A Novel Transgenic Technique That Allows Specific Marking ... · strated that a subset of migrating neural crest cells and a wide variety of cells in the neural crest cell lineage

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Developmental Biology 212, 191–203 (1999)Article ID dbio.1999.9323, available online at http://www.idealibrary.com on

A Novel Transgenic Technique That Allows SpecificMarking of the Neural Crest Cell Lineage in Mice

Yasutaka Yamauchi,*,†,‡ Kuniya Abe,* Akio Mantani,*asuyuki Hitoshi,* Misao Suzuki,§ Fumitaka Osuzu,†higeru Kuratani,¶ and Ken-ichi Yamamura*,1

*Department of Developmental Genetics, Institute of Molecular Embryology and Genetics, and§Center for Animal Resources and Development, Kumamoto University School of Medicine,4-24-1 Kuhonji, Kumamoto 862-0976, Japan; ¶Department of Biology, Okayama University,3-1-1 Tsushimanaka, Okayama 700-8530, Japan; †The First Department of Internal Medicine,National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama 359-0042, Japan; and‡Armed Forces Kumamoto Hospital, 15-1 Higashi Hon-Machi, Kumamoto 862-0902, Japan

Neural crest cells are embryonic, multipotent stem cells that give rise to various cell/tissue types and thus serve as a goodmodel system for the study of cell specification and mechanisms of cell differentiation. For analysis of neural crest celllineage, an efficient method has been devised for manipulating the mouse genome through the Cre–loxP system. Weenerated transgenic mice harboring a Cre gene driven by a promoter of protein 0 (P0). To detect the Cre-mediated DNAecombination, we crossed P0-Cre transgenic mice with CAG-CAT-Z indicator transgenic mice. The CAG-CAT-Z Tg linearries a lacZ gene downstream of a chicken b-actin promoter and a “stuffer” fragment flanked by two loxP sequences, so

that lacZ is expressed only when the stuffer is removed by the action of Cre recombinase. In three different P0-Cre linescrossed with CAG-CAT-Z Tg, embryos carrying both transgenes showed lacZ expression in tissues derived from neuralcrest cells, such as spinal dorsal root ganglia, sympathetic nervous system, enteric nervous system, and ventral craniofacialmesenchyme at stages later than 9.0 dpc. These findings give some insights into neural crest cell differentiation inmammals. We believe that P0-Cre transgenic mice will facilitate many interesting experiments, including lineage analysis,purification, and genetic manipulation of the mammalian neural crest cells. © 1999 Academic Press

Key Words: Cre recombinase; protein zero; chicken b-actin promoter; lacZ; neural crest; mouse embryo; HoxB5; tissue
culture.
atisB

INTRODUCTION

Stem cells have attracted intense and increasing interestbecause of their biological significance and potential medi-cal importance. The specification and expansion of diversecell lineages from a few stem cell populations is fundamen-tal for the generation of complex, multicellular organisms.The differentiation of these cells among so many distinctpathways is an essential problem in developmental biology(Anderson, 1989; Bronner-Fraser, 1992). These processes arethought to be controlled by a hierarchy of genes encodingfactors that act in both cell-autonomous and non-cell-

1 To whom correspondence should be addressed at 4-24-1 Ku-
honji, Kumamoto 862-0976, Japan. Fax: 181-96-373-5321. E-mail:[email protected].
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utonomous manners. For example, a number of transcrip-ion factors and cell–cell signaling molecules are involvedn the restriction of multipotent stem cells to variousublineages (Stemple and Anderson, 1992; Anderson, 1997;aker et al., 1997). Neural crest cells are embryonic, mul-

tipotent stem cells which give rise to various cell/tissuetypes and thus represent a good model system for studies ofstem cell biology (Bronner-Fraser and Fraser, 1988; Sellecket al., 1993).

The study of neural crest development at the cellular levelhas focused on how environmental influences control thegeneration of different cell types in this cell lineage (Shah etal., 1996; Lahav et al., 1996). For such studies, many experi-mental procedures have been applied including quail–chick
chimeras (Le Douarin, 1973), retroviral-mediated gene transfer(Cepko, 1988; Murphy et al., 1991), and cell marking using a
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vital dye such as DiI (Serbedzija et al., 1989, 1992) or usingNC-1 or HNK-1 antibodies (Tucker et al., 1984). These at-tempts have greatly contributed to the understanding of thedifferentiation pathway of neural crest cells and resultantderivatives, although mostly in the avian system because ofthe ease of embryonic manipulation in this system (Le Doua-rin, 1982). In contrast, cell lineage analysis or direct analysis ofthe neural crest cell differentiation at the molecular level hasbeen hampered in mammals by lack of reagents and embryo-logical techniques that allow specific and comprehensivecharacterization of neural crest cells. Microsurgical manipu-lation and exo utero culture of embryos have been formidabletasks in most mammals. In addition, a “pan”-neural crest cellmarker such as the HNK-1 (Tucker et al., 1984) is notavailable in mice.

In order to overcome these difficulties, we took advantage ofthe Cre–loxP DNA recombination system. Cre recombinasefound in bacteriophage P1 carries out site-specific DNA re-combination at a specific 34-bp sequence called loxP (Kilby etal., 1993). If there are two loxP sites tandemly located in thesame orientation, the Cre enzyme catalyzes an intramolecularrecombination reaction, resulting in excision of one circularDNA molecule with a loxP site from the original DNA. It hasbeen shown that this Cre-mediated site-specific DNA recom-bination can take place in mammalian cells (Orban et al.,1992). When Cre recombinase is introduced into cells thathave genomic DNA with two properly oriented loxP sites,genomic DNA flanked by the two loxP sites is cleaved out.Therefore, if we can make the Cre work specifically inpluripotent stem cells carrying the genome with two loxPsites, we could introduce an “unerasable” mark into thegenome of all stem cell descendants.

In the present study, we established and characterizedtransgenic mouse lines expressing the Cre enzyme in neuralcrest cells. To detect and visualize the Cre-mediated DNArecombination in neural crest cells and derivatives, we uti-lized another transgenic (Tg) line with the CAG-CAT-Z indi-cator construct (Araki et al., 1995). This Tg line carries a lacZreporter gene downstream of a chicken b-actin promoter and a“stuffer” fragment flanked by two loxP sequences. The lacZ isexpressed only when the stuffer is removed by the action ofCre recombinase. Using these transgenic mice, we demon-strated that a subset of migrating neural crest cells and a widevariety of cells in the neural crest cell lineage could be markedby lacZ expression. We believe that the use of this systemfacilitates many interesting experiments, including lineageanalysis, purification, and manipulation of the mammalianneural crest cells. Also, this cell-type-specific transgenesissystem should facilitate functional analysis of genes of inter-est in neural crest cell lineage.

MATERIALS AND METHODS

Experimental Design

The experimental design is outlined in Fig. 1. An indicatortransgenic line carries a lacZ reporter gene downstream of the

Copyright © 1999 by Academic Press. All right

hicken b-actin promoter and a stuffer CAT gene fragment inetween. This CAT transcription unit flanked by two loxP se-uences prevents the actin promoter-driven lacZ expression (Fig.). Therefore, lacZ is expressed only when the stuffer is removed byhe action of the Cre recombinase (Fig. 1). Once the recombinationccurs in a particular set of cells, the lacZ gene is continuously

expressed in these cells as well as their descendants, therebyenabling specific marking of a given cell lineage.

Plasmid Construction and Productionof Transgenic Mice

The Cre expression vector pP0-Cre was constructed by fusingthe 1.2-kb XhoI–MluI fragment of the cre gene (Sauer and Hender-son, 1990; gift from B. Sauer) to the 1.1-kb protein 0 (P0) promotersequence (Lemke and Axel, 1985; Messing et al, 1992; gift from G.Lemke) (Fig. 1). A transgene construct, pCAG-CAT-Z (Araki et al.,1995; gift from K. Araki), containing a chicken b-actin gene (CAG)promoter-loxP-CAT gene–loxP-lacZ region (Miyazaki et al., 1989;

uckow and Schutz, 1987; Sauer and Henderson, 1988), was alsosed for microinjection to establish an indicator transgenic line. Toonitor activity of the CAG promoter in embryos, we removed the

tuffer fragment by injecting the pCAGGS-Cre (Niwa et al., 1991)expression vector into eggs from a CAG-CAT-Z 34 3 B6D2F1

mating. The plasmid pCAGGS-Cre was purified by NACS PREPACand suspended in 1 mM Tris–HCl (pH 7.5/0.1 mM EDTA) at aconcentration of 5 mg/ml.

Genotyping by Southern Blot Analysis and PCR

Genotypes of transgenic embryos/animals were scored by South-ern analysis and PCR. For PCR analysis, DNAs (0.2–0.5 mg) wereubjected to 28 amplification cycles (1 min at 94°C, 2 min at 65°Cor Cre primers or 60°C for AG-2 primers, and 2 min at 72°C) on ahermal cycler. The 59 and 39 primers used for detection were forhe CAT gene, CAT2 (59-CAGTCAGTTGCTCAATGTACC-39)nd CAT3 (59-ACTGGTGAAACTCACCCA-39), for the lacZene, Z1 (59-GCGTTACCCAACTTAATCG-39) and Z2 (59-GTGAGCGAGTAACAACC-39), and for the Cre gene, Cre1 (59-GACATGTTCAGGGATCGCCAGGCG-39) and Cre2 (59-CATAACCAGTGAAACAGCATTGCTG-39). Primers for

etection of the junction between CAG promoter and lacZ wereG-2 (59-CTGCTAACCATGTTCATGCC-39) and Z3 (59-GCCTCTTCGCTATTACG-39).

Northern Blot Analysis and RT-PCR

Total RNA was extracted from various tissues by the guani-dinium thiocyanate procedure. The total RNAs (20 mg) wereubjected to electrophoresis on a 1% formaldehyde agarose gel.fter transfer to nylon membranes, (Hybond-N1; Amersham),

amples were hybridized with the 32P-labeled BamHI fragment ofhe Cre gene. For RT-PCR, 5 mg of total RNA was reverse

transcribed (RT) using SuperScript RT (SuperScript Preamplifica-tion System; BRL) and then subjected to 28 amplification cycles (1min at 94°C, 2 min at 65°C, and 2 min at 72°C). Cre1 and Cre2 wereused for detection of Cre gene expression.

Detection of b-Galactosidase (lacZ) Activities

Whole embryos were stained for b-galactosidase activity accord-ing to the method of Allen et al. (1988). Samples were fixed for 30

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193A Genetic Marker for Neural Crest Cell Lineage

to 60 min at 4°C in 1% formaldehyde, 0.2% glutaraldehyde, and0.02% NP-40 in phosphate buffer (PB). Fixed samples were washedtwo times in PB and incubated overnight at 30°C in stainingsolution, 5 mM potassium ferricyanide, 5 mM potassium ferrocya-nide, 2 mM MgCl2, 0.5% X-gal in PB. Samples were rinsed twice in

B and postfixed in 4% paraformaldehyde.

Preparation for Cryostat Sectioning

For frozen sectioning, the stained embryos were sequentiallysoaked in a graded series of 6, 10, and 17% sucrose (w/v) in PB whilebeing gently agitated on a shaking platform. Embryos were thenwarmed to 37°C and incubated in Tris-buffered saline containing0.5 M sucrose and 7.5% (w/v) gelatin (300 Bloom; Sigma) for 1–3 h.They were then embedded in gelatin compound, chilled on ice for1 h, and frozen in dry ice-chilled n-pentane for 12 min. Sectionswere cut at 15-mm thickness and mounted on gelatin-coated slides.Sections were counterstained with Nuclear Fast red after dehydra-tion when necessary.

Neural Crest Cell Culture Methods

Neural crest culture was performed as previously described(Huszar et al., 1991). Mouse embryos carrying transgenes wereisolated at 9.0 dpc. The embryos were freed of placenta and fetalmembranes using fine forceps under a dissecting microscope. Theywere incubated for 12 min at 37°C in PB containing 5% pancreatin(GIBCO BRL, Grand Island, NY). The enzyme action was stopped

FIG. 1. The Cre transient expression system. Schematic diagraeneration of transgenic mice; the light gray boxes represent the P0ransgenes. Black arrow indicates loxP sequence and An, a polyxpression system removes the loxP-surrounded CAT reporter gen

chicken b-actin promoter; CAT, chloramphenicol acetyltransferas

by transferring the embryos to Dulbecco’s modified Eagle’s me-dium (DMEM) supplemented with 10% fetal calf serum (HyClone


Laboratories, Inc.). With fine tungsten needles, somites and sur-rounding tissues were removed to obtain neural tube segments.These segments were washed in a serum-free medium and trans-ferred to a fibronectin-coated-cellware 24-well plate (Falcon) con-taining DMEM supplemented with 5% fetal calf serum and 5%horse serum (GIBCO). Each sample was incubated at 36.6°C in 5%CO2 for 36 to 48 h after explantation and examined microscopi-ally.

Immunocytochemistry

HoxB5. Immunocytochemistry was performed essentially aspreviously described (Kuratani and Wall, 1992). Frozen sectionswere placed at room temperature and washed in Tris-buffered-saline (pH 8.0, 150 mM NaCl; TS) at 37°C for 10 min to removegelatin. The slides were blocked with 0.3% hydrogen peroxide inmethanol at room temperature for 20 min. After being blockedwith 5% dry nonfat milk, 1% Triton X-100 in TS (TSTM), and 1%periodic acid for 10 min, the slides were incubated for 90 min atroom temperature with anti-HoxB5 antiserum diluted 1:100 inTSTM. Slides were washed in TS 1 1% Triton-X 100 (TST) for 5min and then incubated for 60 min at room temperature withbiotinylated goat anti-rabbit IgG diluted 1:200 in 5% milk/TST.After a wash in TST, streptolysin–avidin diluted 1:100 in TSTMwas added and incubated for 60 min at room temperature. Peroxi-dase reaction was performed by adding TS containing 0.03%diaminobenzidine (DAB) and 0.03% hydrogen peroxide for 5 min atroom temperature.

f the P0-Cre and CAG-CAT-Z transgene cassettes used in theoter and the CAG promoter; the open boxes, Cre, CAT, and lacZ

ylation signal. If the P0 promoter is working, the Cre transientus, the recombination results in b-galactosidase expression. CAG,

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Islet-1. Immunocytochemistry was performed essentially aspreviously described (Ericson et al., 1992). Two types of Islet-1


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(motor neurons and neuronal neural crest marker) mouse mono-clonal supernatants (4D5 and 2D6) were obtained from the Devel-opmental Studies Hybridoma Bank (University of Iowa, Iowa City,IA). The slides were blocked with 0.3% hydrogen peroxide inmethanol at room temperature for 20 min. Antibody solution wasapplied to each section and incubated for 60 min at room tempera-ture with anti-Islet-1 antiserum diluted 1:10 with 3% BSA in PBS.Slides were washed in PBS for 5 min and then incubated for 30 minat room temperature with biotinylated goat anti-mouse IgG diluted1:100 in 3% BSA/PBS. After a wash in PBS, avidin D and biotinyl-ated horseradish peroxidase were added and incubated for 30 min atroom temperature. Peroxidase reaction was performed by addingPBS containing DAB and 0.03% hydrogen peroxide for 5 min atroom temperature. Each section was counterstained with hema-toxylin and eosin after dehydration.

RESULTS

Production of Transgenic Mice CarryingP0-Cre Gene

Transgenic founder mice were identified by PCR andSouthern hybridization of genomic DNA prepared from tailbiopsies taken at the time of weaning. Of 181 pups, 34 hadtransgene integration. In 6 of these 34 founder mice, Po-Cre2, 3, 8, 60, 94, and 105, we were able to detect Cre geneexpression in RNA isolated from tail by both RT-PCR andNorthern analysis. We eventually established three lines,Po-Cre 3, 8, and 94. These three lines showed essentiallysimilar expression patterns and therefore we chose thePo-Cre 94 line for most of the subsequent analysis.

In order to examine Cre-mediated DNA recombination inthese Tg mice, we crossed P0-Cre mice with the CAG-CAT-Z indicator transgenic mice. Since P0 is supposed tobe expressed in Schwann cells of the peripheral nervoussystem (Trapp et al., 1981; Giese et al., 1992), we firstchecked the lacZ expression and DNA recombination inthe peripheral nervous system. Tails, sciatic nerves, andtrigeminal ganglia of mice carrying both transgenes, i.e.,P0-Cre and CAG-CAT-Z, were fixed and stained with X-gal.In sections of the stained tissues, small nerve fascicles werespecifically stained (data not shown). Genomic DNA wasisolated from the doubly hemizygous transgenic animalsand subjected to PCR and Southern analysis. Both analysesdemonstrated that the stuffer fragment was removed fromthe CAG-CAT-Z construct in vivo, leading to lacZ expres-sion in a specific cell population. Such restricted lacZexpression or site-specific DNA recombination was neverobserved in normal mice or in transgenic mice carryingonly one type of transgene, suggesting that the recombina-tion is specific and mediated by Cre recombinase.

Marking of Neural Crest Cell Lineages by theCre-Mediated lacZ Expression duringEmbryonic Development

To trace back to when the recombination occurs duringembryonic development, we examined lacZ expression in


mbryos derived from P0-Cre 3 CAG-CAT-Z mating. Un-xpectedly, lacZ expression was first apparent even in.0-dpc embryos, in which Schwann cells are not yetormed. The expression pattern of the reporter gene wasolocalized with the expected distribution pattern of crestells (Fig. 2A), i.e., ventral craniofacial mesenchyme in theharyngeal arches, the frontonasal region, and the cranialensory ganglion primordia were positive at 9.5 dpc. Thoseells, however, did not cover the entire expected area of therest-derived tissues, as was observed in chick embryos (Leouarin, 1982). Nevertheless, in the sectioned specimen,

he positive cells within trunk somites were localized alonghe ventrolateral migration pathway of the crest cells, theite of spinal dorsal root ganglia development. The cells inistal areas, such as those in pharyngeal arches or aroundhe dorsal aorta where crest cells form the sympatheticrunk, were always positive (Fig. 3C).

Most of the positive cells were restricted to the mesen-hyme in the pharyngeal arch except for a few positive cellsn the pharyngeal ectoderm and endodermal pharyngealouch (Fig. 3C). The notochord, which is not derived fromhe neural crest, also showed positive staining.

In 10.5-dpc embryos, the X-gal staining pattern wasroadened into almost the entire expected area, to whichhe crest cells migrate (Fig. 2B). For example, neural crestells originating in the midbrain move primarily as a broad,nsegmented sheet of cells under the ectoderm. Theyontribute to the periocular skeleton and connective tissuesf the ventral face, part of the trigeminal ganglia, andchwann cells (Le Douarin, 1982). The b-galactosidase

activity was detected in these regions at high levels (Fig.2B). Moreover, neural crest cells from the hindbrain regionare known to migrate as three broad streams of cells. Thefirst group of cells will populate the trigeminal ganglia andmandibular arch, the second populates the hyoid arch, andthe third migrates into the third and more caudal branchialarches and peripheral ganglia (D’Amico-Martel and Noden,1983; Lumsden et al., 1991). The lacZ expression wasclearly found in these three pathways of the crest cellmigration. The X-gal-positive cells migrating dorsoven-trally were clearly observed as a confluent cell mass be-tween the neural tube and the dermatomyotome, at thelevel of the anterior half of the somite (Fig. 2B).

At 12.5 dpc in the craniofacial region, the positive mes-enchyme extended to the whole area of pharyngeal archesand frontonasal region including the mesenchymal sheathcovering the telencephalon (Figs. 4A and 4B). As shown inFig. 3A, X-gal-positive cells were found along the dorsolat-eral pathway, which migrates through the region betweenthe dermatomyotome and the epidermis (Serbedzija et al.,1989) and in the ventral pathway which emigrates throughthe rostral half of the somites (Fig. 3E) (Rickmann et al.,1985; Krull et al., 1997). In the trunk region, the spinaldorsal root ganglia, sympathetic nervous system, and en-teric nervous system were positive (Fig. 3A). A high level of
b-galactosidase activity was detected in the outer mesen-chymal layer throughout the length of the developing

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gastrointestinal tract, which is known to be innervated bythe neural crest cell-derived enteric nervous system at thisstage (Figs. 3A and 3D). In the trunk, peripheral nervoussystem components, such as the brachial plexus and lumbarplexus, were positive (Figs. 2C and 3A). The staining wasseen not only in the ganglia but also along the axonaldistribution, suggesting that supporting cells and/or sen-sory axons themselves, both derived from the neural crest,expressed lacZ (Figs. 3D and 3F). In a higher magnificationview of the spinal dorsal root ganglia, the b-galactosidase

FIG. 2. Detection of b-galactosidase (lacZ) activity. Temporal anCAG-CAT-Z 34) in whole-mount X-gal-stained mouse embryos. (A(D) 10.5-dpc embryo of the Cre-injected CAG-lacZ transgenic miceoocytes with a circular Cre expression plasmid. (A), (B), and (C) ar

ostly in the area of the neural crest migration. In (D) almost the enm, dermatomyotome; hm, head mesenchyme; st, sympathetic tru

activity was mostly detected in cell bodies of the sensoryneurons (Fig. 3F).


Dissection of a stained embryo revealed that mesenchy-al cells of the skull base primordium rostral to the

ypophyseal foramen were positive (Figs. 4A and 4B). Theb-galactosidase activity was detected in mesenchymal cellsof the frontonasal region and in alisphenoid or basisphenoidprimordia (Fig. 4A). On the other hand, the posterior mes-enchyme, which gives rise to occipital cartilage and verte-brae, was only weakly stained. The proximal ganglia ofcranial nerves V (trigeminal), VII (facial), VIII (acoustic), IX(glosspharyngeal), and X (vagus) were also positive. These

tial expression of lacZ in double-transgenic mice (P0-Cre 94 and, (B) 10.5-, and (C) 12.5-dpc embryos of the double-transgenic mice.e CAT gene between the two loxP sites is removed by injection ofral views demonstrating that b-galactosidase activity is observed

embryo is stained. n.V, trigeminal nerve; DRG, dorsal root ganglia;brpl, brachial plexus.

d spa) 9.5-. The late

ganglia are thought to be derived from neural crest cellsoriginating from rhombomere 1 (r1) to r7 within the hind-


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of the aortic arch artery. nc, nasal capsule; ns, nasal septum; as, alisphenoid; bs, basisphenoid; sm, scleral mesenchyme; V, trigeminalganglia; VIII, acoustic ganglia; mn, meninges; AO, aorta; oft, outflow tract; PA, pulmonary artery; H, heart.


FIG. 3. b-Galactosidase activity in histological cryostat sections at 9.5 and 12.5 dpc. (A) Transverse section through the trunk of a 12.5-dpcouble transgenic embryo displays lacZ expression in the dorsal root ganglia, sympathetic trunk ganglia, brachial plexus, vagal nerve, andelanocyte. (B) Transverse control section through the trunk of a 9.5-dpc embryo of the CAG-lacZ transgenic mice. Blue staining was

bserved in all the tissues, but a particularly high level of expression was seen in cardiac muscle primordium, myotome, and nervousystem. (C) Transverse section through the pharynx of a 9.5-dpc embryo, with b-galactosidase activity staining in the mesenchyme in the

pharyngeal arch. (D) Higher magnification view of the enteric nervous system of 12.5-dpc embryo displays lacZ expression. (E) A sagittalsection through the trunk of a 12.5-dpc double-transgenic embryo; the b-galactosidase activity was observed in the spinal dorsal rootganglia. (F) Higher magnification view of the spinal dorsal root ganglia shown in (E). DRG, dorsal root ganglia; sg, sympathetic ganglia; brpl,

FIG. 4. X-gal staining of sections of double-transgenic embryo. Each section was stained with X-gal and counterstained with Nuclear Fastred. (A) Transverse section through the cranial region of a 12.5-dpc embryo. (B) Sagittal section through the cranial region of a 12.5-dpcembryo. Dissection of a stained embryo revealed that mesenchymal cells of the skull base primordium rostral to the hypophyseal foramenare positive. (C) Transverse section through the outflow tract of a 12.5-dpc embryo. (D) Sagittal section through the heart of a 12.5-dpcembryo. Positive cells are contiguous with mesenchyme located within the outflow tract of the heart and are also found within the wall

brachial plexus; m, melanocyte; NC, notochord; n.X, vagal nerve; H, heart; aa, aortic arch; pa, pharyngeal arch; en, enteric nervous system;ie, intestinal epithelium.

Copyright © 1999 by Academic Press. All rights of reproduction in any form reserved.

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198 Yamauchi et al.

brain (Figs. 4A and 4B). Positive cells were also foundwithin the wall of the aortic arch artery. These positivecells expanded ventrally and were contiguous with astrongly positive mesenchyme located within the outflowtract of the heart (Figs. 4C and 4D). Non-crest-derivedstructures found to be positive at previous stages keptexpressing lacZ at this stage as well (Fig. 3A).

To rule out the possibility that the obtained stainingpattern was due to a property of the CAG promoter, wegenerated mice that constitutively express lacZ under thecontrol of the same promoter. The Cre-expression vectorwas injected into eggs of the CAG-CAT-Z 34 indicator lineso that the stuffer was excised at the very beginning ofdevelopment, and transgenic lines were established fromthose embryos. Embryos collected from the transgenic linewere examined at various stages of development. Bluestaining was observed in almost all the tissues, but aparticularly high level of expression was seen in the cardiacmuscle primordium, myotome, and nervous system (Figs.2D and 3B). This pattern of expression is qualitatively andquantitatively different from the staining pattern seen inembryos of P0-Cre/CAG-CAT-Z double-transgenic mice.These results suggest that the specific staining patternrestricted to neural crest cell lineage is not due to the CAGpromoter activity itself. Rather, the above results clearlyshowed that the Cre gene was turned on in a subset ofmigrating neural crest cells, resulting in removal of thestuffer fragment, and thereby allowing lacZ expression inthese cells. In fact, whole-mount in situ analysis of Creexpression in the transgenic embryos showed that Cresignals were detected in neural crest-derived tissues such asthe ventral craniofacial mesenchyme, which was positivefor lacZ (data not shown). Since all the descendants of thesemigrating neural crest cells carry and inherit the modifiedtransgene, the reporter is continuously expressed in thesecells irrespective of P0-Cre transgene expression.

Neural Crest Cell Differentiation in Vitro

It is well known that neural crest cells will emigrate fromneural tube explants under appropriate culture conditions(Boisseau and Simonneau, 1989). To examine whether theP0-Cre-mediated DNA recombination and the specificmarking of the neural crest cells occur in vitro, neural tubes

FIG. 5. Neural crest cell outgrowth from mouse 9.0-dpc neural tubin culture. (B) Higher magnification view of the dorsal side of thestaining with X-gal is shown at higher magnification. In the cumicrographs show that subpopulations of migrating neural crest ceimmunostained with anti-HoxB5 and stained with X-gal. (C) Transof the expected neuronal precursors of the enteric nervous system.while HoxB5 was found within the nucleus. Section of the neuronaX-gal and anti-Islet-1. (E) Sagittal section through the spinal dorsal
the cytoplasm of the same cell, lacZ expression was clearly observed (aand (F) were counterstained with hematoxylin and eosin.

solated from the transgenic embryos were cultured underhe conditions described by Huszar et al. (1991). Afterpproximately 6 h in culture on a fibronectin-coated plate,eural crest cells started migration from the neural tubexplant. Photographs shown in Figs. 5A and 5B were takenfter 36 h of incubation. Subpopulations of cells emigratingrom the explant were certainly stained positive. This resultndicates that the Cre-mediated recombination did notappen in premigrating neural crest cells, but occurred in aubset of cells that emigrated from the explant in vitro,hich is compatible with the results obtained from in vivo

tudies. The neural tube was devoid of positive staining,hile the migrating cell population was positively stained

Fig. 3A).

Double Labeling of Neural Crest Derivatives withKnown Markers and X-gal in the Transgenic Mice

The above results strongly suggested that P0-Cre-mediated DNA recombination occurred in the neural crestcells and specifically marked the neural crest cell lineages.To confirm this point in detail, we used neural crest markerantibodies. Since no ubiquitous neural crest markers suchas HNK-1 are available for mice, we first used a polyclonalantibody against HoxB5 protein. HoxB5 is expressed invagal crest-derived cells (Wall et al., 1992; Kuratani andWall, 1992) that enter the mesenchymal gut wall andeventually give rise to neuronal and glial cells of entericganglia (Le Douarin and Teillet, 1973; Allan and Newgreen,1980). As shown in Figs. 5C and 5D, immunoreactivity foranti-HoxB5 was found in the nuclei of the cells in theenteric ganglia of the 13.5-dpc embryo, which were alsopositive for lacZ.

We also used another neuronal neural crest marker, a LIMhomeodomain protein, Islet-1. Islet-1 is expressed in neu-ronal neural crest derivatives, i.e., neural crest-derivedsensory, sympathetic, or branchial neurons (Yamada et al.,1993; Ikeya et al., 1997). We used two types of monoclonalantibodies against Islet-1 and obtained essentially similarresults. As shown in Figs. 5E and 5F, subsets of the spinaldorsal root ganglia, sympathetic ganglia (data not shown),and trigeminal ganglia of 12.5-dpc embryo were positive foranti-Islet-1. Islet-1 immunoreactivity was observed in thenuclei (arrowhead) of these cells, while lacZ expression was

er 36 h in culture. (A) A neural tube that had been growing for 36 hnted neural tube. A region of neural crest cells after fixation andneural crest cells can be identified along the neural tube. The

xpressed lacZ. Transverse section of the gut of a 13.5-dpc embryosection at the level of the foregut. (D) Higher magnification viewcytoplasm of the same cell, lacZ expression was clearly observed,

ral crest derivatives of a 12.5-dpc embryo with doubly stained withganglia. (F) Higher magnification view of the trigeminal ganglia. In

e aftexplalture,lls everseIn thel neuroot

rrow), while Islet-1 was found within the nucleus (arrowhead). (E)



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seen in the cytoplasm (arrow) of the same cell. These resultsstrongly support the assumption that the transgene isactivated in subsets of the neural crest cell lineage inyounger embryos and that the expression is maintained inboth neural and nonneuronal neural crest cell derivatives atlater stages.

DISCUSSION

One of the most significant findings in this paper is thata wide variety of neural crest cell derivatives could bemarked by reporter lacZ expression in P0-Cre/CAG-CAT-Zdouble-transgenic mice and, for the first time in mice, thisreporter expression can be utilized as an extensive neuralcrest cell lineage marker. This specific marking must bedue to an “unerasable” mark introduced into the genome ofthe migrating neural crest cells by Cre-mediated DNArecombination. This fact in turn suggests that, although theP0 promoter is not necessarily active in all the neural crestderivatives, it should be operative in a subset of migratingneural crest cells. The P0 glycoprotein is a cell adhesionmolecule of the immunoglobulin superfamily that consti-tutes myelin sheaths in the peripheral nervous system(Martini et al., 1988; Lemke et al., 1988). Lemke ando-workers (Messing et al., 1992) generated P0-hGH trans-enic mice and reported that the transgene is active inyelinating Schwann cells. The Schwann cells first synthe-

ize detectable levels of P0 only after birth. Akagi et al.(1997) produced a series of transgenic lines with constructssimilar to those used in the present study. They describedthe expression of the reporter in the adult peripheral ner-vous system, but did not mention lacZ expression duringmbryonic development. Since we obtained essentiallydentical results with three independent lines, we suspectedhat the P0 promoter used in the present study must bective already at early stages of neural crest cell differentia-ion. Observations previously made in other species suggesthat P0 is expressed in a subpopulation of neural crest cellsr in nonmyelinating as well as myelinating Schwann cellsBarbu, 1990; Bhattacharyya et al., 1991; Zhang et al., 1995;heng and Mudge, 1996). Our analyses with both HoxB5nd Islet-1 antibodies clearly demonstrated that lacZ geneas co-expressed with these neural crest cell markers ofifferent lineages. Therefore, the results strongly suggesthat Cre recombinase is functioning within the multipoten-ial, early neural crest cells. Moreover, our whole-mount initu hybridization analysis of P0-Cre Tg with the Cre probendicated that the Cre gene was in fact expressed in neuralrest cells during early development (data not shown). Atny rate, the results presented in the present paper stronglyuggested that Cre-mediated recombination occurred in aubset of migrating neural crest cells, but not before detach-ng from the neuroepithelium. Both observations on devel-
ping embryos and the cultured neural tube explants lendupport to this notion. To our knowledge, there have been

o such markers specifically turned on in neural crest cellsfter emigration from the neural tube in mammals, furtheralidating the utility of this system.Our observations clearly indicated that lacZ expression

ccurs in almost all the cells/tissues of neural crest cellrigin. However, we found that one non-crest-derived or-an, the notochord, was positive for the X-gal staining.lthough the notochord influences formation and differen-

iation processes of somites as well as the neural tubePourquie et al., 1993), cells comprising the notochord doot migrate toward these tissues. Notochordal cells stay asrod-shaped structure underneath the neural tube and

ventually become surrounded by vertebrate columns (Ju-and, 1974). We therefore believe that these positive noto-hordal cells do not contribute to the formation of theeural crest-derived tissues and would not compromise thenterpretation of results obtained through our transgenicystem. Expression in the notochord is probably affected byhe character of the P0 promoter used. Such an expressionattern is not unprecedented: genes such as Int6 (Diella etl., 1997) or qkI (K. Abe, unpublished observations) arexpressed in both neural crest derivatives and notochord.Several different approaches have been used to study the

eural crest cell lineage. For example, injection of theipophilic dye, DiI, into the lumen of the neural tube orirectly into neural folds has been used for this purposeith much success (Serbedzija et al., 1989, 1991; Fraser andronner-Fraser, 1991; Sechrist et al., 1993, 1994; Osumi-amashita et al., 1997). Le Douarin and colleagues (1982)tilized interspecific chimeras between quail and chickmbryos and precisely documented the full range of neuralrest derivatives arising from different regions of theeuraxis. However, these approaches have not been widelysed in mice, because of difficulties in culturing embryos initro and in microsurgical operations. Another problemnherent to the DiI labeling is that the dye will not stay forong period of development. It is therefore necessary tostablish a novel method for lineage analysis of neural crestells in mice, since genetic manipulation techniques areell advanced in mice and mutations affecting neural crestifferentiation are already known.Our transgenic system seems to have a great utility for

elineating neural crest cell diversification in mice asxemplified by the following findings. In mammals, it is notell documented which skeletal elements in the skull aref neural crest origin (Le Douarin, 1982). In this context, its of special interest that, in the present study, the mesen-hyme covering the forebrain was X-gal positive at 12.5 dpcnd at older stages. This part of the mesenchyme gives riseo the meninges and neurocranium. Of the meninges, theura mater is regarded as a mesodermal structure since it isocated just lateral to the neural tube. The prechordal braintelencephalon), however, is thought to be covered by crest-erived meninges since this area is devoid of mesodermalesenchyme (Couly et al., 1993). The b-gal-positive men-

ingeal anlage described in the present study is restricted tothe prechordal brain, a part of a “New Head” as defined by


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Gans and Northcutt (1983). The presence of crest-derivedneurocranial elements in the mammalian embryo is alsosuggested by Otx2 mutation in which putative trabecularcartilage derivatives were affected in some heterozygousembryos (Matsuo et al., 1995). The latter phenomenon isalso consistent with the distribution of b-gal-positive cellsin the cranial base restricted rostral to the hypophysealregion (Figs. 2C and 4B). Although further studies arerequired for describing which skeletal elements in theskull, including meningeal structures, are derived from theneural crest, our transgenic mice should be useful for thispurpose.

Patterning of specific regions of the developing heart alsodepends on the neural crest derivative; in the chick, thesecrest cells give rise to the ectomesenchyme in the heartwhere it serves for aortic–pulmonary septation (Kirby andWaldo, 1995). In the present study, we showed that cellsaround the aortic arch arteries and in the outflow tract ofthe heart were positive for X-gal staining (Figs. 4C and 4D),indicating that these cells are crest-derived also in mice. Inthis context, it is interesting that targeted disruptions ofmurine ActRIIB (Oh and Li, 1997), dHAND (Strivastava etal., 1997), eHAND (Riley et al., 1998), HoxA3 (Chisaka andCapecchi, 1991), NF-1 (Brannan et al., 1994), NT-3 (Dono-an et al., 1996), ETA receptor (Clouthier et al., 1998), andax3 (Conway et al., 1997) genes affect the neural crest cellsnd cause defects in cardiogenesis. However, early embry-nic lethalities found in these cases may preclude thenalysis of the contribution of neural crest cells to cardio-enesis. Use of our P0-Cre lines in conjunction with aoxed targeted allele of those genes should facilitate thenalysis of this subject, and one such project is currentlynder way. Studies of this sort should shed light on thenvolvement of cardiac crest cells in the cardiac morpho-enesis as well as in the pathology of human congenitaleart diseases such as CATCH 22 (Wilson et al., 1993) andelocardiofacial syndrome, known to be related to abnor-al development of the cardiac crest (Shprintzen et al.,

978).The enteric nervous system (ENS) has been found, in

vian embryos, to be of the vagal crest origin (Le Douarinnd Teillet, 1973; Allan and Newgreen, 1980). Doubleabeling with anti-HoxB5, a marker for murine entericanglia (Wall et al., 1992; Kuratani and Wall, 1992), and-gal clearly showed double-positive cells in the ENS ofmbryos derived from P0-Cre 3 CAG-CAT-Z mating, sup-orting the assumption that the vagal crest-derived cellsontribute to the mammalian enteric ganglia as well.In addition to lineage analysis, combination of the P0-Creice and other transgenic mice should facilitate many

nteresting experiments which were previously impossibleo perform in mice. Taking advantage of the reporterxpression, it is possible to isolate a pure population ofeural crest cells and their derivatives (Abe et al., 1996)uring the entire course of the differentiation processes.
hese purified cells may be subjected to direct character-
zation at either the cellular or the molecular level, includ-


ng transplantation of the cells into mutant animals orther species or construction of cDNA libraries from eachtage. Also, instead of using the reporter gene, any gene cane inserted between the two loxP sequences, thereby en-bling a directed expression of genes of interest in theeural crest cell lineage. Generation of transgenic linesarrying genes involved in cell-signaling or gene expressionegulation is currently under way. Also, the P0-Cre line iseing utilized to achieve neural crest cell-specific geneisruption experiments. We believe that our transgenicines involving the Cre-loxP system should be useful forunctional analysis of genes of interest in specific cell types,nd its use will open up a new field in the biology of neuralrest cell development in mammals.

ACKNOWLEDGMENTS

The authors thank Drs. T. Kaname, K. Araki, and T. Sekimotofor their invaluable advice on neural crest cell culture techniquesand whole-mount in situ hybridization. The expert technicalassistance of Mrs. Kawasaki was gratefully acknowledged. Thiswork was supported in part by Grants-in Aid from the Ministry ofEducation, Science and Culture; the Science and TechnologyAgency; the Yamanouchi Foundation for Research on MetabolicDisorders; and the Osaka Foundation for Promotion of ClinicalImmunology.

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Received for publication November 18, 1998
Revised March 23, 1999Accepted April 19, 1999

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A Novel Transgenic Technique That Allows Specific Marking ... · strated that a subset of migrating neural crest cells and a wide variety of cells in the neural crest cell lineage