DEVELOPMENTAL DYNAMICS 194222-230 (1992)
Id Expression During Mouse Development: A Role in Morphogenesis YAOQI WANG, ROBERT BENEZRA, AND DAVID A. SASSOON Department of Biochemistry, Boston University School o f Medicine, Boston, Massachusetts 02118 (Y W , D A S ) and Cell Brology and Genetics Program, Memorial Sloan Kettering, N e w York, N e w York 10021 (R B )
ABSTRACT We have characterized the spatial and temporal pattern of Id transcription during mouse embryogenesis. The Id gene encodes a helix-loophelix (HLH) protein which can heterodimerize with the ubiquitously expressed HLH protein products of the E2A gene, and prevent them from binding DNA either alone or as a heterodimer with tissue specific HLH transcription factors such as the muscle determination gene, MyoDl (Benezra et al., 1990 Cell 61:49-59). Since Id has been shown to be down-regulated during induced differentiation in several cell lines, it has been postulated that Id plays a general inhibitory role in cell differentiation (Benezra et al., 1990).In situ analysis of Id mRNA expression in the mouse embryo was performed in order to determine whether the pattern of Id expression is consistent with this postulate. A detailed study throughout the entirety of mouse postimplantation development reveals that Id is expressed upon gastrulation at very high levels in almost all regions of the mouse embryo and expression declines as embryogenesis proceeds. In skeletal muscle, in which the inhibitory action of Id has been established in tissue culture models (Benezra et al., 19901, Id and the HLH myogenic factors are expressed in a mutually exclusive manner suggesting that myogenic precursors do not express both types of HLH gene products. In addition, Id colocalizes both spatially and temporally with Hox7.1, a murine homeobox gene which is associated with regions of high cell proliferation and positional fate assignment. 0 1992 Wiley-Liss, Inc. Key words: Embryogenesis, Myogenic factors, Hox-7.1, Muscle INTRODUCTION The cloning and characterization of the muscle determination genes (myogenic factors) have been significant steps toward understanding muscle cell commitment and differentiation a t the molecular level. At present, it is unclear whether cell commitment and subsequent differentiation during embryogenesis involve a simple regulation of one or several of the myogenic determination genes, or whether these processes are under more subtle molecular control (Olson, 1990; Weintraub et al., 1991). Each member of the myogenic factor gene family shares two functional motifs, the C 1992 WILEY-LISS, INC.
helix-loophelix domain (HLH) involved in dimerization and a cluster of basic amino acids involved in DNA binding (Davis et al., 1990). Although the four currently identified genes MyoDl (Davis et al., 19871, myogenin (Wright et al., 1989; Edmondson and Olson, 1989), MRF4 (also known as herculin, myf6; Rhodes and Konieczny, 1989; Miner and Wold, 1990; Braun et al., 19901, and myf5 (Braun et al., 1989) can all stably convert many nonmuscle cell types to muscle following transfection, each gene shows a unique and specific pattern of expression throughout embryogenesis (Sassoon et al., 1989; Ott et al., 1991; Bober et al., 1991; Hinterberger et al., 1991) suggesting nonredundant roles for these proteins. Nonetheless, the myogenic factors are most likely positive regulators of muscle determination and differentiation. The role of negative regulators may be equally important for the development of committed cell lineages in the embryo. One such negative regulator, Id, is a helix-loophelix (HLH) protein which lacks a DNA binding motif and has been shown to antagonize the activity of MyoD1 in vivo (Benezra et al., 1990; Jen et al., 1992). Id transcription has been detected in a number of tissue and cell types, however, suggesting that its regulatory role is not unique to skeletal muscle (Benezra et al., 1990). The high level of Id expression in proliferative and undifferentiated cells suggests a potential key function during embryonic growth. Our in situ hybridization results are consistent with a general role for Id in the developing embryo. Prior to gastrulation, Id expression is detected primarily in parietal endoderm and extraembryonic cell layers. Soon after, Id expression is found in most cell types in the embryo. Id levels decrease in a temporal and tissuespecific pattern with levels first declining in the forming cardiac mesoderm and CNS. In contrast, levels are maintained in cells that are proliferative and also undergoing rapid morphogenesis such as the limb, neural folds, visceral arches, frontonasal processes, and migrating neural crest. We note that not all regions of the embryo undergoing rapid proliferation are positive for Id, indicating that the Id protein is not uniquely re-
Received July 2, 1992. Address reprint requestsicorrespondence to David A. Sassoon, Department of Biochemistry, Boston University School of Medicine, 80 E. Concord Street, Boston, MA 02118.
Id EXPRESSION DURING MOUSE DEVELOPMENT
quired for cell division. We observe that myogenic precursor cells, which express extremely high levels of Id, first down-regulate Id in these cells before they accumulate one or more of the currently identified myogenic factors. Finally, we have observed a high concordance of Id expression with a murine homeobox containing gene, Hox-7.1 in specific regions of the mouse embryo including the limbs, visceral arches (and facial primordial, heart, and neural folds (Robert et al., 1989; Hill et al., 1989; Mackenzie et al., 1991). The coordinate expression of Id and Hox-7.1 suggests that Id plays a complex role during mouse embryogenesis and is associated with morphogenic centers of development such as the early limb.
MATERIALS AND METHODS Staged embryos were obtained from timed breedings of CD-1 mice (Charles River) and the morning of vagin*l plugs was counted as 0.5 days postcoitum (p.c.1. Embryos from 6.5 days p.c. to birth were removed from the surrounding decidua and fixed in freshly prepared cold 4% paraformaldehyde in phosphate-buffered saline for 16 hr. At least three embryos were examined for each stage and representative examples are presented here. Tissue was then slowly dehydrated and embedded into paraffin following standard procedures as described by Sassoon et al. (1988). To date, we have used in situ hybridization to analyze a number of important developmental gene transcripts during mouse embryogenesis and have refined the resolution to detect transcripts a t the intracellular level (Fontaine et al., 1988; Sassoon e t al., 1988, 1989). Serial sections (5-7 Fm) are collected individually on glass microscope “subbed’ slides (Gall and Pardue, 1971). The procedures used for section treatment, hybridization, and washings are based on those used by Wilkinson et al. (1987) with modifications described by Sassoon et al. (1988). Hybridization was carried out a t 52°C for 16 hr in 50% deionized formamide, 0.3 M NaC1,ZO mM Tris-HC1 (pH 7.4), 5 mM EDTA, 10 mM NaPO, (pH 8),10% dextran sulfate, 1x Denhardt’s, 50 pg/ml total yeast RNA with 75,000 dpm/pl 35S-labeled RNA probe under siliconized coverslips. Probes were generated using T7 polymerase on a Bluescribe recombinant and [a-35slu*tP (>1,000 Ciimmol, New England Nuclear). Coverslips were then floated off in 5 x SSC (1x SSC is 0.15 M NaC1, 15 mM sodium citrate) 10 mM dithiothreitol (DTT), at 50°C and then a stringent wash at 65°C in 50% formamide, 2 x SSC, 0.1 M DTT. Slides were then rinsed in washing buffer and treated with RNase A (20 pgiml; Sigma) and washed a t 37°C in 2 x SSC and 0.1 x SSC for 15 min, respectively. Slides were then dehydrated rapidly and processed for standard autoradiography using NTB-2 Kodak emulsion and exposed for 7-9 days at 4°C. Analysis was carried out using both light and darkfield optics on a Leitz Orthoplan microscope. The probe used for Id was a cDNA fragment from position 5 to 927 described by Benezra et al. (1990)
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subcloned into Bluescript minus (Stratagene). The plasmid was linearized with BamHI and antisense riboprobe was obtained using T7 polymerase. Hox-7.1 probe used in this study was identical to that previously described by Robert et al. (1989). An EcoRI fragment of Hox-7.1 was subcloned into Bluescribe (Stratagene), linearized with Sac1 and transcribed with T7 polymerase. The probe used for myogenin was identical to that previously described by Wright et al. (1989). A cDNA corresponding to myogenin was subcloned into Bluescribe minus (Vector cloning systems) linearized with Hind111 followed by transcription with T7 polymerase. The probe used for myf5 was identical to that previously described by Ott et al. (1991). A BuZIiApeLI cDNA fragment was subcloned into Bluescript minus and linearized with Hind111 followed by transcription with T7 polymerase.
RESULTS Id Expression Initiates During Gastrulation in the Embryonic Ectoderm The first stage examined in this study was 6.5 days postcoitum (P.c.) which corresponds to early gastrulation in the mouse embryo. At this stage, Id transcripts can be easily detected over all extraembryonic tissues except the ectoplacental cone, however it is notably absent in the primitive embryonic ectoderm which will eventually give rise to the embryo (Fig. 1A; Rugh, 1990). By mid to late gastrulation (7.5 days P.c.), Id is detected in all embryonic cell layers; however the ectoplacental cone remains negative (Fig. 1B). This situation continues throughout the end of gastrulation (Fig. lC), but by the time the heart is formed and the neural tube is already well established though largely undifferentiated, a marked decrease in Id levels is observed in the heart and CNS (Fig. 1D-E). In contrast, the cephalic and neural folds show a particularly high accumulation of Id transcripts a s well a s the neural crest and visceral arches (Fig. 3A-D) and frontonasal processes (Figs. 1D-E and 3) which contain cells derived from the neural crest. Id Expression Becomes Restricted as Development Progresses Fetal development is marked by a n overall decrease in Id transcription. By 14.5 days P.c., Id transcription is limited to the remaining unfused neural folds, specific regions of the CNS including the ventricular cells of the spinal cord, the cranial facial structures, and limbs (Figs. 1, 3, and 4). Expression of Id is observed in specific cell types of developing organs such as the interstitial cells of the developing lungs (Fig. 1H) and the condensing vertebrae (Fig. 1F) whereas it is notably absent in developing liver and skeletal muscle. One of the most striking regions of Id localization is the developing mouse limb. By 13.5 days P.c., the limb shows clear digit formation which is accompanied by cell death in the interdigital zones. The interdigital mesenchyme, which consists primarily of undifferentiated
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cells, shows strong expression of Id (Fig. 4G). Although Id decreases to undetectable levels in most tissues by 16 days P.c., certain tissues such as heart valves and limb digit tips and the inner lining of the gut are strongly positive for Id (Fig. 11). The heart, which is among the first tissues to differentiate, shows a marked absence of Id signal early in development. At later stages, however, we observe that the endocardial cells which give rise to the endocardial cushions and ultimately the valves and outflow tract of the heart show maintained high levels of Id expression (Figs. 1E and 4A-D). We also note that the blood islands, which are a prominent feature of the early embryonic hematopoietic system (Wilkinson et al., 1987; Rugh, 19901, also show a detectable signal for Id (data not shown). This signal remains clearly detectable until about 10.5 days p.c. of development. Expression is very high in the developing meninges of the CNS as well as the pericardium (Figs. 1G and 3) but absent in immediately adjacent tissues.
bud corresponding to the rapidly dividing cells of the “progress zone” (Summerbell et al., 1973).
Id and Hox-7.1 Show a High Concordance of Expression The expression of Id is associated with regions of the embryo such as the limb bud progress zone which are sites of proliferation coupled with positional fate assignment. We had previously reported the identification of a murine homeobox containing gene, Hox-7.1 (Robert et al., 1989; Hill et al., 1989) which is also associated with these regions. Therefore we performed additional Hox-7.1 hybridizations on adjacent sections to investigate this further. Our results indicate that while the two genes do not have identical distributions in all regions there is a striking correspondence of the expression of both genes noted in the vast majority of regions, most notable the developing limbs, visceral arches, heart valves, and CNS associated tissues. For instance, as presented in Figure 3, by 9.75 days P.c., when Id expression shows a marked decrease in overall Id Expression Is Not Detected in All expression, high levels for Id and Hox-7.1 are noted in Proliferative Regions the frontonasal processes (Fig. 3A,B), visceral arches It is clear from our results that Id is not expressed in (Fig. 3C,D), and specific regions of the CNS correspondall proliferative regions of the embryo (Figs. l A , 3, and ing to either migrating neural crest and presumptive 4). For example, Id is not expressed in the early 6.5 day meninges (Fig. 3E,F). This correspondence is even p.c. embryonic ectoderm and in the numerous regions more striking in later development when both Id and in the embryo present from 9.5 days p.c. and later Hox-7.1 levels are more spatially restricted throughout which are undergoing rapid proliferation. In addition, the embryo. In the developing endocardial cushions we observe that only specific regions of the developing that later give rise to the atrial-ventricular valves, CNS such as the roof of the mesencephalon (Fig. 3E), strong expression for both genes is detected (Fig. 4Athe back wall of the telencephalon at 13 days P.c., or the neural folds of the spinal cord and brain (see Fig. 1). Id is not expressed at uniformly high levels throughout the ventricular lining of the neural tube as might be Fig. 1. Id expression during postimplantation development-darkfield expected if Id was merely associated with proliferation. photomicrographs. (A) Parasagittal section of a 6.5 day p.c. embryo. As mentioned, a notable lack of Id transcripts is ob- Accumulation of Id transcripts is detected over the extraernbryonic tisserved in the developing liver, which is clearly under- sues (x) whereas the parietal endoderm (p), ectoplacental cone (c), and embryonic ectoderm and egg cylinder (e) do not show detectable levels. going a high degree of proliferation. Scale bar = 100 Km. (B) Parasagittal section of a 7.5 day p.c. embryo. Id and the bHLH Myogenic Factors Are Not Coexpressed We compared the expression patterns of the myogenic factors and Id. Adjacent serial sections were hybridized with myf5 and myogenin [which are the first factors to appear during the course of development (Sassoon et al., 1989; Ott et al., 199111 and labeling patterns were compared t o those obtained for Id. As can be seen in Figure 2A-C, the myotomal compartments are strongly labeled for myf5 and myogenin whereas strong Id transcription is absent in the same region. A similar pattern of mutually exclusive expression of Id and myogenic factors is also observed elsewhere in the embryo. For instance, a s limb development progresses, the first cells to become positive for the myogenic factors are present in the proximal portion of the 10.5-11 days p.c. limb buds (Sassoon et al., 1989; Beddington and Martin, 1989). During this stage, Id transcription becomes more localized to the distal portion of the limb
All embryonic tissues including ectoderm (e) and amnion (a) are labeled whereas the ectoplacental cone remains negative (c). Scale same as A. (C) Horizontal section through an 8.5 day p.c. embryo showing strong labelling in all embryonic tissues. Scale same as A. (D) Horizontal section through a 9.75 day p.c. embryo showing strong accumulation in the neural folds (nf), limb buds (Ib), cephalic neural folds (cf), and frontonasal processes (v) whereas the heart (h) and brain (between cf and v) are essentially negative for Id. Scale bar = 0.5 mm. (E) Parasagittal section through a 10.5 day p.c. embryo showing strong accumulation of Id in the mandibular processes (m), frontonasal processes (f), and sclerotome derived mesoderm throughout the embryo. Telencephalon (t) and heart (h) remain negative for Id. Scale same as D. (F) Parasagittal caudal view of a 12.5 day p.c. embryo showing accumulation of Id transcripts in the neural folds (nf) and underlying spinal cord, sclerotome (s) and condensing vetebrae (c) and genital eminence (9). Scale same as D. ( G ) 13.5 day p.c. embryo (head only). Parasagittal view showing strong labeling in the Also note that the hind wall facial structures including the olfactory pit (0). of the telencephalon (th) is labeled whereas the rest of the telencephalon (t) is not labeled. (H) High magnification view of the lung bud of a 13.5 day p.c. embryo showing diffuse labeling throughout except for the inner cells of the lung (I) which are distinctly negative for Id. Scale bar = 30 km. (I) High magnification of the gut of a 16.5 day p.c. embryo. At this stage, very few structures are positive for Id however the epithelial lining of the gut (e) is strongly labeled. Scale same as H.
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D). In the genital eminence (which will give rise to the external genitalia, Rugh, 1990) and developing limbs which show marked similarities during development, a concordant expression of both genes is noted (Fig. 4EH). Just prior to birth, levels of Id and Hox-7.1 are greatly decreased with the exception of the heart valves, digit tips, and meninges of the brain suggesting that the two gene products participate in related cellular processes. DISCUSSION Id and the HLH Myogenic Factors Are Not Coexpressed An inhibitory role for Id has been postulated in skeletal muscle, B cell maturation, and neural development based on tissue culture models and genetic studies in Drosophila (Benezra et al., 1990; Pongubala and Atchinson, 1991; Cordle et al., 1991; Ellis et al., 1990; Garrell and Modolell, 1990). The ability to follow both the temporal and spatial patterns of gene transcription during embryogenesis provides a descriptive picture for which subsequent functional experiments can be designed. In this study, we demonstrate that the pattern of Id transcription in the early embryo is consistent with its postulated role as a n inhibitor of differentiation in multiple cell lineages. Id expression is widespread during early development and becomes restricted as development proceeds. However, the fact that Id is not detected in the embryonic ectoderm prior to gastrulation nor in the developing liver, both of which are highly proliferative, suggests that Id plays a role specific to certain developmental programs. With respect to skeletal muscle differentiation, observations in tissue culture cells indicate that Id and myogenic regulatory factors are coexpressed. I t has been proposed that Id prevents the onset of terminal differentiation in these cells by associating with products of the E2A gene (a ubiquitously expressed bHLH protein) and preventing the formation of active myogenic factoriE2A heterooligomeric transcription complexes (Benezra et al., 1990; J e n et al., 1992; Sun et al., 1991). In the embryo, however, we observe that coordinate expression of myogenic factors and Id does not occur to any significant extent. This may well be explained by the levels of Id expression and a hierarchy of Id interactions with other HLH proteins. In two independent studies i t has been shown that Id associates more tightly with E2A proteins than with MyoD (Benezra et al., 1990; Sun et al., 1991). One could imagine therefore that only when cells express high levels of Id (as, for example, in the embryo) can Id associate with MyoD and prevent MyoD from activating its own transcription. At intermediate Id levels (present in proliferating tissue culture cells) Id/E2A complexes predominate and MyoD is free to activate its own transcription. Consistent with this model is the observation that overexpression of Id in tissue culture cells significantly reduces the levels of steady-state MyoD mRNA (Jen et al., 1992). Also it is possible that the interme-
diate levels of Id present in proliferating myoblast cultures may simply be due to growth selection, i.e., high levels of Id expression have been shown to be growth suppressive in tissue culture cells (Jen et al., 1992) and
Fig. 2. Parasagittal section through the somites of a 9.75 day p.c. mouse embryo. (A) The brightfield view indicating the myotorne (Mj, sclerotome (S), and dermotome (D). The dashed line indicates one boundary between the sclerotome and myotorne. (6) Darkfield of an immediately serial section to A hybridized with a “co*cktail” of rnyf5 and myogenin showing strong accumulation of silver grains over the myotomes. (C) A darkfield view of A which was hybridized for Id. Note the strong signal in the sclerotome and weaker although clearly detectable signal over the dermatome. We note that Id is not detected in the rnyotome compartment (see dashed line in A, 6,and C for comparison). Scale bar = 30 pm.
Fig. 3. Detailed parasagittal sections of 9.5 day p.c. embryo hybridized for Id (A,C,E) and Hox-7.1 (B,D,F). (A) Id labeling of the frontonasal mass as shown in Figure 1D. (B) An section adjacent to (A) hybridized for Hox-7.1. (C) Section showing Id accumulation in the visceral arches (rn) and cleft (c) as well as the pericardial tissues lining the ventricle (v) and atria (a). (D) Parallel section to (C) showing a similar distribution of Hox-
7.1. We note that the distribution is not exactly that shown for Id. (E) Forebrain showing Id accumulation in the dorsal portion of the telencephalon (t) and mesencephalon (m) as well as the developing choroid structures (c). (F) Parallel section to (E) hybridized with Hox-7.1 showing a similar distribution in the same structures. Scale bar = 0.05 mm.
low levels of Id would be expected to lead to terminal differentiation. The observed down-regulation of Id transcription by differentiating myoblasts in vitro would imply that differentiated muscle in vivo would have either low or undetectable levels of Id. This prediction is true, and in
addition we note that all mesodermal cells in regions of differentiating muscle, including chondroblasts, endothelial cells, and connective tissue, down-regulate Id in a coordinate manner. We have observed a striking colocalization of transcripts of Id and Hox-7.1 throughout mouse
Fig. 4.
Id EXPRESSION DURING MOUSE DEVELOPMENT
embryogenesis. We had postulated that Hox-7.1 may be involved in repressing the myogenic program based upon several observations. First, Hox-7.1 and the myogenic factors are expressed in a mutually exclusive manner, and expression of Hox-7.1 is limited to undifferentiated tissue in the embryo (Y. Wang and D. Sassoon, in preparation; Robert e t al., 1989). Second, forced expression of Hox-7.1 in myogenic cell lines delays or abolishes the capacity to terminally differentiate (Song et al., 1992). It is unlikely that coexpression in such a wide variety of cell types (CNS, heart valves, limb and visceral arch mesenchyme, etc.) is purely coincidental and reflects functionally unrelated events. During the entirety of mouse embryogenesis, the only regions of Id expression that do not correspond to Hox-7.1 expression are the dermatome and sclerotome of the somites and the lining of the gut and cells in the interstitial cells of the testis. We propose that Hox-7.1 related genes, or other Hox genes not currently isolated or characterized may also participate in negative regulation in these tissue types. A recently characterized homeobox-containing gene, S8 (Opstelten et al., 1991), is also expressed in a pattern reminiscent of Hox-7.1. Although its pattern of expression is not identical to Hox-7.1, it is expressed in the sclerotome and dermatome of the somites in a pattern coincident with Id. Another helix-loophelix gene with a similar distribution to Id and Hox-7.1 is the murine twist gene (Perrin-Schmitt, 1991), which is expressed in the somites but not in the myotome. The observation that several regulatory genes are coexpressed during development strongly suggests that they participate in a common regulatory pathway. The coordinate down-regulation of Id in the limb suggests a regulatory mechanism that is not unique to skeletal myoblasts; instead, a regional control of Id expression must be invoked for the embryo. Our results reveal that Id is regulated regionally in entire structures such as the limb bud and therefore may not be regulated by a n autonomous cell program. Homeobox Fig. 4. Parasagittal sections hybridized with Id (A,C,E,G) and adjacent sections hybridized with Hox-7.1 (B,D,F,H). (A) 10.5 day p.c. heart showing the endocardial cushion (ec) which are the only structure in the heart positive for Id. Also labeling is present in the mandibular arch (m). (B) Adjacent section to (A) hybridized with Hox-7.1 showing accumulation in the endocardial cushions and the mandibular arch. (C) Section through a 13.5 day p.c. heart showing the ventricle (ve), atria (a), and ventricular valve (v). The valve shows a clear accumulation of Id transcripts whereas the heart is negative. (D)Adjacent section to (C) hybridized for Hox-7.1 showing a virtually identical distribution of transcripts as observed with Id. (E) Higher magnification of genital eminence (9) of a 12.5 day p.c. embryo (see Fig. 1F) showing Id distribution in the distal portion. (F) Adjacent section to (E) hybridized for Hox-7.1 showing a similar distribution as seen for Id. This pattern is also highly reminiscent for that seen in early limb bud progress zones. (G) Section hybridized for Id showing 13.5 day p.c. hindlirnb (digits numbered 1-5). Note that Id transcripts accumulate primarily in the interdigital regions and digit tips during this stage of limb development. (H) Adjacent section to (G) hybridized with Hox-7.1. Again, a very similar distribution of Hox-7.1 and Id transcripts is noted. (A-F) Scale bar = 0.05 mm. (G,H) Scale bar = 0.4 mm.
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genes are strong candidates as regional regulators of differentiation and proliferation during development, since they are expressed in regional domains that follow either rostral-caudal or proximal-distal boundaries but are not restricted to specific cell lineages (Davidson, 1991).
Id Expression Is Not Uniquely Associated With Proliferating Cells The observation that Id is down-regulated in a number of cell lines a s differentiation proceeds and cells exit the cell cycle has suggested that Id may play a role in cell proliferation (Benezra et al., 1990). Our in situ hybridization results clearly indicate that Id expression is not a prerequisite for cell proliferation. For instance, Id is not expressed prior to gastrulation in the embryonic ectoderm during a period of time when cells are rapidly proliferating. In addition, if Id was necessary for cell proliferation, we would expect a wider distribution throughout the embryo including the developing liver, which is negative for Id. Furthermore, the ventricles of the brain and spinal cord are labeled for Id only during discrete periods of development whereas proliferation occurs in these zones for longer periods of time. In agreement with these observations is the fact that several immortalized cell lines show very little Id mRNA accumulation (Benezra e t al., 1990). It is possible, however, that down-regulation of Id expression is necessary to allow cells to withdraw from the cell cycle. This may be a result of Id antagonizing the proposed growth suppressive property of MyoD or other tissue specific b-HLH proteins (Sorrentino et al., 1990; Crescenzi et al., 1990). Consistent with this notion is the observation that Id expression is not observed in terminally differentiated cells and is downregulated in fibroblasts and myoblasts that have entered Go (Benezra et al., 1990; Christy et al., 1991). This study demonstrates that Id expression is found primarily in undifferentiated cells undergoing positional fate assignment, such as the visceral arches, genital eminence, and most notably the limb progress zone (Wedden et al., 1988). It has been recently reported that Hox-7.1 transcription is similarly maintained in the limb progress zone by interactions with the limb apical ectoderm (Davidson et al., 1991). Id is highly associated with Hox-7.1 expression in many regions of the embryo. We suggest that Id and Hox-7.1 may be involved in related developmental processes. Current studies indicate that like Id, forced expression of Hox-7.1 can delay or block differentiation of myoblasts (Song et al., 1992). We note that two additional Id-like genes, Id2 (Sun et al., 1991) and HLH462 (Id3; Christy et al., 1991) have been recently isolated and can both be demonstrated to functionally inhibit DNA binding of other HLH DNAbinding proteins. Thus, i t will be of interest to determine if they are codistributed with Id or are expressed in complementary regions during development.
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ACKNOWLEDGMENTS The authors wish to thank Esfir Slonimsky and Anna Pavlova for technical assistance, advice, and support. We also with to thank Drs. Nadia Rosenthal, Debby Dobson, Steve Farmer, and Margaret Buckingham for helpful discussion and criticism a s well a s Kening Song and Xun Weng. DS is supported in part by a grant from The Council for Tobacco Research, U S A . and American Cancer Society. We acknowledge Dr. Hans Arnold for the cDNA probe for myf5. REFERENCES Beddington, R.S.P., and Martin, P. (1989) An in situ transgenic enzyme marker to monitor migration of cells in the mid-gestation mouse embryo:somite contribution to the early forelimb bud. Mol. Biol. Med. 6:263-274. Benezra, R., Davis, R.L., Lockshon, D., Turner, D.L., and Weintraub, H. (1990) The protein Id: A negative regulator of helix-loop-helix DNA binding proteins. Cell 61:49-59. Bober, E., Lyons, G., Braun, T., Cossu, G., Buckingham, M., and Arnold, H. (1991) The muscle regulatory gene, Myf-6, has a biphasic pattern of expression during early mouse development. J . Cell Biol. 113:1255-1265. Braun, T., Buschhausen-Denker, G., Bober, E., Tannich, E., and Arnold, H.H. (1989) A novel human muscle factor related to but distinct from MyoDl induces myogenic conversion in 10T112 fibroblasts. EMBO J. 8:701-709. Braun, T., Bober, E., Winter, B., Rosenthal, N., and Arnold, H.H. (1990) Myf-6, a new member of the human gene family of myogenic determination factors: Evidence for a gene cluster on chromosome 12. EMBO J. 9521-831. Christy, B.A., Sanders, L.K., Lau, L.F., Copeland, N.G., Jenkins, N.A. and Nathans, D. (1991) An Id-related helix-loop-helix protein encoded by a growth factor-inducible gene. Proc. Natl. Acad. Sci. U.S.A. 88:1815-1819. Cordle, S.R., Henderson, E., Masuoka, H., Weil, P.A. and Stein, R. (1991) Pancreatic b-cell-type-specific transcription of the insulin gene is mediated by basic helix-loop-helix DNA-binding proteins. Mol. Cell. Biol. 11:1734-1738. Crescenzi, M., Fleming, T.P., Lassar, A.B., Weintraub, H., and Aaronson, S.A. (1990) MyoD induces growth arrest independent of differentiation in normal and transformed cell. Proc. Natl. Acad. Sci. U.S.A. 87:8442-8446. Davidson, D.R., Crawley, A,, Hill, R.E., andTickle, C. (1991) Positiondependent expression of two related homeobox genes in developing vertebrate limbs. Nature (London) 352:429-431. Davidson, E.H. (1991) Spatial mechanisms of gene regulation in metazoan embryos. Development 113:l-26. Davis, R.L., Cheng, P.-F., Lassar, A.B., and Weintraub, H. (1990) The MyoD DNA binding domain contains a recognition code for musclespecific gene activation. Cell 60:733-746. Davis, R.L., Weintraub, H., and Lassar, A.B. (1987) Expression of a single transfected cDNA converts fibroblasts to myoblasts. Cell 51: 987-1000. Edmondson, D.G., and Olson, E.N. (1989) A gene with hom*ology to the myc similarity region of myoDl is expressed during myogenesis and is sufficient to activate the differentiation program. Genes Dev. 3:628-640. Ellis, H.M., Spann, D.R., and Posakony, J.W. (1990) Extramachrochaete, a negative regulator of sensory organ development in Drosophila, defines a new class of helix-loop-helix proteins. Cell 61:27-38. Fontaine, B., Sassoon, D., Buckingham, M., and Changeux, J-P. (1988) Detection of the nicotinic acetylcholinereceptor a-subunit mRNA by in situ hybridization at neuromuscular junctions of 15day-old chick striated muscles. EMBO J. 7(3):603-609. Gall, J.G., and Pardue, M.L. (1971) Nucleic acid hybridization in cytological preparation. Methods Enzymol. 21:470-480. Garrell, J., and Modolell, J . (1990)The Drosophila extramachrochaete locus, an antagonist of proneural genes that, like these genes, encodes a helix-loop-helix protein. Cell 61:39-48. Hill, R.E., Jones, P.F., Rees, A.R., Sime, C.M.,Justice, M.J., Copeland, N.G., Jenkins, N.A., Graham, E., and Davidson, D.R. (1989) A new
family of mouse homeobox-containing genes: Molecular structure, chromosomal location, and developmental expression of Hox7.1. Genes Dev. 3:26-37. Hinterberger, T., Sassoon, D., Rhodes, S., and Koneiczny, S. (1991) Expression of the muscle regulatory factor MRF4 during somite and skeletal myofiber development. Dev. Biol. 147:144-156. Jen, Y., Weintraub, H., and Benezra, R. (1992) Overexpression of Id protein inhibits the muscle differentiation program: In vivo association of Id with E2A proteins. Genes Dev., 6:1466-1479. Mackenzie, A,, Leeming, G.L., Jowett, A.K., Ferguson, M.W.J., and Sharpe, P.T. 11991)The homeobox gene Hox7.1 has specific regional and temporal expression patterns during early murine craniofacial embryogenesis, especially tooth development in uiuo and in uitro. Development 111:269-285. Miner, J.H., and Wold, B. (1990) Herculin, a fourth member of the myoD family of myogenic regulatory genes. Proc. Natl. Acad. SEi. U.S.A. 87:1089-1093. Olson, E.N. (1990) MyoD family: A paradigm for development? Genes Dev. 4:1454-1461. Opstelten, D.J.E., Vogels, R., Robert, B., Kalkhoven, E., Zwartkruis, F. Laaf, L., Destree, O.H., Deschamps, J., Lawson, K.A., and Meijlink, K. (1991) The mouse homeobox gene, S8, is expressed during embryogenesis predominantly in mesenchyme. Mech. Dev. 34:2942. Ott, M-0, Bober, E., Lyons, G., Arnold, H., and Buckingham, M. (1991) Early expression of the myogenic regulatory gene, myf-5, in precursor cells of skeletal muscle in the mouse embryo. Development 111:1097-1107. Perrin-Schmitt, F. (1991) The m-twist gene of Mus is expressed in subsets of mesodermal cells and is closely related to the Xenopus X-twz and Drosophila twist genes. Dev. Biol. 143:363-373. Pongubala, J.M., and Atchinson, M.L. (1991) Functional characterization of the developmentally controlled immunoglobulin kappa 3’ enhancer: Regulation by Id, a repressor of helix-loop-helix transcription factors. Mol. Cell. Biol. 11:1040-1047. Rhodes, S.J., and Konieczny, S.F. (1989) Identification of MRF4: A new member of the muscle regulatory factor gene family. Genes Dev. 3:2050-2061. Robert, B., Sassoon, D., Jacq, B., Gehring, W., and Buckingham, M. (1989) Hox-7, a mouse homeobox gene with a novel pattern of expression during embryogenesis. EMBO J . 8:91-100. Rugh, R. (1990) “The Mouse: Its Reproduction and Development.” Oxford: Oxford Univ. Press. Sassoon, D., Garner, I., and Buckingham, M. (1988) Transcripts of a-cardiac and a-skeletal actin are early markers for myogenesis in the mouse embryo. Development 104:155-164. Sassoon, D., Lyons, G., Wright, W.E., Lin, V., Lassar, A,, Weintraub, H., and Buckingham, M. (1989) Expression of two myogenic regulatory factors myogenin and myoDl during mouse embryogenesis. Nature (London) 341:303-307. Song, K., Wang, Y., and Sassoon, D. (1992) Expression of Hox7.1 in myoblasts inhibits terminal differentiation and induces cell transformation. Nature (London), in press. Sorrentino, V., Pepperkok, R., Davis, R.L., Ansorge, W., and Philipson, L. (1990) Cell proliferation inhibited by MyoDl independently of myogenic differentiation. Nature (London) 345:813-815. Summerbell, D., Lewis, J.H., and Wolpert, L. (1973) Positional information in chick limb morphogenesis. Nature (London)244:492-496. Sun, X-H., Copeland, N., Jenkins, N.A., and Baltimore, D. (1991) Id proteins Id1 and Id2 selectively inhibit DNA binding by one class of helix-loop-helix proteins. Mol. Cell Biol. 11:5603-5611. Wedden, S.E., Ralphs, J.R., and Tickle, C. (1988)Pattern formation in the facial primordia. Development (supplement) 103:31-40. Weintraub, H., Davis, R., Tapscott, S., Thayer, M., Krause, M., Benezra, R., Blackwell, T.K., Turner, D., Rupp, R., Hollenberg, S., Zhuang, Y., Lassar, A. (1991) The MyoD gene family: Nodal point during specification of the muscle cell lineage. Science 251:761-766. Wilkinson, D.G., Bailes, J.A., Champion, J.E., and McMahon, A.P.(19871 A molecular analysis of mouse development from 8-10 day post coitum detects changes only in embryonic globin expression. Development 99:493-500. Wright, W.E., Sassoon, D.A., and Lin, V.K. (1989) Myogenin, a factor regulating myogenesis, has a domain hom*ologous to MyoD1. Cell 56:607-617.
