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Fate Maps of Neural Crest and Mesoderm in theMammalian Eye

Philip J. Gage,1,2 William Rhoades,1 Sandra K. Prucka,3 and Tord Hjalt4

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

METHODS. Complementary binary genetic approaches wereused to mark indelibly the neural crest and mesoderm in thedeveloping eye. Component one is a transgene expressing Crerecombinase under the control of an appropriate tissue-spe-cific promoter. The second component is the conditional Crereporter R26R, which is activated by the Cre recombinaseexpressed from the transgene. Lineage-marked cells werecounterstained for expression of key transcription factors.

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

CONCLUSIONS. The data provide essential new information to-ward understanding the complex interactions required for nor-mal development and function of the mammalian eye. Theresults also underscore the importance of confirming neuralcrest and mesoderm fates in a model mammalian system. Thecomplementary systems used in this study should be useful forstudying the respective cell fates in other organ systems. (In-vest Ophthalmol Vis Sci. 2005;46:42004208) DOI:10.1167/iovs.05-0691

The vertebrate eye is constructed during development fromthree general embryonic precursor sources: neural ecto-derm, surface ectoderm, and a loose array of cells termed theperiocular mesenchyme. A primary function of the periocular

mesenchyme is to provide multiple mature cell lineages thatare necessary for normal ocular development and vision, in-cluding the corneal endothelium and stroma, trabecular mesh-work, Schlemms canal, sclera, ciliary body muscles, irisstroma, extraocular muscles, and ocular blood vessels. A sec-ond essential function of the periocular mesenchyme duringocular development is to provide essential signals for pattern-ing of ocular ectoderm primordia, including induction of lac-rimal glands from the surface ectoderm, specification of retinalpigmented epithelium from the optic cup, and differentiationof the optic stalk from the neural ectoderm.13 Genetic oracquired defects in development or function of the periocularmesenchyme result in ocular disease and vision loss, includingparticularly glaucoma.4,5

Historically, the periocular mesenchyme was thought toarise developmentally from the mesoderm. However, fatemaps developed in birds by using quail chick chimeras, vitaldye labeling, or neural crest-specific antibodies have demon-strated that periocular mesenchyme actually receives initialcontributions from both neural crest and mesoderm.610 Thesestudies further established that the corneal endothelium andstroma, trabecular meshwork, most of the sclera, and theciliary muscles in birds are derived solely from the neural crest.In contrast, all vascular endothelium, the caudal region of thesclera, Schlemms canal, and extraocular muscles are derivedfrom the mesoderm. These results have generally served wellas the model for all vertebrates, including mammals. However,the documentation of important developmental differencesbetween birds and mammals has become increasingly com-mon.11,12 In the neural crest, these include differences in thetiming of neural crest migration, the migratory pathways taken,and their ultimate fates,11 making it essential to uncover anyspecies differences that may exist in mammals to accountaccurately for the embryonic origins of each mature structure.Several key regulators of periocular mesenchyme developmenthave recently been identified.2,1317 Determining the neuralcrest 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 mechanismsby which these genes function during ocular development.The major impediment to addressing any of these questionshas been the lack of a reliable strategy for quantitative, long-term labeling of neural crest and mesoderm in a mammalianeye. Fortunately, the use of two complementary Cre-lox basedapproaches has recently provided the necessary breakthrough.

A binary transgenic system has been developed that allowsfor indelible, permanent labeling of early presumptive neuralcrest and all subsequent progeny cells.18 The first componentof this system is a Wnt1-Cre transgene, which expresses Crerecombinase under the control of the promoter for the Wnt1gene. Like Wnt1 itself, Cre expression from the transgene istransient and limited to a 24-hour period in early presumptiveneural crest cells before their emigration from the dorsal neuraltube.18 The second component of the system is the conditionalCre reporter allele, R26R.19 Cre-mediated activation of -galac-tosidase (-gal) expression from the R26R reporter allele byDNA recombination provides for indelible, long-term labelingof all cells derived from the neural crest.19 This system has

From the Departments of 1Ophthalmology and Visual Sciences,2Cell and Developmental Biology, and 3Human Genetics, University ofMichigan Medical School, Ann Arbor, Michigan; and the 4Departmentof Cell and Molecular Biology, Lund University, Lund, Sweden.

Supported by a University of Michigan UROP Summer BiomedicalResearch Fellowship (WR), the Glaucoma Research Foundation (PJG),National Eye Institute Grants EY014126 and EY07003 (PJG), Researchto Prevent Blindness (PJG), the Swedish Science Council (TH), andNational Institute of Child Health and Development Grant 34283 toSally 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 pagecharge payment. This article must therefore be marked advertise-ment in accordance with 18 U.S.C. 1734 solely to indicate this fact.

Corresponding author: Philip J. Gage, Department of Ophthalmol-ogy and Visual Sciences, University of Michigan Medical School, 350Kellogg Eye Center, 1000 Wall Street, Ann Arbor, MI 48105;philgage@umich.edu.

Investigative Ophthalmology & Visual Science, November 2005, Vol. 46, No. 114200 Copyright Association for Research in Vision and Ophthalmology

been used to map neural crest fates in cardiovascular, cranio-facial, and skull vault development.2022

The mouse glycoprotein hormone -subunit gene (GSU) isexpressed initially in Rathkes pouch, the oral-ectodermde-rived primordium of the anterior pituitary gland, and subse-quently in multiple lineages of the mature anterior pituitarygland.23 GSU expression has also been reported in mesodermof the early ocular primordium and in the olfactory epithe-lium.24 The promoter regulatory elements required to recapit-ulate this expression pattern have been identified and used toexpress Cre recombinase in transgenic mice.25 Mice doublytransgenic for GSU-Cre and a Cre-responsive reporter cassetteexhibit indelible expression of -gal in a pattern consistentwith expression of endogenous GSU itself.25 GSU-Cre hasalso been used successfully to generate a lineage-specific geneknockout of the transcription factor gene Dax1 in the anteriorpituitary gland.26,27

In the current study, we used the complementary Wnt1-Cre/R26R and GSU-Cre/R26R labeling systems to establishfor the first time the long-term fates of neural crest and meso-derm, respectively, in a model mammalian eye. The fates weresimilar to but not identical with those previously reported inbirds, with the most significant differences occurring in theanterior segment. We also demonstrated that five known orpotential transcriptional regulators of periocular mesenchymehave unique expression patterns in the neural crest and meso-derm during early ocular development. Finally, the data alsoimply that the mechanism of activation for the homeobox genePitx2 in the neural crest and mesoderm is likely to be distinct.These findings have important implications for ocular develop-ment and function and may provide a model for understandinginteractions between the neural crest and mesoderm in otherorgan systems.


Animals and Isolation of Lineage-Marked Mice

Wnt1-Cre mice transmit a transgene consisting of a cDNA cassetteexpressing Cre protein placed under the transcriptional control of themurine Wnt1 promoter.18 Gsu-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 Creprotein under the transcriptional control of the pituitary glycoproteinsubunit promoter.25 R26R mice were purchased from Jackson Lab-oratories and transmit a -galactosidase reporter allele that is geneti-cally activated in vivo by Cre recombinase activity.19 All proceduresusing mice were approved by the University of Michigan Committeeon Use and Care of Animals. All experiments were conducted inaccordance with the principles and procedures outlined in the NIHGuidelines for the Care and Use of Experimental Animals and in theARVO Statement for the Use of Animals in Ophthalmic and VisionResearch.

Timed pregnancies were produced by mating homozygous R26Rfemales either with Wnt1-Cre t