Eric C. Swindell

Neuroretina specification in mouse embryos requires Six3-mediated suppression of Wnt8b in the anterior neural plate

Retinal degeneration causes vision impairment and blindness in humans. If one day we are to harness the potential of stem cell-based cell replacement therapies to treat these conditions, it is imperative that we better understand normal retina development. Currently, the genes and mechanisms that regulate the specification of the neuroretina during vertebrate eye development remain unknown. Here, we identify sine oculis-related homeobox 3 (Six3) as a crucial player in this process in mice. In Six3 conditional-mutant mouse embryos, specification of the neuroretina was abrogated, but that of the retinal pigmented epithelium was normal. Conditional deletion of Six3 did not affect the initial development of the optic vesicle but did arrest subsequent neuroretina specification. Ectopic rostral expansion of Wnt8b expression was the major response to Six3 deletion and the leading cause for the specific lack of neuroretina, as ectopic Wnt8b expression in transgenic embryos was sufficient to suppress neuroretina specification. Using chromatin immunoprecipitation assays, we identified Six3-responsive elements in the Wnt8b locus and demonstrated that Six3 directly repressed Wnt8b expression in vivo. Our findings provide a molecular framework to the program leading to neuroretina differentiation and may be relevant for the development of novel strategies aimed at characterizing and eventually treating different abnormalities in eye formation.

Unique domain appended to vertebrate tRNA synthetase is essential for vascular development

New domains were progressively added to cytoplasmic aminoacyl transfer RNA (tRNA) synthetases during evolution. One example is the UNE-S domain, appended to seryl-tRNA synthetase (SerRS) in species that developed closed circulatory systems. Here we show using solution and crystal structure analyses and in vitro and in vivo functional studies that UNE-S harbours a robust nuclear localization signal (NLS) directing SerRS to the nucleus where it attenuates vascular endothelial growth factor A expression. We also show that SerRS mutants previously linked to vasculature abnormalities either deleted the NLS or have the NLS sequestered in an alternative conformation. A structure-based second-site mutation, designed to release the sequestered NLS, restored normal vasculature. Thus, the essential function of SerRS in vascular development depends on UNE-S. These results are the first to show an essential role for a tRNA synthetase-associated appended domain at the organism level, and suggest that acquisition of UNE-S has a role in the establishment of the closed circulatory systems of vertebrates.

Carboxypeptidase Z(CPZ) modulates Wnt signaling and regulates the development of skeletal elements in the chicken

Carboxypeptidase Z (CPZ) is a secreted Zn-dependent enzyme whose biological function is largely unknown. CPZ has a bipartite structure consisting of an N-terminal cysteine-rich domain (CRD) and a C-terminal catalytic domain. In the early chicken embryo CPZ is initially expressed throughout the somites and subsequently becomes restricted to the sclerotome. To initiate a functional analysis of CPZ, a CPZ producing retroviral vector was applied to the presomitic mesoderm at the level of the future wing. This resulted in a loss of the scapular blade and of rostral ribs. Such dysmorphogenesis is preceded by ectopic Pax3 expression in the hypaxial part of the dermomyotome, a region from which the blade of the scapula normally derives. A mutant CPZ, lacking a critical active site glutamate, fails to induce Pax3 expression and does not cause skeletal defects. The induction of Pax3, a Wnt-responsive gene in somites, and the presence of a CRD prompted us to examine whether CPZ affects Wnt signaling. In an in vitro assay we found that CPZ, but not its inactive mutant form, enhances the Wnt-dependent induction of the homeobox gene Cdx1. In addition, immunoprecipitation experiments suggest that the CRD of CPZ acts as a binding domain for Wnt. Taken together these data provide the first evidence for CPZ playing a role in Wnt signaling.

MAT2A Mutations Predispose Individuals to Thoracic Aortic Aneurysms

Up to 20% of individuals who have thoracic aortic aneurysms or acute aortic dissections but who do not have syndromic features have a family history of thoracic aortic disease. Significant genetic heterogeneity is established for this familial condition. Whole-genome linkage analysis and exome sequencing of distant relatives from a large family with autosomal-dominant inheritance of thoracic aortic aneurysms variably associated with the bicuspid aortic valve was used for identification of additional genes predisposing individuals to this condition. A rare variant, c.1031A>C (p.Glu344Ala), was identified in MAT2A, which encodes methionine adenosyltransferase II alpha (MAT IIα). This variant segregated with disease in the family, and Sanger sequencing of DNA from affected probands from unrelated families with thoracic aortic disease identified another MAT2A rare variant, c.1067G>A (p.Arg356His). Evidence that these variants predispose individuals to thoracic aortic aneurysms and dissections includes the following: there is a paucity of rare variants in MAT2A in the population; amino acids Glu344 and Arg356 are conserved from humans to zebrafish; and substitutions of these amino acids in MAT Iα are found in individuals with hypermethioninemia. Structural analysis suggested that p.Glu344Ala and p.Arg356His disrupt MAT IIα enzyme function. Knockdown of mat2aa in zebrafish via morpholino oligomers disrupted cardiovascular development. Co-transfected wild-type human MAT2A mRNA rescued defects of zebrafish cardiovascular development at significantly higher levels than mRNA edited to express either the Glu344 or Arg356 mutants, providing further evidence that the p.Glu344Ala and p.Arg356His substitutions impair MAT IIα function. The data presented here support the conclusion that rare genetic variants in MAT2A predispose individuals to thoracic aortic disease.

Rax is a selector gene for mediobasal hypothalamic cell types.

The brain plays a central role in controlling energy, glucose, and lipid homeostasis, with specialized neurons within nuclei of the mediobasal hypothalamus, namely the arcuate (ARC) and ventromedial (VMH), tasked with proper signal integration. Exactly how the exquisite cytoarchitecture and underlying circuitry becomes established within these nuclei remains largely unknown, in part because hypothalamic developmental programs are just beginning to be elucidated. Here, we demonstrate that the Retina and anterior neural fold homeobox (Rax) gene plays a key role in establishing ARC and VMH nuclei in mice. First, we show that Rax is expressed in ARC and VMH progenitors throughout development, consistent with genetic fate mapping studies demonstrating that Rax+ lineages give rise to VMH neurons. Second, the conditional ablation of Rax in a subset of VMH progenitors using a Shh::Cre driver leads to a fate switch from a VMH neuronal phenotype to a hypothalamic but non-VMH identity, suggesting that Rax is a selector gene for VMH cellular fates. Finally, the broader elimination of Rax throughout ARC/VMH progenitors using Six3::Cre leads to a severe loss of both VMH and ARC cellular phenotypes, demonstrating a role for Rax in both VMH and ARC fate specification. Combined, our study illustrates that Rax is required in ARC/VMH progenitors to specify neuronal phenotypes within this hypothalamic brain region. Rax thus provides a molecular entry point for further study of the ontology and establishment of hypothalamic feeding circuits.

PALS1 Is Essential for Retinal Pigment Epithelium Structure and Neural Retina Stratification

The membrane-associated palmitoylated protein 5 (MPP5 or PALS1) is thought to organize intracellular PALS1-CRB-MUPP1 protein scaffolds in the retina that are involved in maintenance of photoreceptor–Müller glia cell adhesion. In humans, the Crumbs homolog 1 (CRB1) gene is mutated in progressive types of autosomal recessive retinitis pigmentosa and Leber congenital amaurosis. However, there is no clear genotype–phenotype correlation for CRB1 mutations, which suggests that other components of the CRB complex may influence the severity of retinal disease. Therefore, to understand the physiological role of the Crumbs complex proteins, especially PALS1, we generated and analyzed conditional knockdown mice for Pals1. Small irregularly shaped spots were detected throughout the PALS1 deficient retina by confocal scanning laser ophthalmoscopy and spectral domain optical coherence tomography. The electroretinography a- and b-wave was severely attenuated in the aged mutant retinas, suggesting progressive degeneration of photoreceptors. The histological analysis showed abnormal retinal pigment epithelium structure, ectopic photoreceptor nuclei in the subretinal space, an irregular outer limiting membrane, half rosettes of photoreceptors in the outer plexiform layer, and a thinner photoreceptor synaptic layer suggesting improper photoreceptor cell layering during retinal development. The PALS1 deficient retinas showed reduced levels of Crumbs complex proteins adjacent to adherens junctions, upregulation of glial fibrillary acidic protein indicative of gliosis, and persisting programmed cell death after retinal maturation. The phenotype suggests important functions of PALS1 in the retinal pigment epithelium in addition to the neural retina.

FOXE3 mutations predispose to thoracic aortic aneurysms and dissections.

The ascending thoracic aorta is designed to withstand biomechanical forces from pulsatile blood. Thoracic aortic aneurysms and acute aortic dissections (TAADs) occur as a result of genetically triggered defects in aortic structure and a dysfunctional response to these forces. Here, we describe mutations in the forkhead transcription factor FOXE3 that predispose mutation-bearing individuals to TAAD. We performed exome sequencing of a large family with multiple members with TAADs and identified a rare variant in FOXE3 with an altered amino acid in the DNA-binding domain (p.Asp153His) that segregated with disease in this family. Additional pathogenic FOXE3 variants were identified in unrelated TAAD families. In mice, Foxe3 deficiency reduced smooth muscle cell (SMC) density and impaired SMC differentiation in the ascending aorta. Foxe3 expression was induced in aortic SMCs after transverse aortic constriction, and Foxe3 deficiency increased SMC apoptosis and ascending aortic rupture with increased aortic pressure. These phenotypes were rescued by inhibiting p53 activity, either by administration of a p53 inhibitor (pifithrin-α), or by crossing Foxe3-/- mice with p53-/- mice. Our data demonstrate that FOXE3 mutations lead to a reduced number of aortic SMCs during development and increased SMC apoptosis in the ascending aorta in response to increased biomechanical forces, thus defining an additional molecular pathway that leads to familial thoracic aortic disease.

Development and plasticity of outer retinal circuitry following genetic removal of horizontal cells.

The present study examined the consequences of eliminating horizontal cells from the outer retina during embryogenesis upon the organization and assembly of the outer plexiform layer (OPL). Retinal horizontal cells exhibit a migration defect in Lim1-conditional knock-out (Lim1-CKO) mice and become mispositioned in the inner retina before birth, redirecting their dendrites into the inner plexiform layer. The resultant (mature) OPL, developing in the absence of horizontal cells, shows a retraction of rod spherules into the outer nuclear layer and a sprouting of rod bipolar cell dendrites to reach ectopic ribbon-protein puncta. Cone pedicles and the dendrites of type 7 cone bipolar cells retain their characteristic stratification and colocalization within the collapsed OPL, although both are atrophic and the spatial distribution of the pedicles is disrupted. Developmental analysis of Lim1-CKO retina reveals that components of the rod and cone pathways initially co-assemble within their normal strata in the OPL, indicating that horizontal cells are not required for the correct targeting of photoreceptor terminals or bipolar cell dendrites. As the rod spherules begin to retract during the second postnatal week, rod bipolar cells initially show no signs of ectopic growth, sprouting only subsequently and continuing to do so well after the eighth postnatal week. These results demonstrate the critical yet distinctive roles for horizontal cells on the rod and cone pathways and highlight a unique and as-yet-unrecognized maintenance function of an inhibitory interneuron that is not required for the initial targeting and co-stratification of other components in the circuit.

Role of adenylate cyclase 1 in retinofugal map development

The development of topographic maps of the sensory periphery is sensitive to the disruption of adenylate cyclase 1 (AC1) signaling. AC1 catalyzes the production of cAMP in a Ca2+/calmodulin‐dependent manner, and AC1 mutant mice (AC1−/−) have disordered visual and somatotopic maps. However, the broad expression of AC1 in the brain and the promiscuous nature of cAMP signaling have frustrated attempts to determine the underlying mechanism of AC1‐dependent map development. In the mammalian visual system, the initial coarse targeting of retinal ganglion cell (RGC) projections to the superior colliculus (SC) and lateral geniculate nucleus (LGN) is guided by molecular cues, and the subsequent refinement of these crude projections occurs via an activity‐dependent process that depends on spontaneous retinal waves. Here, we show that AC1−/− mice have normal retinal waves but disrupted map refinement. We demonstrate that AC1 is required for the emergence of dense and focused termination zones and elimination of inaccurately targeted collaterals at the level of individual retinofugal arbors. Conditional deletion of AC1 in the retina recapitulates map defects, indicating that the locus of map disruptions in the SC and dorsal LGN of AC1−/− mice is presynaptic. Finally, map defects in mice without AC1 and disrupted retinal waves (AC1−/−;β2−/− double KO mice) are no worse than those in mice lacking only β2−/−, but loss of AC1 occludes map recovery in β2−/− mice during the second postnatal week. These results suggest that AC1 in RGC axons mediates the development of retinotopy and eye‐specific segregation in the SC and dorsal LGN. J. Comp. Neurol. 520:1562–1583, 2012.

Genetic ablation of Pals1 in retinal progenitor cells models the retinal pathology of Leber congenital amaurosis

Mutation of the polarity gene Crumbs homolog 1 (CRB1) is responsible for >10% of Leber congenital amaurosis (LCA) cases worldwide; LCA is characterized by early-onset degenerative retinal dystrophy. The role of CRB1 in LCA8 pathogenesis remains elusive since Crb1 mouse mutants, including a null allele, have failed to mimic the early-onset of LCA, most likely due to functional compensation by closely related genes encoding Crb2 and Crb3. Crb proteins form an evolutionarily conserved, apical polarity complex with the scaffolding protein associated with lin-seven 1 (Pals1), also known as MAGUK p55 subfamily member 5 (MPP5). Pals1 and Crbs are functionally inter-dependent in establishing and maintaining epithelial polarity. Pals1 is a single gene in the mouse and human genomes; therefore, we ablated Pals1 to establish a mouse genetic model mimicking human LCA. In our study, the deletion of Pals1 leads to the disruption of the apical localization of Crb proteins in retinal progenitors and the adult retina, validating their mutual interaction. Remarkably, the Pals1 mutant mouse exhibits the critical features of LCA such as early visual impairment as assessed by electroretinogram, disorganization of lamination and apical junctions and retinal degeneration. Our data uncover the indispensible role of Pals1 in retinal development, likely involving the maintenance of retinal polarity and survival of retinal neurons, thus providing the basis for the pathologic mechanisms of LCA8.