|Molecular Vision 2001;
Received 6 April 2001 | Accepted 6 August 2001 | Published 11 August 2001
Programmed cell death in extraocular muscle tendon/sclera precursors
Kathleen K. Sulik,1,2 Deborah
B. Dehart,1,2 Corey S. Johnson,1,2 S. Leigh
Ellis,1,2 Shao-Yu Chen,1,2 William C. Dunty, Jr.,1,2
Robert M. Zucker3
1Department of Cell and Developmental Biology and 2Bowles Center for Alcohol Studies, The University of North Carolina, Chapel Hill, NC; 3Reproductive Toxicology Division, National Health Effects and Environmental Research Laboratory, United States Environmental Protection Agency, Research Triangle Park, NC
Correspondence to: Kathleen K. Sulik, Ph.D., CB 7178, Bowles Center for Alcohol Studies, The University of North Carolina, Chapel Hill, NC 27599; Phone (919) 966-3208; FAX (919) 966-5679; email: firstname.lastname@example.org
Purpose: This study was designed to examine the occurrence of natural cell death in the periocular mesenchyme of mouse embryos.
Methods: Vital staining with LysoTracker Red and Nile blue sulfate as well as terminal nick end labeling (TUNEL) were utilized to identify apoptotic cell death in whole and histologicaly sectioned gestational day 10.5 to 14 mouse embryos. Laser scanning confocal microscopy was used to provide a three dimensional representation of the cell death pattern. Immunohistochemical staining for neural crest and myoblast populations was utilized to indicate the cell population undergoing apoptosis.
Results: Programmed cell death was evident in the developing rectus muscle tendons/sclera on gestational days 11 through 12.5 (corresponding to the weeks 5-6 of human development). Although each of these peripheral periocular condensations has readily apparent amounts of apoptosis, the pattern of cell death varied among them. Cell death was most apparent in the superior rectus tendon primordium, while that for the lateral rectus had the least evidence of apoptosis.
Conclusions: Although apoptosis was readily evident in the periocular mesenchyme in distinct regions located medial and distal to the developing rectus muscles, programmed cell death in these sites has not previously been reported. New imaging techniques coupled with stains that evidence apoptotic cell death have made it possible to define this tissue as a prominent region of programmed cell death. Although neuronal tissues, including particular regions of the developing eye, are well recognized as sites of programmed cell death, description of this phenomenon in the extraocular tendon/sclera precursors is novel.
Dr. Perry Gilbert's 1947 and 1957 publications detailing the development of the extrinsic ocular muscles in the cat and human include the first description of the presence of four mesenchymal condensations located at the periphery of the developing optic cup immediately below the surface ectoderm [1,2]. These peripheral condensations were identified in human embryos belonging to horizons xv, xvi, and xvii, that is, at approximately 33-41 days after fertilization (from mid week 5 to the middle of the sixth week of human development). It was noted that each condensation is cone-shaped with the apex directed toward the optic stalk and is approximately one fourth the size of the lens vesicle. The condensations are equally spaced and positioned with one just inferior to the choroid fissure (the inferior peripheral condensation, ipc), one directly opposite the choroid fissure at the superior rim of the optic cup (the superior peripheral condensation, spc), and the others are located at the medial (medial peripheral condensation, mpc; depending on species, this position may also be considered anterior or rostral), and lateral margins (lateral peripheral condensation, lpc) of the optic cup (Figure 1A). The superior peripheral condensation (spc) becomes apparent first, followed by the inferior then the medial and lateral condensations. As the remainder of the periocular mesenchyme condenses, the peripheral condensations become incorporated in it, contributing to the formation of the scleral coat of the eye and marking the insertion points on the sclera of the four rectus muscles. In 6 week human embryos, before the scleral coat becomes a dense layer, the anlage of the rectus muscles have extended toward the optic cup, making contact with the outer margin of the peripheral condensations.
Work in this laboratory directed toward understanding the role of apoptosis in normal and abnormal ocular morphogenesis resulted in our "rediscovery" of the peripheral condensations. A number of techniques allow identification of apoptotic cell death. These include terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) of DNA fragments and vital staining with chemicals such as Nile blue sulfate or LysoTracker Red that become entrapped in membrane-bound, acidic apoptotic bodies or in the lysosomes of cells that take up apoptotic debris [3-5]. As described herein, laser scanning confocal microscopic imaging provided a three dimensional perspective of these condensations, identifying them as locations of intense naturally occurring (programmed) cell death. Additionally, pilot studies of ethanol-induced teratogenesis have suggested that, as in other locations of programmed cell death, teratogen exposure increases the amount of apoptosis in these sites . Nile blue sulfate (NBS) vital staining of alcohol-exposed embryos readily illustrates the position of the peripheral condensations (Figure 1B). Although programmed cell death has long been recognized as essential for normal embryonic development of the lens, optic cup, and optic stalk [7,8], to our knowledge it has not been previously reported in extraocular tissues.
Recently, considerable progress has been made in identifying progenitor populations and gene expression patterns pertinent to developing ocular and extraocular tissues. Notable is work identifying the extraocular mesenchyme that gives rise to the sclera as well as the corneal stroma and endothelium as originating from neural crest cells . Neural crest cells also contribute the connective tissues for the periocular muscles [10,11], while the myoblasts for these muscles are of mesodermal origin [9,12]. Much of the work detailing the origin of ocular tissues has been conducted using avian species, due in large part to accessibility of the embryos for cell labeling and grafting procedures. Currently available molecular probes have allowed extension of these analyses as exemplified by a recent publication by Noden and co-workers  that describes the spatial and temporal patterns of gene expression in developing chick extraocular muscles and comparison to other craniofacial muscles. Although there is slight temporal variation in the maturation of each of the extraocular muscles, by stage 24 (corresponding to approximately day 12.5 in the mouse and horizon xvi in the human) they, like the first branchial arch-associated muscles, have all initiated myosin production. Until recently, molecular probes for identification of neural crest cells (esp HNK-1) have also been most successfully used in the chick [14-16]. Although not exclusive to neural crest, the transcription factor AP-2 is expressed in mammalian cranial neural crest cells and has been used to identify this cell population in the developing face and eye of mouse embryos [17,18].
The current investigation was directed toward defining the spatial and temporal pattern of apoptosis in the periocular peripheral condensations of mouse embryos. For this purpose, routine light microscopy, vital staining as observed with a dissecting microscope and with confocal laser microscopy, and TUNEL staining of histological sections were used. In addition, immunohistochemistry was employed to aid in discriminating the cell type involved.
C57BL/6J mice were housed and cared for according to AAALAC specifications. They were mated for 1 h and subsequently observed for copulation plugs. The time the plug was noticed was considered gestational day (GD) 0. Pregnant mice were killed at 0.5 day intervals from GD10.5 to GD14. The embryos were dissected free of extraembryonic tissues in 37 °C Ringer's solution and subsequently processed for histological sectioning or whole mount vital staining.
Vital staining and imaging
For initial estimates of cell death, NBS supravital staining was utilized. Whole embryos were incubated at 37 °C for 30 min. in 1:50,000 NBS/Ringer's solution (0.002% (W/V); The Coleman & Bell Co., Norwood, OH) then transferred to fresh Ringer's solution, visualized and immediately photographed using a dissecting photomicroscope.
For confocal microscopic visualization of cell death patterns, a previously published procedure for Lysotracker Red staining developed by Zucker et al.  was used. Stained specimens were cleared in a solution of 1:2 (V/V) benzyl alcohol and benzyl benzoate, then sealed in depression slides or specially made aluminum slides. They were imaged using a Leica laser scanning confocal microscope (TCS-SP) using 5x-20x objectives. The LysoTracker dye was excited using the 568 nm laser line and the emission fluorescence was measured between 580-630 nm. Specimens were approximately 1.2 mm thick and were analyzed at 20 μm intervals. Using Leica software, data were prepared for presentation as a maximum projection, a gallery of images, and a movie. For the movie, gamma was raised to 2.14 to provide clear anatomical references.
Terminal nick end labeling and visualization
Embryos at specific developmental stages were removed from all extraembryonic membranes, fixed in 4% paraformaldehyde, and processed for paraffin embedding. They were serially sectioned at 5 μm in a plane parallel to the equator of the eye. Apoptotic cells were visualized in these sections using the TACSTM In Situ Apoptosis Detection Kit (Trevigen®, Gaithersburg, MD) with slight modifications. Tissue sections were subjected to Proteinase-K treatment for no more than 2 min and labeled for 1 h at 37 °C in a humidified chamber using the TdT dNTP solution. Sections were counterstained for 20-30 s with methyl green to aid in morphological identification, then examined and photographed using a Nikon photomicroscope.
Indirect immunohistochemistry was used to detect the presence of MyoD, myogenin and AP-2 protein expression in histological sections of GD 11.5, 12, and 12.5 embryos. Polyclonal antibodies (Myo D, catalog number SC-760; AP-2, catalog number SC 184; myogenin, catalog number SC-576; Santa Cruz Biotechnology Inc., Santa Cruz, CA) were applied to 4% paraformaldehyde-fixed paraffin-embedded sections. AP-2 labeling was performed at a dilution of 1:500, while MyoD and myogenin dilutions were 1:100. All labeling was done at room temperature for 1 h. All three antibodies were detected using steam antigen retrieval , with a vegetable steamer as the steam source and a commercially available kit (Antigen Retrieval Citra, BioGenex, San Ramon, CA). Following indirect avidin/biotin-immunoperoxidase reactions (Vector Laboratories, Burlingame, CA), antigens were detected using diaminobenzidine as a substrate (Innovex Biosciences, Richmond, CA).
Vital staining showed no apparent naturally occurring (physiological) cell death in the periocular region in gestational day (GD) 10.5 mouse embryos. It first became evident on GD 11 and remained through GD12.5 in the peripheral periocular condensations. NBS-stained apoptotic cell death appeared first in the superior condensation (spc) in embryos having approximately 40 somite pairs (Figure 2A). By GD 11.5, a small amount of staining was also observed in the medial condensation. AP-2 labeling of sections made in a plane parallel to the equator of the optic cup illustrated that at this stage (GD 11.5), cells of the trigeminal ganglion labeled intensely as did cells of the lens and vitreous, as well as the surface ectoderm around the eye. Labeling was also apparent in the periocular mesenchyme, with the most intense mesenchymal AP-2 staining located medial and inferior to the optic cup and including the medial and inferior periocular condensations (Figure 2B,C).
By GD12, apoptosis was evident at the site of all four periocular condensations. NBS (Figure 3A,B) and LysoTracker Red uptake (Figure 3C,D and Figure 4) was notably heaviest in the superior condensation and lightest in the lateral condensation. Cuts made through the ocular region with a scalpel (Figure 3B), as well as optical sections made with a confocal microscope (Figure 3C,D and Figure 4), illustrated the position and extent of vital dye uptake in the periocular mesenchyme. In the most proximal region of the orbit that was imaged (at a position just behind the lens), the medial and inferior condensations exhibited a moderate amount of cell death, the lateral had a small amount, and the superior had the least. More distal (superficial) optical sections showed an increased amount of cell death in the inferior and medial condensations, accompanied by a decrease in the lateral condensation. The intensity of dye uptake in the superior condensation also increased from proximal to distal. In the most superficial optical sections examined (that is, at a position near the front of the lens), the superior condensation had a very high level of staining while that in the other three condensations was reduced. Naturally occurring cell death was also evident in the choroid fissure (Figure 3C) and optic stalk (Figure 3B). The position of TUNEL-positive cells (Figure 5A,B) as observed in histological sections corresponds to that of vital dye uptake. In GD 12 embryos, the periocular mesenchyme continued to label with AP-2 antibody (Figure 5C,D) with least intense labeling in the position of the lateral condensation (Figure 5C).
Gestational day 12.5 was the final time at which cell death in the periocular mesenchyme was readily notable. Although present, but diminishing in all of the condensations (Figure 6A,B), examination of a series of sections indicated that the superior condensation remained the one with the most and the lateral condensation the one with the least apoptotic death. Figure 6B illustrates a few TUNEL-positive cells in the ipc. At this stage of development, AP-2 labeled cells surround the eye. Labeling was least intense in the superior periocular condensation (Figure 6C,D). Muscle-specific Myo D and myogenin antibody labeling in histological sections made in a plane just behind the lens of the eye was positive in the facial muscles, but not in the periocular region (Figure 6E,F,I,J). Deeper into the tissue, at a level at which the vitreous is no longer present, Myo D and myogenin labeling was present in the extraocular muscles and also could be seen in the facial muscles. The extraocular muscle fibers are located peripheral to, and in contact with the periocular condensations (Figure 6G,H,K,L).
This work illustrates physiologically-occurring cell death in peripheral periocular condensations of developing mouse embryos. Although the amount of programmed cell death in this tissue is remarkably extensive, to our knowledge, it has not previously been reported.
The peripheral periocular condensations in mouse embryos are comparable in position to that described over 50 years ago by Gilbert for the cat and human. Additionally, the developmental stages at which we observed these structures in mice correspond to the previous findings; gestational days 11 and 12 in mice being roughly equivalent to the end of the fifth and beginning of the sixth week of human in utero development.
Although from routine histological analyses it had previously been concluded that the peripheral periocular condensations provide attachment for the rectus muscles, new probes have provided the opportunity to distinguish cell types and make more definitive conclusions. The absence of MyoD and myogenin labeling in the peripheral condensations at developmental stages when the facial muscles and extraocular muscles label positively for these muscle markers establishes the fact that, indeed, the cells of the periocular condensations are not myogenic. Additionally, the results of this study indicate that the mesenchyme comprising the condensations is of neural crest origin as had been shown by Johnston et al.  for the sclera.
AP-2 protein immunostaining was used to localize neural crest cells in this study. These proteins act as transcription factors and have previously been localized to ocular and periocular tissue . The AP-2 antibody used in this investigation (SC-184) is pan-specific, recognizing the three different AP-2 proteins, AP-2α, AP-2β, and AP-2βγ (AP-2.2). Interestingly, Moser et al, [20,21] have illustrated that AP-2 may act as a survival factor, inhibiting naturally occurring cell death during embryogenesis Consistent with this suggestion are our results showing that the superior periocular condensation which had the most apoptosis also appeared to have the least amount of AP-2 labeling. Additionally, the cells of each individual condensation are not uniformly AP-2 positive. Noteworthy are mutations involving AP-2α in mice that present with ocular abnormalities including corneal-lenticular adhesion. Additionally, the human AP-2α gene, TFAP2, lies within a region of chromosome 6 that has been associated with a diverse array of human ocular disorders including Peter's anomaly and Rieger's syndrome , conditions that are commonly classified as neurocristopathies.
Relatively little attention has been paid to the molecular mechanisms responsible for tendon development. However recent studies by Xu et al  report that homologues of the Drosophila eyes absent (eya) gene participate in the patterning of the distal tendons of the limb. Azuma et al  have subsequently shown that mutation of the human EYA1 gene occurs in patients with congenital cataracts and anterior segment anomalies. Studies of mouse embryos have shown that Eya2 is detectable in the sclera and cornea , and Eya4 is reported as being expressed primarily in the craniofacial mesenchyme . Mutation of another Drosophila gene, Pitx2, also results in anterior segment anomalies (Rieger's syndrome) in humans . In mice, this gene is expressed in the periocular mesenchyme as well as in the region of the extraocular muscles. Pitx2 knockout yields absence of the extraocular muscles and excessive amounts of mesenchyme between the lens and surface ectoderm . It will be of interest to determine if these mutations affect the patterns or amount of naturally occurring cell death in the periocular condensations.
Clues relative to the developmental role of apoptosis in the peripheral periocular condensations may be provided by understanding quantitative differences among the four condensations and the temporal pattern of cell death with respect to other developmental events. The techniques utilized in this study to examine apoptosis effectively provided a comprehensive picture of the spatio-temporal pattern. Nile blue sulfate staining proved particularly useful as a quick method for analyses of large numbers of specimens and as a rough indicator of quantity and pattern of apoptosis. Optical sectioning and reconstruction of LysoTracker Red-stained specimens provided a more comprehensive result. As expected, the pattern of labeling with the TUNEL procedure was consistent with that of the vital dyes. This study showed that the lateral periocular condensation exhibits minimal cell death relative to the others while the superior condensation is the site of the most intense apoptosis. Cell death in the peripheral condensations occurs at approximately the same developmental stage as their contact proximally with the extraocular myoblasts. A possible explanation for the cell death may involve requirements for survival factors provided by the myoblasts, much as neurons require nerve growth factor supplied by their targets for survival. Supporting this idea is the demonstration that although tendons initially develop in muscleless limbs, the tendons are not maintained and degenerate . Future experiments directed toward modifying the amount or timing of this cell death and subsequent developmental analyses promise to provide information of importance for our understanding of both normal and abnormal genesis of periocular structures.
The information in this document has been funded in part by the U. S. Environmental Protection Agency. It has been subjected to review by the National Health and Environmental Effects Research Laboratory and approved for publication. Approval does not signify that the contents reflect the views of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use.
Supported by NIH (AA11605). We thank Heather Baudet for her assistance with illustrations and Dave Cowan for his expert advice regarding immunohistochemical procedures.
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