Conditional knockdown of DNA methyltransferase 1 reveals a key role of retinal pigment epithelium integrity in photoreceptor outer segment morphogenesis.

Dysfunction or death of photoreceptors is the primary cause of vision loss in retinal and macular degenerative diseases. As photoreceptors have an intimate relationship with the retinal pigment epithelium (RPE) for exchange of macromolecules, removal of shed membrane discs and retinoid recycling, an improved understanding of the development of the photoreceptor-RPE complex will allow better design of gene- and cell-based therapies. To explore the epigenetic contribution to retinal development we generated conditional knockout alleles of DNA methyltransferase 1 (Dnmt1) in mice. Conditional Dnmt1 knockdown in early eye development mediated by Rx-Cre did not produce lamination or cell fate defects, except in cones; however, the photoreceptors completely lacked outer segments despite near normal expression of phototransduction and cilia genes. We also identified disruption of RPE morphology and polarization as early as E15.5. Defects in outer segment biogenesis were evident with Dnmt1 exon excision only in RPE, but not when excision was directed exclusively to photoreceptors. We detected a reduction in DNA methylation of LINE1 elements (a measure of global DNA methylation) in developing mutant RPE as compared with neural retina, and of Tuba3a, which exhibited dramatically increased expression in mutant retina. These results demonstrate a unique function of DNMT1-mediated DNA methylation in controlling RPE apicobasal polarity and neural retina differentiation. We also establish a model to study the epigenetic mechanisms and signaling pathways that guide the modulation of photoreceptor outer segment morphogenesis by RPE during retinal development and disease.

Multiple requirements of the focal dermal hypoplasia gene porcupine during ocular morphogenesis.

Wnt glycoproteins control key processes during development and disease by activating various downstream pathways. Wnt secretion requires post-translational modification mediated by the O-acyltransferase encoded by the Drosophila porcupine homolog gene (PORCN). In humans, PORCN mutations cause focal dermal hypoplasia (FDH, or Goltz syndrome), an X-linked dominant multisystem birth defect that is frequently accompanied by ocular abnormalities such as coloboma, microphthalmia, or even anophthalmia. Although genetic ablation of Porcn in mouse has provided insight into the etiology of defects caused by ectomesodermal dysplasia in FDH, the requirement for Porcn and the actual Wnt ligands during eye development have been unknown. In this study, Porcn hemizygosity occasionally caused ocular defects reminiscent of FDH. Conditional inactivation of Porcn in periocular mesenchyme led to defects in mid- and hindbrain and in craniofacial development, but was insufficient to cause ocular abnormalities. However, a combination of conditional Porcn depletion in optic vesicle neuroectoderm, lens, and neural crest-derived periocular mesenchyme induced severe eye abnormalities with high penetrance. In particular, we observed coloboma, transdifferentiation of the dorsal and ventral retinal pigment epithelium, defective optic cup periphery, and closure defects of the eyelid, as well as defective corneal morphogenesis. Thus, Porcn is required in both extraocular and neuroectodermal tissues to regulate distinct Wnt-dependent processes during morphogenesis of the posterior and anterior segments of the eye.

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.

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.

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.

COUP-TFs regulate eye development by controlling factors essential for optic vesicle morphogenesis.

Transcriptional networks, which are initiated by secreted proteins, cooperate with each other to orchestrate eye development. The establishment of dorsal/ventral polarity, especially dorsal specification in the optic vesicle, is poorly understood at a molecular and cellular level. Here, we show that COUP-TFI (Nr2f1) and COUP-TFII (Nr2f2) are highly expressed in the progenitor cells in the developing murine eye. Phenotype analysis of COUP-TFI and COUP-TFII single-gene conditional knockout mouse models suggests that COUP-TFs compensate for each other to maintain morphogenesis of the eye. However, in eye-specific COUP-TFI/TFII double-knockout mice, progenitor cells at the dorso-distal optic vesicle fail to differentiate appropriately, causing the retinal pigmented epithelium cells to adopt a neural retina fate and abnormal differentiation of the dorsal optic stalk; the development of proximo-ventral identities, neural retina and ventral optic stalk is also compromised. These cellular defects in turn lead to congenital ocular colobomata and microphthalmia. Immunohistochemical and in situ hybridization assays reveal that the expression of several regulatory genes essential for early optic vesicle development, including Pax6, Otx2, Mitf, Pax2 and Vax1/2, is altered in the corresponding compartments of the mutant eye. Using ChIP assay, siRNA treatment and transient transfection in ARPE-19 cells in vitro, we demonstrate that Pax6 and Otx2 are directly regulated by COUP-TFs. Taken together, our findings reveal novel and distinct cell-intrinsic mechanisms mediated by COUP-TF genes to direct the specification and differentiation of progenitor cells, and that COUP-TFs are crucial for dorsalization of the eye.

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.

The role of the visceral mesoderm in the development of the gastrointestinal tract.

The gastrointestinal (GI) tract forms from the endoderm (which gives rise to the epithelium) and the mesoderm (which develops into the smooth muscle layer, the mesenchyme, and numerous other cell types). Much of what is known of GI development has been learned from studies of the endoderm and its derivatives, because of the importance of epithelial biology in understanding and treating human diseases. Although the necessity of epithelial-mesenchymal cross talk for GI development is uncontested, the role of the mesoderm remains comparatively less well understood. The transformation of the visceral mesoderm during development is remarkable; it differentiates from a very thin layer of cells into a complex tissue comprising smooth muscle cells, myofibroblasts, neurons, immune cells, endothelial cells, lymphatics, and extracellular matrix molecules, all contributing to the form and function of the digestive system. Understanding the molecular processes that govern the development of these cell types and elucidating their respective contribution to GI patterning could offer insight into the mechanisms that regulate cell fate decisions in the intestine, which has the unique property of rapid cell renewal for the maintenance of epithelial integrity. In reviewing evidence from both mammalian and nonmammalian models, we reveal the important role of the visceral mesoderm in the ontogeny of the GI tract.

Pitx3 controls multiple aspects of lens development.

The transcription factor Pitx3 is critical for lens formation. Deletions in the promoter of this gene cause abnormal lens development in the aphakia (ak) mouse mutant, which has only rudimentary lenses. In this study, we investigated the role of Pitx3 in lens development and differentiation. We found that reduced expression of Pitx3 leads to changes in the proliferation, differentiation and survival of lens cells. The genetic interactions between Pitx3 and Foxe3 were investigated, as these two transcription factors are expressed at the same time in lens development and their absence has similar consequences for lens development. We found no evidence that these two genes genetically interact. In general, our study shows that the abnormal phenotype of the ak lenses is not due to just one molecular pathway, rather in the absence of Pitx3 expression multiple aspects of lens development are disrupted.

The transcription factor Maz is essential for normal eye development.

Wnt/ß-catenin signaling has an essential role in eye development. Faulty regulation of this pathway results in ocular malformations, owing to defects in cell-fate determination and differentiation. Herein, we show that disruption of Maz, the gene encoding Myc-associated zinc-finger transcription factor, produces developmental eye defects in mice and humans. Expression of key genes involved in the Wnt cascade, Sfrp2, Wnt2b and Fzd4, was significantly increased in mice with targeted inactivation of Maz, resulting in abnormal peripheral eye formation with reduced proliferation of the progenitor cells in the region. Paradoxically, the Wnt reporter TCF-Lef1 displayed a significant downregulation in Maz-deficient eyes. Molecular analysis indicates that Maz is necessary for the activation of the Wnt/ß-catenin pathway and participates in the network controlling ciliary margin patterning. Copy-number variations and single-nucleotide variants of MAZ were identified in humans that result in abnormal ocular development. The data support MAZ as a key contributor to the eye comorbidities associated with chromosome 16p11.2 copy-number variants and as a transcriptional regulator of ocular development.

Frizzled 5 signaling governs the neural potential of progenitors in the developing Xenopus retina.

Progenitors in the developing central nervous system acquire neural potential and proliferate to expand the pool of precursors competent to undergo neuronal differentiation. The formation and maintenance of neural-competent precursors are regulated by SoxB1 transcription factors, and evidence that their expression is regionally regulated suggests that specific signals regulate neural potential in subdomains of the developing nervous system. We show that the frizzled (Fz) transmembrane receptor Xfz5 selectively governs neural potential in the developing Xenopus retina by regulating the expression of Sox2. Blocking either Xfz5 or canonical Wnt signaling within the developing retina inhibits Sox2 expression, reduces cell proliferation, inhibits the onset of proneural gene expression, and biases individual progenitors toward a nonneural fate, without altering the expression of multiple progenitor markers. Blocking Sox2 function mimics these effects. Rescue experiments indicate that Sox2 is downstream of Xfz5. Thus, Fz signaling can regulate the neural potential of progenitors in the developing nervous system.

Eye formation in the absence of retina.

Eye development is a complex process that involves the formation of the retina and the lens, collectively called the eyeball, as well as the formation of auxiliary eye structures such as the eyelid, lacrimal gland, cornea and conjunctiva. The developmental requirements for the formation of each individual structure are only partially understood. We have shown previously that the homeobox-containing gene Rx is a key component in eye formation, as retinal structures do not develop and retina-specific gene expression is not observed in Rx-deficient mice. In addition, Rx-/- embryos do not develop any lens structure, despite the fact that Rx is not expressed in the lens. This demonstrates that during normal mammalian development, retina-specific gene expression is necessary for lens formation. In this paper we show that lens formation can be restored in Rx-deficient embryos experimentally, by the elimination of beta-catenin expression in the head surface ectoderm. This suggests that beta-catenin is involved in lens specification either through Wnt signaling or through its function in cell adhesion. In contrast to lens formation, we demonstrate that the development of auxiliary eye structures does not depend on retina-specific gene expression or retinal morphogenesis. These results point to the existence of two separate developmental processes involved in the formation of the eye and its associated structures. One involved in the formation of the eyeball and the second involved in the formation of the auxiliary eye structures.

Mutant neurogenin-3 in congenital malabsorptive diarrhea.

BACKGROUND: Neurogenin-3 (NEUROG3) is expressed in endocrine progenitor cells and is required for endocrine-cell development in the pancreas and intestine. The NEUROG3 gene (NEUROG3) is therefore a candidate for the cause of a newly discovered autosomal recessive disorder characterized by generalized malabsorption and a paucity of enteroendocrine cells. METHODS: We screened genomic DNA from three unrelated patients with sparse enteroendocrine cells for mutations of NEUROG3. We then tested the ability of the observed mutations to alter NEUROG3 function, using in vitro and in vivo assays. RESULTS: The patients had few intestinal enteroendocrine cells positive for chromogranin A, but they had normal numbers of Paneth's, goblet, and absorptive cells. We identified two homozygous mutations in NEUROG3, both of which rendered the NEUROG3 protein unable to activate NEUROD1, a downstream target of NEUROG3, and compromised the ability of NEUROG3 to bind to an E-box element in the NEUROD1 promoter. The injection of wild-type but not mutant NEUROG3 messenger RNA into xenopus embryos induced NEUROD1 expression. CONCLUSIONS: A newly discovered disorder characterized by malabsorptive diarrhea and a lack of intestinal enteroendocrine cells is caused by loss-of-function mutations in NEUROG3.

Expression of complement components coincides with early patterning and organogenesis in Xenopus laevis.

The complement system is the central component of innate immunity and an important player in the adaptive immunity of vertebrates. We analyzed the expression patterns of several key members of the complement cascade during Xenopus development. We found extensive expression of these molecules already during gastrula/early neurula stage. Remarkably, several genes also showed an organ-specific expression pattern during early organogenesis. Early expression is notable for two different expression patterns in the neuroectoderm. In one group, there is early strong neural plate and neural precursor expression. This is the case of properdin, C1qA, C3 and C9. The second pattern, seen with C1qR and C6, is noteworthy for its expression at the periphery of the neural plate, in the presumptive neural crest. Two genes stand out for their predominantly mesodermal expression. C3aR, the message for the cognate receptor for C3 in the complement cascade, is expressed at the same time as C3, but in a complementary, reciprocal pattern in the mesoderm. C1qA expression also predominates in somites, pronephros, visceral mesoderm and ventral blood islands. Finally, several genes are characterized by later expression in developing organs. C1qR displays a reticular pattern consistent with expression in the developing vasculature. The late expression of C1qA and C3bC4b is strongest in the pronephros. Finally, the expression of properdin in the hindbrain and in the developing lens are novel findings. The expression patterns of these molecules suggest that these components of the complement system may have in Xenopus a so far undefined developmental role.

Regulation and function of foxe3 during early zebrafish development.

In this article, we investigate the expression, regulation, and function of the zebrafish forkhead gene foxe3. In wild type embryos, foxe3 is first expressed in a crescent-shaped area at the anterior end of the prechordal plate, corresponding to the polster. At later stages, the hatching gland, the lens, and the anterior pituitary express this gene. Using morpholinos against the zinc finger Kruppel-like factor 4 (KLF4) we show that foxe3 is regulated differently in the polster and in the lens. In the absence of KLF4, expression of foxe3 in the polster is not activated, whereas in the lens placode the expression of KLF4 is not required for the transcription of foxe3. The expression of foxe3 is also regulated by the hedgehog and nodal signaling pathways. foxe3 expression is altered in the hedgehog pathway mutants iguana and you-too and the nodal pathway mutant cyclops. foxe3 function is necessary for the execution of lens-specific gene expression and lens morphogenesis, as the knockdown of foxe3 results in a loss of platelet-derived growth factor receptor alpha (pdgfralpha) expression and in the vacuolization of the lens.

Severe defects in proliferation and differentiation of lens cells in Foxe3 null mice.

During mouse eye development, the correct formation of the lens occurs as a result of reciprocal interactions between the neuroectoderm that forms the retina and surface ectoderm that forms the lens. Although many transcription factors required for early lens development have been identified, the mechanism and genetic interactions mediated by them remain poorly understood. Foxe3 encodes a winged helix-forkhead transcription factor that is initially expressed in the developing brain and in the lens placode and later restricted exclusively to the anterior lens epithelium. Here, we show that targeted disruption of Foxe3 results in abnormal development of the eye. Cells of the anterior lens epithelium show a decreased rate of proliferation, resulting in a smaller than normal lens. The anterior lens epithelium does not properly separate from the cornea and frequently forms an unusual, multilayered tissue. Because of the abnormal differentiation, lens fiber cells do not form properly, and the morphogenesis of the lens is greatly affected. The abnormally differentiated lens cells remain irregular in shape, and the lens becomes vacuolated. The defects in lens development correlate with changes in the expression of growth and differentiation factor genes, including DNase II-like acid DNase, Prox1, p57, and PDGFalpha receptor. As a result of abnormal lens development, the cornea and the retina are also affected. While Foxe3 is also expressed in a distinct region of the embryonic brain, we have not observed abnormal development of the brain in Foxe3(-/-) animals.

Expression of FoxP2 during zebrafish development and in the adult brain.

Fox (forkhead) genes encode transcription factors that play important roles in the regulation of embryonic patterning as well as in tissue specific gene expression. Mutations in the human FOXP2 gene cause abnormal speech development. Here we report the structure and expression pattern of zebrafish FoxP2. In zebrafish, this gene is first expressed at the 20-somite stage in the presumptive telencephalon. At this stage there is a significant overlap of FoxP2 expression with the expression of the emx homeobox genes. However, in contrast to emx1, FoxP2 is not expressed in the pineal gland or in the pronephric duct. After 72 hours of development, the expression of zebrafish FoxP2 becomes more complex in the brain. The developing optic tectum becomes the major area of FoxP2 expression. In the adult brain, the highest concentrations of the FoxP2 transcript can be observed in the optic tectum. In the cerebellum, only the caudal lobes show high levels of Foxp2 expression. These regions correspond to the vestibulocerebellum of mammals. Several other regions of the brain also show high levels of Foxp2 expression.

Xenopus laevis FoxE1 is primarily expressed in the developing pituitary and thyroid.

The members of the FoxE subfamily of Fox (forkhead) genes are expressed in the developing pituitary, thyroid and lens. Mammalian Foxe1 is expressed primarily in the developing pituitary and thyroid gland, Foxe3 is expressed in the developing lens, while Xenopus FoxE4 is expressed in the developing lens and thyroid. Here we report the identification of Xenopus FoxE1, a gene that is primarily expressed in the developing pituitary and thyroid.

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.

Identification and developmental expression of Xenopus paraxis.

During vertebrate embryogenesis, the paraxial mesoderm becomes segmented in a rostro-caudal progression and gives rise to the somites. In this paper we report the isolation of a Xenopus orthologue of paraxis, a member of a family of basic helix-loop-helix proteins, which has been suggested to play a role in paraxial mesoderm development. Xenopus paraxis is initially expressed in the presomitic paraxial mesoderm and later in the dorsal portion of the developing somites. Finally, paraxis expression becomes restricted to the most dorso-lateral region of mature somites.