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Mechanisms of Development, 37 (1992) 111-120 © 1992 Elsevier Scientific Publishers Ireland, Ltd. 0925-4773/92/$05.00 MOD00085
Expression and modification of Hox 2.1 protein in mouse embryos Nancy A. Wall, C. Michael Jones, Brigid L.M. Hogan and Christopher V.E. Wright Department of Cell Biology, Vanderbilt University, Nashville, TN 37232, U.S.A. (Received 4 October 1991; revision received 9 December 1991; accepted 19 December 1991)
A polyclonal antibody, txHox 2.1a, has been generated and used to immunolocalize Hox 2.1 protein in mouse embryos. Protein is present in nuclei of all tissues previously shown to express Hox 2.1 RNA. In addition, protein is seen in somites and proximal regions of the limb buds, tissues in which Hox 2.1 RNA expression was not clearly detected previously by in situ hybridization. At the 7 somite stage, protein is detectable in the neural tube up to the level of somite 1, but later retracts to a more posterior position. Immunoblot, in vitro translation, and immunoprecipitation experiments were carried out to characterize the Hox 2.1 protein. The results show that the Hox 2.1 gene produces at least two related phosphorylated proteins present in different proportions in different tissues. Mouse embryo; Hox 2.1; Antibody; Phosphorylation; Immunohistochemistry
Introduction
Homeobox containing genes have been shown, either by mutation, overexpression, or deletion, to be critical to normal development in species ranging from Drosophila to mouse (Gehring et al., 1990; Kenyon, 1986; Wright et al., 1989a; Cho et al., 1991; Kessel et al., 1990; Li et al., 1990; Chisaka and Cappechi, 1991; Lufkin et al., 1991). There is good genetic and biochemical evidence that homeodomain proteins are DNA binding proteins (selector molecules) which regulate the transcription of downstream effector genes in developing embryos (Li et al., 1990; Gehring et al., 1990; Kissinger et al., 1990). One important method for investigating the specific function(s) of homeobox genes during development is to determine their temporal and spatial patterns of expression at the single cell and intracellular level, and to correlate these patterns with those of other genes expressed in both normal and mutant animals. Although in situ hybridization has been used to localize transcripts of many homeobox genes in embryonic tissues, demonstration that the protein product is present is necessary to confirm and extend the functional significance of any transcription pattern.
Correspondence to: C.V.E. Wright, Department of Cell Biology, Vanderbilt University, Nashville, TN 37232, U.S.A.
Immunological techniques also provide methods for determining, for example, the DNA sequence(s) these proteins bind to in vivo (Gould et al., 1990). Additionally, by employing immunoprecipitation, in vitro translation, and two-dimensional gel electrophoresis, resolution of different homeobox containing proteins a n d / o r modified forms of these proteins can be achieved. Using these techniques in Drosophila embryos, fushi tarazu ( ftz ) and Ultrabithorax ( Ubx ) have been shown to produce multiple protein isoforms (Krause et al., 1988; Gavis and Hogness, 1991), while Voss et al. (1991) have demonstrated multiple translation start sites and proteins for Pit-1. In spite of the advantages of studying homeobox genes with antibodies (Oliver et al., 1988; Wright et al., 1989b; Patel et al., 1989; Mahaffey et al., 1989; Sundin and Eichele, 1990), very few have been reported for mouse Hox gene products. Awgulewitsch and Jacobs (1990) used peptide antibodies to discern specific cell types that express Hox 3.1 in mouse spinal cord, and Davis et al. (1991) used fusion protein antibodies to demonstrate the localization of engrailed protein in mouse, as well as in chicken and Xenopus embryos, We report here the generation of an antibody specific for the mouse homeoprotein Hox 2.1. This antibody has been used in whole mount immunohistochemistry to provide data that extend the in situ hybridization patterns already reported for this gene (Holland and Hogan, 1988). We report the presence of Hox 2.1
112
Lu
protein in regions where previously it either had not been seen or was at the limits of detection using m R N A probes, particularly during initial phases of expression at early somite stages. Western blotting, immunoprecipitation, and in vitro translation of poly A + selected R N A reveal that at least two phosphorylated forms of Hox 2.1 protein are present in developing mouse embryos. This antibody, and the data reported here, will make it possible to better characterize Hox gene expression in mice in which Hox genes have been deleted or overexpressed.
Lb
Asc
Fb
46kDa
Results
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Synthesis and modification of Hox 2.1
Fig. 1. Western immunoblot with aHox 2.1a of protein from tissue extracts of 12.5d mouse lung (Lu), limb bud (Lb), anterior spinal cord (Asc), and forebrain (Fb). Lines represent Mr markers of 46,000 and 30,000. 5 /zg of total protein (determined by Bradford assay) was loaded per lane.
We generated an affinity purified rabbit polyclonal antibody, completely specific for Hox 2.1 (see Materials and Methods for antibody production and specificity), using the glutathione-S-transferase fusion protein vector p G E X . Previously, in situ hybridization (Holland and Hogan, 1988) and Northern analysis (Krumlauf et al., 1987) using 12.5-14.5 day old (12.5d-14.5d) mouse embryos had shown that Hox 2.1 is expressed at high levels in the anterior spinal cord and lung. Extracts of these tissues taken from 12.5d mouse embryos, along with forebrain and limb bud which do not show Hox 2.1 R N A expression by in situ analysis or immunostaining, were used in Western immunoblot analysis. Fig. 1 shows that a H o x 2.1a specifically recognizes at least two proteins, one of M r ~ 40,000 and another at M r ~ 38,000. We occasionally detect an M r ~ 37,500 species that could be a third Hox 2.1 species or a degradation product. Although Krumlauf et al. (1987) predicted a M r of 29,469 for the Hox 2.1 protein, its migration with a higher apparent M r is easily explained by post-translational modification a n d / o r anomalous migration often encountered during S D S - P A G E analysis. Lungs and anterior spinal cord from 12.5d mouse embryos were explanted and metabolically labelled with
Lu
Asq
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NRS Ab NRS Ab
A
Ab
B
[35S]methionine or [32p]orthophosphate, and Hox 2.1 protein was immunoprecipitated from tissue extracts. Fig. 2A ([35S]methionine labelled) and Fig. 2B ([32p]orthophosphate labelled) show that a H o x 2.1a specifically recognizes two newly synthesized proteins of M r ~ 40,000 and ~ 38,000 and that both are labelled with [32p]orthophosphate. Both Western immunoblot and immunoprecipitation experiments show that the ratio of 40 k D a / 3 8 kDa proteins is consistently higher in anterior spinal cord than in the lung, indicating that different tissues contain different proportions of Hox 2.1 proteins. We next extracted poly A ÷ R N A from 12.5d mouse lung and anterior spinal cord, performed in vitro translation using rabbit reticulocyte lysate, and examined the products by immunoprecipitation. Fig. 2C shows that both the M r ~ 40,000 and M r ~ 38,000 Hox 2.1 proteins are produced. There is no apparent difference in the M r of the in vitro translated proteins and those
Asc NRS
Ab
NRS
Lu
C
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i
Fig. 2. Immunoprecipitation of in vivo and in vitro translated protein with aHox 2.1a. (A) [35S]methionine labelled protein from 12.5d mouse lung (Lu) and anterior spinal cord (Asc) with NRS or antibody (Ab). (B) [32p]orthophosphate labelled protein. (C) [35S]methionine labelled protein from 12.5d mouse lung (Lu) and anterior spinal cord (Asc) and in vitro translated protein from 12.5d mouse embryo poly A + RNA from lung (Lu*) and anterior spinal cord (Asc*). Line represents Mr marker of 30,000.
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Fig. 3. Whole embryos immunostained wilh a H o x 2.1a. (A) Dorsal view of a 7 somite mouse embryo demonstrating differential anterior limits of Hox 2.1 expression in the neural tube and somites. T h e first 5 somites are indicated by n u m b e r e d arrowheads. T h e arrow indicates anterior limit of expression in the neural tube. Bar = 250 p.m. (B) Higher magnification of (A). Bar = 250/xm. (C) Dorsal view of a 9 somite mouse embryo demonstrating retraction of the anterior limit of expression in the neural tube. N u m b e r e d arrowheads indicate somites 3 and 4. Arrow indicates anterior limit of expression in the neural tube. Bar = 250 ~ m . (D) Higher magnification of a different 9 somite mouse embryo demonstrating staining in the posterior portion of somite 5 (arrow). Bar = 100/xm. ot, otic vesicle; nc, neural crest; ep, ectodermal placode.
114 immunoprecipitated from fresh tissue. There is a slight increase in the ratio of the 40 kDa/38 kDa species in lung in vitro translated products compared to in vivo lung extracts, but when compared to anterior spinal cord in vitro translated products it still seems that the anterior spinal cord protein ratio is quantitatively different.
Hox 2.1 protein expression in whole embryos We could not detect Hox 2.1 protein before the 7 somite stage ( ~ 8.0d) of development by our immunostaining methods (data not shown). We examined ten embryos by whole mount immunohistochemistry at a stage when 7-9 somites could be distinguished by light
Fig. 4. Whole embryos immunostained with a H o x 2.1a. (A) 9.0d embryo demonstrating that anterior limits of expression in neural tube and L P M are now apparently in register. Arrowhead indicates the otic vesicle. Bar = 500 /zm. (B) 9.5d embryo showing staining in neural crest cells (arrow). Arrowhead indicates the otic vesicle. Bar = 500 izm. (C) 10.5d embryo, Arrow indicates neural crest. Bar = 500/~m. (D) Magnification of neural crest in B (arrowheads). Bar = 125 /~m. (E) Magnification of neural crest from a 10.0d embryo when the ectodermal placode first exhibits expression of Hox 2.1 protein. Arrows indicate the ectodermal placode. Bar = 125 /zm. (F) Magnification of neural crest in C. Ectodermal placode cells (arrow) and neural crest (arrowheads) stain positive. Bar = 250 /.Lm. ot, otic vesicle; nc, neural crest; ep, ectodermal placode.
115 microscopy. Those at the 7 somite stage clearly showed Hox 2.1 staining in the posterior half of somite 5 and in all cells of more caudal somites (Fig. 3). In contrast, expression in the neural tube extends more anteriorly, and can be clearly detected up to the level of somite 1 (Fig. 3A,B). At the 9 somite stage, Hox 2.1 protein is still present in the posterior of somite 5. However, in the neural tube expression has retracted posteriorly to the level between somites 3 and 4 (Fig. 3C). By 9.0d (~ 12 somites) the anterior border of expression in the neural tube and the anterior limit of expression in the
,a.
B
C
somites and lateral plate mesoderm appear to be in register (Fig. 4A). The 9.5d ( ~ 22 somites) embryos show relatively high levels of Hox 2.1 protein in the neural tube and the lateral plate mesoderm, but expression in the somites seems to be decreased (Fig. 4B). Also at 9.5d, the lung bud and gut are undergoing organogenesis and react positively for Hox 2.1. Staining is also seen in the anterior proximal region of the forelimb buds (Fig. 5C) an area where Hox 2.1 had not been detected previously in the mouse by in situ hybridization, although this result is not surprising since similar expression of the presumed 2.1 homolog, Ghox 2.1, has been demonstrated in the developing chick limb bud (Wedden and Eichele, 1989). Of particular interest is the appearance in 9.5d embryos of a streak of positively staining cranial neural crest cells located posterior and lateral to the otic vesicle at about the level of the heart, and lying at a dorsoanterior to ventroposterior angle (Fig. 4D). At 10.0d (27 somites) a new area of positive staining is noticeable near the center of this streak of neural crest cells. This indention of ectoderm represents the ectodermally derived cells that contribute to the inferior nodose ganglion of the Xth cranial (or vagus) nerve (Fig. 4E). Although barely discernible at 10.0d, Hox 2.1 immunoreactivity now appears in the metanephric tubules and undifferentiated nephrogenic cord (data not shown). Hox 2.1 is still detected in the neural tube, somites (at decreased levels), the anterior proximal region of forelimb buds, and the mesenchyme of the developing lung and gut. In 10.5d (~ 31 somites) embryos, immunostaining in the metanephric tubules is much more noticeable (data not shown). The lateral plate mesoderm is clearly expressing Hox 2.1 protein and somite expression is still detectable, but at a reduced level (Fig. 4C). Neural crest cells are still present in the aforementioned streak but appear to be condensing around the nodose ganglion placodes (Fig. 4F). Positively staining cells are also seen in the third and fourth branchial arches at this stage, probably representing migrating cranial neural crest (data not shown). Hox 2.1 protein in sectioned and whole mount gut
Fig. 5. 12.5d whole gastrointestinal and respiratory tracts immunostained with a H o x 2.1a. (A) Positive staining is seen in the larynx (la), lungs (lu), stomach (st), and hindgut (i) but is absent in the trachea and esophagus (open arrow). Bar = 1 mm. (B) Magnification of the area indicated by the closed arrow in A (structure is rotated 90 ° counterclockwise relative to (A)). Staining is restricted to individual cells of the enteric plexus (arrow) and ends in the region which is herniated at this stage. Bar = 100 Izm. (C) Dorsal view of staining in 10.5d forelimb bud. Arrows indicate region of positive staining, a, anterior; p, posterior. Bar ~ 100/xm.
Immunohistochemical staining of both whole mount gut and sectioned ll.5d-14.5d embryos shows that Hox 2.1 protein is strongly expressed in mesoderm, but not endodermal epithelium, of the developing gastrointestinal and respiratory tracts, in an on-off pattern. Some cells of the larynx stain positively for Hox 2.1 protein (Fig. 5A). At present, we cannot tell if these cells are derived from neural crest or mesoderm. Tracheal and esophageal mesenchyme do not express Hox 2.1, but the mesoderm of the lung, stomach, and proximal portion of the small intestine react positively with
116 Discussion
Distribution of Hox 2.1 protein in embryos
Fig. 6. 12.5d parasagittal sections immunostained with aHox 2.1a. Sections are oriented so that dorsal is top and anterior is left. (A) Cross-section through part of the hindgut. Cells of the enteric plexus show positive staining (arrow). Bar = 100 /~m. (B) Section of the stomach demonstrating staining in mesoderm. The expression pattern is restricted to the dorsal, anterior and posterior regions of the stomach as it is positioned in the embryo at this stage. Arrow indicates area of most intense staining. Bar = 100 ~m. (C) Positive staining is restricted to the ventral regions of the somites (arrows). Bar = 100/zm.
a H o x 2.1a. Fig. 6B demonstrates that Hox 2.1 protein is expressed more strongly anteriorly than posteriorly in the stomach of 12.5d embryos. Although positive staining of gut mesoderm is seen in the proximal region of the small intestine, immunostaining does not extend throughout the entire hindgut. Instead, in the distal hindgut, Hox 2.1 expression is restricted to numerous individual cells that most likely represent cells of the enteric parasympathetic nervous system (enteric plexus), which is derived from the migrating vagal neural crest. This is demonstrated in Fig. 6A which shows that the cells expressing Hox 2.1 are arranged somewhat symmetrically in a ring within the mesoderm. Whole mounts at all stages examined clearly show that the posterior limit of Hox 2.1 expression in the enteric plexus of the gut is at the point of herniation through the body wall (Fig. 5B).
The localization of Hox 2.1 protein observed with antibody staining is, for the most part, entirely consistent with that reported for Hox 2.1 transcripts by in situ hybridization (Holland and Hogan, 1988). Results reported in this paper corroborate previously published data for Hox 2.1 and also throw light on the dynamics of protein expression in the early somite stage embryo when regional specification is probably being defined. When 7 somites are visible Hox 2.1 is clearly present in the posterior half of somite 5 (the first cervical somite) and all ceils of more caudal somites. Gaunt et al. (1990) reported expression of Hox 2.1 R N A in the posterior of the second cervical prevertebra (somite 6) at 12.5d. However, given the low level of transcripts they detected, and our finding that expression in somites declines with development, we consider that our immunostaining technique more accurately reflects the in vivo localization than in situ hybridization. An important conclusion from this work is that Hox 2.1 expression in the neural tube initially extends anteriorly as far as the level of somite 1, and then gradually retracts towards the junction between the hindbrain and spinal cord. By 9.0d the anterior limits of expression in the neural tube and the paraxial mesoderm appear to have become aligned, due to a combination of the expansion of somites including, and posterior to, somite 5, and the receding expression in the neural tube. Unfortunately, at the 7 somite stage in mouse it is not possible to localize the initial anterior limit of Hox 2.1 expression with respect to either rhombomere boundaries or the otic vesicles, which are not yet delimited. However, in the chick embryo, somite 1 is at the level of rhombomere 7 (Keynes and Lumsden, 1990) and this co-localization can presumably be extrapolated to the mouse embryo. In support of our finding is the observation by Wedden et al. (1990) that in the stage 17 chick embryo the anterior border of Ghox 2.1 R N A is "at the level of the fourth ventricle". In addition, Hox 2.1 expression in the mouse neural tube at the level of rhombomere 7 would be consistent with Hox 2.1 expression in neural crest derived cells in the nodose ganglion. The nodose ganglion is the inferior ganglion of the X (vagus) nerve, which is thought to be derived at least in part from neural crest cells arising in rhombomere 7 and not from neural crest arising near the spinal cord-hindbrain junction (Keynes and Lumsden, 1990). It remains to be seen whether our finding that the initial expression of Hox 2.1 protein in the neural tube, which is more anterior than previously found by in situ hybridization, can be extrapolated to other homeobox genes in the hindbrain. If so, it has important implications for current models of the inter-
117 actions of these genes in pattern formation in the vertebrate head (Hunt et al., 1991). Immunohistochemistry also reveals the presence of Hox 2.1 protein in areas that previously had been shown to express Ghox 2.1 RNA in chick (Wedden et al., 1989) but were probably not seen in the mouse because of low expression (e.g. somites and at 9.5d10.5d but not 12.5d, proximo-anterior limb buds). Since the regions exhibiting Hox 2.1 protein expression do not include tissues in which Hox 1.3 and 3.4 (the two Hox genes most similar to 2.1) are specifically expressed (e.g., Hox 1.3 is expressed in the trachea (Dony and Gruss, 1987) and Hox 3.4 is expressed in the esophagus (Gaunt et al., 1990)), cross-reactivity between the products of these or other closely related genes and the affinity purified antibody is not a concern.
Hox 2.1 in the neural crest Cranial ganglia are derived from cells from both neural crest and ectodermal placodes (LeDouarin et al., 1986) and our data provides evidence that Hox 2.1 protein is expressed in both of these components at early stages. In 9.5d whole mount embryos, bilateral streaks of neural crest cells stain positive for Hox 2.1 and expression in these cells persists through at least 10.5d. At 10.0d an indentation of ectoderm (the nodose placode) in close proximity to the Hox 2.1 expressing neural crest cells is also expressing this protein. Localization of Hox 2.1 in the nuclei of ectodermal cells has been confirmed by staining of mouse embryo sections (Shigeru Kuratani, Baylor College of Medicine, personal communication). As discussed above, it is generally believed that the neural crest cells of the nodose ganglion are derived from the neural tube at the level of rhombomere 7. This is the level at which Hox 2.1 protein is observed transiently at the 7 somite stage ( ~ 8.0d). It is of interest that Hox 2.1 protein expression is not detected by immunohistochemistry in the neural crest of whole mount embryos until approximately 9.5d, by which time Hox 2.1 expression in the neural tube has retracted to a more posterior position. One interpretation of these results is that Hox 2.1 expression is part of a pre-specification program initiated before the neural crest cells leave the tube at the level of rhombomere 7. Hox 2.1 expression would then be down regulated during migration until the cells approach the end of their migration pathway when Hox 2.1 expression is reactivated. An alternative explanation is that the migrating neural crest cells receive a positional signal when they arrive in the region of the developing nodose ganglion. Full interpretation of our results must await detailed identification of the Hox 2.1 expressing cell types in the circumpharyngeal region and nodose ganglion, and the
relative contribution of neural crest and placode derived cells. Such experiments are underway.
Evidence for post-translational modification of Hox 2.1 protein Although there appears to be three Hox 2.1 proteins by Western blot analysis, the M r ~ 37,500 protein was not consistently detected by immunoprecipitation of tissue extracts or in vitro translation of poly A + RNA from the same tissues. For this reason we think (but cannot conclusively demonstrate) that this protein is a partially degraded form of the M r ~ 40,000 a n d / o r ~ 38,000 proteins. Further analysis will need to be performed before a final determination can be made. In the following discussion we will consider that there are two Hox 2.1 proteins. There are several possible explanations for the recognition by aHox 2.1a of more than one protein species in tissue extracts by Western immunoblot and immunoprecipitation techniques. For example, both lung and anterior spinal cord contain multiple Hox 2.1 transcripts (Krumlauf et al., 1987), which could represent mRNAs alternatively spliced within the coding region similar to reports for the X1Hbox 1 (the Hox 3.3 homolog) gene (Cho et al., 1988). However, there is at present no direct evidence for such alternative splicing of Hox 2.1. An alternative explanation for multiple isoforms is that there are different post-translational modifications of Hox 2.1 protein. For example, Krause et al. (1988) and Gavis and Hogness (1991) have shown multiple isoforms of the homeoproteins ftz and Ubx in Drosophila embryos, probably differing by phosphorylation. Both the M r ~ 40,000 and M r ~ 38,000 species of Hox 2.1 are phosphorylated, but at present we have not identified which residues are involved. Since the Hox 2.1 protein sequence is 16.4% serine, and Gay et al. (1991) have shown that Drosophila en is phosphorylated on serine, this residue could be the target of an undetermined kinase. Multiple phosphorylated forms of homeoproteins could represent intermediates in the post-translational modification process or, alternatively, these different phosphorylated states may be associated with changes in the activity of the protein at the level of DNA binding or transactivation potential (Binetruy et al., 1991; Boyle et al., 1991; Tanaka and Herr, 1990). A third explanation for multiple forms of Hox 2.1 protein is the use of alternative translational start sites in the mRNA, as demonstrated for the homeobox containing gene Pit-1 (Voss et al., 1991). Immunoprecipitation of proteins translated in vitro from poly A ÷ RNA reveals that at least two Hox 2.1 proteins are made. Are these proteins translated from the same transcript, or separate transcripts? On the one hand, comparison of the isoform ratios in immunoprecipita-
118 tions from tissues and in vitro translations shows that the tissue differences are slightly reduced in vitro, indicating that the two isoforms could be translated from the same transcript, for the following reasons. First, if the two proteins derive from separate transcripts, the ratios seen in vivo should be maintained in vitro, since both transcripts would be present in poly A + RNA. Second, the M r ~ 40,000 species, representing translation from the first initiator methionine, is the predominant species made in vitro. Another methionine codon, within a very good Kozak consensus sequence (Kozak, 1987) (8 of 10 nucleotides), lies 39 amino acids downstream of the presumed initiator methionine. Initiation at this second methionine could account for the difference in mobility of the two Hox 2.1 proteins. On the other hand, if there is a single Hox 2.1 transcript, then in vitro translation of purified poly A + RNA should produce similar Hox 2.1 protein patterns regardless of the tissue from which it is prepared. However, our results show that the 40/38 kDa ratio in lung is consistently lower than the ratio in anterior spinal cord, both in vivo and in vitro. Thus, we conclude that we cannot presently distinguish between alternative transcripts or differential translational initiation. We have shown that at least two isoforms of Hox 2.1 protein, which are phosphorylated, are present in different proportions in different tissues. Differential tissue distribution and post-translational modification of Hox 2.1 homeoproteins may well be important in regulation of their individual activities and add to what is known about the mechanisms utilized by this class of gene in embryonic development.
Materials and Methods
Antigen production A glutathione-S-transferase (GST)/Hox 2.1 fusion protein was made using the pGEX system described by Smith and Johnson (1988). Using the mouse Hox 2.1 cDNA designated c2.1A (Krumlauf et al., 1987), a 309 bp XholI/XhoI fragment (nucleotides 259 to 568) from the region 5' of the homeobox and omitting the homeopeptide coding region was inserted into the BamHI site at the 3' end of the GST coding region in the pGEX 3X vector. This region of c2.1A was chosen because it encodes the part of the Hox 2.1 protein that is most highly diverged between the genes Hox 1.3 and Hox 3.4. These genes occupy homologous positions in the Hox 1 and 3 clusters, and in other regions of the proteins exhibit extensive homology with Hox 2.1. Comparison of the predicted amino acid sequence of Hox 1.3 (Fibi et al., 1988) and Hox 2.1 in the region used for the GST/Hox 2.1 fusion protein (Krumlauf et
al., 1987) reveals only 16.6% (17 of 102 amino acids) identity in the region used for the GST/Hox 2.1 fusion protein, with the only area of concentration of identical amino acids being six of the first eight residues. There is even less homology in this region between Hox 3.4 and Hox 2.1, with only 9 of 103 amino acids being identical (P. Sharpe, Univ. of Manchester, personal communication). This construct was sequenced, transformed into JM109 cells, and GST/Hox 2.1 fusion protein was induced by adding isopropylthio-/3-D-galactopyranoside (IPTG) to 1 mM final concentration. After sonication the fusion protein was affinity purified by incubation with glutathione-agarose beads (Sigma G-4510), eluting with 5 mM glutathione (Sigma G-4251) in 0.05 M Tris-HCl, pH 8.0. The protein was then dialyzed against 10 mM Tris, pH 7.4 with 150 mM NaCI (TBS) and stored in aliquots at -70°C.
Antibody preparation and specificity Female New Zealand white rabbits initially were injected subcutaneously and intramuscularly in a total of four sites with a total of 750 /zg of GST/Hox 2.1 fusion protein emulsified in Freund's complete adjuvant. Approximately 28% of this total represents Hox 2.1 protein. Subsequent 750/zg boosts were in Freund's incomplete adjuvant. Antibody was purified by two subtractive depletions to remove antibodies against GST and any contaminating bacterial proteins, and then affinity purified. Subtractive depletion matrix was made using extract from JM109 cells transfected with the pGEX 3X vector alone which were induced to produce GST. Whole cell extract was prepared by French press and coupled to CNBr-activated Sepharose (Pharmacia 17-0430-01) following manufacturer's instructions to produce a depletion matrix. Affinity matrix was made using purified GST/Hox 2.1 fusion protein coupled to CNBr-activated Sepharose. aHox 2.1a serum was incubated twice with separate 2 ml aliquots (settled volume) of depletion matrix, then tested for absence of antibodies against GST or contaminating bacterial proteins by Western blot analysis on GST-containing whole bacterial cell extract. Many proteins of varying molecular weight, including GST protein, were detected with aHox 2.1a before incubation with subtractive matrix, but none were detected after incubation. Depleted serum was then further purified by binding to 0.5 ml of an affinity matrix made with the GST/Hox 2.1 fusion protein. After thorough washing with 10 mM Tris, pH 8.0, 150 mM NaC1, 0.05% Tween 20 (TBST), the aHox 2.1 antibodies were eluted in 0.2 ml fractions with 0.15 M glycine, pH 2.5, directly into 0.2 ml aliquots of 2 M Tris, pH 8.0. Each fraction was then tested for reactivity with GST/Hox 2.1 fusion protein blotted onto nitrocellu-
119 lose strips. Fractions showing recognition of the ~ 30 kDa GST/Hox 2.1 fusion protein were pooled and used directly for immunoblotting and immunohistochemistry. In order to demonstrate that aHox 2.1a reacts specifically with the Hox 2.1 portion of the fusion protein, test strips made with another GST fusion protein, GST/Vgr-1 132 (to be described elsewhere), were used, and these showed no reactivity with the antibodies. aHox 2.1a was also shown not to cross-react in other species. Sections of chick embryos, at stages previously reported to show mRNA expression of the Hox 2.1 homologue, Ghox 2.1 (Wedden et al., 1989), do not react with aHox 2.1a. The same is true for sections of Xenopus embryos of appropriate stages. These findings indicate that aHox 2.1 is species specific for mouse, and suggest strongly that the antibody we describe here is entirely specific for Hox 2.1 protein.
In vitro translation Poly A ÷ RNA was isolated from either 12.5d mouse lung or anterior spinal cord by standard methods (Auffray and Rougeon, 1988) and 1 /zg was heated at 65°C for 10 min, cooled and then incubated at 30°C for 1 h with 1/zl RNase inhibitor (Boehringer Mannhiem 1175 025), 35/zl rabbit reticulocyte lysate (Promega L4210), 1/.d 1 mM amino acid mix minus methionine (Promega L4210), and 5/zl all-trans [35S]methionine (ICN 51006). The translation mix wag then used in immunoprecipitation experiments.
Immunoprecipitation Embryonic tissues were dissected in Dulbecco's modified Eagle medium (DMEM) without methionine or without phosphate, depending upon the radiolabel to be used, containing 50/zg/ml penicillin and streptomycin. Lungs and anterior spinal cord tissue from 10-14 embryos were incubated at 37°C in 1 ml of medium without methionine with 100 /xCi all-trans [35S]methionine or in 1 ml of medium without phosphate with 1 mCi [32p]orthophosphate for 4 h followed by three washes in phosphate buffered saline (PBS), pH 7.4. Ten volumes of 400 mM NaC1, 50 mM Tris, pH 8.0, 5 mM EDTA, and 1% NP-40 (NET buffer) with 1% sodium dodecyl sulfate (SDS) was added, followed by vortexing which sufficed to completely lyse the tissues. Samples were then microfuged and 20 /zl of supernatant was incubated with either 3 /zl of normal rabbit serum (NRS) or 5/zl of aHox 2.1a in 1 ml NET buffer for 1.5 h at room temperature. Antigen-antibody complex was bound to Protein-A sepharose, prewashed with NET buffer (Sigma P-3391), and analyzed by SDS-PAGE and autoradiography.
Western blot analysis Dissected tissues (lungs, anterior spinal cord, limb buds and forebrain) were placed directly into 10 volumes of sample buffer containing 5% /3-mercaptoethanol, 2.3% SDS, 62 mM Tris-HC1 pH 6.8 and 10% glycerol, then boiled for 5 min and either used immediately or stored at -20°C. SDS polyacrylamide gel electrophoresis (SDS-PAGE) was performed using a 12% separating gel, and proteins were then electroblotted to a nylon filter (Amersham RPN.303E). The filter was blocked with 5% fetal calf serum (FCS), 1% bovine serum albumin (BSA), 0.5% Tween 20, 0.1 M MgC12, 0.01 M Tris-HC1 pH 7.4. Hox 2.1 protein was detected by incubation with affinity purified primary antibody diluted 1:200 in TBST and subsequently with a 1 : 6000 dilution of anti-rabbit IgG conjugated to alkaline phosphatase (Boehringer Mannheim 605 230). Nitro Blue Tetrazolium/5-Bromo-4-Chloro3-Indolyl Phosphate (NBT/BCIP) was used as the chromagen for alkaline phosphatase.
Embryos and fixation ICR females were mated with Swiss Webster males and noon on the day of plug was defined as 0.5d post coitum (p.c.). Embryos used for section staining were treated with Bouin's fixative, embedded in paraffin wax, sectioned at 7/zm and mounted following standard procedures. Embryos used for whole mount immunostaining were fixed in methanol/DMSO (4:1) overnight at 4°C, treated to inhibit endogenous peroxidases with methanol/DMSO/30% H 2 0 2 (4:1 : 1) for 5-10 h at room temperature, and then placed in 100% methanol and used immediately for staining or stored at - 20°C.
Irnmunohistochemical staining of sections Sections were rehydrated through xylene and graded alcohols following standard procedures, then blocked with 5% FCS, 1% BSA, 0.5% Tween 20, 0.1 M MgCI2, 0.01 M Tris pH 7.4. Sections were incubated with affinity purified primary antibody at a 1 : 200 dilution in TBST followed by a 1 : 1000 dilution of anti-rabbit IgG conjugated with alkaline phosphatase (Boehringer Mannheim 605 230) in TBST. NBT/BCIP was used as substrate for the alkaline phosphatase. The reaction was stopped in 20 mM Tris with 5 mM EDTA, pH 8.0, then mounted in Crystal Mount (Fisher). All washes were in TBST.
Immunohistochemical staining of whole embryos Embryos were placed in Eppendorf tubes and all washes and incubations were done on a slowly rocking
120 platform. Rehydration was through a graded methanol series to PBS, pH 7.4, followed by washing in 2% Carnation dry milk powder and 0.5% Triton X-100 in PBS, pH 7.4 (PBSMT) and then overnight incubation at 4°C with affinity purified primary antibody diluted 1:200 in TBST. Embryos were washed five times, 1 h each, with PBSMT and then incubated overnight at 4°C with anti-rabbit IgG conjugated to horseradish peroxidase (HRP) (Boehringer Mannheim 605 220) diluted 1:500 in TBST. Embryos were washed as before with an additional 30 min wash in PBS with 2% BSA and 0.05% Triton X-100 (PBT). Diaminobenzidine (DAB) was used as the substrate for HRP at a concentration of 0.6 mg/ml with 0.06% NiC12 in PBT. H20 2 was added to 0.03% and the reaction stopped by rinsing twice in PBT. Embryos were then dehydrated through a methanol series to a clearing agent, benzyl alcohol/benzyl benzoate, 1 : 2 (BABB).
Acknowledgements The authors would like to thank Alex Joyner for providing the protocol used for whole mount immunostaining, Olof Sundin for helpful discussion of immunostaining technique, and Paul Sharpe for providing the coding sequence of mouse Hox 3.4. Also, we thank Margaret Kirby and Shigeru Kuratani for helpful discussion and information about structures derived from neural crest.
References Auffray, C. and Rougeon, F. (1980) Eur. J. Biochem. 107, 303-314. Awgulewitsch, A. and Jacobs, D. (1990) Development 108, 411-420. Binetruy, B., Smeal, T. and Karin, M. (1991) Nature 351, 122-127. Boyle, W.J., Smeal, T., Defize, L.H.K., Angel, P., Woodgett, J.R., Karin, M. and Hunter, T. (1991) Cell 64, 573-584. Chisaka, O. and Capecchi, M.R. (1991) Nature 350, 473-479. Cho, K., Goetz, J., Wright, C.V.E., Fritz, A., Hardwicke, J. and DeRobertis, E.M. (1988) EMBO J. 7, 2139-2149. Cho, K.W.Y., Morita, E.A., Wright, C,V.E. and DeRobertis, E.M. (1991) Cell 65, 55-64.
Davis, C.A., Holmyard, D.P., Millen, K.J. and Joyner, A.L. (1991) Development 111,287-298. Dony, C. and Gruss, P. (1987) EMBO J. 6, 2965-2975. Fibi, M., Zink, B., Kessel, M., Colberg-Poley, A.M., Labeit, S., Lehrach, H. and Gruss, P. (1988) Development 102, 349-359. Gaunt, S.J., Coletta, P.L., Pravtcheva, D. and Sharpe, P.T. (1990) Development 109, 329-339. Gavis, E.R. and Hogness, D.S. (1991) Development 112, 1077-1093. Gay, N.J., Poole, S.J. and Kornberg, T.B. (1988) Nucleic Acids Res. 16, 6637-6647. Gehring, W.J., Muller, M., Affolter, M., Percival-Smith, A., Billeter, M., Qian, Y.Q., Otting, G. and Wuthrich, K. (1990) Trends Genet. 6, 323-329. Gould, A.P., Brookman, J.J., Strutt, D.I. and White, R.A.H. (1990) Nature 348, 308-312. Holland, P.W. and Hogan, B.L.M. (1988) Development 102, 159-174. Hunt, P., Gulisano, M., Cook, M., Sham, M., Faiella, A., Wilkinson, D., Boncinelli, E. and Krumlauf, R. (1991) Nature 353, 861-864. Kenyon, C. (1986) Cell 46, 477-487 Kessel, M., Bailing, R. and Gruss, P. (1990) Cell 61, 301-308. Keynes, R. and Lumsden, A. (1990) Neuron 4, 1-9. Kissinger, C.R., Beishan, L., Martin-Blanco, E., Kornberg, T.B., and Pabo, C.O. (1990) Cell 63, 579-590. Kozak, M. (1987) Nucleic Acids Res. 15, 8125-8148. Krause, H.M., Klemenz, R. and Gehring, W.J. (1988) Genes and Development 2, 1021-1036. Krumlauf, R., Holland, P.W.H., McVey, J.H. and Hogan, B.L.M. (1987) Development 99, 603-617. LeDouarin, N.M., Fontaine-Perus, J. and Couly, G. (1986) Trends Neurosci. 9, 175-180. Li, S., Crenshaw, III, E.B., Rawson, E.J., Simmons, D.M., Swanson, L.W. and Rosenfeld, M.G, (1990) Nature 347, 528-533. Lufkin, T.A., Dierech, M., LeMeur, M. Mark and Chambon, P. (1991) Cell 66, 1105-1119. Mahaffey, J.W., Diederich, R.J. and Kaufman, T.C. (1989) Development 105, 167-174. Oliver, G., Wright, C.V.E., Hardwicke, J. and DeRobertis, E.M. (1988) EMBO J. 7, 3199-3209. Patel, N.H., Martin-Blanco, E., Coleman, K.G., Poole, S.J., Ellis, M.C., Kornberg, T.B. and Goodman, C.S. (1989) Cell 58, 955-968. Smith, D.B. and Johnson, K.S. (1988) Gene 67, 31-40. Sundin, O.H. and Eichele, G. (1990) Genes Dev. 4, 1267-1276. Tanaka, M. and Herr, W. (1990) Cell 60, 375-386. Voss, J.W., Yao, R.P. and Rosenfeld, M.G. (1991) J. Biol. Chem. 266, 12832-12835. Wedden, S.E., Pang, K. and Eichele, G. (1989) Development 105, 639-650. Wright, C.V.E., Cho, K.W., Hardwicke, J., Collins, R.H. and DeRobertis, E.M. (1989a) Cell 59, 81-93. Wright, C.V.E., Schnegelsberg, P. and DeRobertis, E.M. (1989b) Development 105, 787-794.
Mechanisms of Development, 37 (1992) 111-120 © 1992 Elsevier Scientific Publishers Ireland, Ltd. 0925-4773/92/$05.00 MOD00085
Expression and modification of Hox 2.1 protein in mouse embryos Nancy A. Wall, C. Michael Jones, Brigid L.M. Hogan and Christopher V.E. Wright Department of Cell Biology, Vanderbilt University, Nashville, TN 37232, U.S.A. (Received 4 October 1991; revision received 9 December 1991; accepted 19 December 1991)
A polyclonal antibody, txHox 2.1a, has been generated and used to immunolocalize Hox 2.1 protein in mouse embryos. Protein is present in nuclei of all tissues previously shown to express Hox 2.1 RNA. In addition, protein is seen in somites and proximal regions of the limb buds, tissues in which Hox 2.1 RNA expression was not clearly detected previously by in situ hybridization. At the 7 somite stage, protein is detectable in the neural tube up to the level of somite 1, but later retracts to a more posterior position. Immunoblot, in vitro translation, and immunoprecipitation experiments were carried out to characterize the Hox 2.1 protein. The results show that the Hox 2.1 gene produces at least two related phosphorylated proteins present in different proportions in different tissues. Mouse embryo; Hox 2.1; Antibody; Phosphorylation; Immunohistochemistry
Introduction
Homeobox containing genes have been shown, either by mutation, overexpression, or deletion, to be critical to normal development in species ranging from Drosophila to mouse (Gehring et al., 1990; Kenyon, 1986; Wright et al., 1989a; Cho et al., 1991; Kessel et al., 1990; Li et al., 1990; Chisaka and Cappechi, 1991; Lufkin et al., 1991). There is good genetic and biochemical evidence that homeodomain proteins are DNA binding proteins (selector molecules) which regulate the transcription of downstream effector genes in developing embryos (Li et al., 1990; Gehring et al., 1990; Kissinger et al., 1990). One important method for investigating the specific function(s) of homeobox genes during development is to determine their temporal and spatial patterns of expression at the single cell and intracellular level, and to correlate these patterns with those of other genes expressed in both normal and mutant animals. Although in situ hybridization has been used to localize transcripts of many homeobox genes in embryonic tissues, demonstration that the protein product is present is necessary to confirm and extend the functional significance of any transcription pattern.
Correspondence to: C.V.E. Wright, Department of Cell Biology, Vanderbilt University, Nashville, TN 37232, U.S.A.
Immunological techniques also provide methods for determining, for example, the DNA sequence(s) these proteins bind to in vivo (Gould et al., 1990). Additionally, by employing immunoprecipitation, in vitro translation, and two-dimensional gel electrophoresis, resolution of different homeobox containing proteins a n d / o r modified forms of these proteins can be achieved. Using these techniques in Drosophila embryos, fushi tarazu ( ftz ) and Ultrabithorax ( Ubx ) have been shown to produce multiple protein isoforms (Krause et al., 1988; Gavis and Hogness, 1991), while Voss et al. (1991) have demonstrated multiple translation start sites and proteins for Pit-1. In spite of the advantages of studying homeobox genes with antibodies (Oliver et al., 1988; Wright et al., 1989b; Patel et al., 1989; Mahaffey et al., 1989; Sundin and Eichele, 1990), very few have been reported for mouse Hox gene products. Awgulewitsch and Jacobs (1990) used peptide antibodies to discern specific cell types that express Hox 3.1 in mouse spinal cord, and Davis et al. (1991) used fusion protein antibodies to demonstrate the localization of engrailed protein in mouse, as well as in chicken and Xenopus embryos, We report here the generation of an antibody specific for the mouse homeoprotein Hox 2.1. This antibody has been used in whole mount immunohistochemistry to provide data that extend the in situ hybridization patterns already reported for this gene (Holland and Hogan, 1988). We report the presence of Hox 2.1
112
Lu
protein in regions where previously it either had not been seen or was at the limits of detection using m R N A probes, particularly during initial phases of expression at early somite stages. Western blotting, immunoprecipitation, and in vitro translation of poly A + selected R N A reveal that at least two phosphorylated forms of Hox 2.1 protein are present in developing mouse embryos. This antibody, and the data reported here, will make it possible to better characterize Hox gene expression in mice in which Hox genes have been deleted or overexpressed.
Lb
Asc
Fb
46kDa
Results
30kDa
Synthesis and modification of Hox 2.1
Fig. 1. Western immunoblot with aHox 2.1a of protein from tissue extracts of 12.5d mouse lung (Lu), limb bud (Lb), anterior spinal cord (Asc), and forebrain (Fb). Lines represent Mr markers of 46,000 and 30,000. 5 /zg of total protein (determined by Bradford assay) was loaded per lane.
We generated an affinity purified rabbit polyclonal antibody, completely specific for Hox 2.1 (see Materials and Methods for antibody production and specificity), using the glutathione-S-transferase fusion protein vector p G E X . Previously, in situ hybridization (Holland and Hogan, 1988) and Northern analysis (Krumlauf et al., 1987) using 12.5-14.5 day old (12.5d-14.5d) mouse embryos had shown that Hox 2.1 is expressed at high levels in the anterior spinal cord and lung. Extracts of these tissues taken from 12.5d mouse embryos, along with forebrain and limb bud which do not show Hox 2.1 R N A expression by in situ analysis or immunostaining, were used in Western immunoblot analysis. Fig. 1 shows that a H o x 2.1a specifically recognizes at least two proteins, one of M r ~ 40,000 and another at M r ~ 38,000. We occasionally detect an M r ~ 37,500 species that could be a third Hox 2.1 species or a degradation product. Although Krumlauf et al. (1987) predicted a M r of 29,469 for the Hox 2.1 protein, its migration with a higher apparent M r is easily explained by post-translational modification a n d / o r anomalous migration often encountered during S D S - P A G E analysis. Lungs and anterior spinal cord from 12.5d mouse embryos were explanted and metabolically labelled with
Lu
Asq
Lu
NRS Ab NRS Ab
A
Ab
B
[35S]methionine or [32p]orthophosphate, and Hox 2.1 protein was immunoprecipitated from tissue extracts. Fig. 2A ([35S]methionine labelled) and Fig. 2B ([32p]orthophosphate labelled) show that a H o x 2.1a specifically recognizes two newly synthesized proteins of M r ~ 40,000 and ~ 38,000 and that both are labelled with [32p]orthophosphate. Both Western immunoblot and immunoprecipitation experiments show that the ratio of 40 k D a / 3 8 kDa proteins is consistently higher in anterior spinal cord than in the lung, indicating that different tissues contain different proportions of Hox 2.1 proteins. We next extracted poly A ÷ R N A from 12.5d mouse lung and anterior spinal cord, performed in vitro translation using rabbit reticulocyte lysate, and examined the products by immunoprecipitation. Fig. 2C shows that both the M r ~ 40,000 and M r ~ 38,000 Hox 2.1 proteins are produced. There is no apparent difference in the M r of the in vitro translated proteins and those
Asc NRS
Ab
NRS
Lu
C
Lu*
Asc
Asc*
i
Fig. 2. Immunoprecipitation of in vivo and in vitro translated protein with aHox 2.1a. (A) [35S]methionine labelled protein from 12.5d mouse lung (Lu) and anterior spinal cord (Asc) with NRS or antibody (Ab). (B) [32p]orthophosphate labelled protein. (C) [35S]methionine labelled protein from 12.5d mouse lung (Lu) and anterior spinal cord (Asc) and in vitro translated protein from 12.5d mouse embryo poly A + RNA from lung (Lu*) and anterior spinal cord (Asc*). Line represents Mr marker of 30,000.
113
Fig. 3. Whole embryos immunostained wilh a H o x 2.1a. (A) Dorsal view of a 7 somite mouse embryo demonstrating differential anterior limits of Hox 2.1 expression in the neural tube and somites. T h e first 5 somites are indicated by n u m b e r e d arrowheads. T h e arrow indicates anterior limit of expression in the neural tube. Bar = 250 p.m. (B) Higher magnification of (A). Bar = 250/xm. (C) Dorsal view of a 9 somite mouse embryo demonstrating retraction of the anterior limit of expression in the neural tube. N u m b e r e d arrowheads indicate somites 3 and 4. Arrow indicates anterior limit of expression in the neural tube. Bar = 250 ~ m . (D) Higher magnification of a different 9 somite mouse embryo demonstrating staining in the posterior portion of somite 5 (arrow). Bar = 100/xm. ot, otic vesicle; nc, neural crest; ep, ectodermal placode.
114 immunoprecipitated from fresh tissue. There is a slight increase in the ratio of the 40 kDa/38 kDa species in lung in vitro translated products compared to in vivo lung extracts, but when compared to anterior spinal cord in vitro translated products it still seems that the anterior spinal cord protein ratio is quantitatively different.
Hox 2.1 protein expression in whole embryos We could not detect Hox 2.1 protein before the 7 somite stage ( ~ 8.0d) of development by our immunostaining methods (data not shown). We examined ten embryos by whole mount immunohistochemistry at a stage when 7-9 somites could be distinguished by light
Fig. 4. Whole embryos immunostained with a H o x 2.1a. (A) 9.0d embryo demonstrating that anterior limits of expression in neural tube and L P M are now apparently in register. Arrowhead indicates the otic vesicle. Bar = 500 /zm. (B) 9.5d embryo showing staining in neural crest cells (arrow). Arrowhead indicates the otic vesicle. Bar = 500 izm. (C) 10.5d embryo, Arrow indicates neural crest. Bar = 500/~m. (D) Magnification of neural crest in B (arrowheads). Bar = 125 /~m. (E) Magnification of neural crest from a 10.0d embryo when the ectodermal placode first exhibits expression of Hox 2.1 protein. Arrows indicate the ectodermal placode. Bar = 125 /zm. (F) Magnification of neural crest in C. Ectodermal placode cells (arrow) and neural crest (arrowheads) stain positive. Bar = 250 /.Lm. ot, otic vesicle; nc, neural crest; ep, ectodermal placode.
115 microscopy. Those at the 7 somite stage clearly showed Hox 2.1 staining in the posterior half of somite 5 and in all cells of more caudal somites (Fig. 3). In contrast, expression in the neural tube extends more anteriorly, and can be clearly detected up to the level of somite 1 (Fig. 3A,B). At the 9 somite stage, Hox 2.1 protein is still present in the posterior of somite 5. However, in the neural tube expression has retracted posteriorly to the level between somites 3 and 4 (Fig. 3C). By 9.0d (~ 12 somites) the anterior border of expression in the neural tube and the anterior limit of expression in the
,a.
B
C
somites and lateral plate mesoderm appear to be in register (Fig. 4A). The 9.5d ( ~ 22 somites) embryos show relatively high levels of Hox 2.1 protein in the neural tube and the lateral plate mesoderm, but expression in the somites seems to be decreased (Fig. 4B). Also at 9.5d, the lung bud and gut are undergoing organogenesis and react positively for Hox 2.1. Staining is also seen in the anterior proximal region of the forelimb buds (Fig. 5C) an area where Hox 2.1 had not been detected previously in the mouse by in situ hybridization, although this result is not surprising since similar expression of the presumed 2.1 homolog, Ghox 2.1, has been demonstrated in the developing chick limb bud (Wedden and Eichele, 1989). Of particular interest is the appearance in 9.5d embryos of a streak of positively staining cranial neural crest cells located posterior and lateral to the otic vesicle at about the level of the heart, and lying at a dorsoanterior to ventroposterior angle (Fig. 4D). At 10.0d (27 somites) a new area of positive staining is noticeable near the center of this streak of neural crest cells. This indention of ectoderm represents the ectodermally derived cells that contribute to the inferior nodose ganglion of the Xth cranial (or vagus) nerve (Fig. 4E). Although barely discernible at 10.0d, Hox 2.1 immunoreactivity now appears in the metanephric tubules and undifferentiated nephrogenic cord (data not shown). Hox 2.1 is still detected in the neural tube, somites (at decreased levels), the anterior proximal region of forelimb buds, and the mesenchyme of the developing lung and gut. In 10.5d (~ 31 somites) embryos, immunostaining in the metanephric tubules is much more noticeable (data not shown). The lateral plate mesoderm is clearly expressing Hox 2.1 protein and somite expression is still detectable, but at a reduced level (Fig. 4C). Neural crest cells are still present in the aforementioned streak but appear to be condensing around the nodose ganglion placodes (Fig. 4F). Positively staining cells are also seen in the third and fourth branchial arches at this stage, probably representing migrating cranial neural crest (data not shown). Hox 2.1 protein in sectioned and whole mount gut
Fig. 5. 12.5d whole gastrointestinal and respiratory tracts immunostained with a H o x 2.1a. (A) Positive staining is seen in the larynx (la), lungs (lu), stomach (st), and hindgut (i) but is absent in the trachea and esophagus (open arrow). Bar = 1 mm. (B) Magnification of the area indicated by the closed arrow in A (structure is rotated 90 ° counterclockwise relative to (A)). Staining is restricted to individual cells of the enteric plexus (arrow) and ends in the region which is herniated at this stage. Bar = 100 Izm. (C) Dorsal view of staining in 10.5d forelimb bud. Arrows indicate region of positive staining, a, anterior; p, posterior. Bar ~ 100/xm.
Immunohistochemical staining of both whole mount gut and sectioned ll.5d-14.5d embryos shows that Hox 2.1 protein is strongly expressed in mesoderm, but not endodermal epithelium, of the developing gastrointestinal and respiratory tracts, in an on-off pattern. Some cells of the larynx stain positively for Hox 2.1 protein (Fig. 5A). At present, we cannot tell if these cells are derived from neural crest or mesoderm. Tracheal and esophageal mesenchyme do not express Hox 2.1, but the mesoderm of the lung, stomach, and proximal portion of the small intestine react positively with
116 Discussion
Distribution of Hox 2.1 protein in embryos
Fig. 6. 12.5d parasagittal sections immunostained with aHox 2.1a. Sections are oriented so that dorsal is top and anterior is left. (A) Cross-section through part of the hindgut. Cells of the enteric plexus show positive staining (arrow). Bar = 100 /~m. (B) Section of the stomach demonstrating staining in mesoderm. The expression pattern is restricted to the dorsal, anterior and posterior regions of the stomach as it is positioned in the embryo at this stage. Arrow indicates area of most intense staining. Bar = 100 ~m. (C) Positive staining is restricted to the ventral regions of the somites (arrows). Bar = 100/zm.
a H o x 2.1a. Fig. 6B demonstrates that Hox 2.1 protein is expressed more strongly anteriorly than posteriorly in the stomach of 12.5d embryos. Although positive staining of gut mesoderm is seen in the proximal region of the small intestine, immunostaining does not extend throughout the entire hindgut. Instead, in the distal hindgut, Hox 2.1 expression is restricted to numerous individual cells that most likely represent cells of the enteric parasympathetic nervous system (enteric plexus), which is derived from the migrating vagal neural crest. This is demonstrated in Fig. 6A which shows that the cells expressing Hox 2.1 are arranged somewhat symmetrically in a ring within the mesoderm. Whole mounts at all stages examined clearly show that the posterior limit of Hox 2.1 expression in the enteric plexus of the gut is at the point of herniation through the body wall (Fig. 5B).
The localization of Hox 2.1 protein observed with antibody staining is, for the most part, entirely consistent with that reported for Hox 2.1 transcripts by in situ hybridization (Holland and Hogan, 1988). Results reported in this paper corroborate previously published data for Hox 2.1 and also throw light on the dynamics of protein expression in the early somite stage embryo when regional specification is probably being defined. When 7 somites are visible Hox 2.1 is clearly present in the posterior half of somite 5 (the first cervical somite) and all ceils of more caudal somites. Gaunt et al. (1990) reported expression of Hox 2.1 R N A in the posterior of the second cervical prevertebra (somite 6) at 12.5d. However, given the low level of transcripts they detected, and our finding that expression in somites declines with development, we consider that our immunostaining technique more accurately reflects the in vivo localization than in situ hybridization. An important conclusion from this work is that Hox 2.1 expression in the neural tube initially extends anteriorly as far as the level of somite 1, and then gradually retracts towards the junction between the hindbrain and spinal cord. By 9.0d the anterior limits of expression in the neural tube and the paraxial mesoderm appear to have become aligned, due to a combination of the expansion of somites including, and posterior to, somite 5, and the receding expression in the neural tube. Unfortunately, at the 7 somite stage in mouse it is not possible to localize the initial anterior limit of Hox 2.1 expression with respect to either rhombomere boundaries or the otic vesicles, which are not yet delimited. However, in the chick embryo, somite 1 is at the level of rhombomere 7 (Keynes and Lumsden, 1990) and this co-localization can presumably be extrapolated to the mouse embryo. In support of our finding is the observation by Wedden et al. (1990) that in the stage 17 chick embryo the anterior border of Ghox 2.1 R N A is "at the level of the fourth ventricle". In addition, Hox 2.1 expression in the mouse neural tube at the level of rhombomere 7 would be consistent with Hox 2.1 expression in neural crest derived cells in the nodose ganglion. The nodose ganglion is the inferior ganglion of the X (vagus) nerve, which is thought to be derived at least in part from neural crest cells arising in rhombomere 7 and not from neural crest arising near the spinal cord-hindbrain junction (Keynes and Lumsden, 1990). It remains to be seen whether our finding that the initial expression of Hox 2.1 protein in the neural tube, which is more anterior than previously found by in situ hybridization, can be extrapolated to other homeobox genes in the hindbrain. If so, it has important implications for current models of the inter-
117 actions of these genes in pattern formation in the vertebrate head (Hunt et al., 1991). Immunohistochemistry also reveals the presence of Hox 2.1 protein in areas that previously had been shown to express Ghox 2.1 RNA in chick (Wedden et al., 1989) but were probably not seen in the mouse because of low expression (e.g. somites and at 9.5d10.5d but not 12.5d, proximo-anterior limb buds). Since the regions exhibiting Hox 2.1 protein expression do not include tissues in which Hox 1.3 and 3.4 (the two Hox genes most similar to 2.1) are specifically expressed (e.g., Hox 1.3 is expressed in the trachea (Dony and Gruss, 1987) and Hox 3.4 is expressed in the esophagus (Gaunt et al., 1990)), cross-reactivity between the products of these or other closely related genes and the affinity purified antibody is not a concern.
Hox 2.1 in the neural crest Cranial ganglia are derived from cells from both neural crest and ectodermal placodes (LeDouarin et al., 1986) and our data provides evidence that Hox 2.1 protein is expressed in both of these components at early stages. In 9.5d whole mount embryos, bilateral streaks of neural crest cells stain positive for Hox 2.1 and expression in these cells persists through at least 10.5d. At 10.0d an indentation of ectoderm (the nodose placode) in close proximity to the Hox 2.1 expressing neural crest cells is also expressing this protein. Localization of Hox 2.1 in the nuclei of ectodermal cells has been confirmed by staining of mouse embryo sections (Shigeru Kuratani, Baylor College of Medicine, personal communication). As discussed above, it is generally believed that the neural crest cells of the nodose ganglion are derived from the neural tube at the level of rhombomere 7. This is the level at which Hox 2.1 protein is observed transiently at the 7 somite stage ( ~ 8.0d). It is of interest that Hox 2.1 protein expression is not detected by immunohistochemistry in the neural crest of whole mount embryos until approximately 9.5d, by which time Hox 2.1 expression in the neural tube has retracted to a more posterior position. One interpretation of these results is that Hox 2.1 expression is part of a pre-specification program initiated before the neural crest cells leave the tube at the level of rhombomere 7. Hox 2.1 expression would then be down regulated during migration until the cells approach the end of their migration pathway when Hox 2.1 expression is reactivated. An alternative explanation is that the migrating neural crest cells receive a positional signal when they arrive in the region of the developing nodose ganglion. Full interpretation of our results must await detailed identification of the Hox 2.1 expressing cell types in the circumpharyngeal region and nodose ganglion, and the
relative contribution of neural crest and placode derived cells. Such experiments are underway.
Evidence for post-translational modification of Hox 2.1 protein Although there appears to be three Hox 2.1 proteins by Western blot analysis, the M r ~ 37,500 protein was not consistently detected by immunoprecipitation of tissue extracts or in vitro translation of poly A + RNA from the same tissues. For this reason we think (but cannot conclusively demonstrate) that this protein is a partially degraded form of the M r ~ 40,000 a n d / o r ~ 38,000 proteins. Further analysis will need to be performed before a final determination can be made. In the following discussion we will consider that there are two Hox 2.1 proteins. There are several possible explanations for the recognition by aHox 2.1a of more than one protein species in tissue extracts by Western immunoblot and immunoprecipitation techniques. For example, both lung and anterior spinal cord contain multiple Hox 2.1 transcripts (Krumlauf et al., 1987), which could represent mRNAs alternatively spliced within the coding region similar to reports for the X1Hbox 1 (the Hox 3.3 homolog) gene (Cho et al., 1988). However, there is at present no direct evidence for such alternative splicing of Hox 2.1. An alternative explanation for multiple isoforms is that there are different post-translational modifications of Hox 2.1 protein. For example, Krause et al. (1988) and Gavis and Hogness (1991) have shown multiple isoforms of the homeoproteins ftz and Ubx in Drosophila embryos, probably differing by phosphorylation. Both the M r ~ 40,000 and M r ~ 38,000 species of Hox 2.1 are phosphorylated, but at present we have not identified which residues are involved. Since the Hox 2.1 protein sequence is 16.4% serine, and Gay et al. (1991) have shown that Drosophila en is phosphorylated on serine, this residue could be the target of an undetermined kinase. Multiple phosphorylated forms of homeoproteins could represent intermediates in the post-translational modification process or, alternatively, these different phosphorylated states may be associated with changes in the activity of the protein at the level of DNA binding or transactivation potential (Binetruy et al., 1991; Boyle et al., 1991; Tanaka and Herr, 1990). A third explanation for multiple forms of Hox 2.1 protein is the use of alternative translational start sites in the mRNA, as demonstrated for the homeobox containing gene Pit-1 (Voss et al., 1991). Immunoprecipitation of proteins translated in vitro from poly A ÷ RNA reveals that at least two Hox 2.1 proteins are made. Are these proteins translated from the same transcript, or separate transcripts? On the one hand, comparison of the isoform ratios in immunoprecipita-
118 tions from tissues and in vitro translations shows that the tissue differences are slightly reduced in vitro, indicating that the two isoforms could be translated from the same transcript, for the following reasons. First, if the two proteins derive from separate transcripts, the ratios seen in vivo should be maintained in vitro, since both transcripts would be present in poly A + RNA. Second, the M r ~ 40,000 species, representing translation from the first initiator methionine, is the predominant species made in vitro. Another methionine codon, within a very good Kozak consensus sequence (Kozak, 1987) (8 of 10 nucleotides), lies 39 amino acids downstream of the presumed initiator methionine. Initiation at this second methionine could account for the difference in mobility of the two Hox 2.1 proteins. On the other hand, if there is a single Hox 2.1 transcript, then in vitro translation of purified poly A + RNA should produce similar Hox 2.1 protein patterns regardless of the tissue from which it is prepared. However, our results show that the 40/38 kDa ratio in lung is consistently lower than the ratio in anterior spinal cord, both in vivo and in vitro. Thus, we conclude that we cannot presently distinguish between alternative transcripts or differential translational initiation. We have shown that at least two isoforms of Hox 2.1 protein, which are phosphorylated, are present in different proportions in different tissues. Differential tissue distribution and post-translational modification of Hox 2.1 homeoproteins may well be important in regulation of their individual activities and add to what is known about the mechanisms utilized by this class of gene in embryonic development.
Materials and Methods
Antigen production A glutathione-S-transferase (GST)/Hox 2.1 fusion protein was made using the pGEX system described by Smith and Johnson (1988). Using the mouse Hox 2.1 cDNA designated c2.1A (Krumlauf et al., 1987), a 309 bp XholI/XhoI fragment (nucleotides 259 to 568) from the region 5' of the homeobox and omitting the homeopeptide coding region was inserted into the BamHI site at the 3' end of the GST coding region in the pGEX 3X vector. This region of c2.1A was chosen because it encodes the part of the Hox 2.1 protein that is most highly diverged between the genes Hox 1.3 and Hox 3.4. These genes occupy homologous positions in the Hox 1 and 3 clusters, and in other regions of the proteins exhibit extensive homology with Hox 2.1. Comparison of the predicted amino acid sequence of Hox 1.3 (Fibi et al., 1988) and Hox 2.1 in the region used for the GST/Hox 2.1 fusion protein (Krumlauf et
al., 1987) reveals only 16.6% (17 of 102 amino acids) identity in the region used for the GST/Hox 2.1 fusion protein, with the only area of concentration of identical amino acids being six of the first eight residues. There is even less homology in this region between Hox 3.4 and Hox 2.1, with only 9 of 103 amino acids being identical (P. Sharpe, Univ. of Manchester, personal communication). This construct was sequenced, transformed into JM109 cells, and GST/Hox 2.1 fusion protein was induced by adding isopropylthio-/3-D-galactopyranoside (IPTG) to 1 mM final concentration. After sonication the fusion protein was affinity purified by incubation with glutathione-agarose beads (Sigma G-4510), eluting with 5 mM glutathione (Sigma G-4251) in 0.05 M Tris-HCl, pH 8.0. The protein was then dialyzed against 10 mM Tris, pH 7.4 with 150 mM NaCI (TBS) and stored in aliquots at -70°C.
Antibody preparation and specificity Female New Zealand white rabbits initially were injected subcutaneously and intramuscularly in a total of four sites with a total of 750 /zg of GST/Hox 2.1 fusion protein emulsified in Freund's complete adjuvant. Approximately 28% of this total represents Hox 2.1 protein. Subsequent 750/zg boosts were in Freund's incomplete adjuvant. Antibody was purified by two subtractive depletions to remove antibodies against GST and any contaminating bacterial proteins, and then affinity purified. Subtractive depletion matrix was made using extract from JM109 cells transfected with the pGEX 3X vector alone which were induced to produce GST. Whole cell extract was prepared by French press and coupled to CNBr-activated Sepharose (Pharmacia 17-0430-01) following manufacturer's instructions to produce a depletion matrix. Affinity matrix was made using purified GST/Hox 2.1 fusion protein coupled to CNBr-activated Sepharose. aHox 2.1a serum was incubated twice with separate 2 ml aliquots (settled volume) of depletion matrix, then tested for absence of antibodies against GST or contaminating bacterial proteins by Western blot analysis on GST-containing whole bacterial cell extract. Many proteins of varying molecular weight, including GST protein, were detected with aHox 2.1a before incubation with subtractive matrix, but none were detected after incubation. Depleted serum was then further purified by binding to 0.5 ml of an affinity matrix made with the GST/Hox 2.1 fusion protein. After thorough washing with 10 mM Tris, pH 8.0, 150 mM NaC1, 0.05% Tween 20 (TBST), the aHox 2.1 antibodies were eluted in 0.2 ml fractions with 0.15 M glycine, pH 2.5, directly into 0.2 ml aliquots of 2 M Tris, pH 8.0. Each fraction was then tested for reactivity with GST/Hox 2.1 fusion protein blotted onto nitrocellu-
119 lose strips. Fractions showing recognition of the ~ 30 kDa GST/Hox 2.1 fusion protein were pooled and used directly for immunoblotting and immunohistochemistry. In order to demonstrate that aHox 2.1a reacts specifically with the Hox 2.1 portion of the fusion protein, test strips made with another GST fusion protein, GST/Vgr-1 132 (to be described elsewhere), were used, and these showed no reactivity with the antibodies. aHox 2.1a was also shown not to cross-react in other species. Sections of chick embryos, at stages previously reported to show mRNA expression of the Hox 2.1 homologue, Ghox 2.1 (Wedden et al., 1989), do not react with aHox 2.1a. The same is true for sections of Xenopus embryos of appropriate stages. These findings indicate that aHox 2.1 is species specific for mouse, and suggest strongly that the antibody we describe here is entirely specific for Hox 2.1 protein.
In vitro translation Poly A ÷ RNA was isolated from either 12.5d mouse lung or anterior spinal cord by standard methods (Auffray and Rougeon, 1988) and 1 /zg was heated at 65°C for 10 min, cooled and then incubated at 30°C for 1 h with 1/zl RNase inhibitor (Boehringer Mannhiem 1175 025), 35/zl rabbit reticulocyte lysate (Promega L4210), 1/.d 1 mM amino acid mix minus methionine (Promega L4210), and 5/zl all-trans [35S]methionine (ICN 51006). The translation mix wag then used in immunoprecipitation experiments.
Immunoprecipitation Embryonic tissues were dissected in Dulbecco's modified Eagle medium (DMEM) without methionine or without phosphate, depending upon the radiolabel to be used, containing 50/zg/ml penicillin and streptomycin. Lungs and anterior spinal cord tissue from 10-14 embryos were incubated at 37°C in 1 ml of medium without methionine with 100 /xCi all-trans [35S]methionine or in 1 ml of medium without phosphate with 1 mCi [32p]orthophosphate for 4 h followed by three washes in phosphate buffered saline (PBS), pH 7.4. Ten volumes of 400 mM NaC1, 50 mM Tris, pH 8.0, 5 mM EDTA, and 1% NP-40 (NET buffer) with 1% sodium dodecyl sulfate (SDS) was added, followed by vortexing which sufficed to completely lyse the tissues. Samples were then microfuged and 20 /zl of supernatant was incubated with either 3 /zl of normal rabbit serum (NRS) or 5/zl of aHox 2.1a in 1 ml NET buffer for 1.5 h at room temperature. Antigen-antibody complex was bound to Protein-A sepharose, prewashed with NET buffer (Sigma P-3391), and analyzed by SDS-PAGE and autoradiography.
Western blot analysis Dissected tissues (lungs, anterior spinal cord, limb buds and forebrain) were placed directly into 10 volumes of sample buffer containing 5% /3-mercaptoethanol, 2.3% SDS, 62 mM Tris-HC1 pH 6.8 and 10% glycerol, then boiled for 5 min and either used immediately or stored at -20°C. SDS polyacrylamide gel electrophoresis (SDS-PAGE) was performed using a 12% separating gel, and proteins were then electroblotted to a nylon filter (Amersham RPN.303E). The filter was blocked with 5% fetal calf serum (FCS), 1% bovine serum albumin (BSA), 0.5% Tween 20, 0.1 M MgC12, 0.01 M Tris-HC1 pH 7.4. Hox 2.1 protein was detected by incubation with affinity purified primary antibody diluted 1:200 in TBST and subsequently with a 1 : 6000 dilution of anti-rabbit IgG conjugated to alkaline phosphatase (Boehringer Mannheim 605 230). Nitro Blue Tetrazolium/5-Bromo-4-Chloro3-Indolyl Phosphate (NBT/BCIP) was used as the chromagen for alkaline phosphatase.
Embryos and fixation ICR females were mated with Swiss Webster males and noon on the day of plug was defined as 0.5d post coitum (p.c.). Embryos used for section staining were treated with Bouin's fixative, embedded in paraffin wax, sectioned at 7/zm and mounted following standard procedures. Embryos used for whole mount immunostaining were fixed in methanol/DMSO (4:1) overnight at 4°C, treated to inhibit endogenous peroxidases with methanol/DMSO/30% H 2 0 2 (4:1 : 1) for 5-10 h at room temperature, and then placed in 100% methanol and used immediately for staining or stored at - 20°C.
Irnmunohistochemical staining of sections Sections were rehydrated through xylene and graded alcohols following standard procedures, then blocked with 5% FCS, 1% BSA, 0.5% Tween 20, 0.1 M MgCI2, 0.01 M Tris pH 7.4. Sections were incubated with affinity purified primary antibody at a 1 : 200 dilution in TBST followed by a 1 : 1000 dilution of anti-rabbit IgG conjugated with alkaline phosphatase (Boehringer Mannheim 605 230) in TBST. NBT/BCIP was used as substrate for the alkaline phosphatase. The reaction was stopped in 20 mM Tris with 5 mM EDTA, pH 8.0, then mounted in Crystal Mount (Fisher). All washes were in TBST.
Immunohistochemical staining of whole embryos Embryos were placed in Eppendorf tubes and all washes and incubations were done on a slowly rocking
120 platform. Rehydration was through a graded methanol series to PBS, pH 7.4, followed by washing in 2% Carnation dry milk powder and 0.5% Triton X-100 in PBS, pH 7.4 (PBSMT) and then overnight incubation at 4°C with affinity purified primary antibody diluted 1:200 in TBST. Embryos were washed five times, 1 h each, with PBSMT and then incubated overnight at 4°C with anti-rabbit IgG conjugated to horseradish peroxidase (HRP) (Boehringer Mannheim 605 220) diluted 1:500 in TBST. Embryos were washed as before with an additional 30 min wash in PBS with 2% BSA and 0.05% Triton X-100 (PBT). Diaminobenzidine (DAB) was used as the substrate for HRP at a concentration of 0.6 mg/ml with 0.06% NiC12 in PBT. H20 2 was added to 0.03% and the reaction stopped by rinsing twice in PBT. Embryos were then dehydrated through a methanol series to a clearing agent, benzyl alcohol/benzyl benzoate, 1 : 2 (BABB).
Acknowledgements The authors would like to thank Alex Joyner for providing the protocol used for whole mount immunostaining, Olof Sundin for helpful discussion of immunostaining technique, and Paul Sharpe for providing the coding sequence of mouse Hox 3.4. Also, we thank Margaret Kirby and Shigeru Kuratani for helpful discussion and information about structures derived from neural crest.
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