Neuron, Vol. 19, 761–772, October, 1997, Copyright 1997 by Cell Press

Specification of Mouse Telencephalic andMid-Hindbrain Progenitors Following HeterotopicUltrasound-Guided Embryonic Transplantation

Martin Olsson,*†‡ Kenneth Campbell,*‡ experiments likely do not reveal the full developmentalpotential of these progenitors. In fact, transplantation ofand Daniel H. Turnbull*§

midgestationstriatal precursors into the forebrain ventricle*Skirball Institute of Biomolecular Medicineof similar stage embryonic hosts in utero has clearlyNew York University Medical Centerdemonstrated a greater developmental potential of theseNew York, New York 10016telencephalic progenitors than was previously observed†Wallenberg Neuroscience Centerby transplantation into neonatal or adult hosts (Fishell,Department of Physiology and Neuroscience1995; Brustle et al., 1995; Campbell et al., 1995). Further-University of Lundmore, recent studies have demonstrated that transplanta-Solvegatan 17tion into neonatal or adult brain regions that undergo pro-S-223 62 Lundtracted neurogenesis, such as the hippocampus or theSwedensubependymal zone, can result in the respecification ofneonatally or adult-derived neural progenitors (Vicario-Abejon et al., 1995; Suhonen et al., 1996).Summary

Although embryonic transplantation reveals a greaterdevelopmental potential of progenitor populations, tel-We have demonstrated the utility of ultrasound back-encephalic progenitors derived from the lateral gangli-scatter microscopy for targeted intraparenchymal in-onic eminence (LGE) were observed to preferentiallyjections into embryonic day (E) 13.5 mouse embryos.incorporate in regionally restricted patterns after injec-This system has been used to test the degree of com-tion into the embryonic forebrain ventricle (Campbell etmitment present in neural progenitors from the embry-al., 1995). These cells wereconsistently seen to integrateonic ventral telencephalon and mid-hindbrain region.homotopically into the striatum and, to a lesser extent,Many E13.5 ventral telencephalic progenitors were ob-heterotopically into distinct forebrain and midbrain nu-served to integrate and adopt local phenotypes follow-clei. The transplanted cells were also observed to un-ing heterotopic transplantation into telencephalic ordergo site-specific differentiation, whether incorporatedmid-hindbrain targets, whereas mid-hindbrain cells ofhomotopically or heterotopically into the telencephalonthe same stage were unable to integrate and change(Fishell, 1995; Brustle et al., 1995; Campbell et al., 1995).fate in the telencephalon. In contrast, many mid-hind-LGE progenitors appear to be positionally specified atbrain cells from an earlier developmental stage (E10.5)least in part by common adhesive mechanisms, sincewere capable of integrating and adopting a forebrainhomotopic incorporation of LGE cells requires intact cellphenotype after grafting into the telencephalon, sug-surface molecules (e.g., adhesion molecules), and theirgesting that mouse mid-hindbrain progenitors be-removal results in widespread incorporation (Olsson etcome restricted in their developmental potential be-al., 1997b). Not only do LGE cells incorporate with re-tween E10.5 and E13.5.gional specificity after transplantation into the forebrainventricle, but progenitors derived from the developingIntroductionmid-hindbrain region also display a consistent pattern ofintegration, with cellsobserved in posterior diencephalicTransplantation experiments offer a unique tool to in-and midbrain nuclei but rarely in the telencephalonvestigate the degree to which progenitors at certain de-(Campbell et al., 1995; Olsson et al., 1997b). Since cellsvelopmental stages are committed to generate specificinjected into the embryonic forebrain ventricle do notneuronal phenotypes, as well as the role that the localconsistently incorporate into all regions of the brain,environment plays in this process. Heterotopic trans-this transplantation model is likely to test the degree ofplantation of midgestation embryonic telencephalic pro-positional specification (e.g., adhesive properties) pres-genitors into adult hosts hasrevealed that the transplantedent in neural progenitors at different stages of develop-cells differentiate largely into phenotypes appropriatement more accurately than their true potential to respec-to the cellular site of origin rather than those typical ofify after placement in ectopic regions.their new location (e.g., Olsson et al., 1995). On the other

In order to more fully test the degree of specificationhand, the neonatal environment has been shown to effi-present in neural progenitors derived from the ventralciently redirect the fate of cortical progenitors taken attelencephalon and mid-hindbrain region, we have devel-the beginning of neurogenesis, but this is not the caseoped an ultrasound-guided in utero grafting paradigm,for cells from later stages of neurogenesis (Barbe andwhich allows for intraparenchymal injections into dis-Levitt, 1991, 1992, 1995; Cohen-Tannoudji et al., 1994).crete developing regions of embryonic day (E) 13.5Since neurogenesis is largely a prenatal event, many ofmouse embryos utilizing high resolution ultrasoundthe instructive cues normally involved in the specifica-backscatter microscopy (Turnbull et al., 1995b). Disso-tion of progenitors may be lacking in these regions ofciated cells derived from the E13.5 LGE or medial gangli-neonatal and later hosts. Thus, these transplantationonic eminence (MGE), as well as the E13.5 or E10.5 mid-hindbrain region, were injected both homotopically and‡Present address: Wallenberg Neuroscience Center, Departmentheterotopically into mouse embryos and traced by eitherof Physiology and Neuroscience, University of Lund, Solvegatan 17,genetic labeling (i.e., ROSA26 cells expressing lacZ;S-223 62 Lund, Sweden.

§To whom correspondence should be addressed. Friedrich and Soriano, 1991) or by the lipophilic dye

Neuron762

PKH26. The present results demonstrate that manyE13.5 ventral telencephalic progenitors are capable ofintegrating and acquiring phenotypes of adjacent telen-cephalic structures as well as of the mid-hindbrain re-gion, whereas E13.5 mid-hindbrain cells transplantedisochronically into either the MGE or the LGE fail tointegrate and do not adopt forebrain gene expression.In contrast, mid-hindbrain progenitors from an earlierstage (E10.5) implanted into the E13.5 telencephalonare observed to disperse and adopt local telencephalicphenotypes, suggesting that between E10.5 and E13.5these cells become restricted in their developmentalpotential.

Results

Ventral Telencephalic ProgenitorsCan Be Respecified by AdjacentTelencephalic EnvironmentsPrevious studies (Fishell, 1995; Brustle et al., 1995;Campbell et al., 1995) have shown that at least a portionof ventral telencephalic progenitors (derived from eitherthe MGE or LGE) are capable of integrating and adoptinglocal phenotypes in heterotopic regions of the telen-cephalon, following intraventricular embryonic injec-tions. These studies do not address whether the majorityof the injected cells can respecify in response to theectopic environment or if a small subpopulation of pro-genitors selectively integrates and respecifies. To testthis, we have used ultrasound-guided injections to per-form heterotopic transplantation into adjacent telence-phalic regions, since the intraventricular injection tech-nique does not allow for specific placement of the cellsin the embryonic brain.

As a baseline for studying the heterotopic telence-phalic grafts, we have performed homotopic trans-plantation of E13.5 LGE and MGE cells. Using a coronalor horizontal ultrasound image, E13.5 LGE cells fromROSA26 mice (Friedrich and Soriano, 1991) were in-jected homotopically into the germinal zones of theE13.5 LGE (n 5 5), with the cortex demarcating thelateral boundary and the fissure between the LGE andMGE the medial boundary (Figure 1A). X-Gal-reacted

Figure 1. Ultrasound Backscatter Microscopy-Guided Injections striatal sections from these animals at E17.5 generallyinto the E13.5 Mouse LGE, MGE, and Mid-Hindbrain Region contained transplanted cells throughout the developing(A, D, and G) Coronal images produced by ultrasound backscatter striatum (Figures 1B and 1C). The injected cells weremicroscopy of the E13.5 telencephalon ([A] and [D]) ormid-hindbrain found as dispersed single cells or in small clusters thatregion (G). The levels in (A) and (D) correspond approximately to

had migrated away from the injection site. The injectionpage 290, and those in (G) to page 316, of Altman and Bayer (1995).site was most often detectable as a subtle scar in theIn (A), the tip (arrow) of the microcapillary is located in the LGE, in

(D) it is positioned in the MGE, and in (G) the microcapillary is parenchyma and/or as an aggregate of grafted cells.situated in the cerebellar anlage. The transplanted LGE cells wereconfined to thestriatum(B, E, and H) Schematic representation showing the distribution of in all animals, with a few cells also observed in thethe homotopically injected E13.5 LGE (B), MGE (E), and mid-hind- cortex; however, these cells were only found along thebrain (H) cells (blue dots), 4 days after injection (i.e., E17.5).

presumed injection tract (two out of five animals). The(C, F, and I) High power photomicrographs from the boxed areasmigration pattern of homotopically transplanted LGEin (B), (E), and (H), respectively, displaying transplanted cells from

ROSA26 mice detected by b-galactosidase activity. Most trans- cells support previous findings that the LGE representsplanted ROSA26 cells exhibited granular staining, and only a few the major source of striatal neurons (Deacon et al., 1994;showed extensive cytoplasmic staining. Homotopically injected LGE Olsson et al., 1995).cells ([B] and [C]) were dispersed throughout but remained largelywithin the developing striatum (Stm), whereashomotopically graftedMGE cells were observed to integrate into the globus pallidus (GP)and striatum (Stm; [E] and [F]). Mid-hindbrain progenitors were dis- ventricle; 3v, third ventricle; aq, aqueduct; 4v, fourth ventricle; Tect,persed within the developing cerebellar (Cb) hemisphere following tectum. Scale bars in (A), (D), and (G), 500 mm; scale bar in (I), 200homotopic transplantation ([H] and [I]). Abbreviations: lv, lateral mm for (C), 120 mm for (F), and 95 mm for (I).

Specification of Neural Progenitors763

Figure 2. Progenitors from the E13.5 MGEand LGE Integrate and Adopt Regional Phe-notypes after Grafting into Adjacent VentralTelencephalic Regions

(A and B) Coronal sections showing telence-phalic expression of the TTF-1 protein atE13.5 (A) and E17.5 (B) as detected by theTTF-1 antibody (Lazzaro et al., 1991). AtE13.5, most cells in the MGE as well as scat-tered cells in the basal forebrain expressTTF-1; however, no expression was detectedin the germinal layers of the LGE. At E17.5,most TTF-1-expressing cells are observed inthe developing globus pallidus (GP), butsome are also seen scattered in the devel-oping striatum.(C) Following homotopic injections, X-Gal-positive MGE cells derived from ROSA26mice were detected in the developing globuspallidus at E17.5, and z14% of the graftedcells displayed TTF-1 expression (brown re-action product; arrows).(D) Fewer MGE cells injected heterotopicallyinto the LGE and detected in the developingstriatum expressed TTF-1 at E17.5 (z21% ofthe number of homotopically injected MGEcells). MGE cells grafted to the LGE were dis-persed throughout the striatum in a similarmanner as homotopically injected LGE cells.(E and F) PKH26-labeled LGE cells injectedinto the MGE display integration in the globuspallidus (GP), with cells migrating toward andinto the septum (sept) and striatum (Stm; [F]).LGE cells were also observed in the basalforebrain, and a few examples in the thala-mus, in a similar pattern as homotopically in-jected MGE cells.(G) LGE were also seen to express TTF-1(arrows; z43% of number of cells seen inhomotopic MGE transplants) 4 days after in-jection into the MGE. The white dotted line in(E) indicates the ventricular border; lv, lateralventricle. Scale bar in (G), 210 mm for (A), 250mm for (B), 20 mm for (C), 20 mm for (D), 100mm for (E), 30 mm for (F), and 20 mm for (G).

Progenitors derived from the MGE of E13.5 ROSA26 later, when our analysis was performed (i.e., E17.5), en-dogenous TTF-1 expression was detected predominantlymice (n 5 5) or from Engrailed-1 lacZ knock-in (En-1lki)

mice that express lacZ from En-1 regulatory elements in postmitotic cells in the developing globus pallidusbut also in cells scattered throughout the developing(Hanks et al., 1995; Matise and Joyner, 1997; n 5 4)

were injected into the germinal zones of the MGE, using striatum, septum, and basal forebrain (Figure 2B). Inter-estingly, the distribution pattern of homotopically in-the fissure between the LGEand MGE as a lateral bound-

ary and the third ventricle as a medial boundary (Figure jected MGE cells was similar to the endogenous patternof TTF-1 expression. Indeed, following homotopic injec-1D). In all animals, the majority of the transplanted MGE

cells at E17.5 were identified in the developing globus tions into the MGE, z14% 6 1% (mean 6 SEM; n 5 3)of the cells expressed detectable levels of TTF-1 (4 dayspallidus as dispersed clusters of cells or single cells

(Figures 1E and 1F). It should be noted that in animals after injections; Figure 2C; Table 1). While this repre-sents a lower proportion of TTF-1-expressing cells thancontaining the highest number of injected MGE cells,

scattered cells were also observed in the striatum (Fig- that seen in the cell suspension (z50%) before grafting,it is unlikely that all of these cells continue to expressures 2E and 2F), septum, basal forebrain, and thalamus

(see Figure 1E). We have used an antibody to Thyroid this marker as they become postmitotic. In fact, theproportion of TTF-1 expressing cells in the E17.5 ventralTranscription Factor-1 (TTF-1, the protein product of the

TTF-1 gene, also known as Nkx2.1 and T/Ebp; Lazzaro telencephalon (which contains more post mitotic cellsthan the E13.5 brain; Figure 2B) was notably lower thanet al., 1991) as a marker of MGE cells. At E13.5, TTF-1

is expressed in many MGE cells and clearly marks the in the E13.5 ventral telencephalon (Figure 2A), whichcontains predominantly progenitors. Injected MGE cellsborder between MGE and LGE (Figure 2A). Four days

Neuron764

expressing TTF-1 were found predominantly in the glo-bus pallidus; however, examples of cells were also seenin the septum, basal forebrain, and striatum (data notshown). The expression of TTF-1 in transplanted striatalcells, however, appears to be a characteristic of homo-topically grafted MGE cells, since in nocase were homo-topically injected LGE cells found in the striatum double-stained with TTF-1 (n 5 5).

E13.5 MGE cells were injected into the LGE (n 5 5)of isochronic hosts in order to determine their potentialto integrate and adopt local phenotypes in this adjacenttelencephalic region. MGE cells heterotopically injectedinto the LGE were dispersed predominantly in the devel-oping striatum, as were the homotopically injected LGEcells, although a few cells were observed in the globuspallidus region (data not shown). Comparisons weremade between heterotopic and homotopic levels ofTTF-1 expression, normalizing the mean percent num-ber of heteropically injected (MGE toLGE) cells express-ing TTF-1 to the control level or the mean percent num-ber of homotopically injected (MGE to MGE) cellsexpressing TTF-1 (Table 1). The transplanted MGE cellsin the striatum displayed TTF-1 expression at only z21%of the control level (Figure 2D; Table 1).

E13.5 LGE cells were injectedectopically into the MGE(n 5 7) in order to determine what proportion of thesecells are capable of undergoing site-specific differentia-tion in this structure. As was the case for the homotopi-cally injected MGE cells, the LGE progenitors injectedinto the MGE were predominantly observed in the globuspallidus region (Figure 2E), with subpopulations of thegrafted cells migrating in streams toward and into thestriatum (Figure 2F), septum, basal forebrain, and thala-mus. At the time of dissection of the LGE (E13.5) fortransplantation, there is no TTF-1 expression in this ger-minal zone (Figure 2A). Following injections into theMGE, transplanted LGE cells were found double-stainedwith TTF-1, which is not the case when they are homo-topically grafted into the LGE (Table 1). These double-labeled cells were found predominantly in the globuspallidus, but examples were also seen in the striatumand septum (Figure 2G). The population of grafted LGEcells expressing TTF-1 after MGE placement was 43%of that seen in homotopic MGE transplants (Table 1).These results support previous findings on the plasticityof ventral telencephalic precursors (Fishell, 1995; Brus-tle et al., 1995; Campbell et al., 1995) and further showthat many cells in the precursor pool can respecify bydown-regulation and induction of region-specific mark-ers in response tosignals present in heterotopic telence-phalic regions.

Ventral Telencephalic Progenitors Can Respecifyafter Ectopic Transplantation intothe Mid-Hindbrain RegionPrevious studies have shown that telencephalic precur-sors can integrate into nontelencephalic regions of thedeveloping brain, including the mid-hindbrain region(Brustle et al., 1995; Campbell et al., 1995). We were thusinterested in testing what proportion of these precursorscan respecify in a nontelencephalic site, such as theEn-1-expressing mid-hindbrain region. In the E13.5 mid-hindbrain region, En-1 is expressed in the posterior re-

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Specification of Neural Progenitors765

Figure 3. Mid-Hindbrain Expression of En

(A) Mid-sagittal section through the mid-hind-brain region of an E13.5 En-1lki embryo re-acted with X-Gal, demonstrating En-1 ex-pression predominantly in the caudal part ofthe midbrain, dorsally, and in the medial cere-bellar anlage. The dissections were madefrom the dorsal aspect of the midbrain-hind-brain, including the caudal (arrow) tectum(Tect; i.e., En-1-expressing area) and thecerebellar (Cb) anlage. At this developmentalstage En-2 expression in the cerebellar an-lage is much broader than En-1 expression(Davis and Joyner, 1988); however, theEnhb-1 antiserum used for double labelingdetects both the En-1 and En-2 antigens.(B) Coronal section through the mid-hind-brain at E17.5 demonstrating En expressionin subsets of cells in the developing midbrainand cerebellum (Cb), as detected by Enhb-1antibodies.(C) Following transplantation of E13.5 mid-hindbrain progenitors into their site of origin,z20% of the grafted cells express En atE17.5, as revealed by double-labeled b-gal-expressing ROSA26 cells (blue) and immuno-reactivity for the Enhb-1 antibody (brown;arrows) at E17.5. Scale bar in (C), 350 mm for(A), 180 mm for (B), and 25 mm for (C).

the anterior hindbrain, including the cerebellum (Figure Heterotopic injections of E13.5 MGE (n 5 8) or LGE(n 5 9) cells into the posterior midbrain or cerebellar3A). This general expression pattern is maintained over

the following days; however, by E17.5, En (both En-1 anlage regions resulted in extensive integration of thedonor cells (Figures 4A and 4D). Ventral telencephalicand En-2) expression becomes restricted to subsets of

cells in the midbrain and cerebellum (Figure 3B). TTF-1 cells were observed to have migrated from the injectionsite in a pattern indistinguishable from that seen whenexpression is never observed in the mid-hindbrain re-

gions during CNS development (Lazzaro et al., 1991). mid-hindbrain progenitors were injected into the poste-rior midbrain or cerebellum. The MGE-derived cells thatIn order to determine the potential for respecification

of ventral telencephalic precursors placed into the mid- integrated into the mid-hindbrain region displayed acomplete lack of TTF-1 expression (Figure 4A), whereashindbrain region, we first characterized transplanted

mid-hindbrain cells after homotopic placement into the many cells that had leaked into the cerebral aqueductmaintained TTF-1 expression (Figure 4A, inset). In addi-posterior midbrain or cerebellum. E13.5 cells from the

En-1 expressing midbrain-hindbrain region, including tion, integrated MGE cells were observed to have initi-ated expression of the region-specific marker En (Davisthe caudal tectum and the cerebellar anlage (Figure 3A),

of En-1lki (n 5 5) or ROSA26 mice (n 5 2) were injected et al., 1991) at z50% the level of mid-hindbrain progeni-tors grafted into their region of origin (Figure 4B; Tableinto the developing cerebellum (Figure 1G), tectum, or

tegmentum using the cerebral aqueduct and the fourth 1). Ectopically integrated LGE cells were observed toexpress En at z25% of the control level (Figures 4E andventricle as landmarks. Since the developing tissue in

these regions at E13.5 is very thin, the tip of the micro- 4F; Table 1). Most of these double-labeled cells weredetected in regions with a high proportion of endoge-capillary was first inserted into the ventricle and then

withdrawn into the tissue to assure injections into the nous En-expressing cells. Some of the MGE cells foundin cellular aggregates in the cerebral aqueduct also ex-brain parenchyma. Graftedcells were found dispersed in

the developing cerebellum (Figures 1H and 1I) following hibited En expression (Figure 4C). Five embryos re-ceived MGE cells prelabeled with BrdU 12 hr and 6 hrinjections at E13.5 into this structure. Likewise, when

the injection site was located within the tectum or teg- before dissection. In these animals, no BrdU positivecells were identified in the parenchyma, whereas a num-mentum, cells were observed to have dispersed in the

colliculi or ventral midbrain, respectively. In addition, ber of cells in the ventricles contained BrdU labeling4 days after injection. This result suggests that cellsgrafted cells were consistently observed as aggregates

in the cerebral aqueduct as well as in the parenchyma dispersed in the parenchyma had continued to prolifer-ate following transplantation and thereby diluted outclose to the injection site. When mid-hindbrain cells

were transplanted into their region of origin, 20% 6 1% the mitotic marker (data not shown). Thus, at E13.5, aconsiderable population of progenitors in both the MGE(mean 6 SEM; n 5 3) of these cells were observed to

express En, as detected by double staining with the and LGE can respond to local cues in the developingmid-hindbrain and adopt appropriate phenotypes, al-Enhb-1 antibody (recognizing both En-1 and En-2; Davis

et al., 1991; Figure 3C) or by b-galactosidase activity in though LGE cells appear to be more restricted in theircapacity.the case of En-1lki donor cells (Table 1).

Neuron766

Figure 4. Progenitors from the E13.5 MGEand LGE Integrate and Adopt Regional Phe-notypes after Grafting into the Mid-Hindbrain

(A) MGE cells heterotopically injected into thetectum were detected as integrated cellsthroughout the developing inferior colliculus.No MGE cells were observed to expressTTF-1 ectopically in the tectal parenchyma,whereas a few of the cells accidentally in-jected into the aqueduct (aq) displayed TTF-1expression (arrow in inset).(B) b-gal-expressing (ROSA26) MGE cells in-tegrated inthe tectum expressedEn immuno-positivity as detected by Enhb-1 antibodies(arrows; z50% of the level of mid-hindbrainprogenitors transplanted into their region oforigin).(C) Examples of cells in the intraventricularaggregates had also induced En expression(arrows).(D) Following injection into the En-expressingmid-hindbrain regions, such as the tegmen-tum (Teg), PKH26-labeled LGE cells werefound dispersed and integrated.(E) In addition, PKH26-labeled LGE cells (red)were double stained with the Enhb-1 anti-body (green), resulting in yellow cells (arrow;z25% of the level of mid-hindbrain progeni-tors transplanted into their region of origin).(F) The induction of a mid-hindbrain pheno-type was also observed in LGE cells fromEn-1lki mice (Hanks et al., 1995; Matise andJoyner, 1997) reacted with X-Gal, demon-

strating En-1-expressing LGE cells in the tegmentum region. The border between the intraventricular aggregate and the host tectal parenchymais indicated by a black dotted line in (C). The white dotted line in (D) indicates the ventricular border; aq, aqueduct. Scale bar in (F), 85 mmfor (A), 20 mm for (A, inset), 20 mm for (B), 25 mm for (C), 80 mm for (D), 25 mm for (E), and 25 mm for (F).

E13.5 Mid-Hindbrain Progenitors Do Not Integrate either Enhb-1 double staining or b-galactosidase immu-nocytochemistry of En-1lki cells (Figure 5B; Table 1). Noand Fail to Adopt Local Phenotypes after

Ectopic Injections into the Forebrain injected cells were observed to express the regionalmarker TTF-1 following injections into the MGE, despiteWe have previously observed that progenitors derived

from the mid-hindbrain region are relatively inefficient the fact that the aggregates were located in the globuspallidus region where a large proportion of the endoge-at incorporating into the telencephalon but are capable

of integrating into more caudal regions of the developing nous cells express TTF-1 (Figure 2B). The lack of migra-tion or TTF-1 expression was not due to cessation inneuraxis, after injection into the embryonic forebrain

ventricle (Campbell et al., 1995; Olsson et al., 1997b). proliferation of these progenitors after heterotopictransplantation, since BrdU injections 2–3 days afterThe lack of extensive incorporation of these cells in the

telencephalon, however, does not exclude the possibil- grafting (n 5 6) resulted in double-labeled grafted cells(data not shown). In any case, mid-hindbrain cellsclearlyity that at least a portion of these cells may respecify if

forced into telencephalic regions. The potential of E13.5 do not possess the same degree of plasticity as theirventral telencephalic counterparts at E13.5.mid-hindbrain cells from the En-1-expressing region to

express forebrain phenotypes was investigated by in-jecting ROSA26 cells or PKH26-labeled cells from En- E10.5 Mid-Hindbrain Progenitors Can Integrate

and Respecify after Ectopic Injections1lki mice into the isochronic LGE (n 5 7) or MGE (n 5

10). The injected mid-hindbrain cells were typically ob- into the ForebrainIncreasing evidence suggests that as neural develop-served as aggregates of cells in the MGE or LGE, with

little or no integration and/or dispersion (Figures 5A and ment proceeds, progenitors become progressively re-stricted in their developmental potential, in order to gen-5B). This was in stark contrast to the extensive integra-

tion of telencephalic progenitors into the mid-hindbrain erate specificneuronal cell types (Anderson, 1989; Levittet al., 1993; Hatten and Heintz, 1995; Barbe, 1996). Weregion. In addition, cells that had leaked into the ventri-

cles were also observed to form aggregates rather than were thus interested in determining whether cells froman earlier stage in mid-hindbrain development couldincorporating across the ventricular wall. Cells in these

aggregates (located either in the parenchyma or ventri- change their fate when placed in a telencephalic envi-ronment. Since E13.5 represents a relatively late stagecle) were observed to express the mid-hindbrain marker

En at z55% (LGE) and z60% (MGE) of the number seen of neurogenesis in the mid-hindbrain region, we tookprogenitors from the E10.5 mid-hindbrain and injectedin homotopic mid-hindbrain grafts, as demonstrated by

Specification of Neural Progenitors767

Figure 5. E10.5 but Not E13.5 Mid-HindbrainProgenitors Can Integrate and Adopt Re-gional Phenotypes Following Transplantationinto the E13.5 Telencephalon

(A) E13.5 mid-hindbrain progenitors fromROSA26mice, injected into theMGE and sub-sequently visualized by X-Gal histochemistry,display limited integration with the majority ofthe injected cells appearing in an aggregateconfined to the injection site.(B) b-gal-expressing E13.5 mid-hindbrain En-1lki cells in an aggregate, following injectioninto the E13.5 LGE(z55% as compared to thenumber of coexpressing cells in homotopicmid-hindbrain transplants).(C) Unlike their E13.5 counterparts, PKH26-labeled E10.5 mid-hindbrain progenitors wereobserved to have migrated away from the in-jection site and were found in the striatum(Stm; inset) in addition to the globus pallidus(GP) at E17.5.(D) Additionally, the E10.5 mid-hindbrain cellscoexpressed the TTF-1 antigen (green) andthe PKH26 label (red), resulting in double-stained yellow cells (arrow; z64% of the ho-motopically injected MGE cells).(E) A few of these cells also coexpressedEnhb-1 (green) and PKH26 (red), again de-tected as yellow cells (arrows; z7% of thegrafted cells), similar to the E13.5 mid-hind-brain progenitors. Scale bar in (E), 250 mm for(A), 30 mm for (B), 130 mm for (C), 20 mm for(C, inset), 25 mm for (D), and 15 mm for (E).

them heterochronically into the E13.5 MGE (Figure 5C; Discussionn 5 12). A subpopulation of the E10.5 mid-hindbrain

This is the first study to demonstrate the utility of ultra-progenitors, only z35% of the number of homotopicallysound backscatter microscopy (Turnbull et al., 1995b)injected E13.5 mid-hindbrain progenitors, maintainedfor targeted injections of dissociated cells into specificEn expression predominantly in cells close to the injec-regions of the embryonic mouse brain. This approachtion site (Figure 5E; Table 1). In contrast to the E13.5was used to test, more rigorously than was possiblecells, many of the injected E10.5 mid-hindbrain cellspreviously, the degree of specification present in neuralwere found integrated and dispersed, predominantly inprogenitors from a particular developmental stage. Withthe globus pallidus (Figure 5C) and the striatum (Figurethis technique, a large proportion of E13.5 ventral telen-5C, inset), but some cells were also observed in thecephalic progenitors was seen to integrate and adoptseptum and basal forebrain in a pattern similar to thatlocal phenotypes after transplantation into adjacent tel-seen with MGE progenitors injected homotopically. In-

terestingly, a number of these integrated cells, 64% of encephalic structures or mid-hindbrain structures ofisochronic mouse hosts. This potential to respecify wasthe number of homotopically injected MGE cells, ex-

pressed the MGE marker TTF-1 (Figure 5D; Table 1) in not observed for mid-hindbrain cells of the same devel-opmental stage. However, a large subpopulation of mid-regions with high levels of endogenous expression such

as the globus pallidus. This level of induction is similar hindbrain progenitors derived from earlier embryos(E10.5) was able to integrate and express telencephalicto, if not greater than, that seen with heterotopic grafts

of LGE cells into the MGE (see above). These results phenotypes. These results demonstrate that while E13.5ventral telencephalic progenitors are relatively plasticindicate that at early developmental stages (e.g., E10.5),

many mid-hindbrain progenitors are plastic and can be in terms of their migratory and molecular genetic pheno-types, mid-hindbrain progenitors become restricted toredirected toward forebrain phenotypes.

Neuron768

mid-hindbrain fates between E10.5 and E13.5. These midgestation embryonic stages has not been demon-strated before in any organism, including frog and chick,studies also provide the ground work for future genetic

studies aimed at identifying genes required for the spec- which become opaque and inaccessible to injection ofinternal structures. This technique allows for targetedification of neural progenitors in mice by injecting mutant

cells into normal hosts or vice versa. In these studies, heterotopic injections of neural progenitors into specificregions of the midgestation embryonic brain, thus chal-we have taken advantage of the En-1lki mice, which pro-

vide a lacZ marker for En-1-expressing cells. lenging theirdegree of specification by placing theentireprecursor pool into an environment producing signalsdifferent from their site of origin and not relying on the

Specification of Ventral Telencephalic Progenitors donor cell’s innate ability to integrate across the ventric-Progenitors from the E13.5 mouse LGE or equivalent ular wall.stage rat embryo, transplanted into the striatum of neo- Our observation that many E13.5 ventral telencephalicnatal or adult hosts, produce grafts containing predomi- progenitors can integrate and acquire molecular geneticnantly striatal projection neurons, whereas grafts of and cellular phenotypes of adjacent telencephalic re-MGE or cerebellar cells do not (Pakzaban et al., 1993; gions, as well as downregulate markers specific for theirDeacon et al., 1994; Olsson et al, 1995, 1997a). Trans- site of origin, makes it likely that many LGE or MGE cellsplants of MGE cells contain neuronal phenotypes typical from the general progenitor pool are capable of respeci-of certain striatal interneurons not found in LGE grafts fying at this developmental stage. Most of the heterotopi-(Olsson et al., 1997c). The differentiation of LGE cells cally grafted ventral telencephalic progenitors integratedinto a striatal projection neuron phenotype in the adult and migrated similar to the homotopically injected MGEstriatal environment is not dependent on placement, and LGE cells, and almost half the number of the LGE-since LGE cells transplanted in the cortex or ventral derived cells expressed TTF-1 after transplantation tomesencephalon also differentiate into a striatal projec- the MGE, as compared to homotopic MGE grafts. Intion neuron phenotype (Olsson et al., 1995), indicating addition, most of the MGE cells injected into the LGEthat these cells are specified at E13.5. As projection down-regulated TTF-1 expression; however, a few MGE-neurons are by far the most numerous neuronal cell type derived cells did continue to express TTF-1. These datain the striatum (reviewed by Smith and Bolam, 1990), suggest that some progenitors in the E13.5 LGE andthese results suggest that the LGE represents the major MGE are irreversibly committed toa regional phenotype;source of striatal progenitors. In the above mentioned however, a considerable number remain plastic at thisexperiments, however, the LGE cells were implanted developmental stage.into brain regions in which neurogenesis had ceased Recent studies have shown that Sonic hedgehogand thus were not subject to the normal putative envi- (SHH) is capable of inducing the expression of TTF-1ronmental signals that direct neural progenitor differen- (referred to as Nkx2.1 in these studies) in forebrain re-tiation in the embryonic brain. Indeed, when LGE cells gions (Ericson et al., 1995; Nakagawa et al., 1996), andare injected into the midgestation (E13.5 mouse or it is possible that SHH also functions to maintain TTF-1equivalent stage rat) lateral ventricle they incorporate expression at later stages. Since Shh is expressed inboth homotopically into the striatum and, to a lesser the MGE but not in the LGE (Shimamura et al., 1995;extent, heterotopically into distinct nuclei throughout Platt et al., 1997), MGE cells placed in the LGE may losethe developing brain, and in many telencephalic regions TTF-1 expression due to the fact that the surroundingthey undergo site-specific differentiation (Fishell, 1995; cells do not express SHH. Conversely, some LGE cellsBrustle et al., 1995; Campbell et al., 1995; Olsson et al., placed into the MGE may be receptive to the SHH signal1997b). The interpretation of these studies is compli- and induce TTF-1 expression. Interestingly, mice car-cated by the fact that the extent of heterotopic incorpo- rying a mutation in the TTF-1 gene (referred to as T/Ebpration is influenced by trypsinization, without which only in this study) have recently been made, and among othera small portion of the transplanted cells incorporate brain defects these animals lack medially situated telen-heterotopically and undergo site-specific differentiation. cephalic structures such as the pallidum and the medialThus, it is unclear whether these cells are regionally septum (Kimura et al., 1996). This finding is interestingspecified, and subsequently respecify in the ectopic in relation to our previous study (Campbell et al., 1995),site, or if they represent a small population of unspeci- in which some E13.5–E14 LGE cells heterotopically situ-fied progenitors that are selectively over-represented ated in theseptum were observed to develop into medialby virtue of their extensive developmental potential. septal phenotypes following intraventricular embryonic

The intraventricular transplantation technique in em- injections. The present findings therefore provide a linkbryos is not ideally suited to address this issue, since to this previous result by demonstrating that some ofits purpose is to allow cells access to the entire length the E13.5 LGE cells implanted into the MGE migrateof the neuraxis, via the ventricular system. Moreover, similarly to homotopic MGE transplants and initiatenot all progenitor populations possess the capacity to TTF-1 expression.incorporate across the ventricular wall throughout the In our studies, homotopically injected MGE cells wereneuraxis. Indeed, mid-hindbrain progenitors are rather detected in high numbers in the globus pallidus, andinefficient at incorporating into the telencephalon (Camp- scattered cells were detected in the septum, striatum,bell et al., 1995; Olsson et al., 1997b). High resolution and basal forebrain. Interestingly, this migratory patternultrasound-guided injection offers a unique way to test correlates well with the distribution of cells expressingthe developmental potential of neural progenitors in ec- the MGE marker, TTF-1, in the E17.5 brain. By proximity,

MGE cells have been suggested to contribute cells totopic sites. Image-guided targeted brain injections into

Specification of Neural Progenitors769

both the developing striatum and globus pallidus (Smart The E13.5 mid-hindbrain cells were unable to integrateand remained in large clusters similar to those seen inand Sturrock, 1979); however, a precise fate map for

this structure is not available at present. Our homotopic grafts to the neonatal or adult brain (Olsson et al., 1995,1997a). Furthermore, none of these cells were observedtransplantation studies presented here support this pre-

vious suggestion that progenitors from the MGE contrib- to initiate TTF-1 expression after transplantation intothe MGE, in spite of the presence of SHH. However, itute cells to the globus pallidus and striatum as well as

to the septum and basal forebrain. In fact, the MGE may is not clear whether these cells fully maintained theirspecification, since the forebrain environment appearedrepresent a major source of striatal cholinergic in-

terneurons, since grafts of MGE cells contain cholinergic to be less efficient at maintaining En expression (55%–60% of the homotopic grafts) than the mid-hindbrainneurons while LGE transplants do not (Olsson et al.,

1997c). In support of this, we have recently observed region. It is worth mentioning, however, that the E13.5mid-hindbrain region contains a considerable numberTTF-1 expression in striatal cholinergic interneurons

(Olsson et al., unpublished data). of postmitotic cells (Bayer and Altman, 1995) in additionto proliferating progenitors, especially in the inferior col-While the ability of E13.5 ventral telencephalic progen-

itors to respecify after heterotopic transplantation into liculus and cerebellum (Pierce, 1973), and these postmi-totic cells may influence the differentiation of the pro-either the isochronic MGE orLGE is impressive, theirability

to efficiently integrate into the mid-hindbrain region and genitors in the population.Early commitment of the mid-hindbrain region has beeninduce En expression (a mid-hindbrain phenotype) fol-

lowing injections into the mid-hindbrain region poten- demonstrated in numerous heterotopic chick trans-plantation experiments, in which solid grafts of mid-tially reveals a more extensive respecification. This is

despite the fact that MGE and LGE cells express a num- hindbrain tissue (derived from stage 10 chick embryos,which corresponds roughly to E9 in the mouse) im-ber of the same developmental control genes (e.g., Bf-1

and Dlx family members; Shimamura et al., 1995) but planted into different forebrain regions maintain theirfate and can further redirect some forebrain cells todo not express many genes found in the mid-hindbrain

region. This respecification was observed in progenitors adopt mid-hindbrain phenotypes (reviewed by Alva-rado-Mallart, 1993). The present results, however, dem-from both the E13.5 LGE and MGE, which were as much

ashalf asefficient as homotopically injected mid-hindbrain onstrate that this commitment is not complete at a singlecell level (i.e., in dissociated grafts) at E10.5 in theprogenitorsat expressing En (Table 1). In addition to initiat-

ing En expression, all MGE cells were observed to down- mouse, since many mid-hindbrain progenitors from thisstage were capable of migrating and initiating TTF-1regulate TTF-1 expression in the dorsal mid-hindbrain

parenchyma. These results extend a recent study by expression when transplanted into the E13.5 MGE. Themigration pattern observed with E10.5 mid-hindbrainBrustle et al. (1995), where it was shown that some MGE

cells injected into the lateral ventricle could incorporate progenitors grafted to the E13.5 MGE was similar to thatof the homotopically injected MGE cells, showing thatinto the inferior colliculus and acquire a differentiated

morphology typical of inferior collicular neurons, by these early progenitors can also respond to local cuesregulating cell migration. A number of the E10.5 trans-showing that many E13.5 MGE cells have this potential.

Since En-1 has been shown tobe required for the normal planted cells also maintained En expression and did notmigrate out after placement in the MGE. Thus, alreadydevelopment of the inferior colliculus (as this structure

fails to develop in mice deficient for this gene; Wurst et at E10.5, a portion of the mid-hindbrain cells lacks theability to adopt a forebrain phenotype after placemental., 1994), the present results provide a possible molecu-

lar link to the study of Brustle et al. (1995) and suggest in the telencephalon, as is the case for the E13.5 mid-hindbrain cells.that respecification of ventral telencephalic progenitors

into mid-hindbrain phenotypes requires the induction of These results suggest that between E10.5 and E13.5in the mouse, mid-hindbrain cells become restricted inEn. This induction of En is likely mediated by diffusable

factors present in the mid-hindbrain region, such as their developmental potential, such that they are unableto integrate and adopt local phenotypes following het-FGF-8, a molecule known to induce mid-hindbrain phe-

notypes in certain forebrain regions (Crossley et al., erotopic placement. This fits well with the view that thegeneration of neuronal diversity in the nervous system1996; Lee et al., 1997).results from the progressive specification of neural pro-genitors, leading ultimately toward their commitment to

Specification of Mid-Hindbrain Progenitors a specific neural fate (Anderson, 1989; Levitt et al., 1993;We have previously demonstrated that mouse mid-hind- Hatten and Heintz, 1995; Barbe, 1996). In contrast, thebrain cells from either the ventral mesencephalon (E12) E13.5 ventral telencephalic cells were rather plastic inor the cerebellar primordium (E13.5) integrate in a re- their ability to respecify, indicating that they possessedgionally restricted pattern following intraventricular em- a similar levelof specificationas the E10.5 mid-hindbrainbryonic injections into the E15 rat (corresponding to progenitors. At later developmental stages, ventral tel-the E13.5 mouse; Campbell et al., 1995; Olsson et al., encephalic progenitors likely also become more restricted1997b). These cells are not efficient at incorporating into in their developmental potential. Indeed, a recent studytelencephalic regionsbut do integrate into the dienceph- by Frantz and McConnell (1996) using ferrets has shownalon and mid-hindbrain. This is despite the fact that they that unlike early cortical progenitors, those taken fromare injected into the forebrain ventricle. Using ultra- later stages in neurogenesis are restricted to fates typi-sound-guided injections, we have forced such mid-hind- cal of late-born cortical cells, even if transplanted back

into earlier environments. The mechanisms involved inbrain cells into the ventral telencephalic environment.

Neuron770

Figure 6. Setup for Ultrasound-Guided In Utero Injections

(A) The anesthetized mouse lies in the lower level of a two-level stage with a modified petri dish pinned over her abdomen. After laparotomy,part of the uterus (u) is pulled through a slit in a thin rubber membrane (r) stretched across a hole in the bottom of a fluid-filled petri dish,exposing the uterus to the UBM imaging transducer (t), which scans over the uterus to image the E13.5 embryo inside. Using the real-timeUBM images for guidance, the injection microcapillary (arrow) is inserted through the uterus and into the predetermined target region of theembryonic brain.(B) Closeup view shown of an E13.5 uterus with a single exposed conceptus.

surgery. Samples from the E13.5 MGE and mid-hindbrain cell sus-the specification of neural progenitors are, at present,pensions were plated onto culture dishes and cultured for about 3not fully understood. However, it is likely to depend onhr before staining with TTF-1 and En antibodies, respectively (seeboth cell-autonomous mechanisms (e.g., expression ofbelow for details). Approximately 53% of the plated MGE cells ex-

developmental control genes) and local environmental pressed TTF-1, whereas z34% of the cells in the mid-hindbrain cellcues that are changing during the course of neurogen- suspension expressed En.esis. The ultrasound-guided injection method providesan approach for directly testing the requirement of spe- Ultrasound Backscatter Microscopy Injection System

Ultrasound backscatter microscopy (UBM) is a high frequency (40–cific genes in various developmental decisions by utiliz-100MHz) ultrasound imaging method resulting inhigh spatial resolu-ing existing mutant mice.tion (90 mm at 40 MHz) over a limited penetration depth (7–10 mmat 40 MHz; Turnbull et al, 1995a, 1995b). In this study, 8 mm 3 8 mmExperimental Procedures(512 3 512 pixels) UBM images were produced by a mechanicallyscanned, focused 40 MHz transducer at image frame rates of fourIsolation of Neural Progenitorsor eight images per second. Images were captured either in directEmbryonic donor cells were obtained from E13.5 or E10.5 mousedigital format into an IBM-compatible PC computer or onto a VCRembryos (the day of plug detection was designated E0.5) generatedfrom the video output of the scanner. A commercial UBM systemby mating wild-type CD1 mice (Charles River, NY) with homozygous(Ultrasound Biomicroscope Model 840; Humphrey Instruments, SanTgR ROSA26 (ROSA26) transgenic mice (Jackson Laboratory, ME;Leandro, CA) has also been used to guide injections into mouseFriedrich and Soriano, 1991) or by mating heterozygous En-1lki miceembryos. This scanner also operates at 40 MHz, producing 5 mm 3with lacZ inserted into the En-1 locus (Hanks et al., 1995; Matise5 mm images at a frame rate of eight images per second, with imageand Joyner, 1997) and selecting heterozygous embryos by X-Galquality comparable to the prototype UBM described above.histochemistry. En-1lki homozygous mutant embryos, identified by

To facilitate in utero injections, the mechanical probe of the UBMa deletion in the mid-hindbrain region, were not used. The ROSA26was mounted on a motorized three-axis positioning stage, and finestrain contains a gene trap construct, with the lacZ gene in anXYZ positioning of the UBM image plane was maintained duringunknown locus, and expresses lacZ ubiquitously in developing tis-the injection procedure using a joystick controller (Newport-Klinger,sue (Friedrich and Soriano, 1991). Cells derived from either strainIrvine, CA). Injections were performed as described below, usingof mouse displayed similar distribution patterns and showed noa three-axis micromanipulator (Narishige, Tokyo) to position thedifference in their differentiation capacity. Nine pregnant mice wereinjection needle. Injection needles were made from glass micropi-injected with BrdU (25 mg/kg; Sigma) 12 hr and 6 hr before dissec-pettes pulled to produce a long taper and broken under a dissectiontion. The germinal zones of the lateral (LGE) and the medial gangli-microscope at an inner diameter of 30–50 mm. The glass microcapil-onic eminence (MGE) were dissected from the E13.5 embryos aslaries were sharpened to produce a bevel angle between 258 anddescribed previously (Olsson et al., 1995), and the dorsal mid-hind-358. An oil-filled manual microsyringe pump (Stoelting, Wood Dale,brain region, including the caudal part of the tectum and the entireIL) with a 25 ml Hamilton syringe was used to draw cell suspensionscerebellar anlage (corresponding to the En expressing domain; Fig-into the injection microcapillary and to inject a precise volume ofure 3A), was dissected at E10.5 and E13.5. The dissected tissuecell suspension into each embryo.pieces were dissociated mechanically in Dulbecco’s modified Ea-

gle’s medium (DMEM) with 0.05% DNase generating single cellsuspensions. Cell suspensions from the En-1lki embryos were la- Ultrasound-Guided In Utero Transplantation

Timed pregnant CD1 mice (Charles River, NY) with embryos at abeled with the membrane-bound lipophilic dye PKH26 (Sigma; 2–4ml/ml of the PKH26 dye in dilutent C for 3–5 min). The labeling gestational age of E13.5 were anesthetized with equithesin (0.2 ml/

35 g body weight). The abdomen was wet shaved and a 2 cm midlinereaction was stopped by washing the cell suspensions twice inDMEM containing 10% fetal bovine serum, and the cells were resus- laparotomy was performed. Each uterine horn was carefully taken

out individually and one side was chosen for injection. The uterinepended in serum-free DMEM (containing 0.05% DNase), generatinga cell suspension of z100,000 cells/ml, which was kept on ice during horns were repositioned in the abdomen with the embryo closest

Specification of Neural Progenitors771

to the ovary of the side to be injected left outside the abdomen. et al., 1991), and b-galactosidase (1:500; 59-39, Incorporated) ormonoclonal antibodies detecting BrdU (1:250 dilution; Sigma). Bio-The pregnant mouse was then placed in the lower level of a two-level

wooden stage (Figure 6A). Petri dishes were modified by punching a tinylated goat anti-rabbit or mouse antibodies were used as second-ary antibodies for DAB double labeling, while FITC-conjugated goat25 mm diameter hole in the bottom of each dish and covering the

hole with a thin rubber membrane. The petri dish was mounted with anti-rabbit antibodies were used for double labeling of specimenscontaining PKH26-labeled cells. Sections incubated with biotinyl-two pins to the top level of the wooden stage, over the mouse’s

abdomen, and the part of the uterus containing the first embryo ated antibodies were processed using the ABC method (VectorLabs) with DAB as the final chromogen. Transplanted cells doublewas gently pulled through a slit in the rubber membrane (Figure 6B).

Fluid coupling between the tissue and transducer was maintained stained with either the TTF-1 or En antibody were counted from twoto five sections (due to variability in the number and dispersion ofby filling the petri dish over the mouse with sterile PBS containing

CaCl2 and MgCl2. Using UBM guidance, embryos were positioned the transplanted cells) from selected animals in all groups.so as to yield a coronal or horizontal ultrasound image. After positiveidentification of the LGE (Figure 1A); the MGE (Figure 1D; forebrain Acknowledgmentstargets); or the cerebellar anlage (Figure 1G), tectum, or tegmentum(mid-hindbrain targets), the glass microcapillary was inserted This research was supported by a grant from the Whitaker Founda-through the uterine wall, and 0.5–1 ml of cell suspension was injected tion. We thank Alexandra Joyner for helpful suggestions andencour-into each site. The tip of the microcapillary could be identified as a agement throughout these studies and for supplying the En antibodybright dot on the screen displaying the ultrasound image (Figures and En-1lki mice. We also thank Roberto Di Lauro for providing the1A, 1D, and 1G). After injecting one embryo, the next embryo was TTF-1 antibody and Anders Bjorklund and Michael Matise for criticalgently pulled through the slit in the rubber membrane and positioned review of this manuscript. M. O. was supported by the Swedishfor injection, while the first embryo was placed back into the abdomi- Medical Research Council. K. C. was supported by the Medicalnal cavity. Between four and ten embryos were injected in every Research Council of Canada and the Swedish Institute. D. H. T. ispregnant mouse, and surgery was limited to 1 hr for each mouse an Investigator of the American Heart Association/New York Cityto optimize survival. The position of each injected embryo along Affiliate.the uterine horn was registered for identification at sacrifice. Afterinjections, the uterine horn was placed back into the abdomen and Received May 23, 1997; revised August 11, 1997.the mother was sutured. Twelve of the mothers carrying injectedembryos were injected with BrdU (25 mg/kg; Sigma) 2–3 days later References(E15.5–E16.5).

All embryos were sacrificed 4 days after injection (E17.5). Approxi- Altman, J., and Bayer, S.A. (1995). Atlas of Prenatal Rat Brain Devel-mately 35% of the injected animals survived until sacrifice (186 out opment (Boca Raton, FL: CRC Press).of 528). Of the surviving embryos, 61% (113 out of 186) contained Alvarado-Mallart, R.M. (1993). Fates and potentialities of the aviandetectable injected cells (PKH26- or X-Gal-positive), and out of mesencephalic/metencephalic neuroepithelium. J. Neurobiol. 24,those, 68% (77 out of 113) contained injected cells in the appropriate 1341–1355.target structure. The injected cells were most often observed in the

Anderson, D.J. (1989). The neural crest lineage problem: neuro-ventricular system if they were not identified in the appropriate targetpoiesis? Neuron 3, 1–12.structure; however, the brains were not examined in regions far fromBarbe, M.F. (1996). Tempting fate and commitment in the developingthe target site.forebrain. Neuron 16, 1–4.

Barbe, M.F., and Levitt, P. (1991). The early commitment of fetalImmunocytochemistry and X-Gal Histochemistryneurons to the limbic cortex. J. Neurosci. 11, 519–533.The embryonic heads were immersion fixed in 4% paraformalde-

hyde in 0.1 M phosphate buffer for 4 hr at 48C and then placed into Barbe, M.F., and Levitt, P. (1992). Attraction of specific thalamicinput by cerebral grafts depends on the molecular identity of the30% sucrose in 0.1 M phosphate buffer for 48–72 hr at 48C before

sectioning. All brains were sectioned in the coronal plane at 12 mm implant. Proc. Natl. Acad. Sci. USA 89, 3706–3710.thickness using a cryostat. Embryos injected into the forebrain (LGE Barbe, M.F., and Levitt, P. (1995). Age-dependent specification ofand MGE) were sectioned and analyzed from the start until the end the corticocortical connections of cerebral grafts. J Neurosci. 15,of the lateral ventricles, whereas embryos injected into the mid- 1819–1834.hindbrain region were sectioned and analyzed from the anterior Bayer, S.A., and Altman, J. (1995). Neurogenesis and neuronal mi-portion of the tectum through the posterior extent of the cerebellar gration. In The Rat Nervous System, G. Paxinos, ed. (San Diego,anlage. Injected ROSA26 and En-1lki mouse cells were identified CA: Academic Press), pp. 1041–1078.using X-Gal histochemistry. Briefly, sections were permeabilized in

Brustle, O., Maskos, U., and McKay, R.D.G. (1995). Host-guidedwash buffer containing 0.1 M phosphate buffer, 2 mM MgCl2, andmigration allows targeted introduction of neurons into the embryonic0.02% Nonidet P-40, followed by incubation in 5 mM K3Fe(CN)6, 5brain. Neuron 15, 1275–1285.mM K4Fe(CN)6·3H2O, and 1 mg/ml X-Gal (Sigma) in wash buffer atCampbell, K., Olsson, M.,and Bjorklund, A. (1995). Regional incorpo-378C for 8–10 hr. The X-Gal reaction generally produced labeledration and site-specific differentiation of striatal precursors trans-grains within the cytoplasm of donor cells and stained the entireplanted to the embryonic forebrain ventricle. Neuron 15, 1259–1273.cytoplasm in only a few cells. Specimens containing b-gal-express-

ing cells were double labeled with antibodies and visualized with Cohen-Tannoudji, M., Babinet, C., and Wassef, M. (1994). Early de-DAB (brown reaction product), whereas sections containing PKH26- termination of a mouse somatosensory cortex marker. Nature 368,labeled cells (visualized with a rhodamine filter) were double stained 460–463.with fluoroscein isothiocyanate-conjugated (FITC-conjugated) sec- Crossley, P.H., Martinez, S., and Martin, G.R. (1996). Midbrain devel-ondary antibodies. Specimens to be reacted with the antibody to opment induced by Fgf8 in the chick embryo. Nature 380, 66–68.BrdU were preincubated in 2N HCl for 40 min. Sections containing

Davis, C.A., and Joyner, A.L. (1988). Expression patterns of thePKH26-labeled cells were incubated for 10 min in 0.1% Triton X-100,homeobox-containing genes En-1 andEn-2 andthe proto-oncogeneand endogenous peroxidase activity was inactivated in sections toint-1 diverge during mouse development. Genes Dev. 2, 1736–1744.be processed with DAB using 3% H2O2 for 10 min. All sections wereDavis, C.A., Holmyard, D.P., Millen, K.J., and Joyner, A.L. (1991).preincubated for 1 hr in 10% normal goat serum and 0.25% TritonExamining pattern formation in mouse, chicken and frog embryosX-100 (DAB double labeling) followed by incubation in 2% normalwith an En-specific antiserum. Development 111, 287–298.goat serum and 0.25% Triton X-100 (DAB double labeling) and the

respective primary polyclonal antibodies recognizing the following Deacon, T.W., Pakzaban, P., and Isacson, O. (1994). The lateralganglionic eminence is the origin of cells committed to striatal phe-proteins, Thyroid Transcription Factor-1 (TTF-1, the protein product

of the TTF-1 gene, also known as Nkx2.1 and T/Ebp; 1:5000 dilution; notypes: neural transplantation and developmental evidence. BrainRes. 668, 211–219.Lazzaro et al., 1991), En-1 and En-2 (aEnhb-1; 1:500 dilution, Davis

Neuron772

Ericson, J., Muhr, J., Placzek, M., Lints, T., Jessel, T.M., and Edlund, Smart, I.H.M., and Sturrock, R.R. (1979). Ontogeny of the neostria-tum. In The Neostriatum, I. Divac and R.G.E. Oberg, eds. (Oxford:T. (1995). Sonic hedgehog induces the differentiation of ventral fore-

brain neurons: a common signal for ventral patterning within the Pergamon Press), pp. 127–146.neural tube. Cell 81, 747–756. Smith, A.D., and Bolam, J.P. (1990). The neural network of the basal

ganglia as revealed by the study of synaptic connections of identi-Fishell, G. (1995). Striatal precursors adopt cortical identities in re-sponse to local cues. Development 121, 803–812. fied neurons. Trends Neurosci. 13, 259–265.

Suhonen, J.O., Peterson, D.A., Ray, J., and Gage, F.H. (1996). Differ-Friedrich, G., and Soriano, P. (1991). Promoter traps in embryonicstem cells: a genetic screen to identify and mutate developmental entiation of adult hippocampus-derived progenitors into olfactory

neurons in vivo. Nature 383, 624–627.genes in mice. Genes Dev. 5, 1513–1523.

Hanks, M., Wurst, W., Anson-Cartwright, L., Auerbach, A.B., and Turnbull, D.H., Starkoski, B.G., Harasiewicz, K.A., Semple, J.L.,Joyner, A.L. (1995). Rescue of the En-1 mutant phenotype by re- From, L., Gupta, A.K., Sauder, D.N., and Foster, F.S. (1995a). Aplacement of En-1 with En-2. Science 269, 679–682. 40–100 MHz B-scan ultrasound backscatter microscope for skin

imaging. Ultrasound Med. Biol. 21, 79–88.Hatten, M.E., and Heintz, N. (1995). Mechanisms of neural patterningand specification in the developing cerebellum. Annu. Rev. Neu- Turnbull, D.H., Bloomfield, T., Baldwin, H.S., Foster, F.S., androsci. 18, 385–408. Joyner, A.L. (1995b). Ultrasound backscatter microscope analysis

of early mouse embryonic brain development. Proc. Natl. Acad. Sci.Kimura, S., Hara, Y., Pineau, T., Fernandez-Salguero, P., Fox, C.H.,USA 92, 2239–2243.Ward, J.M., and Gonzalez,F.J. (1996). The T/ebp null mouse: thyroid-

specific enhancer-binding protein is essential for the organogenesis Vicario-Abejon, C., Cunningham, M.G., and McKay, R.D.G. (1995).of the thyroid, lung, ventral forebrain, and pituitary. Genes Dev. 10, Cerebellar precursors transplanted to the neonatal dentate gyrus60–69. express features characteristic of hippocampal neurons. J. Neu-

rosci. 15, 6351–6363.Lazzaro, D.M., Price, M., De Felice, M., and Di Lauro, R. (1991). Thetranscription factor TTF-1 is expressed at the onset of thyroid and Wurst, W., Auerbach, A.B., and Joyner, A.L. (1994). Multiple develop-lung morphogenesis and in restricted regions of the foetal brain. mental defects in Engrailed-1 mutant mice: an early mid-hindbrainDevelopment 113, 1093–1104. deletion and patterning defects in forelimbs and sternum. Develop-

ment 120, 2065–2075.Lee, S.M.K., Danielian, P.S., Fritzsch, B., and McMahon, A.P. (1997).Evidence that FGF8 signaling from the mid-hindbrain junction regu-lates growth and polarity in the developing midbrain. Development124, 971–982.

Levitt, P., Ferri, R.T., and Barbe, M.F. (1993). Progressive acquisitionof cortical phenotypes as a mechanism for specifying the developingcerebral cortex. Perspect. Dev. Neurobiol. 1, 65–74.

Matise, M.P., and Joyner, A.L. (1997). Expression patterns of devel-opmental control genes in normal and Engrailed-1 mutant mousespinal chord reveal early diversity in developing interneurons. J.Neurosci. 17, in press.

Nakagawa, Y., Kaneko, T., Ogura, T., Suzuki, T., Torii, M., Kaibuchi,K., Arai, K., Nakamura, S., and Nakafuku, M. (1996). Roles of cell-autonomous mechanisms for differential expression of region-spe-cific transcription factors in neuroepithelial cells. Development 122,2449–2464.

Olsson, M., Campbell, K., Wictorin, K., and Bjorklund, A. (1995).Projection neurons in fetal striatal transplants are predominantlyderived from the lateral ganglionic eminence. Neuroscience 69,1169–1182.

Olsson, M., Bentlage, C., Wictorin, K., Campbell, K., and Bjorklund,A. (1997a). Extensive migration and target innervation by striatalprecursors after grafting into the neonatal striatum. Neuroscience79, 57–78.

Olsson, M., Bjerregaard, K., Winkler, C., Gates, M., Bjorklund, A.,and Campbell, K. (1997b). Incorporation of neural progenitors trans-planted into the embryonic forebrain is developmentally regulatedand dependent on regional and adhesive properties. Eur. J. Neu-rosci., in press.

Olsson, M., Bjorklund, A., and Campbell, K. (1997c). Early specifica-tion of striatal projection neurons and interneuron subtypes in thelateral and medial ganglionic eminence. Neuroscience, in press.

Pakzaban, P., Deacon, T.W., Burns, L.H., and Isacson, O. (1993).Increased proportion of acetylcholinesterase-rich zones and im-proved morphological integration in host striatum of fetal graftsderived from the lateral but not medial ganglionic eminence. Exp.Brain. Res. 97, 13–22.

Pierce, E.T. (1973). Time of origin of neurons in the brain stem ofthe mouse. Prog. Brain Res. 40, 53–65.

Platt, K.A., Michaud, J., and Joyner, A. (1997). Expression of themouse Gli and Ptc genes is adjacent to embryonic sourcesof hedge-hog signals, suggesting a conservation of pathways between fliesand mice. Mech. Dev. 62, 121–135.

Shimamura, K., Hartigan, D.J., Martinez, S., Puelles, L., and Ru-benstein, J.L.R. (1995). Longitudinal organization of the anterior neu-ral plate and neural tube. Development 121, 3923–3933.