The mouse anterior chamber angle and trabecular meshwork develop without cell death

Abstract

The iridocorneal angle forms in the mammalian eye from undifferentiated mesenchyme between the root of the iris and cornea. A major component is the trabecular meshwork, consisting of extracellular matrix organized into a network of beams, covered in trabecular endothelial cells. Between the beams, channels lead to Schlemm's canal for the drainage of aqueous humor from the eye into the blood stream. Abnormal development of the iridocorneal angle that interferes with ocular fluid drainage can lead to glaucoma in humans. Little is known about the precise mechanisms underlying angle development. There are two main hypotheses. The first proposes that morphogenesis involves mainly cell differentiation, matrix deposition and assembly of the originally continuous mesenchymal mass into beams, channels and Schlemm's canal. The second, based primarily on rat studies, proposes that cell death and macrophages play an important role in forming channels and beams. Mice provide a potentially useful model to understand the origin and development of angle structures and how defective development leads to glaucoma. Few studies have assessed the normal structure and development of the mouse angle. We used light and electron microscopy and a cell death assay to define the sequence of events underlying formation of the angle structures in mice. The mouse angle structures and developmental sequence are similar to those in humans. Cell death was not detectable during the period of trabecular channel and beam formation. These results support morphogenic mechanisms involving organization of cellular and extracellular matrix components without cell death or atrophy.

Background

The iridocorneal angle forms in the mammalian eye from undifferentiated mesenchyme between the root of the iris and cornea. A major component is the trabecular meshwork, consisting of extracellular matrix organized into a network of beams, covered in trabecular endothelial cells. Between the beams, channels lead to Schlemm's canal for the drainage of aqueous humor from the eye into the blood stream. Abnormal development of the iridocorneal angle that interferes with ocular fluid drainage can lead to glaucoma in humans. Little is known about the precise mechanisms underlying angle development. There are two main hypotheses. The first proposes that morphogenesis involves mainly cell differentiation, matrix deposition and assembly of the originally continuous mesenchymal mass into beams, channels and Schlemm's canal. The second, based primarily on rat studies, proposes that cell death and macrophages play an important role in forming channels and beams. Mice provide a potentially useful model to understand the origin and development of angle structures and how defective development leads to glaucoma. Few studies have assessed the normal structure and development of the mouse angle. We used light and electron microscopy and a cell death assay to define the sequence of events underlying formation of the angle structures in mice.

Results

The mouse angle structures and developmental sequence are similar to those in humans. Cell death was not detectable during the period of trabecular channel and beam formation.

Conclusions

These results support morphogenic mechanisms involving organization of cellular and extracellular matrix components without cell death or atrophy.

Background

]. Primary access of aqueous to the uveoscleral route is likely deep in the angle recess at the iridocorneal junction. The resistance to aqueous flow presented by the tissues of the TM, SC, and likely uvea and sclera are important determinants of the rate of aqueous outflow and IOP.

]. Additionally, however, they demonstrate the presence of cranial paraxial mesoderm-derived cells in this tissue. Thus, aberrations of both neural crest and mesoderm cell migration or differentiation may contribute to anterior segment dysgenesis and glaucoma.

].

]. It is not clear if different mechanisms are important in rodents as compared to these other species, if there is something unusual about the studied rat strain, or if cell death occurs in the other species but was not detected due to inadequate tissue sampling or the stages analyzed.

]. The aims of this work were to determine the developmental profile of the mouse iridocorneal angle to its mature form and to assess the role of cell death in modeling the angle recess and TM. We present a light and electron microscopic (EM) evaluation of iridocorneal angle development in staged embryos and through eight postnatal weeks, when the angle structures have reached full maturity. The mouse and human TM and SC have similar structures, and the developmental progression is similar except for the accelerated time frame in mice. Extensive use of light microscopy, EM and a cell death assay (on sections spanning complete eyes) failed to identify cell death at all tested ages in various mouse strains. These results substantiate models of iridocorneal angle mesenchymal differentiation and modeling that involve organization of cellular and extracellular matrix components without cell death or atrophy, and they suggest a conservation of developmental mechanisms between mice and non-rodent mammals.

Prenatal development

].

Postnatal development

).

).

). At P12 either endothelial-lined vessels or a more mature SC were present in most sections.

).

Other strains

). The biggest difference between the studied backgrounds was a consistently more robust ciliary muscle in the 129BS mice.

Electron microscopy

), consistent with its likely derivation from coalescing venules.

, SC is represented by a vascular channel (vc) adjacent to the differentiating TM (tm). The endothelial cells (arrowheads) lining this channel are less attenuated than in the adult SC and giant vacuoles are absent. Bars 1 μm.

) In the anterior TM, the beams are more delicate, and contain less extracellular matrix (arrow). A portion of the anterior iris (i) is present in this image. Bars 1 μm.

) located near the inner wall of SC close to its posterior termination were first noted at P14. The major developmental changes had occurred by P18, with subsequent maturation primarily involving final enlargement of spaces in the posterior TM.

).

Absence of cell death in angle development

) were frequently identified (often 2 or more apoptotic cells in a section) during the established period of developmental ganglion cell death (assessed between P10 and P21) and less abundantly afterwards. Testis sections served as additional positive controls with each batch of processed slides, and abundant apoptotic cells were always detected.

) Morphologic features of cell death were absent in the TM of a P10, B6 mouse. The trabecular cells demonstrate normal nuclei and normal cytoplasmic morphology. The same was true in many sections of eyes of different ages and strains. The iris (i) is resting against the inner edge of this central portion of the TM. A small lymphocyte (arrowhead) lies in the space between two trabecular beams. Bar 1 μm.

, which respectively lack functional FAS and FASL. The eyes of mice lacking functional FAS or FASL were similar in appearance to eyes from age-matched B6 mice (not shown). This indicates that these pro-apoptotic molecules are not required for normal iridocorneal angle development.

Sequence and timing of iridocorneal angle morphogenesis

].

Briefly, in mice, migrating mesenchyme begins to fill the space between the anterior edge of the optic cup, the surface ectoderm and the lens vesicle at E11 to E12. Anlage formation appears complete by P4 to P6. Cell differentiation within the anlage has started by P8. Trabecular beams are recognizable but not fully developed at P10. SC is first evident around P10 and appears structurally mature around P14. Although SC is functional at this age, giant vacuoles are rare. By P18 to P21, the major developmental changes have occurred, and intertrabecular spaces have enlarged to adult size in the anterior TM and some parts of the posterior TM. Giant vacuoles become more abundant as spaces between the trabecular beams increase and are abundant at P18 to P21. After P18-P21, maturation primarily involves enlargement of spaces in the posterior TM.

Participation of cell death in iridocorneal angle morphogenesis is

controversial

]), the role of cell death or atrophy is controversial.

]. Together, these observations suggest that macrophage induced cell death may be important in angle morphogenesis.

]. The reason for these differing results is still unclear, and may reflect factors such as the age of tissue sampled or the amount of tissue available for study.

No evidence for cell death during mouse angle development

] is not required for TM channel formation. Based on these observations, we conclude that neither apoptosis nor necrosis are important mechanisms in development of the mouse TM and iridocorneal angle. Our data, together with the rare occurrence of cell death in studies of various mammalian species including humans, suggests that this is true for mammals in general.

Possible explanations for conflicting results between various

studies

]. In the current investigation, we observed macrophages in the anterior chamber between the iris and cornea, and associated with the pupillary membrane between P6 and P10. Thus, we suggest that the macrophages previously reported in TM of mice (and possibly some other species) were involved in the process of pupillary membrane regression and were sometimes deposited in the TM but were not significant for TM development. That TM cell death was not recorded in both studies supports this.

]. Although the spontaneous axonal regeneration in SD rats is consistent with atypical macrophage activity, further experiments are needed to test this.

Conclusions

]. In general, however, previous mouse studies have not examined the effects of mutations on the TM and SC. This is partly due to limited documentation of the sequence of events underlying iridocorneal angle development and limited documentation of the mature angle structures in mice. The current study provides important baseline information for mechanistic studies of angle development in the existing mouse models of anterior segment dysgenesis. Additionally, it will facilitate experiments with mutant mice to determine how newly identified genes function in angle development and how the pathways in which they participate overlap or interact with each other. These experiments will enhance understanding of the developmental processes involved in anterior segment formation, and glaucomas associated with anterior segment dysgenesis.

Light microscopy

].

], plastic embedded, sectioned at 1.5 μm thickness and stained with hematoxylin and eosin. For both paraffin and plastic-embedded B6 eyes, 25 to 40 sections were collected from each of 3 different ocular locations, using the lens as a landmark, resulting in 75 to 120 sections per eye. Collected regions included the lens periphery, central lens, and a region halfway between the center of the lens and the lens periphery. Iridocorneal angle development is somewhat variable both temporally and spatially within a single eye and between eyes. This necessitated careful scanning of all sections. The eyes of other strains were processed identically, except that 30 to 40 sections through the pupil and optic nerve were typically collected and analyzed. This also was true for some of the adult eyes from B6 mice that were P60 or older. Developmental changes had to be consistently present in multiple sections from the same region to be regarded as real, and conclusions were drawn only from high quality sections. This approach guarded against the potential for distortion artifacts in the delicate tissues analyzed.

Electron microscopy

]. Tissue blocks from 6 to 8 different locations around the eye were sectioned and analyzed for each eye.

Fluorescent programmed cell death (PCD) assays

]. Samples were analyzed with a confocal microscope and cells were identified as apoptotic only when they were double labeled. The occurrence of PCD was evaluated in the iris, ciliary body and TM.

Acknowledgments

mice. Supported in part by CORE grant CA34196. SWMJ is an Assistant Investigator of The Howard Hughes Medical Institute.