Fate Maps of Neural Crest and Mesoderm in the Mammalian Eye Philip J. Gage,1,2 William Rhoades,1 Sandra K. Prucka,3 and Tord Hjalt4 PURPOSE. Structures derived from periocular mesenchyme arise mesenchyme is to provide multiple mature cell lineages that by complex interactions between neural crest and mesoderm. are necessary for normal ocular development and vision, in- Defects in development or function of structures derived from cluding the corneal endothelium and stroma, trabecular mesh- periocular mesenchyme result in debilitating vision loss, in- work, Schlemm’s canal, sclera, ciliary body muscles, iris cluding glaucoma. The determination of long-term fates for stroma, extraocular muscles, and ocular blood vessels. A sec- neural crest and mesoderm in mammals has been inhibited by ond essential function of the periocular mesenchyme during the lack of suitable marking systems. In the present study, the ocular development is to provide essential signals for pattern- first long-term fate maps are presented for neural crest and ing of ocular ectoderm primordia, including induction of lac- mesoderm in a mammalian eye. rimal glands from the surface ectoderm, specification of retinal METHODS. Complementary binary genetic approaches were pigmented epithelium from the optic cup, and differentiation used to mark indelibly the neural crest and mesoderm in the of the optic stalk from the neural ectoderm.1–3 Genetic or developing eye. Component one is a transgene expressing Cre acquired defects in development or function of the periocular recombinase under the control of an appropriate tissue-spe- mesenchyme result in ocular disease and vision loss, including cific promoter. The second component is the conditional Cre particularly glaucoma.4,5 reporter R26R, which is activated by the Cre recombinase Historically, the periocular mesenchyme was thought to expressed from the transgene. Lineage-marked cells were arise developmentally from the mesoderm. However, fate counterstained for expression of key transcription factors. maps developed in birds by using quail chick chimeras, vital dye labeling, or neural crest-specific antibodies have demon- RESULTS. The results established that fates of neural crest and strated that periocular mesenchyme actually receives initial mesoderm in mice were similar to but not identical with those 6–10 in birds. They also showed that five early transcription factor contributions from both neural crest and mesoderm. These genes are expressed in unique patterns in fate-marked neural studies further established that the corneal endothelium and crest and mesoderm during early ocular development. stroma, trabecular meshwork, most of the sclera, and the ciliary muscles in birds are derived solely from the neural crest. CONCLUSIONS. The data provide essential new information to- In contrast, all vascular endothelium, the caudal region of the ward understanding the complex interactions required for nor- sclera, Schlemm’s canal, and extraocular muscles are derived mal development and function of the mammalian eye. The from the mesoderm. These results have generally served well results also underscore the importance of confirming neural as the model for all vertebrates, including mammals. However, crest and mesoderm fates in a model mammalian system. The the documentation of important developmental differences complementary systems used in this study should be useful for between birds and mammals has become increasingly com- studying the respective cell fates in other organ systems. (In- mon.11,12 In the neural crest, these include differences in the vest Ophthalmol Vis Sci. 2005;46:4200–4208) DOI:10.1167/ timing of neural crest migration, the migratory pathways taken, iovs.05-0691 and their ultimate fates,11 making it essential to uncover any species differences that may exist in mammals to account he vertebrate eye is constructed during development from accurately for the embryonic origins of each mature structure. Tthree general embryonic precursor sources: neural ecto- Several key regulators of periocular mesenchyme development derm, surface ectoderm, and a loose array of cells termed the have recently been identified.2,13–17 Determining the neural periocular mesenchyme. A primary function of the periocular crest and mesoderm expression patterns, as well as the lineage- specific effects of genetic lesions in these genes, would signif- icantly enhance our understanding of the normal mechanisms From the Departments of 1Ophthalmology and Visual Sciences, by which these genes function during ocular development. 2Cell and Developmental Biology, and 3Human Genetics, University of The major impediment to addressing any of these questions Michigan Medical School, Ann Arbor, Michigan; and the 4Department has been the lack of a reliable strategy for quantitative, long- of Cell and Molecular Biology, Lund University, Lund, Sweden. term labeling of neural crest and mesoderm in a mammalian Supported by a University of Michigan UROP Summer Biomedical eye. Fortunately, the use of two complementary Cre-lox based Research Fellowship (WR), the Glaucoma Research Foundation (PJG), approaches has recently provided the necessary breakthrough. National Eye Institute Grants EY014126 and EY07003 (PJG), Research A binary transgenic system has been developed that allows to Prevent Blindness (PJG), the Swedish Science Council (TH), and for indelible, permanent labeling of early presumptive neural National Institute of Child Health and Development Grant 34283 to 18 Sally Camper. crest and all subsequent progeny cells. The first component Submitted for publication June 2, 2005; revised July 1, 2005; of this system is a Wnt1-Cre transgene, which expresses Cre accepted August 30, 2005. recombinase under the control of the promoter for the Wnt1 Disclosure: P.J. Gage, None; W. Rhoades, None; S.K. Prucka, gene. Like Wnt1 itself, Cre expression from the transgene is None; T. Hjalt, None transient and limited to a 24-hour period in early presumptive The publication costs of this article were defrayed in part by page neural crest cells before their emigration from the dorsal neural charge payment. This article must therefore be marked “advertise- tube.18 The second component of the system is the conditional ment” in accordance with 18 U.S.C. §1734 solely to indicate this fact. Cre reporter allele, R26R.19 Cre-mediated activation of ␤-galac- Corresponding author: Philip J. Gage, Department of Ophthalmol- ␤ ogy and Visual Sciences, University of Michigan Medical School, 350 tosidase ( -gal) expression from the R26R reporter allele by Kellogg Eye Center, 1000 Wall Street, Ann Arbor, MI 48105; DNA recombination provides for indelible, long-term labeling [email protected]. of all cells derived from the neural crest.19 This system has Investigative Ophthalmology & Visual Science, November 2005, Vol. 46, No. 11 4200 Copyright © Association for Research in Vision and Ophthalmology Downloaded from iovs.arvojournals.org on 10/01/2021 IOVS, November 2005, Vol. 46, No. 11 Ocular Mesenchyme Fates in Mammals 4201 been used to map neural crest fates in cardiovascular, cranio- mM potassium ferrocyanide, 0.1% X-gal in wash buffer), rinsed in wash facial, and skull vault development.20–22 buffer, embedded in JB-4 resin, and sectioned at 3 ␮m. Mounted The mouse glycoprotein hormone ␣-subunit gene (␣GSU)is sections were counterstained with nuclear fast red (Sigma-Aldrich, St. expressed initially in Rathke’s pouch, the oral-ectoderm–de- Louis, MO). rived primordium of the anterior pituitary gland, and subse- quently in multiple lineages of the mature anterior pituitary Immunohistochemistry gland.23 ␣GSU expression has also been reported in mesoderm of the early ocular primordium and in the olfactory epithe- Embryos harvested for immunostaining were fixed for 2 to 4 hours lium.24 The promoter regulatory elements required to recapit- with 4% paraformaldehyde in PBS, washed and dehydrated, and em- ulate this expression pattern have been identified and used to bedded in paraffin. Mounted sections were deparaffinized and treated express Cre recombinase in transgenic mice.25 Mice doubly for antigen retrieval by boiling for 10 minutes in citrate buffer (pH 6.0). ␣ Immunostaining was performed according to standard methods. transgenic for GSU-Cre and a Cre-responsive reporter cassette ␤ exhibit indelible expression of ␤-gal in a pattern consistent Briefly, sections were incubated with antibodies directed against -ga- with expression of endogenous ␣GSU itself.25 ␣GSU-Cre has lactosidase (Eppendorf-5Prime, Boulder, CO), FOXC1 or -2 (Abcam, also been used successfully to generate a lineage-specific gene Inc., Cambridge, MA), myogenin (MYOG; clone F5D, developed by knockout of the transcription factor gene Dax1 in the anterior Woodring Wright and obtained from NICHD/Developmental Hybrid- 26,27 oma Studies Bank, Iowa City, IA), PITX1 (a gift from Jacques Drouin, pituitary gland. 28 29 In the current study, we used the complementary Wnt1- Montreal, Canada ) or PITX2 followed by biotinylated species-spe- Cre/R26R and ␣GSU-Cre/R26R labeling systems to establish cific secondary antibodies (Jackson ImmunoResearch, West Grove, for the first time the long-term fates of neural crest and meso- PA). Signals were detected using tyramide signal-amplification kits derm, respectively, in a model mammalian eye. The fates were (PerkinElmer, Boston, MA). similar to but not identical with those previously reported in birds, with the most significant differences occurring in the ESULTS anterior segment. We also