Abstract
Precise spatiotemporal expression of the Nodal-Lefty-Pitx2 cascade in the lateral plate mesoderm establishes the left–right axis, which provides vital cues for correct organ formation and function. Mutations of one cascade constituent PITX2 and, separately, the Forkhead transcription factor FOXC1 independently cause a multi-system disorder known as Axenfeld–Rieger syndrome (ARS). Since cardiac involvement is an established ARS phenotype and because disrupted left–right patterning can cause congenital heart defects, we investigated in zebrafish whether foxc1 contributes to organ laterality or situs. We demonstrate that CRISPR/Cas9-generated foxc1a and foxc1b mutants exhibit abnormal cardiac looping and that the prevalence of cardiac situs defects is increased in foxc1a−/−; foxc1b−/− homozygotes. Similarly, double homozygotes exhibit isomerism of the liver and pancreas, which are key features of abnormal gut situs. Placement of the asymmetric visceral organs relative to the midline was also perturbed by mRNA overexpression of foxc1a and foxc1b. In addition, an analysis of the left–right patterning components, identified in the lateral plate mesoderm of foxc1 mutants, reduced or abolished the expression of the NODAL antagonist lefty2. Together, these data reveal a novel contribution from foxc1 to left–right patterning, demonstrating that this role is sensitive to foxc1 gene dosage, and provide a plausible mechanism for the incidence of congenital heart defects in Axenfeld–Rieger syndrome patients.
Highlights
While it has been suggested that zebrafish foxc1b may represent a functional ortholog of human FOXC2 [66], the amino acid sequences of Foxc1a and Foxc1b are more similar to human FOXC1 than FOXC2 (55% and 53%)
The most common phenotype was isomerism of the liver and / or pancreas (Figure 3B), and overall, these results suggest that cardiac situs is more sensitive to loss of foxc1 than gut situs
In situ analysis ysis of elovl6, an enzyme asymmetrically expressed in lateral plate mesoderm that is reof elovl6, an enzyme asymmetrically expressed in lateral plate mesoderm that is responsive sponsive to left–right organizer (LRO) function [65,80], revealed no changes in foxc1a−/−; foxc1b−/− double homoto LRO function [65,80], revealed no changes in foxc1a−/− ; foxc1b−/− double homozygotes zygotes at the 18 ss. These results suggest that Foxc1 regulates lefty2 rather than induces at the 18 ss. These results suggest that Foxc1 regulates lefty2 rather than induces loss or loss or randomization of all left–right patterning gene expressions, as seen randomization of all left–right patterning gene expressions, as seen when
Summary
The stomach and liver are positioned left and right of the midline, respectively. This normal arrangement is called situs solitus, while the complete reversal of normal organ situs (termed situs inversus) is surprisingly well tolerated [1]. Far more deleterious are partial situs defects, collectively known as heterotaxy [2], characterized by mis-patterning of visceral organs along the left–right axis. These are associated with congenital diseases of the heart, lungs, spleen, stomach, and liver [3,4,5] that may be challenging to treat. Many heterotaxy-associated genes cause isolated congenital heart defects (CHDs) [6], suggesting that a proportion of idiopathic
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