, it is unclear what is behind this association, a pleiotropic effect of common genetic causes of laterality diseases or the impact of abnormal left-right patterning on the downstream cardiovascular development. MEGF8 is a disease gene of Carpenter syndrome characterized by defective lateralization and CHD. Here we performed spatial and temporal deletion to dissect the tissue and time requirements of Megf8 on cardiovascular development. None of conditional deletions in cardiomyocytes, endothelium/endocardium, epicardium, cardiac mesoderm or neural crest cells led to cardiovascular defects. More surprisingly, temporal deletion with a ubiquitous Cre driver at embryonic day 7.5 (E7.5), a time point before symmetry break and cardiogenesis, causes preaxial polydactyly (PPD) and exencephaly, but not laterality and cardiovascular defects. These data suggested that Megf8 was dispensable for cardiac organogenesis. Only with E6.5 deletion, we observed aortic arch artery defects including right aortic arch, an indicator of reversed left-right patterning. The concurrence of laterality and cardiovascular defects in pre-streak stage deletion rather than cardiac organogenesis stage deletion indicates that the laterality defect may directly impact heart development. Interestingly, the latent effect of Megf8 on the left-right patterning suggests that the regulation of laterality may be much earlier than we previously thought.

Introduction

MEGF8 (Multiple epidermal growth factor-like domains protein 8) encodes a high molecular weight protein that is evolutionarily conserved from human to fruit fly(Nakayama et al., 1998). Megf8 is essential for embryonic development. This role is first revealed in an echocardiography-based screening of ENU (N-ethyl-N-nitrosourea)-induced mouse mutants(Aune et al., 2008; Zhang et al., 2009). Megf8C193R/C193R mice have a wide spectrum of defects including heterotaxy, transposition of the great arteries (TGA) and PPD etc. Later on, MEGF8 mutations are found associated with a special subtype of Carpenter syndrome patients(Twigg et al., 2012). These patients have a spectrum of defects similar to mouse mutants, such as laterality defects, TGA and PPD etc.. The role of Megf8 in left-right patterning is highly conserved. Morpholino knockdown of Megf8 in zebrafish causes randomization in both embryonic turning and heart tube looping(Zhang et al., 2009). Interestingly, Megf8 ortholog CG7466 in fruit fly is also essential for instar larvae development(Lloyd et al., 2018). CG7466 knockout leads to disorganized denticle belts and lethality in the 2nd or 3rd instar larvae.

The phenotypic similarity among Megf8, Acvr2b and Cfc1 mutants suggests that Megf8 may mediate Nodal signaling(Gaio et al., 1999; Oh and Li, 1997; Yan et al., 1999). Examination of the left-right axis patterning in Megf8 mutants shows that asymmetric Nodal expression in the node is correctly established but Nodal expression in the lateral plate mesoderm (LPM) fails to initiate, supporting a role of Megf8 in Nodal signaling(Zhang et al., 2009). Besides, Megf8 regulates the growth of trigeminal sensory neuron through mediating Bmp4 signaling(Engelhard et al., 2013). Recently, a screening for Shh signaling modifier has found that Megf8 acts as a negative regulator of Shh signaling through inhibiting Smo accumulation in primary cilia(Pusapati et al., 2018). These data suggest that Megf8 has different roles in modulating intracellular signaling in different cellular contexts.

One of the most salient features of Megf8 mutants is cardiovascular defect. CHD is also the most life-threatening condition in patients with MEGF8 mutation(Twigg et al., 2012). However, it is unclear how Megf8 regulates cardiac development. The aforementioned signaling pathways are all crucial for cardiovascular development. To dissect the molecular pathogenesis underlying cardiovascular defects of Megf8 mutants, we used a “knockout-first” strategy to generate a new Megf8LacZ allele, which enabled us to examine the endogenous Megf8 expression by X-gal staining. After removal of the inserted cassette by Flp-mediated recombination, we obtained conditional Megf8fl allele. X-gal staining results showed that Megf8 was widely expressed during embryogenesis. We used cardiomyocyte-specific, epicardium-specific, endothelium/endocardium-specific, neural crest-specific and cardiovascular mesoderm-specific Cre to map the crucial cell type in which Megf8 functions to regulate cardiac development. However, none of tissue-specific conditional mutants presented cardiovascular defects. Using CAGG-CreERT, we identified that Megf8 was required for cardiac development in a time much earlier than cardiac organogenesis. The close association of the left-right patterning and cardiovascular defects suggests that the left-right patterning defect can impact the downstream cardiac development. In addition, the temporal requirement of Megf8 on the left-right patterning is much earlier than symmetry break, suggesting a latent effect of Megf8 on laterality.

Section snippets

Generation of Megf8lacZ allele

To dissect the tissue-specific role of Megf8 and visualize its endogenous expression pattern, we used a knockout-first strategy(Skarnes et al., 2011) to construct a new Megf8 allele, in which a SAIRESLacZNeo cassette was inserted into intron 1 of Megf8 and exon 2–5 was flanked by two LoxP sites (Fig. 1A). The splicing acceptor (SA) of the cassette would splice IRES-LacZ to Megf8 exon 1, which would disrupt normal Megf8 transcription but enable IRES-LacZ expression to recapitulate the endogenous

Discussion

The overt morphological asymmetry during embryogenesis first appears as the rightward looping of linear heart tube. Cardiac progenitors arise from LPM in which the left-right axis is established. The imprinted left-right asymmetry persists through cardiac morphogenesis and may impact its development. In an echocardiography-based ENU mutagenesis screening, mutations causing congenital heart defects are highly enriched in cilia-related genes(Li et al., 2015). Cilia in embryonic node are essential

Mouse lines and breeding

Animal care and use were in accordance with the guidelines approved by the Institutional Animal Care and Use Committee of Shanghai Children's Medical Center. The following mouse lines have been described previously: Tie2-Cre(Kisanuki et al., 2001), cTnT-Cre(Jiao et al., 2003), Mesp1-Cre(Saga et al., 1999), Wnt1-Cre(Danielian et al., 1998), WT1CreERT(Zhou et al., 2008) and CAGG-CreERT(Hayashi and McMahon, 2002). All the lines were maintained in C57Bl/6 background.

Megf8Lacz/+ allele was generated

Author contributions

W. W., X. Z performed experiments and analyzed data. H. S., J. Y. performed mouse husbandry and genotyping. X. L., Y. W. assisted in experiments. M. Z. did scRNA-seq analysis. Z.Z. conceived the work. W.W, and Z.Z wrote the manuscript.

Declaration of competing interest

None.

Acknowledgements

The work was supported by National Natural Science Foundation of China (81170153, 31371465 and 31771612), Shanghai Municipal Education Commission-Gaofeng Clinical Medicine Grant Support (20171925), Shanghai Municipal Commission of Health and Family Planning (XBR2015), and Professor of Special Appointment (Eastern Scholar) at Shanghai Institutions of Higher Learning to Z. Z.

References (41)

  • R. Argelaguet et al.

    Multi-omics profiling of mouse gastrulation at single-cell resolution

    Nature

    (2019)
  • C.N. Aune et al.

    Mouse model of heterotaxy with single ventricle spectrum of cardiac anomalies

    Pediatr. Res.

    (2008)
  • J. Brennan et al.

    Nodal activity in the node governs left-right asymmetry

    Genes Dev.

    (2002)
  • C. Chazaud et al.

    Retinoic acid is required in the mouse embryo for left-right asymmetry determination and heart morphogenesis

    Development

    (1999)
  • A.J. Copp et al.

    Genetics and development of neural tube defects

    J. Pathol.

    (2010)
  • P.S. Danielian et al.

    Modification of gene activity in mouse embryos in utero by a tamoxifen-inducible form of Cre recombinase

    Curr. Biol.

    (1998)
  • T.Y. de Soysa et al.

    Single-cell analysis of cardiogenesis reveals basis for organ-level developmental defects

    Nature

    (2019)
  • H. Deng et al.

    Genetic basis of human left-right asymmetry disorders

    Expet Rev. Mol. Med.

    (2015)
  • C. Engelhard et al.

    MEGF8 is a modifier of BMP signaling in trigeminal sensory neurons

    Elife

    (2013)
  • T. Fujiwara et al.

    Distinct requirements for extra-embryonic and embryonic bone morphogenetic protein 4 in the formation of the node and primitive streak and coordination of left-right asymmetry in the mouse

    Development

    (2002)
  • Cited by (4)

    • Receptor control by membrane-tethered ubiquitin ligases in development and tissue homeostasis

      2022, Current Topics in Developmental Biology
      Citation Excerpt :

      Conditional disruption of Megf8 in various cardiac lineages using a panel of cre drivers (cTnt-cre, Wt1-cre, Tie2-cre, Wnt1-cre, Mesp1-cre) did not reproduce the heart defects seen in the global Megf8 KO. Timed global deletion of Megf8 at E7.5 also did not reproduce the cardiac defects (Wang et al., 2020). These data suggest that Megf8 is required for cardiac development at a time point earlier than cardiac organogenesis and supports the hypothesis that the heart defects seen in MMM mutant mice are a consequence of disrupted left-right patterning early in development.

    View full text