Cyp26 enzymes are required to balance the cardiac and vascular lineages within the anterior lateral plate mesoderm

Abstract

Normal heart development requires appropriate levels of retinoic acid (RA) signaling. RA levels in embryos are dampened by Cyp26 enzymes, which metabolize RA into easily degraded derivatives. Loss of Cyp26 function in humans is associated with numerous developmental syndromes that include cardiovascular defects. Although previous studies have shown that Cyp26-deficient vertebrate models also have cardiovascular defects, the mechanisms underlying these defects are not understood. Here, we found that in zebrafish, two Cyp26 enzymes, Cyp26a1 and Cyp26c1, are expressed in the anterior lateral plate mesoderm (ALPM) and predominantly overlap with vascular progenitors (VPs). Although singular knockdown of Cyp26a1 or Cyp26c1 does not overtly affect cardiovascular development, double Cyp26a1 and Cyp26c1 (referred to here as Cyp26)-deficient embryos have increased atrial cells and reduced cranial vasculature cells. Examining the ALPM using lineage tracing indicated that in Cyp26-deficient embryos the myocardial progenitor field contains excess atrial progenitors and is shifted anteriorly into a region that normally solely gives rise to VPs. Although Cyp26 expression partially overlaps with VPs in the ALPM, we found that Cyp26 enzymes largely act cell non-autonomously to promote appropriate cardiovascular development. Our results suggest that localized expression of Cyp26 enzymes cell non-autonomously defines the boundaries between the cardiac and VP fields within the ALPM through regulating RA levels, which ensures a proper balance of myocardial and endothelial lineages. Our study provides novel insight into the earliest consequences of Cyp26 deficiency that underlie cardiovascular malformations in vertebrate embryos.

Excessive feedback of Cyp26a1 promotes cell non-autonomous loss of retinoic acid signaling

“…Increases in RA signaling can inhibit cardiomyocyte specification, while decreases in RA signaling can promote cardiomyocyte specification (Keegan et al, 2005; Rydeen and Waxman, 2014; Waxman and Yelon, 2009). Interestingly, examining heart morphology, cell number, and cardiomyocyte marker expression, we found that the hearts of vp-rar mRNA-injected Tg(-5.1myl7:DsRed-NLS) f2 embryos (Mably et al, 2003) were enlarged, had increased cell number, and increased cardiomyocyte marker expression at 48 hpf (Fig.…”

“…6B-B3, D-D3, E), which suggests the vp-rar expression can promote cell non-autonomous loss of RA signaling. Next, to determine if the cell non-autonomous effects of vp-rar expression were dependent on Cyp26a1, we transplanted RA sensor donor cells into WT hosts or hosts co-injected with vp-rar mRNA and previously characterized cyp26a1 MOs (D’Aniello et al, 2013; Rydeen and Waxman, 2014). In contrast to when RA sensor cells were transplanted into vp-rar mRNA injected hosts, we found that depletion of Cyp26a1 in vp-rar mRNA injected host embryos results in RA sensor donor cells with robust GFP expression (Fig.…”

“…Cyp26a1 mRNA was injected at 500pg as previously described (Kudoh et al, 2002). Previously published cyp26a1 MOs were injected using a cocktail of 2–4ng cyp26a1 MO1 and 1–2ng cyp26a1 MO2, as well as 3ng p53 MO to combat non-specific cell death (D’Aniello et al, 2013; Rydeen and Waxman, 2014). …”

“…RT-qPCR was performed as has been previously described (D’Aniello et al, 2013; Rydeen and Waxman, 2014). Whole embryo RNA was obtained from pooled groups of 30 embryos, which were homogenized in Trizol (Ambion) and isolated using Purelink RNA Microkit (Invitrogen).…”

“…Similarly, in zebrafish, Martin and Kimelman (2010) found that Cyp26a1 does not have cell autonomous roles within mesodermal progenitor pool, suggesting non-autonomous functions. Furthermore, recent work from our lab has suggested that loss of Cyp26 enzymes can have cell non-autonomous consequences in patterning the cardiovascular progenitor fields within the anterior lateral plate mesoderm (Rydeen and Waxman, 2014). Additionally, using computational modeling, White et al (2007) proposed a model in zebrafish embryos where the embryonic RA gradient is shaped through self-enhanced degradation by promoting Cyp26a1 expression, in effect arguing that Cyp26a1 may have cell autonomous and cell non-autonomous consequences in shaping the embryonic RA gradient.…”