The Role of Early Lineage in GABAergicand Glutamatergic Cell Fate

Determination in Xenopus laevis

MEI LI, CONOR W. SIPE, KRISTINA HOKE, LISA L. AUGUST,

MELISSA A. WRIGHT, AND MARGARET S. SAHA*

Department of Biology, College of William and Mary, Williamsburg, Virginia 23187

ABSTRACTProper functioning of the adult nervous system is critically dependent on neurons adopting

the correct neurotransmitter phenotype during early development. Whereas the importance ofcell-cell communication in fate determination is well documented for a number of neurotrans-mitter phenotypes, the contributions made by early lineage to this process remain less clear. Thisis particularly true for �-aminobutyric acid (GABA)ergic and glutamatergic neurons, which arepresent as the most abundant inhibitory and excitatory neurons, respectively, in the centralnervous system of all vertebrates. In the present study, we have investigated the role of earlylineage in the determination of these two neurotransmitter phenotypes by constructing a fatemap of GABAergic and glutamatergic neurons for the 32-cell stage Xenopus embryo with the goalof determining whether early lineage influences the acquisition of these two neurotransmitterphenotypes. To examine these phenotypes, we have cloned xGAT-1, a molecular marker for theGABAergic phenotype in Xenopus, and described its expression pattern over the course ofdevelopment. Although we have identified isolated examples of a blastomere imparting a statis-tically significant bias, when taken together, our results suggest that blastomere lineage does notimpart a widespread bias for subsequent GABAergic or glutamatergic fate determination. Inaddition, the fate map presented here suggests a general dorsal-anterior to ventral-posteriorpatterning progression of the nervous system for the 32-cell stage Xenopus embryo. J. Comp.Neurol. 495:645–657, 2006. © 2006 Wiley-Liss, Inc.

Indexing terms: GABAergic; glutamatergic; blastomere; lineage; cell fate; fate map

During the development of the vertebrate central nervoussystem (CNS), a diverse array of neural cell types arises froma field of undifferentiated progenitor cells. The process bywhich a cell acquires a given phenotype and the degree towhich extracellular environment and lineage contribute to adifferentiated phenotype remain central issues currently fac-ing developmental neurobiology (Howard, 2005).

Classically, neurobiologists have divided the interac-tions leading to a differentiated phenotype into two gen-eral categories: those originating from outside the cell, or,conversely, those within the cell itself (Edlund and Jessell,1999). Extrinsic effectors arise from cell-cell interactionsvia direct contact or signaling through diffusible ligandsand have been implicated in nearly every aspect of neuraldetermination studied to date (Helms and Johnson, 2003;Pearson and Doe, 2004; Vanderhaeghen and Polleux,2004; Yang, 2004). Although it is clear that extrinsic fac-tors play the major role in neural fate determination, thecontribution of cell lineages, particularly those estab-lished early in development, to a final neural phenotyperemains unresolved. Studies in the invertebrate CNS haverevealed several mechanisms by which lineage controlscell fate, including conferring different phenotypes to

progeny according to birth order, or the asymmetric dis-tribution of signaling receptors to progeny (Martindaleand Shankland, 1990; Shankland, 1995; Zhong, 2003).

Grant sponsor: National Science Foundation; Grant number: 0088911;Grant sponsor: Commonwealth of Virginia Technology Research Fund;Grant sponsor: Jeffress Memorial Trust; Grant number: J-686.

The first two authors contributed equally to this work.The nucleotide sequence reported in this paper has been submitted to the

GenBank with accession no. AY904365.Dr. Li’s current address is Department of Cardiovascular Medicine,

Johns Hopkins University, Baltimore, MD 21205.Dr. Hoke’s current address is Laboratory for Cell Biology and Genetics,

The Rockefeller University, New York, NY 10021.Dr. Wright’s current address is University of Colorado School of Medi-

cine, Denver, CO 80206.*Correspondence to: Margaret S. Saha, Department of Biology, College

of William and Mary, P.O. Box 8795, Williamsburg, VA 23187.E-mail: mssaha@wm.edu

Received 18 July 2005;Revised 13 September 2005; 25 October 2005.DOI 10.1002/cne.20900Published online in Wiley InterScience (www.interscience.wiley.com).

THE JOURNAL OF COMPARATIVE NEUROLOGY 495:645–657 (2006)

© 2006 WILEY-LISS, INC.

Whether these results will prove applicable to thehighly complex vertebrate nervous system is uncertain,but several lines of evidence have suggested a role forearly cell lineage in acquiring a particular neural pheno-type. For example, Huang and Moody (1995) reported thatthe percentages of dopamine and neuropeptide Y ama-crine cells in the Xenopus retina produced by most blas-tomere progenitors was significantly different from thatpredicted by a blastomere’s overall contribution to theretina. Moreover, they demonstrated a marked asymme-try in the blastomere origins of these two cell types, whichlie in close proximity to one another in the inner nuclearlayer of the retina. A follow-up study revealed anotherinstance of early lineage bias in the development of threedifferent types of serotonin-containing amacrine cells inthe retina, with overlapping but distinct subsets of blas-tomere progenitors giving rise to each subtype (Huangand Moody, 1997). Quite clearly, extrinsic and intrinsicinteractions are not mutually exclusive, but rather act ina complementary fashion to influence a given cell’s fate atdifferent stages of development. For instance, lineage maypreferentially place a given cell in proximity to localizedintercellular cues within a tissue that directly induce aspecific phenotype.

Whereas the acquisition of several neurotransmitter phe-notypes (e.g., serotonergic and catecholaminergic) is rela-tively well understood (Goridis and Rohrer, 2002; Pattyn etal., 2004), the overall processes of fate determination for thetwo most abundant neurotransmitter phenotypes in the ver-tebrate nervous system, �-aminobutyric acid (GABA) andglutamate, remain less certain. Nonetheless, several tran-scription factors have been reported to be involved in regu-lating the fate of these cell types. In mice lacking the pro-neural gene Mash1, a decrease in transcriptional activationleads to a loss of progenitor cells in the ventral telencepha-lon, resulting in fewer GABAergic interneurons in the cortex(Casarosa et al., 1999). A protein coexpressed with Mash1 inthe mouse brain, Heslike, is involved in regulating the tim-ing of GABAergic neurogenesis (Miyoshi et al., 2004). Inaddition, several other factors are implicated in the develop-ment of GABAergic neurons in specific areas of the CNS,including Nkx2.1, Dlx1/2, and Gsh2 (Sussel et al., 1999;Corbin et al., 2000; Letinic et al., 2002; Schuurmans andGuillemot, 2002). In comparison, less is known regarding thedetermination of glutamatergic neurons. Ectopic expressionof the homeobox gene Tlx3 is sufficient to repress GABAergicdifferentiation and induce the formation of glutamatergiccells in specific regions of the spinal cord (Logan et al., 1998;Cheng et al., 2004). To date, there has been no investigationinto the contribution of early lineage in the determination ofeither of these neurotransmitter phenotypes.

In the present study, we have constructed a fate map ofGABAergic and glutamatergic neurons for the 32-cellstage Xenopus embryo with the goal of determiningwhether early lineage influences the acquisition of thesetwo neurotransmitter phenotypes. The Xenopus embryoprovides an ideal experimental system for this work, as itcleaves in stereotypical patterns at blastomere stages,allowing the labeling of identical progenitors in a largenumber of animals (Huang and Moody, 1992). The descen-dants of each blastomere were examined for molecularmarkers of the GABAergic or glutamatergic phenotype,allowing us to test whether a given blastomere contributesa greater proportion of a neurotransmitter phenotypethan would be expected based on the blastomere’s overall

contribution to a given structure and whether a blas-tomere preferentially gives rise to one neurotransmitterphenotype over the other. To examine these phenotypes,we have used xVGlut1 to mark glutamatergic neurons(Gleason et al., 2003) and xGAT-1, a marker we havecloned and characterized to delineate the GABAergic phe-notype in Xenopus. Although we document several exam-ples of blastomere identity influencing phenotype in astatistically significant manner, our overall results sug-gest that blastomere lineage does not impart a widespreadbias for subsequent GABAergic or glutamatergic fate de-termination.

MATERIALS AND METHODS

Cloning of Xenopus GABA transporter-1cDNA

A 0.7-kb fragment of the P. leucopus GABA transporter1 (GAT-1) gene (kind gift of E. Bradley) was radiolabeledand used to screen a Xenopus laevis tadpole brain cDNAlibrary in �ZAPII (kind gift of I. Dawid). Plasmid DNAwas isolated from positive clones according to the manu-facturer’s protocols and sequenced. A 2,451-bp fragment ofthe Xenopus GAT-1 cDNA (GenBank accession no.AY904365) was cloned into pBluescript SK� (Stratagene,La Jolla, CA) to produce the pxGAT1 plasmid used insubsequent procedures.

In situ hybridization (ISH)

Xenopus xGAT-1 RNA probes were transcribed from px-GAT1 and labeled with digoxigenin-UTP. To generate thexGAT-1 antisense probe, pxGAT1 was linearized withBamHI and transcribed with T7 RNA polymerase; the senseprobe was generated by linearizing pxGAT1 with HindIIIand transcribing with T3 RNA polymerase. An 836-bp frag-ment of the Xenopus glutamic acid decarboxylase gene(GAD; GenBank accession no. U38225, bases 454–1,289)was amplified by polymerase chain reaction (PCR) andcloned into pCRII-TOPO for use as an additional marker forGABAergic neurons. To generate the GAD antisense probe,this plasmid was linearized with NotI and transcribed withSP6 RNA polymerase. A 1,638-bp fragment of the Xenopusvesicular glutamate transporter 1 (xVGlut1; GenBank acces-sion no. AF548627, bases 104–1,741) was used as a markerfor glutamatergic neurons in fate mapping experiments(Gleason et al., 2003). Whole mount in situ hybridizationwas performed on staged embryos (Nieuwkoop and Faber,1967) as described in Sive et al. (2000) with modifications asdescribed in Sipe et al. (2004). For histological analysis,embryos were embedded in paraffin, cut into 10-�m sections,and mounted on microscope slides for visualization with anOlympus I � 50 inverted microscope. Images were capturedby using an Evolution MP digital camera (Media Cybernet-ics, Silver Spring, MD) and processed in Adobe (San Jose,CA) Photoshop to adjust brightness and contrast.

Real-time RT-PCR analysis

Real-time PCR primer sets were designed by using Bea-conDesigner 3.0 (Premier Biosoft, Palo Alto, CA) to am-plify short segments of the coding region of the XenopusxGAT-1 (bases 549–677) and histone H4 (GenBank acces-sion X00224; bases 469–585) genes. The authenticity ofthe PCR products generated by these primers was verifiedby melt curve analysis and agarose gel electrophoresis. A

646 M. LI ET AL.

standard curve was generated for both primer sets toensure that PCR efficiencies for both amplicons were nearoptimal. Total RNA from various developmental stages ofXenopus was isolated by using the Qiagen (Chatsworth,CA) RNeasy kit according to the manufacturer’s instruc-tions. cDNA was synthesized from 1 �g total RNA byusing the Bio-Rad (Hercules, CA) iScript kit according tothe manufacturer’s protocols, purified by using a Qiaquickpurification column (Qiagen), and quantified by using aNanodrop spectrophotometer (Nanodrop, Wilmington,DE).

Each 50 �l PCR reaction contained 20 ng of cDNAtemplate, 300 nM forward and reverse primer, and 1� iQSYBR Green Supermix (Bio-Rad). Reactions were cycledin an iCycler iQ system (Bio-Rad) as follows: 95°C for 5minutes, 40 cycles of 95°C for 15 seconds, 60°C for 30seconds, and 72°C for 1 minute. xGAT-1 and histone H4expression levels for a given developmental stage weremeasured in triplicate. Samples lacking reverse transcrip-tase treatment were routinely run to ensure a lack ofgenomic DNA contamination. The resulting CT valueswere normalized to control for cDNA template amountand analyzed for changes in xGAT expression levels rela-tive to the invariable histone H4 control by using the��CT method (Livak and Schmittgen, 2001).

Embryos and microinjection

Embryos used for microinjection were obtained fromhuman chorionic gonadotropin (HCG)-induced naturalmatings as described in Sive et al. (2000). Fertilized eggswere collected, dejellied in a 2% cysteine solution, andmaintained in 0.1� Marc’s Modified Ringers (MMR). Blas-tomere identity was determined by pigmentation differ-ences between the sperm entry point and the location ofthe future Spemann organizer. Those embryos with regu-lar cleavage planes and obvious pigmentation differenceswere selected at the 16- to 32-cell stage and placed in 0.5�MMR with 6% Ficoll for 10 minutes prior to injection.Blastomeres were identified according to the nomencla-ture of Nakamura and Kishiyama (1971) (Fig. 1).

At the 32-cell stage, one blastomere from each embryowas injected with 2.3 nl of 0.5% tetramethylrhodamine-linked dextran (RLDX; Molecular Probes, Eugene, OR) forlineage tracing. Following injection, the embryos wereexamined under a fluorescent microscope to confirm theinjection of the tracer into the proper blastomere. Em-bryos that showed leakage, blebbing, cell death, or irreg-ular cleavage patterns were discarded. After 3 hours, em-bryos were transferred to 0.1� MMR with 6% Ficoll andmaintained at 16°C overnight. The next morning, injectedembryos were transferred to 0.1� MMR and allowed togrow to hatching stages (stage 32–34), when they werefixed in 1� MEMFA and stored in 100% ethanol untilprocessing with in situ hybridization for xGAT-1 or xV-Glut1 signal.

Fate map data analysis

To visualize RLDX and ISH signal, tissue sections wereexamined on an Olympus I � 50 microscope at 40� or100� using a fluorescent filter with sufficient brightfieldillumination to detect ISH signal. Using an optical micro-meter, measurements were made to determine the per-centage of tissue in a structure contributed by each blas-tomere to the forebrain, midbrain, hindbrain, spinal cord,olfactory placodes, pineal gland, lens, notochord, and cra-

nial ganglia. For each embryo, estimations of the levels ofRLDX labeling for each of the above structures were re-corded by using the following notation: ���, �50% of agiven structure was labeled; ��, 26–50% was labeled; �,0–25% was labeled; �, 0% was labeled. The embryos werethen examined to determine the approximate percentageof RLDX-labeled tissue displaying overlap with ISH signalaccording to the following scale: ���, �50% of a givenregion or structure displayed overlap; ��, 26–50% dis-played overlap; �, 0–25% displayed overlap; �, 0% dis-played overlap. Full data tables for individual embryosand structures are presented in supplementary material.Because regions of overlap were irregularly shaped andmicrometer measurements were therefore not precise,these measurements remain semiquantitative. To calcu-late both the average contribution to a structure and theaverage percentage of RLDX and ISH overlap for eachblastomere, the measurements collected from each in-jected embryo were averaged together by using the follow-ing formula: [75 � (no. ��� embryos)] � [37.5 � (no. ��embryos)] � [12.5 � (no. � embryos)]/(total no. embryosanalyzed for a given blastomere).The median values of the approximate range of fluores-cence or overlap for a structure (i.e., 75, 37.5, or 12.5%)from individual embryos were added together and dividedby the total number of embryos, yielding an overall aver-age percentage of RLDX fluorescence or ISH overlap foreach structure. These summary data are presented inTables 1 and 2 and were used to construct Figures 5 and7. An analysis of covariance/comparison of slopes test (us-ing the R software package) was used to test whetherblastomere identity imparts a statistically significant biasto phenotype determination.

Fig. 1. Schematic of the blastomeres of a 32-cell stage Xenopusembryo with each cell labeled according to Nakamura and Kishiyama(1971).

647EARLY LINEAGE IN GABAergic AND GLUTAMATERGIC DETERMINATION

RESULTS

Cloning of Xenopus GABA transporter 1(GAT-1) cDNA

Screening of a Xenopus laevis tadpole brain cDNA li-brary (2.5 � 105 pfu) produced a single hybridizing plaquecontaining a 2,451-bp cDNA clone (GenBank accession no.AY904365), including a 1,799-bp open reading frame thatdisplayed significant identity to the coding regions fromother GAT-1 cDNAs. At the nucleotide level, this clonewas most similar to GAT-1 sequences in rat (81% iden-tity), mouse (81%), and human (80%). The deduced aminoacid sequence predicts a protein of 599 amino acids thatshows 88% identity and 94% similarity to GAT-1 in themammalian species. There is a particularly high degree ofsimilarity (97%) between the Xenopus clone and mamma-lian GAT-1 sequences within 12 highly conserved trans-membrane domains. This degree of identity on both thenucleotide and amino acid levels has led us to designateour clone as xGAT-1.

Developmental expression pattern of xGAT-1

Real-time PCR was used to analyze the temporal ex-pression pattern of xGAT-1 during Xenopus development(Fig. 2). The results obtained from PCR were confirmed inall cases by ribonuclease protection assay (data notshown). Transcripts were first detected at low levels dur-ing late neurula stages. A sharp increase in xGAT-1 ex-pression is observed during tailbud stages, at which levelsreach approximately five times those found at neurulastages. Transcript levels continue to increase dramaticallythrough swimming tadpole stages and into adulthood withlevels in the adult brain fivefold higher than those presentin tailbud stage embryos.

The spatial expression pattern of xGAT-1 within thedeveloping embryo was examined by whole mount in situhybridization by using an antisense RNA probe consistingof a 2,451-bp xGAT-1 cDNA fragment. xGAT-1 expressionis first observed at late neurula stages in the anteriorportion of the developing spinal cord (Fig. 3A). In earlyneural tube stage embryos, xGAT-1 signal has extendedcaudally in the spinal cord and is first observed in thehindbrain and forebrain (Fig. 3B). By tailbud stages, bi-lateral xGAT-1 expression is apparent in all regions of theCNS, including distinct regions of the forebrain, midbrain,hindbrain, and olfactory placodes (Fig. 3C,D,H). A similarpattern is present in larval stage embryos, with xGAT-1signal extending the full length of the spinal cord (Fig.3E,G). Consistent with real-time PCR analysis, xGAT-1mRNA levels continue to increase throughout the CNS inolder swimming tadpoles (Fig. 3F). The xGAT-1 expres-sion pattern is nearly indistinguishable from that of GAD,a marker for presumptive GABAergic neurons, in allstages examined (Fig. 3, insets; see in particular 3H), butxGAT-1 appears to be more abundant.

To examine the xGAT-1 expression pattern in greaterdetail, embryos hybridized with the antisense probe weresubjected to histological analysis. Near the anteriormostaspect of the eye, xGAT-1 is expressed in lateral regions ofthe forebrain midway down its dorsal-ventral axis (Fig.4A), whereas more posterior, in the midbrain, xGAT-1expression is located more ventrally (Fig. 4B). Apparentstaining of tissue immediately adjacent to the centralcanal suggests that some cells contacting the neurocoelexpress xGAT-1 (Fig. 4B, right inset). Expression in the

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649EARLY LINEAGE IN GABAergic AND GLUTAMATERGIC DETERMINATION

anterior hindbrain (at the level of the otic vesicles) be-comes more intermediate and lateral again (Fig. 4C,D),and this pattern is maintained in the hindbrain and an-terior spinal cord (Fig. 4E,F). xGAT-1 expression in theanterior portion of the spinal cord becomes confined to amore distinct area containing presumptive interneurons(Fig. 3G). More posterior sections of the spinal cord dem-onstrate xGAT-1 signal present in the ventral regionsbordering the neurocoel (Fig. 4H) that are populated bymotor neurons and GABA-immunoreactive Kolmer-Agduhr cells (Dale et al., 1987). These patterns remainconsistent in older swimming tadpole stage embryos, withoverall xGAT-1 expression increasing dramatically withage. In addition, xGAT-1 signal intensifies rapidly in theretina at older stages (Fig. 4I).

Histological analysis of GAD expression reveals a par-allel expression pattern with xGAT-1, although somewhatweaker in most regions (Fig. 4, insets for equivalent stagesand regions). An exception is in the retina, where GADexpression is detectable prior to xGAT-1 during tailbudstages (Fig. 4B, inset). Complementary xGAT-1 and GADsense probes were used as a negative control and in nocase showed any staining above background levels (datanot shown).

Lineage analysis

To examine the role of early lineage relationships in thedetermination of the GABAergic and glutamatergic phe-notypes, we have constructed a fate map of the 32-cellstage Xenopus embryo for GABAergic and glutamatergicneurons using the markers xGAT-1 and xVGlut1 (Fig. 5).An estimated percentage of overlap between RLDX andthe in situ hybridization signal of either xGAT-1 or xV-Glut1 in a given structure was used to analyze the fre-quency with which a given blastomere contributed toGABAergic or glutamatergic neurons in the developing

nervous system (Fig. 6). Summaries for each region of theCNS, as well as for the cranial ganglia, are presentedgraphically on the blastomere diagrams in Figure 7 toshow the spatial disposition of the prospective regions inthe 32-cell stage embryo.

Lineage of GABAergic neurons

In the forebrain, the majority of GABAergic neurons aredescended from blastomeres A1 and B1, although A1 con-tributes a relatively smaller proportion of cells (Table 1,Fig. 5A). Blastomeres A2, B2, and C2 also served as pro-genitors to GABAergic neurons, albeit at lower frequency.Midbrain progenitors are similar to those for the fore-brain, with A1 and B1 contributing the majority ofGABAergic neurons. Likewise, A2, B2, C2, and A3 alsocontribute a relatively smaller proportion of cells. Theanteriormost progeny of C1 also give rise to xGAT-1 neu-rons in the ventral midbrain region in 20% of injectedembryos. Hindbrain GABAergic neurons consistently de-rive from three blastomeres: 90% of A1, 75% of B1, and58% of A2 injected blastomeres gave rise to progeny inthese regions. Blastomere B2 also makes a significantcontribution to intermediate hindbrain regions, whereasC1 and C2 give rise to small portions of both regions.Blastomere A3 contributed to the intermediate hindbrainin 80% of injected embryos, although it provided an overallfewer number of cells than major contributors.

In the spinal cord, the major progenitor of GABAergicneurons in intermediate regions is B2, with 80% of in-jected blastomeres giving rise to this tissue. BlastomeresA1, B1, A2, and A3 also consistently provided from 10 to25% of GABAergic neurons in the intermediate spinalcord, and C1, C2, B3, and C3 also contributed a smallnumber of cells at relatively lower frequencies (Table 1).The majority of ventral spinal cord GABAergic neuronsare derivatives of blastomeres A1 and B1, with 80 and60% of injected embryos, respectively, displaying RLDXsignal overlap with xGAT-1 expression. Relatively fewernumbers of GABAergic cells in the ventral spinal cord arederived from A2, B2, and D2. In 60% of A3-injected em-bryos, GABAergic cells were present in the olfactory pla-codes. No xGAT-1-expressing cells were observed in thepineal gland, cranial nerves, lens, or notochord.

Lineage of glutamatergic neurons

Glutamatergic neurons in the forebrain descend largelyfrom the blastomeres A1, B1, A2, and B2 (Table 2, Fig.5B). Of these blastomeres, A1 contributes the greatestproportion of xVGlut1-expressing cells to the dorsal andintermediate forebrain in 80% of the embryos examined.Blastomeres A3 and B3 also give rise to a small percent-age of cells in the dorsal and intermediate forebrain. Theventral forebrain regions are populated most heavily bydescendents of B1, with A1 and B2 contributing cells in 40and 10% of injected embryos, respectively. The majority ofglutamatergic neurons in the midbrain are derived from asimilar set of blastomeres; 100% of A1, 90% of B1, 80% ofB2, and 67% of A2 injected blastomeres gave rise to prog-eny in the intermediate midbrain, whereas ventral re-gions are composed of cells descending solely from A1, B1,and A2. Trace numbers of cells were contributed to theintermediate midbrain by C2 and B3 in a small number ofembryos. The major progenitors of hindbrain glutamater-gic neurons are B1 and A2, which consistently contribute26–50% of intermediate tissue. Additionally, 90% of A1

Fig. 2. Temporal analysis of xGAT-1 expression during Xenopusdevelopment. The graph shows the mean fold change in xGAT-1mRNA expression for the stages indicated as measured using quan-titative real-time PCR. xGAT-1 expression is first detectable abovebackground during late neurula stages (stage 20) and increases dra-matically over the course of development. Threshold cycle values werenormalized to control for input template amount and analyzed asdescribed in Materials and Methods. ue, unfertilized egg.

650 M. LI ET AL.

and B2 injected blastomeres contributed progeny to theintermediate hindbrain. Approximately half of the em-bryos examined for these four blastomeres also gave riseto glutamatergic neurons in the ventral hindbrain. Blas-tomeres A3 and B3 gave rise to small numbers of gluta-matergic neurons in the intermediate hindbrain. Cellsdescended from C2, found in both the intermediate andventral hindbrain, also express xVGlut1.

Unlike other regions of the central nervous system,glutamatergic neurons in the spinal cord as a whole aredescended from a wide range of blastomeres; only D1,C3, A4, and D4 do not contribute xVGlut1-positive cellsto the spinal cord. No single blastomere contributed aclear majority of glutamatergic neurons to the spinalcord in any embryo examined. Blastomeres A2, B2, C2,

A3, B3, C3, and B4 all contribute to dorsal spinal cordtissue in 50% or more of injected embryos. Likewise, A1,B1, A2, B2, C2, and B3 contribute progeny to interme-diate spinal cord tissue in a majority of embryos. Mostglutamatergic neurons in the ventral spinal cord arisefrom a more restricted set of blastomeres, consisting ofA1, B1, A2, and B2.

The olfactory placodes, located anterior to the forebrain,are fated to become the mature olfactory epithelium. Themajority of glutamatergic neurons in this region are de-scended from blastomeres A1 and A2, contributing 26–50% of this tissue in more than 75% of all embryos exam-ined (Table 2). Blastomeres B1, B2, and A3 alsocontributed a relatively smaller proportion of cells in ap-proximately half of the injected embryos.

Fig. 3. Whole mount xGAT-1 expression in the developing Xeno-pus embryo. Insets show a stage-matched embryo hybridized with anantisense GAD probe. A: xGAT-1 expression is first detected in thespinal cord during late neurula stages (stage 21). B: xGAT-1 signal ispresent in the hindbrain and forebrain by stage 23. C: By stage 26,expression increases in the spinal cord and can be observed in allparts of the developing brain. D–F: xGAT-1 expression continues tostrengthen during tailbud stages (D, stage 28; E, stage 32), and by

swimming tadpole stages (F, stage 40) is present at high levelsthroughout the central nervous system and eye. G: Higher magnifi-cation of the head of a stage 33 embryo showing distinct domains ofxGAT-1 expression in the forebrain, midbrain, and hindbrain. H: Dor-sal view of a tailbud stage embryo (stage 32). fb, forebrain; hb, hind-brain; mb, midbrain; ol, olfactory placodes; sc, spinal cord. Scale bar 1 mm in A–H.

651EARLY LINEAGE IN GABAergic AND GLUTAMATERGIC DETERMINATION

Fig. 4. Histological analysis of xGAT-1 mRNA expression. A–H:Serial (anterior to posterior) 10-�m transverse sections of a larvalstage embryo (stage 34). Left insets show a stage-matched embryohybridized with the antisense GAD probe. A: Anteriormost xGAT-1signal is concentrated in the lateral regions of the forebrain. B: In themidbrain, expression is found in more ventral regions. Higher mag-nification (right inset) demonstrates that xGAT-1 signal is presentimmediately adjacent to the neurocoel. C,D: At the level of the hind-brain, xGAT-1 signal is again concentrated in lateral regions midway

down the dorsal-ventral axis of the structure. E,F: This expressionpattern is maintained in the anterior spinal cord. G,H: xGAT-1mRNA becomes localized to more distinct regions of the spinal cordcontaining Kolmer-Agduhr cells and presumptive interneurons. I: Byswimming tadpole stages, xGAT-1 mRNA expression has increaseddramatically throughout the brain, and is detectable in the retina ofthe eye. Arrows indicate areas of xGAT-1 mRNA expression. asterisk,otic vesicle; cg, cement gland; e, eye; gu, gut; no, notochord. Scalebar 0.5 mm in A–I.

652 M. LI ET AL.

Glutamatergic neurons in the pineal gland, another an-terior neural structure closely associated with the fore-brain, share a similar array of progenitors with the olfac-tory placodes; A1 and A2 contribute 26–50% ofglutamatergic pineal tissue in 100% of injected embryos.

Sixty percent of B1, 60% of B2, and 80% of A3 injectedblastomeres supplied 10–25% of glutamatergic tissue inthe gland.

Glutamatergic neurons in the cranial ganglia all gener-ally arise from the same set of blastomeres: only D3, B4,

Fig. 5. Summary of GABAergic and glutamatergic fate mappingexperiments. Blastomere contributions to GABAergic (A) or glutama-tergic (B) cells in specific regions of the nervous system. Major regionsof the CNS are denoted in black as follows: FB, forebrain; MB, mid-brain; HB, hindbrain; SC, spinal cord. The specific regions of theforebrain, midbrain, hindbrain, and spinal cord examined for expres-sion of xGAT-1 or xVGlut1 are indicated by lower case letters: d,dorsal; i, intermediate; v, ventral. The peripheral structures exam-

ined include the olfactory placodes (OL), the pineal gland (PG), andthe cranial ganglia: V, trigeminal; VII, geniculate; VIII, acousticus;IX, glossopharyngeal. The approximate percentage of GABAergic orglutamatergic tissue in a specific CNS region or peripheral structurecontributed by a given blastomere is indicated as follows: boldfacetype, 26–50%; regular type, 10–25%; white type with black outline,0–10%.

Fig. 6. Colocalization of xGAT-1 and xVGlut1 signal with RLDX.Representative sections of a B1-injected embryo demonstrating colo-calization of RLDX with xGAT-1 (A–D) or xVGlut1 (E–H) in situhybridization signals in the hindbrain. A,E: Brightfield images ofxGAT-1 or xVGlut1 expression with arrowheads indicating ISH stain-ing. B,F: RLDX fluorescence distribution in the same section. C,G:

Overlay of the brightfield and fluorescent images reveals overlap ofISH signal with RLDX. D,H: A false-color merged image showing ISHsignal (blue), RLDX-labeled tissue (red), and colocalization of xGAT-1or xVGlut1 with RLDX (yellow). Scale bar 0.25 mm in A (applies toA–D), E (applies to E–H).

653EARLY LINEAGE IN GABAergic AND GLUTAMATERGIC DETERMINATION

D4, and C4 do not contribute to these structures. Thetrigeminal (V) and geniculate (VII) ganglia, which lie inclose proximity to one another, receive a majority of glu-tamatergic cells from A1, B1, C1, A2, B2, and A3, which

contribute progeny in 100% of the embryos examined.These blastomeres also give rise to xVGlut1-positive neu-rons in the acousticus (VIII) nerve in a majority of em-bryos, although only A2 and B2 contribute over 25% oftissue in this structure. Likewise, B2 contributes the larg-est proportion of tissue to the glossopharyngeal (IX) nerve.A single blastomere, D1, contributes a trace amount oftissue to nerves V and VII but not VIII and IX. Consistentwith previous findings, xVGlut1 expression was not ob-served in the lens or notochord.

DISCUSSION

GAT-1 is highly conserved among species

A pairwise comparison of xGAT-1 with other GAT-1sequences demonstrates that it is highly conserved acrossa range of vertebrate and invertebrate species. We haveidentified a partial coding sequence having 97% identity toxGAT-1 in the genome of the sister species, Xenopus tropi-calis (assembly 3.0). Two zebrafish expressed sequencetags (ESTs; GenBank accession nos. CK707536 andCK362328) were aligned to produce a predicted open read-ing frame encoding a protein with 82% identity to xGAT-1,and a recently reported invertebrate homolog in Caeno-rhabditis elegans, snf-11, has 70% similarity to xGAT-1(Jiang et al., 2005). A putative sodium neurotransmittersymporter (snf) domain, common to all reported GAT-1sequences, was identified in xGAT-1 by using CD-search(Marchler-Bauer and Bryant, 2004). Likewise, the posi-tions of three residues in this domain that have beendemonstrated in humans to be involved in the binding ofNa� (Trp68), Cl� (Arg69), and GABA (Tyr140) during neu-rotransmitter transport are located at identical positionsin xGAT-1 (Chen et al., 2004).

xGAT-1 marks GABAergic neurons

We first detected xGAT-1 mRNA expression at late neu-rula stages. This is somewhat earlier than has been pre-viously reported for mammalian GAT-1, which is first seenin the ventral mesencephalon in mice at E12 (Perrone-Capano et al., 1994), although this discrepancy may bedue to the increased sensitivity of our real-time PCR as-say. The observed timing of xGAT-1 mRNA expression isconsistent with the appearance of GABA-immunoreactiveneurons in Xenopus hindbrain by stage 25 (Roberts et al.,1987) and GABA uptake activity in spinal interneuronsaround the same time (Lamborghini and Iles, 1985). Theonset of xGAT-1 expression is also consistent with severalreports indicating that the capability for GABA accumu-lation develops before phenotype maturation (Hollyfield etal., 1979; Lam et al., 1980).

Fig. 7. Summary of GABAergic and glutamatergic fate mappingexperiments by structure and blastomere. Blastomere contributionsto tissue expressing either xGAT-1 (left column) or xVGlut1 (rightcolumn) in specific regions of the nervous system. The approximatepercentage of GABAergic or glutamatergic tissue in a specific struc-ture contributed by a given blastomere is denoted by shades of gray,with darker shades representing a higher percentage contribution.These percentages were calculated by averaging the contributions todorsal, intermediate, and ventral regions (or, in the case of the cranialganglia, each nerve) as described in Materials and Methods. FB,forebrain; MB, midbrain; HB, hindbrain; SC, spinal cord; CG, cranialganglia.

654 M. LI ET AL.

As previously described in the mouse, xGAT-1 expres-sion is restricted to the CNS and is present along the fulllength of the neural tube (Jursky et al., 1994). The strongmedial-ventral pattern of xGAT-1 expression in the devel-oping midbrain is consistent with reports that severalGABAergic components in the rodent brain are concen-trated in periventricular regions (Evans et al., 1996).Many investigators have previously employed GAD, a keycomponent of the GABA synthesizing pathway, to markGABAergic neurons (Wuenschell et al., 1986). Evidencesuggests that GAD and GAT-1, on both the protein andmRNA levels, are found in similar cell populations in thepostnatal rat brain and can be co-expressed in identicalneurons (Augood et al., 1995; Yasumi et al., 1997; Frahmand Draguhn, 2001). However, to date there has been nodirect study of this phenomenon in the embryonic CNS.Our results demonstrate that GAD and xGAT-1 mRNAsare confined to the same populations of cells in the devel-oping Xenopus CNS, although xGAT-1 ISH signal is con-sistently stronger. Given this finding, and the fact thatGAD is known to be expressed in a number of non-neuraltissues (Maddox and Condie, 2001; Geigerseder et al.,2003), xGAT-1 could serve as an alternative molecularmarker of neural GABAergic cell populations.

Comparison with previous fate maps

In order to construct and interpret a fate map forGABAergic and glutamatergic neurons in the 32-cell stageXenopus embryo, the origins of the CNS and peripheralstructures were determined by lineage labeling. Thesedata were systematically compared with that of two pre-viously published fate maps for the 32-cell Xenopus em-bryo (Dale and Slack, 1987; Moody, 1987). Overall, ourresults are consistent with these studies and show thatindividual structures in the nervous system consistentlyderive from multiple blastomeres and that different blas-tomeres preferentially give rise to specific structures. Forexample, all three studies agree that the blastomeres onthe future organizer side of the embryo, particularly A1and B1, give rise to a majority of brain tissue and that theorigins of the spinal cord are more varied, with heavycontributions from A2, B2, and C2.

In nearly all cases, our data were directly comparablewith that of Moody (1987) in terms of both the relativeamount of a given blastomere’s contribution as well as thenumber of injected embryos in which the contribution wasobserved. For instance, we demonstrate that the interme-diate and ventral regions of the forebrain are descendednearly exclusively from A1 (D1.1.1 in Moody’s nomencla-ture), B1 (D1.1.2), A2 (D1.2.1), and B2 (D1.2.2), whereasthe dorsal forebrain has a wider array of progenitors,including C2 (D2.2.2), A3 (V1.2.1), and B3 (V1.2.2); Moodyreports a similar origin for the forebrain and includes aminor contribution by A3 (V1.2.1) to the dorsal forebrain.Thus, the two sets of results are consistent and evenmirror the relatively small contribution of blastomere A3.

In contrast, there are a number of key differences be-tween the data obtained in the present study and the fatemap of Dale and Slack (1987). We believe that these dis-crepancies have led to an overall underestimation of thecontributions of third- and fourth- column blastomeres tothe central and peripheral nervous systems. For instance,Dale and Slack report that A3 and B3 contribute 2 and 0%of the total volume of brain tissue, respectively, and, in-stead, first contribute progeny in the posterior hindbrain

(A3) or in the anterior spinal cord (B3). In contrast, ourfindings, along with those of Moody (1987), demonstratethat these blastomeres contribute a significant portion oftissue in the dorsal forebrain, the hindbrain, and, in thecase of A3, the pineal gland. Likewise, Dale and Slack findno contribution of A4 to neural tissue (at least by stage30), whereas we find that this blastomere contributes asignificant portion of tissue to the cranial ganglia. Thereasons for these discrepancies are not clear, although itshould be noted that Dale and Slack’s analysis examinedembryos younger (stage 30) than those used in the presentstudy and did not focus on the developing nervous system.

The cranial ganglia show two distinctpatterns of origin

We also present the first complete fate map of the tri-geminal (V), geniculate (VII), acousticus (VIII), and glos-sopharyngeal (IX) nerves by using a molecular marker inXenopus. The cranial ganglia, which innervate many pe-ripheral sensory structures, are descended from a heter-ogenous collection of blastomeres, with all first- andsecond-column blastomeres (organizer side) and half ofthe third- and fourth-column blastomeres (contra-organizer side) contributing progeny. These results areremarkably consistent with those reported by Moody(1987), who examined the cranial ganglia as a singlestructure. One exception is the comparably reduced con-tribution of C3 that we observed, which is not surprisinggiven that we did not examine contributions to the entireset of cranial nerves. Our examination of each nerve sep-arately reveals two similar but different patterns of origin.The trigeminal and geniculate nerves descend from thesame set of blastomeres in roughly the same percentage ofembryos. Conversely, the acousticus and glossopharyn-geal nerves descend from a more confined set of progeni-tors, with a number of blastomeres contributing a rela-tively smaller amount of tissue and/or contributing infewer numbers of injected embryos (i.e., A1, C1, D2, andA3). The significance of this division in lineage is notknown; however, the cranial ganglia may present an at-tractive region in which to investigate the role of lineagebias in the future.

Lineage bias

Using the 32-cell stage Xenopus embryo, we set out toinvestigate whether early lineage can bias the determina-tion of the GABAergic and glutamatergic phenotypes bypreferentially giving rise to descendants of one phenotypeover another in regions where both neurotransmitter phe-notypes are expressed. Previous studies of dopamine, se-rotonin, and neuropeptide Y amacrine cells in the Xenopusretina have shown that each phenotype is derived fromdistinct, but overlapping, groups of blastomere progeni-tors (Huang and Moody, 1995, 1997). We did not findevidence that such a bias exists in the GABAergic orglutamatergic lineages; in nearly all cases in which ablastomere gives rise to a region containing both neuro-transmitter phenotypes, blastomere identity did not exerta significant effect on phenotype choice. An isolated excep-tion is blastomere B2, which gives rise to a higher propor-tion of glutamatergic tissue (26–50%, compared with 10–25% GABAergic) in the intermediate forebrain andmidbrain in a higher percentage of injected embryos (P 0.001). Similarly, in the intermediate spinal cord, C2 givesrise to a relatively higher number of injected embryos that

655EARLY LINEAGE IN GABAergic AND GLUTAMATERGIC DETERMINATION

contribute a greater proportion of glutamatergic tissuewhen compared with its GABAergic contribution (P 0.001). These exceptions indicate that blastomere lineagecannot be definitively ruled out as a determinative factorfor phenotype, although in no case did a blastomere reli-ably give rise to one phenotype and not another.

Lineage bias can also be discerned when a given blas-tomere contributes a disproportionate amount of GABAer-gic or glutamatergic tissue compared with its total overallcontribution to a given region. This type of bias has beendemonstrated in the Xenopus retina, where specific blas-tomeres give rise to three serotonin subtypes in a mannernot correlated with the numerical and spatial distributionof each blastomere clone (Huang and Moody, 1997). Over-all, our data do not support such a widespread bias withineither the glutamatergic or GABAergic lineages. However,in the olfactory placodes, A3 contributes significantlymore GABAergic tissue than either A1 or B1, which makeno GABAergic contributions to this structure (P 0.19).Similarly, C2 contributes 10–25% of tissue in the dorsalforebrain but does not give rise to any glutamatergic neu-rons. In contrast, B1 contributes the same proportion oftissue to the dorsal forebrain but also produces glutama-tergic neurons in 60% of injected embryos, a highly signif-icant difference (P 0.001). Taken as a whole, theseresults do not implicate lineage bias as a major determi-native factor for neurotransmitter choice, although spe-cific blastomeres may have the ability to influence fatedetermination in some cases.

Beside the obvious hypothesis that early lineage doesnot have a measurable effect on the acquisition ofGABAergic or glutamatergic phenotypes, there are sev-eral possible explanations for the observed lack of stronglineage bias in either of the examined neurotransmitterphenotypes. The phenotypic effects of blastomere biasmay be too subtle to detect with the current experimentaldesign, and the examination of larger numbers of injectedembryos may be necessary to gain statistical power. More-over, we have examined neurons expressing xGAT-1 andxVGlut1, the principal transporters employed by GABAer-gic and glutamatergic neurons in the CNS. Several othermembers of the GABA and glutamate transporter familieshave been reported in mammals, and it remains a possi-bility that subpopulations within these neurotransmitterphenotypes are biased by early lineage. If different sub-types were produced by different subsets of progenitors,an analysis of the entire class, such as that performed inthe current study, would be unable to reveal fate bias(Moody et al., 2000).

Implications for axial patterning

In addition to addressing the question of glutamatergicand GABAergic phenotype acquisition, our results haveconsequences for axial patterning of the amphibian ner-vous system. Based largely on evidence gathered frommesodermal derivatives, a revised fate map has been pro-posed with a new rostral-caudal axis in the 32-cell Xeno-pus embryo essentially in place of the traditional dorsal-ventral axis, which was shifted 90° to overlap the animal-vegetal axis (Lane and Smith, 1999; Lane and Sheets,2000). In terms of the newly proposed rostral-caudal axis(former dorsal-ventral axis), the lack of a contribution byA3 and B3 to anterior neural structures reported by Daleand Slack was cited as support for a rostral-to-caudalpatterning progression in ectodermal derivatives (Lane

and Sheets, 2002). However, the fate mapping data ob-tained from the present study indicate that a rostral-caudal axis cannot be superimposed on the 32-cell stageembryo. We have shown that significant rostral portions ofthe nervous system are descended from third-column blas-tomeres (i.e., A3 and B3), including the dorsal forebrainand the pineal gland, which is definitively marked byxVGlut1. In addition, A4, a blastomere that would beexpected to contribute to caudalmost structures in therevised axial scheme, does not show any progeny in thespinal cord and instead is a major contributor to the cra-nial ganglia.

In terms of the newly proposed dorsal-ventral axis(former animal-vegetal axis), given that the CNS is adorsal structure, we obviously did not fate map any ven-tral tissues. However, the first-column blastomeres (par-ticularly A1 and B1) contribute to large tracts of the CNS,including more posterior spinal cord regions. Furtheralong the horizontal aspect of the embryo, second-columnblastomeres (i.e., A2, B2, C2) contribute less overall neu-ral tissue but still at relatively high levels. This trendcontinues to the third and fourth columns, where onlyslight contributions to the CNS are made by A4 and B4.Given these results, and that the majority of anterior CNSstructures are contributed by the A1-B1-B2-A2 quadrantof blastomeres, there appears to be a loose dorsal-anteriorto ventral-posterior patterning progression of the nervoussystem extending diagonally from A1 toward D4, similarto that proposed for lithium-treated embryos (Kao andElinson, 1988). Our results do not fully support either thenew or the traditional fate map. It may be more useful toemploy the terminology proposed by Kumano and Smith(2002)—animal-vegetal and organizer-contraorganizer—with the realization that the morphogenetic movementsthat occur later in development during gastrulation andneurulation make it impossible to project exact axes ontothe 32-cell stage embryo based on the final body plan ofthe adult nervous system.

In the present study, we report the cloning of XenopusGAT-1 and analyze its temporal and spatial expressionpattern. In addition, we have demonstrated that the de-termination of the GABAergic and glutamatergic neuralphenotypes is not strongly biased by early lineage. Al-though we cannot rule out the possibility of a more subtlebias imparted by additional blastomeres, it seems likelythat the potential to produce GABAergic or glutamatergicneurons is largely modulated via cell-cell interactions.This investigation forms a basis for further addressinghow a final neurotransmitter phenotype is acquired in thevertebrate central nervous system.

ACKNOWLEDGMENTS

The authors thank George Gilchrist for kind help withstatistical analyses and Eric Bradley for technical assis-tance with embryo microinjections.

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657EARLY LINEAGE IN GABAergic AND GLUTAMATERGIC DETERMINATION