Fate Maps of Neural Crest and Mesoderm in the Mammalian Eye

Abstract

purpose. Structures derived from periocular mesenchyme arise by complex interactions between neural crest and mesoderm. Defects in development or function of structures derived from periocular mesenchyme result in debilitating vision loss, including glaucoma. The determination of long-term fates for neural crest and mesoderm in mammals has been inhibited by the lack of suitable marking systems. In the present study, the first long-term fate maps are presented for neural crest and mesoderm in a mammalian eye.

methods. Complementary binary genetic approaches were used to mark indelibly the neural crest and mesoderm in the developing eye. Component one is a transgene expressing Cre recombinase under the control of an appropriate tissue-specific promoter. The second component is the conditional Cre reporter R26R, which is activated by the Cre recombinase expressed from the transgene. Lineage-marked cells were counterstained for expression of key transcription factors.

results. The results established that fates of neural crest and mesoderm in mice were similar to but not identical with those in birds. They also showed that five early transcription factor genes are expressed in unique patterns in fate-marked neural crest and mesoderm during early ocular development.

conclusions. The data provide essential new information toward understanding the complex interactions required for normal development and function of the mammalian eye. The results also underscore the importance of confirming neural crest and mesoderm fates in a model mammalian system. The complementary systems used in this study should be useful for studying the respective cell fates in other organ systems.

The vertebrate eye is constructed during development from three general embryonic precursor sources: neural ectoderm, surface ectoderm, and a loose array of cells termed the periocular mesenchyme. A primary function of the periocular mesenchyme is to provide multiple mature cell lineages that are necessary for normal ocular development and vision, including the corneal endothelium and stroma, trabecular meshwork, Schlemm’s canal, sclera, ciliary body muscles, iris stroma, extraocular muscles, and ocular blood vessels. A second essential function of the periocular mesenchyme during ocular development is to provide essential signals for patterning of ocular ectoderm primordia, including induction of lacrimal glands from the surface ectoderm, specification of retinal pigmented epithelium from the optic cup, and differentiation of the optic stalk from the neural ectoderm. ^{1 2 3} Genetic or acquired defects in development or function of the periocular mesenchyme result in ocular disease and vision loss, including particularly glaucoma. ^{4 5}  

Historically, the periocular mesenchyme was thought to arise developmentally from the mesoderm. However, fate maps developed in birds by using quail chick chimeras, vital dye labeling, or neural crest-specific antibodies have demonstrated that periocular mesenchyme actually receives initial contributions from both neural crest and mesoderm. ^{6 7 8 9 10} These studies further established that the corneal endothelium and stroma, trabecular meshwork, most of the sclera, and the ciliary muscles in birds are derived solely from the neural crest. In contrast, all vascular endothelium, the caudal region of the sclera, Schlemm’s canal, and extraocular muscles are derived from the mesoderm. These results have generally served well as the model for all vertebrates, including mammals. However, the documentation of important developmental differences between birds and mammals has become increasingly common. ^{11 12} In the neural crest, these include differences in the timing of neural crest migration, the migratory pathways taken, and their ultimate fates, ¹¹ making it essential to uncover any species differences that may exist in mammals to account accurately for the embryonic origins of each mature structure. Several key regulators of periocular mesenchyme development have recently been identified. ^{2 13 14 15 16 17} Determining the neural crest and mesoderm expression patterns, as well as the lineage-specific effects of genetic lesions in these genes, would significantly enhance our understanding of the normal mechanisms by which these genes function during ocular development. The major impediment to addressing any of these questions has been the lack of a reliable strategy for quantitative, long-term labeling of neural crest and mesoderm in a mammalian eye. Fortunately, the use of two complementary Cre-lox based approaches has recently provided the necessary breakthrough. 

A binary transgenic system has been developed that allows for indelible, permanent labeling of early presumptive neural crest and all subsequent progeny cells. ¹⁸ The first component of this system is a Wnt1-Cre transgene, which expresses Cre recombinase under the control of the promoter for the Wnt1 gene. Like Wnt1 itself, Cre expression from the transgene is transient and limited to a 24-hour period in early presumptive neural crest cells before their emigration from the dorsal neural tube. ¹⁸ The second component of the system is the conditional Cre reporter allele, R26R. ¹⁹ Cre-mediated activation of β-galactosidase (β-gal) expression from the R26R reporter allele by DNA recombination provides for indelible, long-term labeling of all cells derived from the neural crest. ¹⁹ This system has been used to map neural crest fates in cardiovascular, craniofacial, and skull vault development. ^{20 21 22}  

The mouse glycoprotein hormone α-subunit gene (αGSU) is expressed initially in Rathke’s pouch, the oral-ectoderm–derived primordium of the anterior pituitary gland, and subsequently in multiple lineages of the mature anterior pituitary gland. ²³ αGSU expression has also been reported in mesoderm of the early ocular primordium and in the olfactory epithelium. ²⁴ The promoter regulatory elements required to recapitulate this expression pattern have been identified and used to express Cre recombinase in transgenic mice. ²⁵ Mice doubly transgenic for αGSU-Cre and a Cre-responsive reporter cassette exhibit indelible expression of β-gal in a pattern consistent with expression of endogenous αGSU itself. ²⁵ αGSU-Cre has also been used successfully to generate a lineage-specific gene knockout of the transcription factor gene Dax1 in the anterior pituitary gland. ^{26 27}  

In the current study, we used the complementary Wnt1-Cre/R26R and αGSU-Cre/R26R labeling systems to establish for the first time the long-term fates of neural crest and mesoderm, respectively, in a model mammalian eye. The fates were similar to but not identical with those previously reported in birds, with the most significant differences occurring in the anterior segment. We also demonstrated that five known or potential transcriptional regulators of periocular mesenchyme have unique expression patterns in the neural crest and mesoderm during early ocular development. Finally, the data also imply that the mechanism of activation for the homeobox gene Pitx2 in the neural crest and mesoderm is likely to be distinct. These findings have important implications for ocular development and function and may provide a model for understanding interactions between the neural crest and mesoderm in other organ systems. 

Materials and Methods

Animals and Isolation of Lineage-Marked Mice

Wnt1-Cre mice transmit a transgene consisting of a cDNA cassette expressing Cre protein placed under the transcriptional control of the murine Wnt1 promoter. ¹⁸ αGsu-Cre transgenic mice (TgN(Cga-cre)S3SAC, cat no. 004426; Jackson Laboratories, Bar Harbor, ME) transmit a construct including the cDNA for a nuclear-localized Cre protein under the transcriptional control of the pituitary glycoprotein subunit α promoter. ²⁵ R26R mice were purchased from Jackson Laboratories and transmit a β-galactosidase reporter allele that is genetically activated in vivo by Cre recombinase activity. ¹⁹ All procedures using mice were approved by the University of Michigan Committee on Use and Care of Animals. All experiments were conducted in accordance with the principles and procedures outlined in the NIH Guidelines for the Care and Use of Experimental Animals and in the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 

Timed pregnancies were produced by mating homozygous R26R females either with Wnt1-Cre transgenic males to produce progeny with marked neural crest or with αGsu-Cre transgenic males to produce progeny with marked mesoderm. The morning a plug was detected was designated as embryonic day (E)0.5. Embryos were collected by cesarian section after the mother was euthanatized. All genetic loci were genotyped by using PCR-based methods with primers for Cre ²⁵ and R26R. ¹⁹  

Detection of β-Galactosidase Activity

Enucleated whole eyes from 6-week-old mice were fixed for 1 to 2 hours at 4°C with 4% paraformaldehyde in phosphate-buffered saline (PBS) and rinsed three times in wash buffer (0.1 M sodium phosphate [pH 8.0], 2 mM MgCl₂, 2% NP-40). Fixed eyes were stained overnight at 37°C in standard staining solution (5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 0.1% X-gal in wash buffer), rinsed in wash buffer, embedded in JB-4 resin, and sectioned at 3 μm. Mounted sections were counterstained with nuclear fast red (Sigma-Aldrich, St. Louis, MO). 

Immunohistochemistry

Embryos harvested for immunostaining were fixed for 2 to 4 hours with 4% paraformaldehyde in PBS, washed and dehydrated, and embedded in paraffin. Mounted sections were deparaffinized and treated for antigen retrieval by boiling for 10 minutes in citrate buffer (pH 6.0). Immunostaining was performed according to standard methods. Briefly, sections were incubated with antibodies directed against β-galactosidase (Eppendorf-5Prime, Boulder, CO), FOXC1 or -2 (Abcam, Inc., Cambridge, MA), myogenin (MYOG; clone F5D, developed by Woodring Wright and obtained from NICHD/Developmental Hybridoma Studies Bank, Iowa City, IA), PITX1 (a gift from Jacques Drouin, Montreal, Canada ²⁸ ) or PITX2 ²⁹ followed by biotinylated species-specific secondary antibodies (Jackson ImmunoResearch, West Grove, PA). Signals were detected using tyramide signal-amplification kits (PerkinElmer, Boston, MA). 

Results

Contributions of Cranial Neural Crest and Anterior Paraxial Mesoderm to the Early Ocular Primordia

We crossed Wnt1-Cre males with R26R females to generate Wnt1-Cre;R26R progeny (neural-crest–marked) with indelibly labeled neural crest by virtue of Cre activation of β-gal reporter expression. In parallel experiments, we crossed αGsu-Cre and R26R mice to generate analogous αGsu-Cre;R26R mice (mesoderm-marked) with β-gal-labeled ocular mesoderm. In either system, the presence and location of lineage-marked β-gal⁺ cells was detected by incubation with X-gal, a chromogenic substrate of β-gal, or by immunohistochemistry with an anti-β-gal antibody. 

X-gal staining of whole embryos from each class at E10.5 demonstrated the distinct labeling specificity of each binary labeling system. The heads of neural-crest–marked embryos were heavily invested with β-gal⁺ cells by this time point, particularly in the branchial arches and trigeminal ganglia (Fig. 1A) . There was also heavy labeling of the midbrain neural ectoderm, as has been reported. ¹⁸ In the trunk, there was substantial staining of the cardiac outflow tract and the dorsal root ganglia along either side of the neural tube in neural-crest–marked embryos (Fig. 1A) . In contrast, head staining in αGsu-Cre;R26R embryos at E10.5 was limited to a wedge of mesenchyme located ventrally and caudally to the optic cup and the oral ectoderm. These positions are analogous to those reported in αGsu-lacZ transgenic mice and in endogenous αGsu expression (Fig. 1B) . ²⁴ αGsu-Cre;R26R embryos also showed heavy staining of the somites, atria and ventricles of the heart, and gut mesentery (Fig. 1B) . In histologic sections, classic neural-crest–derived structures such as the trigeminal ganglia, meninges, and presumptive calvarial vault were heavily stained in neural-crest–marked embryos (Figs. 1C 1E)but were unstained in mesoderm-marked embryos (Figs. 1D 1F) . The dorsal neural tube, which was labeled in neural-crest–marked embryos (Fig. 1E) , was unstained in mesoderm-marked embryos (Fig. 1F) . Collectively, these data are consistent with the Wnt1-Cre;R26R and αGsu-Cre;R26R marking systems labeling mesenchyme derived from neural crest and mesoderm, respectively. 

We next examined each binary system for β-gal labeling in the developing eye at the cellular level. Labeled neural crest cells in Wnt1-Cre;R26R embryos surrounded the optic cup and stalk at E10.5 (Fig. 1G) . The neural crest also occupied the presumptive cornea, beginning immediately after separation of the lens vesicle from the surface ectoderm, and a few cells appeared within the hyaloid space posterior to the lens. Mesenchymal cells located in a morphologically distinct wedge of cells located ventrally and caudally to the optic cup and stalk did not label as neural crest in Wnt1-Cre;R26R embryos, suggesting that these cells were of mesoderm origin (Fig. 1G) . Indeed, the position of these cells corresponds well with that of the previously reported rostral limit of cranial paraxial mesoderm. ^{30 31} Cells in this morphologically distinct wedge are prominently labeled in αGsuCre;R26R embryos (Fig. 1H) . The unique morphology of these cells relative to surrounding mesenchyme, the previous mapping of cranial paraxial mesoderm to this location at this time point, and the absence of labeling in Wnt1-Cre;R26R embryos all argue that the labeled cells in the ocular primordia of αGsuCre;R26R embryos represent cranial paraxial mesoderm. Thus, the two lineage-specific β-gal labeling systems resulted in unique staining patterns in the early ocular primordia that were consistent with the previously reported arrangement of neural crest and mesoderm precursors in the chick. ^{6 7 8 9 10} Occasional labeled mesoderm cells were also present within the presumptive cornea, in the hyaloid space, and adjacent to the optic cup and stalk in αGsuCre;R26R embryos at this early time point (Fig. 1Hand data not shown), an observation that is consistent with previous reports that mesoderm cells have already begun to intermingle with neural crest by this time point. ^{30 31} By E12.5, neural crest and mesoderm were extensively comingled in multiple ocular structures (data not shown and see Figs. 3 4 and 5 ). 

Transcription Factor Expression in Marked Neural Crest and Mesoderm

Molecular markers that are specifically expressed in neural crest or mesoderm precursors have not been described for the mammalian ocular primordia. Therefore, we examined the protein expression patterns of several transcription factors previously implicated in ocular development to determine their expression profiles between the two embryonic precursor pools. The homeodomain transcription factor PITX2 is associated with ocular defects in humans and is essential for ocular development in mice. ^{2 13 32 33} PITX2 expression labeled the neural crest, beginning initially in the presumptive anterior segment and quickly extending to the periphery of the optic cup in Wnt1Cre;R26R embryos by E11.5 (Fig. 2A) . In contrast, PITX2 expression had marked all β-gal-labeled mesoderm in αGsuCre;R26R embryos at E11.5, before these cells entered the ocular field (Fig. 2B) . By E12.5, PITX2 expression had spread to all ocular neural crest (data not shown). The forkhead transcription factor FOXC1 is also essential for POM development in mice and humans. ^{16 34} FOXC1 expression marked the neural-crest–derived cells in the anterior segment and surrounding the optic cup and stalk at E11.5 (Fig. 2C) . Colabeling was also identifiable in a small percentage of mesoderm cells at E11.5 (Fig. 2D) . Therefore, PITX2 and FOXC1 were each expressed in both the neural crest and mesoderm, albeit to very different degrees in the mesoderm. The transcription factor FOXC2 is necessary in periocular mesenchyme for normal anterior segment development in mice, but not in humans. ^{17 35} In contrast to FOXC1, expression of FOXC2 was detectable only in neural-crest–labeled cells and was not present in mesoderm-labeled cells of αGsu-Cre;R26R embryos at E11.5 (Figs. 2E 2F) . The homeodomain protein PITX1 is related to PITX2, and the two genes are often coexpressed and are functionally redundant in other tissues. ^{36 37} However, mesenchymal PITX1 expression in the developing eye was limited to a subset of labeled mesoderm cells and was never observed in neural-crest–labeled cells at E11.5 (Figs. 2G 2H) . Consistent with a previous report, PITX1 expression was also detected in the early lens vesicle (Fig. 2H) . ³⁸ Finally, we examined MYOG as a second marker for mesoderm-derived muscle precursors. MYOG expression was limited to a subset of mesoderm-labeled cells in αGsuCre;R26R embryos and was not expressed in neural-crest–labeled cells at E11.5 (Figs. 2I 2J) . Based on these collective results, particularly the complete absence of FOXC2 colabeling in mesoderm-marked embryos and the specificity of PITX1 and MYOG for subsets of mesoderm cells, we conclude that, in the developing eye, the Wnt1Cre;R26R and αGsuCre;R26R systems specifically mark the neural crest and mesoderm, respectively. 

Fates in the Extraocular Muscles

The early primordia of extraocular muscles were apparent as mesenchymal condensations lateral to the optic cup and stalk by E12.5 (Figs. 3A 3B) . A small number of neural crest and a significant number of mesoderm precursor cells are already present within the presumptive muscle condensations at this early time point (Figs. 3A 3B) . Subsequently, the fate of each embryonic precursor pool was determined based on cellular morphology. Connective fascia cells in perinatal and adult muscles were derived exclusively from the neural crest (Figs. 3C 3E) . In contrast, myotubules in the developing muscle (Fig. 3D)and myofibers in the mature muscle (Fig. 3F)are derived exclusively from mesoderm. No ectopic labeling by either labeling system, such as neural crest labeling of muscle cells or mesoderm labeling of connective fascia cells, was ever observed in multiple specimens examined at any time point. 

Fates in the Ocular Vasculature

The hyaloid vasculature forms posterior to the lens as a transient, embryonic blood system that maintains the posterior lens and developing retina. Mesenchymal precursor cells were present within the hyaloid space by E11.5 and primitive blood vessels were present by E12.5 (Figs. 4A 4B) . Both neural crest and mesoderm-derived cells contributed to the early hyaloid vessels (Figs. 4A 4B) . Subsequently, endothelial cells lining the mature hyaloid blood vessels were derived solely from the mesoderm (Figs. 4D 4F) . Pericytes attaching themselves to the unstained endothelial tube were derived exclusively from the neural crest (Figs. 4C 4E) . Endothelial and smooth muscle cells of the hyaloid artery were derived from the mesoderm and neural crest, respectively (data not shown). 

The choriocapillary bed comprises a network of microvessels that immediately overlie the retinal pigmented epithelial cells and provide vascular functions to the RPE and underlying photoreceptors of the retina. Fates of the embryonic precursor pools in the choroid were identical with those in the hyaloid. Pericytes and other associated cells were derived from the neural crest (Fig. 4G) , whereas endothelial cells were derived from the mesoderm (Figs. 4H) . Endothelial cells of blood vessels within the ciliary body were similarly derived from the mesoderm (Fig. 5H) . 

Fates in Anterior Segment Structures

Multiple cell lineages within the anterior segment of the eye were derived from mesenchyme, including, in approximate order of appearance, the corneal endothelium and stroma, ciliary muscles, ciliary blood vessels, anterior iris, and trabecular meshwork and Schlemm’s canal within the iridocorneal angle. Neural crest cells migrated into the presumptive anterior segment between the anterior face of the newly formed lens vesicle and the surface ectoderm by E10.5 (Fig. 1G) . Mesoderm cells followed 12 to 24 hours later (data not shown). By E14.5, neural-crest–derived cells comprised most of the cells within the endothelium and stroma of the cornea, and the presumptive iridocorneal angle (Fig. 5A) . Although significantly fewer in number, mesoderm-derived cells were also consistently present in the same structures (Fig. 5B) . The presence and relative ratios of the two embryonic cell lineages remain unchanged as these structures proceeded through differentiation (Figs. 5C 5D) . 

To detect β-gal⁺ cells while preserving the integrity of mature anterior segment structures, we stained enucleated adult eyes in X-gal and then processed them for plastic-embedded sections. The relative contributions of neural crest and mesoderm after this processing was unchanged from that observed with fluorescent immunostaining on samples derived from earlier time points, indicating efficient penetration of the stain. Most cells in the endothelium and stroma layers of the mature cornea expressed β-gal in neural-crest–marked animals, establishing a neural crest origin (Fig. 5E) . However, cells that did not express β-gal were also consistently present in both layers of neural-crest–marked embryos (Fig. 5E) , indicating that not all cells in these layers derived from neural crest in mice. A comparable number of β-gal⁺ cells were consistently present in the mature corneal endothelium and stroma of mesoderm-marked mice (Fig. 5F) . The number of β-gal⁺ cells in mesoderm-marked mice parallels the number of unlabeled cells in neural-crest–marked mice. Collectively, these data indicate that most of the corneal endothelial and stromal cells are derived from neural crest but additional cells in both layers are derived from mesoderm. This finding was unexpected, because only cells of neural crest origin have been reported to be present in the corneal endothelium and stroma of birds. ^{6 7 8 9 10}  

Contributions of the neural crest and mesoderm to the limbal region and iridocorneal angle of the mature eye were complex and highly variable. Ciliary muscles were derived from the neural crest but not the mesoderm (Figs. 5I 5J 5K 5L) . The endothelial lining of Schlemm’s canal was derived from the mesoderm (Figs. 5K 5L) . Most cells within the trabecular meshwork labeled as neural crest, but a measurable number did not (Figs. 5I 5J) . Similar to observations in the cornea, labeling of some trabecular meshwork cells in mesoderm-labeled mice indicates that these cells derived from the mesoderm (Figs. 5K 5L) . We observed little to no labeling for neural crest in the iris stroma (Figs. 5M 5O) . In contrast, iris stroma regularly labels as mesoderm (Figs. 5N 5P) . 

Discussion

We used binary transgenic systems based on Cre/loxP technology to label specifically the neural crest or mesoderm in eyes of mouse as a model vertebrate. Early indelible labeling of the respective embryonic precursor cell lineages by the two complementary systems allowed their fates to be followed throughout development and in mature mouse eyes. The data demonstrate that neural crest and mesoderm fates in mammalian eyes are similar to but not identical with those previously reported in birds. ^{6 7 8 9 10} We also report the neural crest and mesoderm expression patterns of several key transcription factors during early ocular development. Collectively, these results have important mechanistic implications for understanding the development and function of structures derived from periocular mesenchyme, and the roles mediated by specific transcription factors in these processes. 

Characterization of the Cell-Labeling Systems

The ability to accurately track cell fates throughout development and into adulthood depends on the fidelity and permanence of the labeling system used. ³⁹ Neural crest and mesoderm fates have been extensively characterized in birds by using quail chick chimeras, vital dye labeling, and antibody staining for neural-crest–specific markers. ^{6 7 8 9 10 40 41} However, these labeling systems are not effective for determining particularly the long-term fates of these lineages in mammals. ³⁹ Activation of the R26R reporter construct by Cre-mediated DNA recombination in the two systems used in the study ensured indelible β-gal labeling of the precursor cells in which Cre was originally expressed, as well as in all subsequent progeny cells. The Wnt1-Cre;R26R labeling system was initially shown to generate specific, quantitative labeling of the midbrain neural ectoderm and all premigratory neural crest. ¹⁸ Subsequent experiments examining cardiovascular, craniofacial, and skull vault development further established that this is a robust approach for quantitative, specific, and long-term fate mapping of neural crest in mice. ^{20 21 22} Our results confirm and extend these conclusions. 

Our data also identify the αGsu-Cre;R26R combination as a similarly robust and specific system for labeling mesenchyme populations derived from mesoderm. The cells marked in αGsu-Cre;R26R embryos are distinct from those in comparable neural crest-labeled Wnt1-Cre;R26R embryos during early ocular development, when the two embryonic precursor populations have not yet mixed. The location of cells labeled in αGsu-Cre;R26R embryos at E11.5 corresponds precisely with the previously established position of cephalic paraxial mesoderm. ^{30 31} The expression patterns of the transcription factors FOXC2, PITX1, and MYOG also distinguish between the β-gal-marked populations in Wnt1-Cre;R26R versus αGsu-Cre;R26R embryos. It is unlikely that there was a previously unrecognized population of neural crest cells that was not marked by β-gal activation in Wnt1-Cre;R26R embryos. Therefore, these results are consistent with the β-gal-marked cells in αGsu-Cre;R26R being of mesoderm origin. Future identification of additional molecular markers is likely to refine our understanding of these cells further. 

The absence of staining in well-established neural-crest–derived tissues, such as the trigeminal ganglia in the head and dorsal root ganglia in the trunk, provides further evidence that the αGsu-Cre;R26R system marks mesoderm and not neural crest elsewhere as well, suggesting that this combination may have further utility in other mesoderm-derived tissues elsewhere in the body. However, further analysis of lineage-specific markers within individual organ systems would be necessary to confirm this preliminary interpretation. Although the source of rare β-gal⁺ cells in lens and surface ectoderm is not clear, PITX1 is capable of transactivating the same αGSU promoter in cell culture and PITX1 expression has been reported in the lens placode and early lens vesicle (Fig. 2Hand Ref. ³⁸ ). Therefore, it may be that expression of PITX1 ectopically activates the αGsu-Cre transgene at a low frequency in lens or the surface ectoderm from which it derives. 

Ocular Fate of Neural Crest and Mesoderm in Birds Versus Mammals

Our results indicate that there is general conservation of neural crest and mesoderm fates in the eye between birds and mammals and that there is frequently a division of labor within individual structures where the neural crest and mesoderm contribute different mature lineages (Fig. 6) . ³⁸ For example, in extraocular muscles the muscle fibers themselves are derived from the mesoderm, whereas connective fascia cells arise from the neural crest. Similarly, the endothelial lining of ocular blood vessels arises from the mesoderm, whereas associated smooth muscle and pericytes derive from the neural crest. Differentiation of ciliary muscles from the neural crest is also conserved between birds and mammals. 

Despite the general conservation of neural crest and mesoderm fates in the eye between birds and mammals, several important differences are apparent (Fig. 6) . Of note, these all occur within anterior segment structures. The endothelial and stromal layers of the cornea, as well as cells of the trabecular meshwork are reported to derive solely from the neural crest in birds. ^{6 7 9 10} Endothelial cells lining Schlemm’s canal are the sole anterior segment lineage reported to derive from the mesoderm in birds. ⁴⁰ Our results indicate that most cells within the corneal endothelial and stroma layers and the trabecular meshwork are also derived from the neural crest in mammals. However, a small population of cells within each of these structures consistently did not express β-gal in neural-crest–labeled animals, strongly suggesting that certain cells in these structures have a different origin. These cells were present by E12.5 and persisted into adulthood. In contrast, a comparable number of cells in each of these structures consistently labeled positively for β-gal in mesoderm-marked animals throughout the same time period, consistent with a mesoderm origin of these cells. Although the possibility of ectopic expression is always a concern when using transgenic mice, the absence of ectopic labeling in any other neural-crest–derived structures or the dorsal neural tube, from which the neural crest arises, strongly argues that these cells were not neural crest. This, together with the demonstration that not all cells in the affected structures labeled as neural crest, leads us to conclude that a minority population of cells within the corneal endothelium and stroma, the limbus, and the trabecular meshwork are derived from the mesoderm. The identification of additional lineage-specific markers that are expressed at later time points is essential for further confirmation of this interpretation and to determine the mature identity of these cells. 

The underlying reason(s) for the surprising presence of mesoderm cells in multiple anterior segment structures in mice, where they had not been reported in birds, remains unknown. One possibility is that these cells are also present in the chick but are not recognized because they represent a relatively small minority of cells in the relevant structures. This seems unlikely, given that multiple laboratories have reported equivalent findings using different labeling approaches. ^{6 7 8 9 10} Therefore, we propose a model wherein the mature corneal endothelial and stromal layers, the mesenchymal layer of the limbus, and the trabecular meshwork in mammals each consist of two mature cell lineages, one of which is derived from the neural crest and the other from the mesoderm. The lineages derived from the mesoderm may not be present in homologous structures in birds. This addition of new cells would represent an evolutionary advance from birds to mammals. Attractive candidates for the identity of at least some of the mesoderm-derived cells are a complex mix of antigen-presenting cells that serve as immune sentinels and are present in mammalian eyes, including murine eyes. In the limbus and peripheral cornea, these include dendritic cells and macrophages. ^{42 43 44} A similar population of cell lineages has recently been demonstrated in the central cornea of mice as well, but the cells in this location are generally immature, lacking the typical dendritic cell morphology and do not express major histocompatibility complex (MHC) class II antigen in normal, uninflamed eyes. ⁴⁵ These cells probably arise from bone marrow, which is consistent with a mesoderm origin. It is intriguing that there have been no published reports of dendritic or macrophage cells’ having been identified in chick eyes, and highly related Langerhans cells are reportedly absent from chick eyes. ⁴⁶ Taken together, these observations suggest that cells within the dendritic and macrophage lineages may account for at least some of the mesoderm-derived cells that we identified in the anterior segment of murine eyes. Recently, lymphohematopoietic lineages have been shown to modulate pathogenicity in a murine model of pigmentary glaucoma. ⁴⁷ Although the significance of these findings remains to be established for human disease, immune surveillance cells in the form of dendritic and Langerhans cells are also found in the anterior segment of human eyes. Therefore, the presence of mesoderm-derived dendritic cells may contribute to the underlying etiology of glaucoma. 

Implications of Transcription Factor Expression Patterns

The ability to identify lineage-specific expression patterns of key regulatory and functional genes in the developing eye is an important advance, because it provides useful insights into potential mechanisms of gene function and interpretation of mutant phenotypes. Demonstration that the five transcription factors we examined were expressed in overlapping but distinct patterns in the neural crest and mesoderm at E11.5 is significant because it suggests that initial molecular patterning of the mesenchymal precursor cells is probably already in place at this early time point, even though morphologic differences were not yet discernible. It is particularly striking that FOXC1 and PITX2 had the most similar expression patterns, because heterozygous mutations in the human FOXC1 or PITX2 genes both result in Axenfeld-Rieger syndrome, an autosomal dominant condition including anterior segment defects and a high risk for glaucoma. ^{33 34} This supports the hypothesis that the two factors probably regulate common downstream targets in cells in which they are coexpressed. Our current demonstration of wider Pitx2 expression in the developing eye is consistent with the demonstration that complete loss of Pitx2 function in mice results in a more severe ocular phenotype than loss of Foxc1. Foxc2 heterozygous mice exhibit ocular phenotypes very similar to the Foxc1 heterozygous phenotype. ¹⁷ This is consistent with the two genes’ being coexpressed in neural crest. 

Pitx2 is essential to specify correctly the multiple lineages derived from the periocular mesenchyme, including the corneal endothelium and stroma and the extraocular muscles. ^{2 13 15 32 48} Our current demonstration that these structures are chimeric, receiving contributions from both the neural crest and mesoderm, raises the intriguing mechanistic question of whether the requirement for Pitx2 function in each affected structure lies in the neural crest or the mesoderm. For example, specification of extraocular muscles may require Pitx2 for an intrinsic function in mesoderm precursors for them to initiate their myogenic program. Alternatively, Pitx2 function may be necessary in the neural crest for expression of an upstream signaling molecule(s) that acts extrinsically on the mesoderm precursor cells. Neural crest- and mesoderm-specific knockouts will ultimately be necessary to address this question in each relevant tissue. 

The distinct timing and location of initial Pitx2 expression in neural crest versus mesoderm prompts intriguing new insights into potential gene function in the developing cornea and provides strong evidence that the mechanism of gene activation is likely to be different between the two embryonic precursor cell populations. Anterior epithelial cells of the lens vesicle act as a signaling center that is needed in multiple steps during development of the anterior segment, including induction of the corneal endothelium and stroma layers. ⁴⁹ Pitx2 expression is activated within neural crest cells immediately after their arrival within the nascent anterior segment. Together with the lack of corneal endothelium and stroma specification in Pitx2-deficient mice, this suggests a model in which Pitx2 itself is an essential early target of the cornea-inducing signal(s) expressed by the anterior lens epithelia. Activation of Pitx2 in wild-type mice, presumably together with additional essential genes, results in the initiation of a genetic cascade leading to specification and differentiation of corneal endothelium and stroma. Pitx2-deficient animals are genetically unable to express PITX2 protein, effectively resulting in uncoupling of the required genetic cascade and agenesis of the cornea. Future extension of this pathway by identification of the upstream inducing signal(s), additional genes coinduced with Pitx2 in the neural crest, and the downstream effector genes will provide essential insights into our understanding of corneal development. In contrast to the neural crest, activation of Pitx2 in the mesoderm occurs before interaction of these cells with the ocular primordia and therefore, presumably, by a distinct mechanism. 

We have illustrated the use of complementary systems for long-term monitoring of neural crest and mesoderm cell fates in the mammalian eye. The subtle but important differences in cell fates between mature chick and mouse eyes underscore the importance of determining whether such variations exist elsewhere in the body where the neural crest and mesoderm interact during development. Differential expression of transcription factors within the two embryonic lineages in wild-type mice provides important new insights into the potential mechanisms of gene function in normal and abnormal ocular development. Determining the lineage-specific expression profiles of additional important genes that are expressed in periocular mesenchyme should provide similarly useful insights. Mutant mice are now available for the genes encoding each of these transcription factors ^{14 16 17} and introduction of the two labeling systems onto each mutant genetic background will allow identification of potential lineage-specific changes in the behavior of embryonic cell types and/or their derivatives. Finally, it will be possible, by using the Wnt1-Cre and αGsu-Cre transgenes, to generate neural-crest–and mesoderm-specific knockouts of relevant genes in the mammalian eye. For genes having complex expression patterns and mutant phenotypes, this will allow parsing of specific features of the phenotype to a requirement for gene function either in neural crest or mesoderm, and to distinguish intrinsic versus extrinsic effects. 

 Supported by a University of Michigan UROP Summer Biomedical Research Fellowship (WR), the Glaucoma Research Foundation (PJG), National Eye Institute Grants EY014126 and EY07003 (PJG), Research to Prevent Blindness (PJG), the Swedish Science Council (TH), and National Institute of Child Health and Development Grant 34283 to Sally Camper.

 Submitted for publication June 2, 2005; revised July 1, 2005; accepted August 30, 2005.

 Disclosure: P.J. Gage, None; W. Rhoades, None; S.K. Prucka, None; T. Hjalt, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Figure 1.

 

Lineage-specific β-galactosidase expression in neural crest and mesoderm of Wnt1-Cre;R26R and αGsu-Cre;R26R embryos. X-gal staining of E10.5 neural crest (A) and mesoderm (B) labeled embryos reveals distinct β-gal expression patterns generated by two binary labeling systems. Strong β-gal expression in the trigeminal ganglion of neural crest (C) but not mesoderm (D) labeled embryos stained with X-gal. The mesoderm-derived endothelial lining of the primary head vein is labeled in mesoderm embryos (D, inset). Strong β-gal expression in the dorsal root ganglion of neural crest (E) but not mesoderm (F) marked embryos stained with X-gal. The dorsal neural tube (★★) is labeled in neural crest (E) but not mesoderm (F) marked embryos. Complementary, nonoverlapping β-gal expression patterns detected by immunohistochemistry in E10.5 ocular primordia of neural crest (G) and mesoderm (H) labeled embryos. X-gal-stained sections are counterstained with nuclear fast red to display all nuclei. TG, trigeminal ganglion; BA, branchial arches; DRG, dorsal root ganglia; C, cornea; CM, cephalic mesoderm; G, gut; H, heart; S, somites.

Figure 2.

 

Coexpression of transcription factors and β-gal in fate-marked embryos. E11.5 Wnt1Cre;R26R and αGsu-Cre;R26R embryos were immunostained for expression of β-gal (green) to display neural crest (A, C, E, G, I) and cephalic mesoderm (B, D, F, H, J), respectively. Sections were counterstained (red) to detect the expression of PITX2 (A, B), FOXC1 (C, D), FOXC2 (E, F), PITX1 (G, H), and MYOG (I, J). Labeling in (A) provides orientation for all panels. NC, neural crest; CM, cephalic mesoderm; C, cornea; L, lens; OC, optic cup; OS, optic stalk.

Figure 3.

 

Detection of β-gal by immunohistochemistry in developing and mature extraocular muscles. β-gal expression (green) in presumptive extraocular muscles (PM) of E12.5 neural crest (A) and mesoderm (B) marked embryos. β-gal expression labels connective fascia cells (arrowheads) in developing (C) and mature (E) extraocular muscles of neural-crest–marked mice. β-gal expression labels myotubules and myofibers (arrows) in developing (D) and mature (F) extraocular muscles of mesoderm marked mice. Adult micrographs (E, F) are presented as the merge of red and green channels to enhance visualization of the myofibers.

Figure 4.

 

Neural crest and mesoderm fates in ocular blood vessels. Immunohistochemical detection of β-gal expression (green) in eyes from neural crest–and mesoderm-marked animals. Neural crest (A) and mesoderm (B) precursor cells are present within the hyaloid space by E12.5. Pericytes (C, E, arrowheads) in mature hyaloid vessels are derived from neural crest, whereas the endothelial lining (D, F, arrows) of mature hyaloid is derived from mesoderm. Neural crest (G, arrowheads) and mesoderm (H, arrows) have analogous fates in the mature choroid vasculature. All micrographs presented as the merge of red and green channels to facilitate visualization of the blood vessels and the location of red blood cells, which are autofluorescent (red/orange). Hy, hyaloid blood vessels; L, lens; R, retina; PE, pigmented epithelium; S, sclera; RBC, red blood cells.

Figure 5.

 

Complex neural crest and mesoderm fates in the ocular anterior segment. Immunohistochemistry (green) and X-gal (blue) were used to detect β-gal expression in neural crest (NC) and mesoderm (M). Neural crest–and mesoderm-derived cells were both present in the developing anterior segment of E14.5 (A, B) and P1 (C, D) eyes. Neural crest and mesoderm contributions to the mature corneal endothelium and stroma (E, F), ciliary body (G, H), iridocorneal angle (I–L), and iris (M–P) of adult eyes. Note β-gal⁻ cells (∧) in corneal stroma and endothelium of neural crest-marked animals (E). Limbus (★), Schlemm’s canal (open arrows), trabecular meshwork (closed arrows), iris stroma (arrowheads). X-gal stained eyes are counterstained with nuclear fast red to display all nuclei. S, corneal stroma; E, corneal endothelium; ICA, iridocorneal angle.

Figure 6.

 

Neural crest and mesoderm fates in bird and mammalian eyes. Cross-sectional diagrams summarizing similarities and differences in contributions of neural crest (red) and mesoderm (blue) to adult chicken and mouse eyes. The major differences occur in the anterior segment. Chicken fates are synthesized from previously published reports. ^{6 7 8 9 10}

The authors thank Andy McMahon and Sally Camper for the gifts of Wnt1-Cre and aGsu-Cre transgenic mice, respectively; Mitchell Gillett for expert technical assistance; David Reed for graphic illustration; Amanda Evans, Sally Camper, Peter Hitchcock, and Anand Swaroop for critical readings of the manuscript; and Sally Camper for support during the early stages of the work.