Mechanisms of Development, 40 (1993) 129-140 0 1993 Elsevier Scientific Publishers Ireland, Ltd. 09254773/93/$06.00

MOD 00139

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The mouse Dk-2 ( Tes-I) gene is expressed in spatially restricted domains of the forebrain, face and limbs in midgestation

mouse embryos

Alessandro Bulfone a,*, Hee-Joong Kim “*, Luis Puelles b, Matthew H. Porteus a, Joseph F. Grippo ’ and John L.R. Rubenstein a

a Neurogenetics Laboratory, Department of Psychiatry and Programs in Neuroscience and Dellelopmental Biology, UCSF, San Francisco, CA 94143, USA, ’ Department of Anatomy, Faculty of Medicine, University of Murcia, Murcia, Spain

and ‘Department of Toxicology and Pathology, Hoffmann-La Roche, Nutley, NJ 07110, USA

(Received 6 October 1992; revision received 13 November 1992; accepted 17 November 1992)

The pattern of RNA expression of the murine DDr-2 (Tes-2) homeobox gene is described in embryos ranging in age from ES.5 through El15 Dlx-2 is a vertebrate homologue of the Drosophila Distal-less (Dll) gene. Dll expression in the Drosophila embryo is principally limited to the primordia of the brain, head and limbs. DLr-2 is also expressed principally in the primordia of the forebrain, head and limbs. Within these regions it is expressed in spatially restricted domains. These include two discontinuous regions of the forebrain (basal telencephalon and ventral diencephalon), the branchial arches, facial ectoderm, cranial ganglia and limb ectoderm. Several mouse and human disorders have phenotypes which potentially are the result of mutations in the DLr genes.

DIX-2; Homeobox gene; Brain; Limb; Branchial arch; Neural crest

Introduction

Candidates for the genes that control vertebrate development are rapidly being identified. Among these, genes encoding homeobox proteins are likely to serve important roles. In invertebrates, homeobox genes con- trol segmentation, specification of segment phenotype and cellular differentiation (Gehring, 1987; McGinnis and Krumlauf, 1992). That these genes serve similar functions in vertebrates can be deduced in at least four ways. First, many, and perhaps most of the invertebrate homeobox-containing genes have been conserved dur- ing vertebrate evolution (McGinnis and Krumlauf, 1992; Murtha et al., 1991). Next, the genomic organiza- tion of some of these genes has been conserved. For instance, the cluster of homeobox genes that specify the identity of segments in Drosophila (the HOM clus-

Correspondence to: J.L.R. Rubenstein, Neurogenetics Laboratory

Department of Psychiatry and Programs in Neuroscience and Devel-

opmental Biology, 401 Parnassus Avenue, UCSF, San Francisco, CA

94143-0984, USA.

* Co-first authors

ter) has been amplified, such that there are four ho- mologous clusters (the Hex clusters) in higher verte- brates (Acampora et al., 1989; Duboule and Doll& 1989; Graham et al., 1989; Kappen et al., 1989). Third, ectopic expression of human and mouse homeobox genes in Drosophila cause phenotypes that are similar to those induced by ectopic expression of the homolo- gous Drosophila genes (Malicki et al., 1990; McGinnis et al., 1990). Finally, mutations in seven vertebrate homeobox-containing genes cause a variety of pheno- types ranging from a homeotic transformation ( HOX-3. I> (Le Mouellic et al., 1992), complicated abnormalities in morphogenesis (Hex-1.5, Hox-1.6, Pax-l, Pax-3 and Pax-61 (Chisaka and Capecchi, 1991; Chisaka et al., 1992; Lufkin et al., 1991; Chalepakis et al., 1991; Ep- stein et al., 1991; Hill et al., 19911, to disruption of terminal differentiation (Pit-l) (Pftiffle et al., 1992; Radovick et al., 1992; Tatsumi et al., 1992).

Recently, the Dlx class of vertebrate homeobox genes has been identified (Price et al., 1991; Porteus et al., 1991; Robinson et al., 1991; Ekker et al., 1992). Robinson and co-workers have evidence that there are four members of this family in mouse. The home- odomain of these genes is homologous to the home-

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odomain of the Drosophila Distal-less (Dll) gene (Cohen et al., 1989). During Drosophila embryogene- sis, DlZ is expressed in the primordia of the limbs (Cohen et al., 1989; Cohen, 19901, in the anlage of facial sensory appendages (within the labral, antennal, maxillary, and labial segments) (Cohen and Jiirgens, 1990), and in the brain (Steve Cohen, personal commu- nication).

Previous studies of Dlx-1 and Dlx-2 (originally re- ported as Tes-I) have focused on their expression pat- tern in the forebrain of mice between E10.5 and post- natal day 7 (Price et al., 1991, 1992; Porteus et al., 1991; Robinson et al., 1991). This work established that within the central nervous system Dlx-1 and Dlu-2 are expressed in the forebrain, and not in more posterior parts of the neural tube. A more recent study describes the expression pattern of the DIx-1 gene outside the central nervous system (Doll6 et al., 1992). The experi- ments reported in this paper describe the expression of DIX-2 from E8.5 through E11.5. Like DLx-1, expression of Dlw-2 is found not only in the forebrain, but also in several other structures including the branchial arches, facial primordia, and the limbs. The correlation be- tween the patterns of expression in Drosophila and in mice is examined.

Results

Expression at E8.5 to E9.0

No expression was detected using whole mount in situ RNA hybridization at E8.5 (Fig. lA), although weak expression was found in the mesenchyme of the head at this stage using tissue sections (data not shown). Strong expression was clearly detected at E8.75. At this stage DIX-2 transcripts were found in several struc- tures: the mesenchyme of the first branchial (maxillary [Mx] and mandibular [Md] branches) and second branchial arches (B2), a restricted domain of ectoderm along the frontonasal prominence, and a patch of cells

dorsal to the first branchial arch in the region of the neural tube; these cells are in the region of the primor- dia of the trigeminal ganglion (TGG), and may repre- sent either placodal and/or neural crest derived cells (Fig. 2A). Expression in the ectoderm of the otic pit was also found (data not shown), and expression in the otic vesicle at E9.75 is shown in Fig. 2E,F. Whole mount in situ RNA hybridization of an E9.0 embryo also showed strong expression in the mesenchyme of the first and second branchial arches, but this method did not detect the expression in any epithelial struc- tures (data not shown). This discrepancy may be due to two possibilities: (1) in situ RNA hybridization to tissue sections is more sensitive then the whole mount proce- dure; (2) proteinase K treatment of the embryos results in the loss of surface ectodermal structures.

Expression at E9.5 to E9.7.5

DLx-2 continued to be expressed in the same struc- tures as on E8.75E9.0, as well as in a number of new structures. Whole mount in situ RNA hybridization shows that DLx-2 is expressed in the branchial arches and in a restricted domain of the diencepbalon (Fig. 1B). A strong signal is seen in the mesenchyme of the maxillary @lx) and mandibular (Md) branches of the first branchial arch. Weaker signals are found in the hyoid arch (second arch [B21), and the third (B3) and fourth arches (B4). Within the forebrain, there is ex- pression along a diagonal band of tissue in the region of the primordia of the ventral thalamus (domain 1).

To obtain results of higher resolution and sensitivity, in situ RNA hybridization to tissue sections was per- formed. These results will be described according to organ system.

Branchial arches and face All four branchial arches express DIX-2 in their

mesenchyme (Figs. lB,F and 2B). Expression in the ectoderm can clearly be detected medially in the first arch (Figs. 1H and 2F,H). There may be ectodermal

Fig. 1. Dir-2 exoression at E8.5 and E9.5 using in situ RNA hybridization to sectioned and whole mount mouse embryos. (A) Whole mount E8.5

embryo. The remainder of the panels show E9.5 embryos. (B) Whole mount embryo. (C,D) Coronal section; (C) is in bright-field, and (D) is in

dark-field. (E,F) Lateral section; (E) is in bright-field, (F) is in dark-field. (G,H) Parasagittal section; (G) is bright-field, (H) is dark-field. Magnification: (A) and (B) have the same magnification; bar in (A) = 500 pm. C-H have the same magnification; bar in (C) = 250 pm. 1, Caudal

domain of Dlu-2 expression containing: VT, PEP, HCC and SCH; 2, rostra1 domain of D/.x-2 expression containing: POA, AEP, MGE, LGE, and

SE; AA, aortic arch; AEP, anterior entopeduncular area; AFG, primordium of the acousticofacial ganglion; B2, branchial arch 2; B3, branchial

arch 3; B4, braichial arch 4; C, primordium of cerebral cortex; Cb, primordium of cerebellum; CGE, caudal ganglionic eminence; D,

diencephalon; DT, dorsal thalamus (alar part of the posterior parencephalon; ~2); E, eye; EC, ectoderm; En, endoderm; EMT, eminentia

thalami; mN, forebrain terminal notch; G, spinal ganglia; HCC, hypothalamic cell cord; H, heart primordium; L, limb bud; LGE, lateral

ganglionic eminence; MGE, medial ganglionic eminence; M, mesencephalon; MB, primordium of the mammillary body; Md, mandibular branch

of first branchial arch; MNC, migrating neural crest; MN, mesonephros; Mp, mesoderm muscle plate; Mx, maxillary branch of first branchial arch; OP, olfactory pit; OV, otic vesicle; OR, optic recess; P, prosencephalon; PEP, posterior entopeduncular area; POA, preoptic area; PSG,

parasympathetic ganglia; PT, pretectum (alar part of the synencephalon; pl); R, rhombencephalon; RCH, retrochiasmatic area; RP, Rathke’s

pouch; SE, septum; SC, spinal cord; SCH, suprachiasmatic area; SP, secondary prosencephalon; T, telencephalon; TGG, primordium of the trigeminal ganglion; VT, ventral thalamus (alar part of the anterior parencephalon; ~3).

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expression in the lateral first arch and in the second arch, but because of the large amount of mesenchymal signal adjacent to the epithelium (Figs. 1F and 2B-D), it is difficult to discern whether the ectoderm also expresses this gene.

In the first arch, there is differential spatial expres- sion of DIX-2 in both mesenchymal and ectodermal compartments. For the mandibular component of the first arch, expression in the ectoderm is limited to a rostro-ventro-medial domain (Figs. 1H and 2E-H); ex- pression in the mesenchyme follows a lateral-to-medial gradient. Laterally, most of the mesenchymal cells ex- press Dh-2 (Figs. 1F and 2B-D), whereas medially there is a dramatic decrease in Dlx-2 expression (Fig. 1H and 2E-HI. Differential spatial expression is also seen for the maxillary component of the first branchial arch. In this case, expression in the ectoderm is ros- trally displaced compared to the expression in the underlying mesenchyme (Fig. 2B,C). There is a core of mesenchymal cells in the mandibular and second arches which do not express Dlx-2; their location is consistent with the mesodermally derived muscle plates (Mp) (Figs. 1F and 2B-D).

Expression in the mesenchyme lines the walls of the aortic arches (AA) (Fig. 2E,F). Dlx-2 expression in the mesenchyme is found in three separate domains (best seen in Fig. 2B). Expression is not seen in the endoder- ma1 components of the branchial apparatus. The dorsal limit of ectodermal expression on the medial aspect of the mandibular arch is in the proper region to delimit the ectodermal-endodermal boundary of the oral cavity (Fig. 2F, see arrow).

Finally, there is a strip of ectoderm underlying the telencephalon that expresses DLx-2 (Figs. 1H and 2E- H). This ectoderm extends rostrally from a region basal to the optic stalk and ends near the apex of the frontonasal prominence. A dorsal portion of the otic vesicle epithelium expresses Dh-2 (Fig. 2E,F); expres- sion of Dir-3 has also been found in the otic vesicle epithelium in zebrafish embryos (Ekker et al., 1992).

Central nervous system Dlx-2 mRNA is detected in the forebrain, which is

consistent with its expression at later developmental stages (Porteus et al., 1991; Robinson et al., 1991; Bulfone, Puelles, Porteus, Frohman, Martin and Rubenstein, manuscript submitted). References for the anatomical terminology of the embryonic forebrain can

be found in Puelles et al. (1987, 1991) and Bulfone, Puelles, Porteus, Frohman, Martin and Rubenstein (manuscript submitted). Forebrain expression begins in the diencephalon (D) within the ventral thalamic (VT) domain at about E9.5 (Fig. lB,H). By N E9.75, the pattern of expression expands rostrally, and spans two discontinuous zones (Fig. 2G,H), separated by a short DZx -negative gap in the region of the optic recess (OR) (Fig. 2G,H). The caudal zone (domain 1) extends from the region of the zona limitans intraparencephalica (which separates the dorsal and ventral thalamus) to the postoptic area (caudal to the optic stalk). The rostra1 zone (domain 2) extends from the preoptic area (rostra1 to the optic stalk) along the floor of the cere- bral vesicle (under the primordia of the medial and lateral ganglionic eminence and septum). Thus, Dir-2 expression in the telencephalon (T) is restricted to the basal region. Furthermore, expression is limited to lateral regions of the forebrain, as medial sections show no evidence of D/X-2 expression (Fig. 2D,E).

Within the wall of the neural tube, some expression is seen in the ventricular zone, although most Dlx-2 expression is in the subventricular zone. A similar pattern is found at later stages of development (Bulfone, Puelles, Porteus, Frohman, Martin and Rubenstein, manuscript submitted).

Cranial neural crest The mesenchyme of the branchial arches is derived

from cranial neural crest as well as from mesoderm (Noden, 1988; Lumsden et al., 1991). Because of the extensive expression of DIx-2 in the mesenchyme of each branchial arch, it is likely that this gene is ex- pressed in neural crest derivatives. Expression is also seen in other groups of cells which are likely to be derived from neural crest. There is strong expression in a cluster of cells just rostra1 to the otic vesicle and dorsal to the second branchial arch (Fig. 2D). These cells may correspond to the primordia of the acoustico- facial ganglion (AFG). Expression in small clusters of cells, which may be neural crest, can also be seen just rostra1 and caudal to the otic vesicle in Fig. 2E and F.

Limbs A restricted pattern of expression is present in the

ectoderm of the nascent forelimb buds (L) (Figs. 1D and 2E,G). Expression extends from the apical ridge to

Fig. 2. Dlx-2 expression at E8.75 and E9.7.5 using in situ RNA hybridization to sectioned mouse embryos. Double exposure photography

simultaneously shows the tissue in blue and the presence of the Dk-2 RNA in red. (A) E8.75 embryo, the remainder are E9.75 embryos. (B) Lateral section. (C) High magnification view of(B). (D) Parasagittal section showing region of the acousticofacial ganglion. (E) Near midsagittal

section. (F) Higher magnification view of(E). (G) Parasagittal section. (H) Higher magnification view of(G). Magnification: the size bar is in (H).

In (G) it corresponds to 400 pm; in (A,B,D,E,H) it corresponds to 200 Km, and in (C,F) it corresponds to 100 pm. The arrow in (F) corresponds

to a boundary of Dir-2 expression which is in the region of the ectodermal-endodermal limit on the mandibular arch.

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the ventral surface. No mesenchymal expression is de- tected.

Body Weak expression can be seen in two columns of cell

clusters (Fig. 2G). The ventral column is in the position of the pronephros and metanephros (MN). The dorsal column is in the position of spinal ganglia (G).

Expression at E10.5

Whole mount in situ RNA hybridization shows strong expression in the forebrain and in the branchial arches (data not shown), which appears very similar to the expression at E9.5. The results with in situ RNA hybridization to tissue sections are reported below.

Branchial arches and face The overall patterns of expression within these tis-

sues are comparable with the patterns at E9.5-E9.75. There continues to be a high level of ectodermal ex- pression in the medial part of the mandibular branch of the first arch (Md) (Fig. 3B-D). Expression in the ectoderm of the maxillary processes also persists and extends into the olfactory pit (OP), where it abruptly ends (Fig. 3A). Ectodermal expression is also seen in a sharply defined domain at the dorsal lip of the olfac- tory pit (Fig. 3A). DLx-2 is not expressed within the invaginating olfactory cavity, except in scattered nests of cells beginning around El25 (Porteus and Ruben- stein, unpublished data).

Brain At E10.5, expression in the forebrain continues to

be constrained within the same two domains as on the previous day (compare Figs. 2H and 3C). The caudal zone includes the ventral thalamus (VT), posterior entopeduncular area (PEP), hypothalamic cell cord (HCC), and the primordia of the suprachiasmatic area (SCH). Expression stops at the boundary of the SCH with the postoptic area. The rostra1 zone extends from the preoptic area (POA) along the floor of the cerebral vesicle under the primordia of the medial and lateral ganglionic eminence (MGE, LGE). There is a short gap in expression in the region of the optic stalk between the POA and SCH (Fig. 3C). The ganglionic eminences have significantly increased in size com- pared to the E9.75 brain. Expression continues ros- trally along the septal area, and ends abruptly before the primordia of the cerebral cortex (0.

Cranial neural crest There is a DLx-2 -positive cluster of cells just dorsal

to the maxillary branch of the first branchial arch (Mx) and just caudal to the eye (E) (Fig. 3A). These cells may correspond to the primordia of trigeminal gan-

glion (TGG). The dorsal-lateral surface of the head is lined with positive cells (Fig. 3A). A similar signal is seen along the dorsal-lateral head at E9.5 (Fig. 2B).

Body Near midsagittal sections show expression in a

metameric structure (Fig. 3B,D); this tissue is likely to be spinal ganglia (G).

Expression at E11.5

No new areas of expression have been detected at this age. Most regions begin to show a decline in their level of RNA. From E11.5 through the rest of develop- ment, the forebrain becomes the predominate domain expressing the DLx-2 gene (Porteus et al., 1991; Robin- son et al., 1991; Bulfone, Puelles, Porteus, Frohman, Martin and Rubenstein, manuscript submitted).

Brain As at earlier stages, D/X-~ expression is limited to

two discontinuous domains in the forebrain (Figs. 3D,E and 4A-H). Laterally, only the rostra1 domain in the telencephalon is present (Fig. 4B). In this tissue section the lateral and medial ganglionic eminence (LGE and MGE) show expression of DIX-2 (Fig. 4B). The caudal boundary is between the MGE and the caudal gan- glionic eminence (CGE); the rostra1 boundary is be- tween the LGE and the cerebral cortex (0.

More medially (Fig. 4D,F) both the rostra1 and caudal Dlx-positive domains are apparent. The rostra1 domain consists of the septum (SE), the medial gan- glionic eminence (MGE), the anterior entopeduncular area (AEP) and preoptic area (POA). The caudal do- main consists of the suprachiasmatic area (SCH), hy- pothalamic cell cord (HCC), posterior entopeduncular area (PEP) and the ventral thalamus (VT). The caudal boundary of the ventral thalamus is at the zona limi- tans (see arrow in Fig. 4D) which separates the ante- rior parencephalon (whose alar region contains the ventral thalamus) from the posterior parencephalon (whose alar region contains the dorsal thalamus) (Puelles et al., 1987, 1991). The Dlx-negative region separating the rostra1 and caudal domains consists of the eminentia thalami (EMT), and several hypothala- mic primordia (Fig. 4D,F). These include the supraop- tic paraventricular area, anterior hypothalamus, and the posterior preoptic area (which contains the optic stalk region).

The most medial section (Fig. 4H), shows expression only in the rostra1 domain (in the MGE). No expres- sion is present in the hypothalamus or dorsalmost ventral thalamus. In this nearly midsagittal section, the basal domain stretching from the mammillary area (MB) to the retrochiasmatic area (RCH) does not express Dlx-2. This demonstrates that expression of

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Dlx-2 is segregated to an alar longitudinal zone across the ventral forebrain.

Discussion

The results described in this paper have implications regarding both mouse developmental biology and the evolutionary conservation of genes that orchestrate de-

velopment of the metazoan body plan. Between ca. E8.75 to E11.5, Dlx-2 is expressed in a discrete set of tissues. These include the forebrain, the primordia of the face and neck, and the ectoderm of the limb buds. The Dlx-I gene is expressed in the same tissues, al- though its onset of expression (Doll6 et al., 1992) lags approximately two days behind the onset of Dh-2 expression. It is remarkable that Dll, the Drosophila

Fig. 3. Dlu-2 expression at El05 and El15 using in situ RNA hybridization to sectioned mouse embryos. Double exposure photography simultaneously shows the tissue in blue and the presence of the Dir-2 RNA in red. (A-D) are El05 embryos, and (E,F) are an E11.5 embryo. (A) Lateral section. (B) Parasagittal section. (C) Higher magnification view of (B). (D) Parasagittal section. (E) Parasagittai section. (F) Higher magnification view of (El. Magnification: the size bar is in (F). In (A,B,D,E) it corresponds to 400 pm; in (C,F) it corresponds to 200 pm. The

asterisk labels the optic recess.

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Fig. 4. DLr-2 expression in the brain at E11.5 using in situ RNA hybridization to the sectioned mouse embryos. (A,C,E,G) are bright-field

photographs of Nissel-stained sections. (B,D,F,H) are dark-field photographs of the same sections. The section in (A) and (B) is the most lateral,

while the section in (G) and (H) is the most medial. All photographs are of the same magnification; the size bar in (A) corresponds to 250 km.

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homologue of the DLx genes, has an analogous pattern of expression in the fly embryo. In Drosophila embryos, DZZ is expressed in the brain (S. Cohen, personal com- munication), in the primordia of the limbs and in the sensory appendages of the following facial structures: maxilla, antenna, labrum and labia (Cohen and Jiirgens, 1990).

Analogous structures for each of these Drosophila appendages can be conceived of in the mammalian embryo on the basis of their function and location within the body plan. The maxillary, labral and labial segments contribute to the development of the mouth. These segments functionally and spatially correspond to the mouth/jaw region of the mouse embryo which is derived from the first branchial arch, a structure with strong Dlw-2 expression. The antenna1 segment forms the antenna, which is a specialized olfactory organ that functionally and spatially corresponds to the epithelia that lines the nasal cavities of vertebrates. Dlx-2 is expressed in the opening of the nasal cavity (Fig. 3A) and in discrete regions of the nasal epithelium (Porteus and Rubenstein, unpublished observations). Similarly, the limbs in flies can be considered as functional analogues to the limbs in mammals, where both the Dll and DLx-2 genes are expressed in the ectoderm. Fi- nally, both genes are expressed in the embryonic brain. Thus, we hypothesize that the DIX-2 and DZl genes are expressed in analogous embryologic regions in the fly and in the mouse.

Expression of Dh-2 is also transiently seen in neu- ral crest derivatives (cranial and peripheral ganglia), nephrogenic tissues (Fig. 2G), and in the genital tuber- cle (Porteus and Rubenstein, unpublished results). Thus, aside from these structures, which perhaps do not have analogues in the fly, the set of tissues which express these genes are strikingly similar. The Dlx-I gene is also expressed in the forebrain, branchial arches and the limbs (Doll& et al., 1991). Other homologous homeobox genes are also expressed in similar parts of vertebrate and invertebrate embryos. The HOX (for a review see McGinnis and Krumlauf, 19921, Em (Simeone et al., 1992aI, and Otx (Simeone et al., 1992b) genes also share similarities in their expression patterns with their Drosophila homologues. These ob- servations suggest that these genes have similar func- tions in controlling the body plan of metazoans.

In the mouse, Dlx-2 is expressed in the limbs, the tip of the tail (Porteus and Rubenstein, unpublished results), the branchial arches and the genital tubercle. The branchial arches and the genital tubercle in the midgestation mouse embryo have a similar appearance to the limb buds, and thus perhaps represent modified forms of appendages. Although this is speculation, it provides a unifying explanation why the DLx genes are expressed in these tissues.

Mutations in the Dll gene show that it is required

for development of the limbs and facial appendages (Cohen and Jiirgens, 1989). It is not known whether brain development is altered in Dff mutants (Steve Cohen, personal communication). Presently, the func- tion of DLr-2 or any of its homologues, DLx-1, DIX-3 and DLx-4, is unknown. DIX-2 maps on mouse and human chromosome 2 near the Hox-4 cluster (Gzcelick et al., 1992). Pulse-field gel electrophoresis shows that Dfx-2 maps within 50 kb of DLx-1 (McGuinness et al., 1992). DLx-1 and DIX-2 also map in the region of a mutation named Ulnaless (IX). Ul is a dominant muta- tion whose phenotype is characterized by abnormalities of the ulna, radius, tibia and fibula (Davisson and Cattanach, 1990). Furthermore, the morphology of the mandible is slightly abnormal. The structure of the brain in this mutant has not been reported. The char- acteristics of mice homozygous for the Ul mutation have not been determined because the males do not breed, for uncertain reasons. Dk-1 andDlw-2 also map near another mutation named First Branchial Arch (Far) (Juriloff and Harris, 1991). Heterozygotes and homozygotes have abnormalities of the first branchial arch. Thus, it is possible that Ul and Far represent mutations in the DLx genes given the proximity of their chromosomal locations and the fact that Dlx-I and Dlw-2 are expressed during the development of the tissues which are disrupted by these mutations. We are presently exploring this hypothesis.

There are at least 18 different human disorders in which there is a combination of facial and limb defects (Smith, 1982). These include Miller and Nager syn- dromes, which are characterized by malar hypoplasia, micrognathia, and postaxial limb deficiencies. Neither the modes of inheritance nor the chromosomal map position of most of these disorders have been proven. The Dlx gene family are good candidates for studies of the genetic basis of these syndromes.

Genes at the 3’ end of the cluster are expressed in the neural ectoderm and neural crest of the hindbrain (Hunt et al., 1991). The hindbrain-derived neural crest migrates ventrally and populates branchial arches 2, 3 and 4 (Lumsden et al., 1991). The ectoderm overlying these arches also expresses the Hex genes. Thus, while the Hex genes contribute to the development of the hindbrain and the neck, as shown by mutations in Hex-1.5 (Chisaka and Capecchi, 1991) and Hox-1.6 (Chisaka et al., 1992; Lufkin et al., 19911, they are probably not involved in the development of more rostra1 parts of the head. Our results show that Dlx-2 is expressed in the ectoderm and mesenchyme of the first branchial arch, in the ectoderm of the frontonasal prominence, and the forebrain. Thus, it is possible that Dlx-2 is part of a hierarchy of transcriptional regula- tors that control patterning of the head. It is intriguing that DLx-1 and DIX-2 map near the Hox-4 cluster. At this point we do not know whether they map at the 3’

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end of the cluster. If they do, it presents the possibility that they have been recruited into the cluster to regu- late development of the head.

Dlw-2 is expressed in two discontinuous zones in the brain. The caudal zone extends rostrally from the zona limitans intrathalamica to the border of the suprachias- matic area (SCH) with the optic stalk. The rostra1 zone extends from the preoptic area (POA) along the basal telencephalon to the primordia of the olfactory bulb. This general pattern of expression is maintained throughout development of the forebrain (Porteus et al., 1991; Robinson et al., 1991; Bulfone, Puelles, Por- teus, Frohman, Martin and Rubenstein, manuscript submitted). Dlx-I also shows this pattern of expression during forebrain development (Price et al., 1991, 1992; Bulfone, Puelles, Porteus, Frohman, Martin and Rubenstein, manuscript submitted). The spatially re- stricted expression patterns of the DLx genes, in con- junction with the expression patterns of another home- obox gene named Gbx-2, and the putative differentia- tion factor Wnt-3, provide evidence for transverse and longitudinal segmentation of the forebrain (Bulfone, Puelles, Porteus, Frohman, Martin and Rubenstein, manuscript submitted). Expression of DLr-2 in the branchial arches is also suggestive of segmentation. Fig. 2B shows expression of Dlx-2 in three separate patches of mesenchyme associated with the first, sec- ond and third arches.

Another interesting feature of branchial arch ex- pression is seen within both branches of the first branchial arch. Here there is a reciprocal pattern of ectodermal and mesenchymal expression. Regions with high expression in the ectoderm have low expression in the mesenchyme (Figs. lF,H and 2C,F). By E12.5, expression in the branchial arches is concentrated in regions where teeth are developing (Bulfone, Puelles, Porteus, Frohman, Martin and Rubenstein, manuscript submitted). Expression in the developing teeth contin- ues throughout prenatal development (Porteus et al., 1991; Robinson et al., 1991). Our earlier studies found that at E18.5 Dlx-2 is expressed in the ectodermally derived preameloblasts (Porteus et al., 19911, whereas at E14.5 Dlx-2 appears to be expressed in the mes- enchymal component, which give rise to the odonto- blasts (Doll6 et al., 1992). Thus, there may be differen- tial spatial expression of Dlx-1 and DIX-2 in the embry- onic teeth, although further work will be needed to establish this.

The Hox-7 and Hex-8 genes are also expressed in the early stages of tooth development, as well as in the branchial arches and limb buds (Robert et al., 1991; MacKenzie et al., 1992). Thus, it is possible that these genes are part of the genetic network which interact with DLW-2 and DLx-1 to control development of these tissues. Homologues of the Dfx, HOX-7 and Hox-8 genes are also expressed in the developing inner ear of

the zebrafish (Ekker et al., 19921, which further sug- gests that these genes interact to orchestrate morpho- genesis and differentiation.

DLY-2 expression is seen in cells that are probably cranial neural crest derivatives. These include the mes- enchyme of the branchial arches (Figs. 1F; 2C,D; 3A,F), as well as two clusters of cells which may part of the trigeminal ganglia (Figs. 2A; 3A) and the acousticofa- cial ganglia (Fig. 2D). The Hox genes are also ex- pressed in cranial neural crest (Hunt et al,, 1990, although they differ from the Dh-2 gene because they are expressed in the overlying neural tube. Present evidence shows that the neural crest for branchial arch 1 comes from the caudal mesencephalon and rhom- bomeres 1 and 2; neural crest for branchial arch 2 derives from rhombomere 4, and neural crest for branchial arch 3 originates in rhombomere 6 (Lumsden et al., 1991). This is paradoxical because we do not detect expression of Dir-2 within the neural tube in any of these regions. There are several possible expla- nations for this observation. First, there may be a transient and/or low level of expression of Dlx-2 in the midbrain and hindbrain. Second, expression of Dir-2 within the cranial neural crest does not begin until the crest cells have left the neural tube. Third, the forebrain gives rise to neural crest cells which migrate caudally. This last hypothesis is the least likely, as none of the studies of neural crest migrations have found any evidence for such a pathway.

Dlx-2 expression in the ectoderm of the limb pri- mordia is restricted to an apical-ventral domain. This differential spatial expression suggests that it may play a role in the patterning of the limb (Figs. 1D; 2B,G). The expression of Dlx-2 in the mandibular branch of the first branchial arch also follows an apical-ventral distribution (Figs. 2F,H; 3B-D). Many other homeobox genes are expressed in the limb bud, including genes of the Hox-1, Hox-3 and Hox-4 clusters (for a review see Izpisua-Belmonte and Duboule, 19921, HOX-7, Hox-8 (Robert et al., 19911, En-l (Davis et al., 1991) and Dlx-1 (DollC et al., 1992). Like DIX-2, expression of En-2 and DLx-1 is also restricted to the limb bud ectoderm, and both Dlw-2 and En-l exhibit a ventral- dorsal gradient.

Functional analysis of the Dlx genes will be neces- sary to determine their role in controlling the develop- ment of the tissues where they are expressed. Expres- sion in a tissue is not evidence that a gene has an important function in these cells, which is clearly shown by the restricted phenotypes of mutations in Pax-6 (Hill et al., 19911, Hox-1.5 (Chisaka and Capecchi, 1990, Hox-I.6 (Chisaka et al., 1992; Lufkin et al., 1991) and several other regulatory genes. This phe- nomenon may be due to redundancy of gene function. Since DLU-2 is part of a family of at least four genes (Robinson et al., 19911, it will not be surprising if

139

mutations in one gene may have no or only subtle developmental defects.

Experimental Procedures

Embryo preparation

Two strains of mice were used: C57Bl/6 (see Figs. 2 and 3) and BALB/C (see Figs. 1 and 4). The C57Bl/6 mice were-set up for mating at 17.00 h and checked for the presence of copulatory plugs at 09.00 h the next morning. The middle of the night cycle, midnight, was defined as day 0 of pregnancy. Timed pregnant inbred BALB/C mice were obtained from Simonsen laborato- ries, Gilroy, CA. The morning on which a copulatory plug was found was considered E0.5. The mothers were sacrificed by cervical dislocation; the embryos were isolated and fixed overnight in 4% paraformalde- hyde in phosphate-buffered saline (PBS), dehydrated and stored at -20°C.

Whole mount in situ RNA hybridization

Whole mount in situ RNA hybridization was carried out as described by Harland (1991) with the following modifications. The embryos were bleached with 3% hydrogen peroxide for 20 min before the proteinase K treatment, and were pre-blocked in 0.5% Blocking Reagent (Boehringer Mannheim) in PBT (PBS + 0.1% Triton X-100) for 60 min. They were then incubated with the digoxigenin antibody coupled to alkaline phos- phatase (Boehringer Mannheim) overnight at 4°C in 0.5% Blocking Reagent (Boehringer Mannheim) in PBT. The digoxigenin labelled DLK-2 probe is de- scribed below.

In situ RNA hybridization using tissue sections

The dehydrated embryos were cleared with toluene, embedded in paraffin, sectioned at 6 pm and mounted on aminoalkylsilane or TESTA (Sigma) treated slides. In situ RNA hybridization was performed according to Wilkinson (Wilkinson et al., 1987) and Frohman (Froh- man et al., 1990). Single-strand 35S-labeled cRNA probes were generated by in vitro transcription accord- ing to the procedure of Zoeller et al. (1989). TheDIx-2 riboprobe was a 700 bp fragment from the 3’ untrans- lated region of the DIX-2 (Tes-I) cDNA (Porteus et al., 1991). Digoxigenin-labeled cRNA probes were ob- tained by labelling the same template described above using digoxigenin-rUTP (Boehringer Mannheim).

Acknowledgements

The authors would like to thank Drew Noden, Lee Niswander, Charles Ordahl, Kathy Sulik, Cynthia Cook,

and Edward Kollar for reviewing our results and pro- viding important anatomical information; Steven Co- hen for discussions about the expression of the Distal- less gene; Melanie Bedolli for assistance with tissue sectioning and microscopy. J.L.R.R. is a Pfizer Scholar, John Merck Scholar, NARSAD Young Investigator, and has an ROl from NIMH (MH49428-01). A.B. was supported by the Italian Association for Cancer Re- search (AIR0 M.H.P. is a member of the Stanford MSTP and was supported by the Merck Corporation.

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