Loss of foxc1 in zebrafish reduces optic nerve size and cell number in the retinal ganglion cell layer.

Mutation of FOXC1 causes Axenfeld-Rieger Syndrome (ARS) with early onset or congenital glaucoma. We assessed retinal ganglion cell (RGC) number in zebrafish due to CRISPR-mediated mutation and antisense inhibition of two-forkhead box transcription factors, foxc1a and foxc1b. These genes represent duplicated homologues of human FOXC1. Using a CRISPR induced null mutation in foxc1b, in combination with antisense inhibition of foxc1a, we demonstrate reduced cell number in the retinal ganglion cell layer of developing zebrafish eyes. As early as 5 days post fertilization (dpf), fewer RGCs are found in foxc1b homozygous mutants injected with foxc1a morpholinos, and a thinner optic nerve results. Our data illustrates that foxc1 is required for the expression of atonal homolog 7 (atoh7), a gene that is necessary for RGC differentiation. As markers of differentiated RGCs (pou4f2) are downregulated in foxc1b-/- mutants injected with foxc1a morpholinos and no cell death is observed, our results are consistent with defects in the differentiation of RGCs leading to reduced cell number, as opposed to increased cell death of RGCs or off targets effects of morpholino injection. Our zebrafish model demonstrates that aberrant regulation of RGC number could act in concert with other known glaucoma risk factors to influence the development of congenital and early onset glaucoma due to FOXC1 mutation.

Angiopoietin-1 is required for Schlemm's canal development in mice and humans.

Primary congenital glaucoma (PCG) is a leading cause of blindness in children worldwide and is caused by developmental defects in 2 aqueous humor outflow structures, Schlemm's canal (SC) and the trabecular meshwork. We previously identified loss-of-function mutations in the angiopoietin (ANGPT) receptor TEK in families with PCG and showed that ANGPT/TEK signaling is essential for SC development. Here, we describe roles for the major ANGPT ligands in the development of the aqueous outflow pathway. We determined that ANGPT1 is essential for SC development, and that Angpt1-knockout mice form a severely hypomorphic canal with elevated intraocular pressure. By contrast, ANGPT2 was dispensable, although mice deficient in both Angpt1 and Angpt2 completely lacked SC, indicating that ANGPT2 compensates for the loss of ANGPT1. In addition, we identified 3 human subjects with rare ANGPT1 variants within an international cohort of 284 PCG patients. Loss of function in 2 of the 3 patient alleles was observed by functional analysis of ANGPT1 variants in a combined in silico, in vitro, and in vivo approach, supporting a causative role for ANGPT1 in disease. By linking ANGPT1 with PCG, these results highlight the importance of ANGPT/TEK signaling in glaucoma pathogenesis and identify a candidate target for therapeutic development.

Congenital glaucoma detected by unilateral megalophthalmos on prenatal sonography.

A 36-week male fetus was noted to have isolated right megalophthalmos on prenatal ultrasound and was found to have buphthalmos and congenital glaucoma at birth. Detection of congenital glaucoma at birth may be occasionally possible if abnormal orbit dimensions are noted on late prenatal sonographic examination. Early neonatal intervention may improve the chances to retain vision. © 2016 Wiley Periodicals, Inc. J Clin Ultrasound 45:499-501, 2017.

[Glaucoma with aniridia and isolated congenital glaucoma in siblings: contribution and limits of genetics]. / Glaucome secondaire à une aniridie et glaucome congénital isolé dans une même fratrie: apports et limites de la génétique.

INTRODUCTION: Congenital glaucoma associated with aniridia and primary congenital glaucoma are regarded as different entities. Indeed, the abnormalities of the angle's structures as well as the genes involved are different. We report the observation of two sisters presenting these two types of glaucoma with particular attention paid to the importance and the difficulty of genetic counseling. OBSERVATIONS: Child L, with no particular family history, had presented bilateral aniridia complicated by bilateral glaucoma since birth. In addition to medical and surgical treatment, general and genetic investigations were undertaken that revealed no abnormalities. No microdeletion of the gene PAX6 responsible for the aniridia was found. Consequently, the genetic advice was in favor of a second pregnancy for this couple. At birth, L's sister also presented bilateral congenital glaucoma, which was isolated, without aniridia. New genetic investigations were carried out but no abnormalities in PAX6, nor in FOXC1 or PITX2 involved in the development of the anterior chamber, were found. Moreover, the haplotypes for aniridia locus AN2 inherited by the two sisters were different, proof that this gene could not be responsible for the glaucoma. DISCUSSION: At L's birth, the hypothesis retained was that she was a sporadic case whose gene mutation could not be identified (which happens in 50% of sporadic cases). The risk for the second pregnancy was negligible, although not null. The primary congenital glaucoma presented by L's sister remains unexplained in the context of aniridia and the role of the PAX6 gene was eliminated. The study of PITX2 and FOXC1 genes involved in anterior segment dysgenesis proved that they were also not involved. Thus, this observation evokes the responsibility of a gene other than PAX6 in aniridia, which could also have a role in isolated congenital glaucoma. CONCLUSION: Analysis of congenital pathologies from a more genetic than clinical point of view seems to progressively break down the barriers established between the various phenotypes of hereditary congenital anomalies. Even if the association of aniridia and primary congenital glaucoma in siblings is reported here for the first time, it does not appear so extraordinary if one considers the complexity of the anterior chamber's development, which involves many genes, most of them still unidentified to date.

Expression patterns of cytochrome P4501B1 (Cyp1b1) in FVB/N mouse eyes.

Homozygous and compound heterozygous mutations in cytochrome P4501B1 (CYP1B1) cause primary congenital glaucoma (PCG) in humans. It is hypothesized that developmental anomalies of the trabecular meshwork prevent appropriate drainage of the aqueous humor and cause PCG in human patients. In this report, we studied the expression patterns of Cyp1b1 in the eye of albino FVB/N mouse at different developmental stages. We isolated a cDNA fragment corresponding to the 3'untranslated region (3'UTR) of the Cyp1b1 gene by PCR and used it to make an (35)S-labelled riboprobe for in situ hybridization. We found that Cyp1b1 is expressed in both anterior and posterior segments of the eye. Anteriorly, the expression is confined to the ciliary body, most likely in the outer/pigmented ciliary epithelial cells. Cyp1b1 mRNA can be detected in these cells at postnatal day 4 (P4) and the expression continues into adulthood. Surprisingly, no above-background levels of Cyp1b1 mRNA were found at or around the trabecular meshwork at all the stages we examined. In the posterior region of the embryonic day 15 (E15) eye, Cyp1b1 is expressed in the retinal neuroepithelium and in the tissues surrounding the optic nerve, but not in the optic nerve itself. In the P7 retina, Cyp1b1 mRNA is found in the inner nuclear layer. Based on our finding that Cyp1b1 is expressed in the developing and mature ciliary body of the mouse eye, we speculate that mutation in this gene can directly contribute to the abnormal elevation of the intraocular pressure (IOP) in the PCG patients or indirectly affect the aqueous outflow by disrupting the proper development of the trabecular meshwork in these patients.

Anterior segment dysgenesis and the developmental glaucomas are complex traits.

Glaucoma refers to a heterogeneous group of disorders that involve retinal ganglion cell death, optic nerve damage, and loss of visual field. Glaucoma is a leading cause of vision loss worldwide, affecting an estimated 67 million people. Elevated intraocular pressure is a major risk factor for glaucoma. Individuals with malformations of structures of the anterior segment of the eye frequently develop elevated intraocular pressure and glaucoma. In this review, we focus on the developmental glaucomas, the subset of glaucomas associated with anterior segment dysgenesis. To minimize overlap with other reviews in this issue and elsewhere, we highlight the complex, multifactorial nature of these diseases and recent advances using mice.

[The Binder syndrome and goniodysgenesis. Apropos of a case]. / Syndrome de Binder et goniodysgénésies. A propos d'un cas.

A 15 years old boy was deferred to our department with growth hormone deficiency, deafness with middle ear bones involvement, and a facial dysmorphy called Binder syndrome. He presented ocular abnormalities with juvenile glaucoma and iris anterior insertion and goniodysgenesis. All these pathologic features are linked with the development of the neural crests. In our knowledge, it is the first so complete clinical association reported, which emphasizes the important role of the neural crest cells in early organogenesis.

Congenital glaucoma.

Congenital and juvenile glaucoma are associated with goniodysgenesis and currently thought to be the result of neural crest cell abnormal terminal induction or migration. Infantile glaucoma may be primary, or may be associated with syndromes such as Sturge-Weber, Rieger's and others. Differentiation from other childhood causes of cloudy cornea such as endothelial dystrophies is essential. Evaluations under anesthesia are often needed and present their own problems in diagnosis.

A common pathway for developmental glaucomas.

In a clinicopathologic study of ten patients, utilizing a modified trabeculectomy technique for acquisition of histologic specimens, a high insertion of the iris was observed in four types of developmental glaucoma. A survey of the literature revealed additional developmental disorders with this abnormality of the anterior chamber angle. The common defect is believed to arise from a developmental arrest during the third trimester of gestation of tissues derived from cranial neural crest cells. The mechanism by which this developmental defect leads to aqueous outflow obstruction may, in some cases, be a paradoxical collapse of the trabecular meshwork and Schlemm's canal in response to contraction of the ciliary musculature, while other patients may have additional developmental abnormalities in the aqueous outflow system as the possible mechanism of glaucoma.

Histopathology of abnormalities of the anterior chamber with glaucoma.

Neural crest cells give rise to all connective tissues anterior to the lens epithelium including corneal stromal cells, corneal and trabecular mesothelial cells, iris stromal cells, and iris melanophores. These cells constitute a continuous layer which extends from the corneal mesothelium to trabecular meshwork mesothelium and onto the anterior surface of the iris. Between the seventh and eighth month of gestation in man, the layer of cells loses its continuity. The present case suggests a defective terminal induction of a particular structural component of the angle derived from neural crest resulting in massive congenital adhesions in the anterior chamber angle. The persistence of a layer of abnormal cells lining the anterior chamber structures further reinforces the presence of defective mesothelial cells. Additional cases having such clinicopathologic correlation may provide further insight into this hypothesis.

Etiology of congenital glaucoma. Genetic and extragenetic factors.

Congenital glaucoma (simple, associated and secondary) has been studied for the frequency of mesodermal anomalies and malformations (skeletal changes, congenital heart diseases, neurological alterations). But congenital glaucoma is not due solely to genetic factors: it is more likely that most cases result from interaction between genetic predisposition and a combination of environmental causes.

Axenfeld-Rieger syndrome: a theory of mechanism and distinctions from the iridocorneal endothelial syndrome.

Twenty-four patients with the diagnosis of Axenfeld's anomaly or Rieger's anomaly or syndrome were the subjects of a clinical study, which included specular microscopy of the corneal endothelium in 16 cases and fluorescein angiography of the iris in 5. Histopathologic material was obtained from ten eyes of eight of these patients (one enucleated eye and nine trabeculectomy specimens) and was studied by light and electron microscopy. The overlapping of ocular and nonocular defects in these patients prevented subclassification according to traditional criteria. Any attempted subdivision appears to have minimal clinical value, and a single classification for the disease spectrum is believed to be more practical. The collective term Axenfeld-Rieger (A-R) syndrome is proposed. A theory of mechanism for the ocular features of the A-R syndrome is postulated which involves a developmental arrest, late in gestation, of tissues derived from neural crest cells. This leads to retention of primordial endothelial tissue on the iris and across the anterior chamber angle, which produces the iridic changes and the peripheral tissue strands. Continued contraction of these membranes after birth explains the progressive changes noted in some patients. This primordial endothelium also produces excessive and atypical basement membrane, especially near the corneolimbal junction, which accounts for the prominent Schwalbe's line. The secondary glaucoma results from arrested development of the anterior chamber angle structures, characterized by incomplete maturation of the trabecular meshwork and Schlemm's canal and a high insertion of the iris. The ICE syndrome may be confused with the A-R syndrome on the basis of certain clinical and histopathologic similarities. Based on available evidence, however, it is postulated that the two entities are distinctly separate, in that the fundamental defect in the ICE syndrome is believed to be an abnormality of the corneal endothelium with secondary proliferation of a tissue layer over the anterior chamber angle and iris, while the A-R syndrome is thought to represent a developmental arrest with retention of a primordial membrane and other developmental defects.

Periods of development of the normal human chamber angle.

Stages of fetal and postnatal development of the normal human chamber angle are reevaluated. Major growth phases can be detected: trabecular anlage formation (around the 15th week), differentiation into definitive structures (around the 24th week), specialisation of the definitive structures (around the 28th week), achievement of final components (birth) and final moulding of the chamber angle and the maturation of its cellular and extracellular constituents (1-8 years). A cleavage or gradual atrophy of chamber angle tissues could not be detected during any of the above phases, nor was a membrane found, which covered the chamber angle recess at any stage of development. In the light of the described periods, potential disturbances of development are discussed, which might be a cause of congenital glaucoma.

The pathogenesis of reversible cupping in congenital glaucoma.

The pathogenesis of reversible cupping of the optic disk in congenital glaucoma was examined by two approaches. Human fetal, neonatal, and adult eyes were examined by histochemistry and electron microscopy to delineate the embryologic development of the optic nerve head. While the neural, glial, and vascular elements of the nerve head attain their adult configuration by midgestation, the connective tissues of the lamina cribrosa are incompletely developed at birth. The response of the optic disk cup to elevated intraocular pressure (IOP) was observed in enucleated infant and adult eyes. While no disk changes were seen in adult eyes subjected to an IOP up to 90 mm Hg for 24 hours, enlargement of the disk cup in infant eyes was documented photographically and histologically after eight to 24 hours of IOP elevations to 50 mg Hg. Reversible cupping in congenital glaucoma can be best explained by compression or posterior movement of optic disk tissues--a result of the incomplete collagenous structural framework of the lamina cribrosa during late gestation and early neonatal life.