kreisler is a recessive mutation resulting in gross malformation of the inner ear of homozygous mice. The defects in the inner ear are related to abnormalities in the hindbrain of the embryo, adjacent to the ear rudiments. At E9.5, the neural tube posterior to the boundary between the third and fourth rhombomeres, r3 and r4, appears unsegmented, and the region that would normally correspond to r4 is unusually thick-walled and contains many dying cells. The absence of morphological segmentation in the posterior hindbrain corresponds to an altered pattern of gene expression in that region, with major abnormalities posterior to the r4/5 boundary and minor abnormalities anterior to it. From the expression patterns at E9.5 of Krox-20, Hoxb-1 (Hox 2.9), Hoxb-2 (Hox 2.8), Hoxa-3 (Hox 1.5), Hoxd-4 (Hox 4.2) and cellular retinoic-acid binding protein I (CRABP I), it appears that the fundamental defect is a loss of r5 and r6. Correspondingly, the glossopharyngeal ganglion and nerve, associated with r6 are missing and the abducens nerve, which originates from r5 and r6, is also absent. Examination of Krox-20 expression at stages as early as E8.5 indicates that Krox-20 fails ever to be expressed in its r5 domain in the homozygous kreisler mutant. The abnormal amount of cell death is seen only later. An interpretation is that the cells that would normally become specified at an early stage as r5 and r6 adopt an r4 character instead, producing an excess of r4 cells that is disposed of subsequently by cell death.

The vertebrate hindbrain became a focus of attention for developmental biologists when studies of HOM/Hox gene expression revealed startling similarities between its anteroposterior patterning and that of the insect body axis (Graham et al., 1989; Duboule and Dollé et al., 1989; Lumsden and Keynes, 1989; Wilkinson et al., 1989b; McGinnis and Krumlauf, 1992). A number of other genes are now known to be expressed in the hindbrain in patterns suggesting that they act in conjunction with the Hox genes as part of a control system that organises the development of this part of the body. The task now is to understand how the control system operates: which genes regulate which others, and how do they govern the anatomical development? For this, we need to identify as many as possible of the genes involved and to analyse their mutant phenotypes both in terms of effects on the expression patterns and activities of other genes and in terms of effects on body structure. In this paper, we examine the role of the kreisler gene.

kreisler, an X-ray-induced recessive mutation, was first described by Hertwig (1944). The homozygous adult is normal in outward appearance, but easily identified by its deafness and abnormal circling behaviour, a consequence of gross malforBmation of its inner ears. There have been several subsequent studies of kreisler development (Ruben, 1973; Van De Water and Ruben, 1974; Li, 1979). In particular, Deol (1964) reported that in the kreisler embryo the earliest abnormalities are to be seen, concurrently, in the positioning of the otic placodes and in the segmentation of the hindbrain. In this paper, it is the abnormalities of the hindbrain that particularly concern us; for Deol’s detailed description suggested that these might reflect a fundamental disturbance of the mechanisms controlling anteroposterior specification.

This hypothesis has been investigated by Frohman et al. (1993), who have analysed the kreisler hindbrain in terms of the expression of five genes – Hoxb-1, Hoxb-3, Hoxb-4, Krox-20 and FGF-3. They report a complex pattern of abnormalities, in which certain regions of the hindbrain express combinations of genes that are never normally encountered. We have independently analysed kreisler using some of the same markers and also several others, including neuroanatomical features not studied by Frohman et al. (1993), and have come to a different and simpler view of the homozygous kreisler phenotype. We find that the major abnormalities are coordinated in a simple way and can be summed up by saying that two rhombomeres, r5 and r6, are missing. We have looked for cell death in the kreisler hindbrain that might explain their absence. We confirm that it occurs to an abnormal extent, but find that it is concentrated at the level normally corresponding to r4. We argue that this can be interpreted as the consequence of an earlier fault in the positional specification of the cells normally destined to form r5 and r6, resulting in a surplus of cells with an r4 character: the increased cell death may reflect the operation of a regulative mechanism for coping with a surplus of r4 cells.

The kreisler mice were from Jackson Laboratories, and the stock was maintained by outcrossing homozygous kreisler males with F1 hybrids between CBA and C3H/He mice. Heterozygous progeny of such matings were mated with one another, or with stud male homozygotes, to produce more homozygotes. (The kreisler mice cannot be maintained as a homozygous stock since homozygous females do not breed, and repeated outcrossing is required to produce stud males that will breed vigorously.) The kreisler (kr) mutation lies on chromosome 2 close to the agouti, Src and wellhaarig loci (Lyon and Searle, 1989), and was kept in linkage with non-agouti (a) and wellhaarig (we) alleles to facilitate selection. Homozygous kreisler embryos were obtained from timed matings between heterozygotes, or between homozygous males and heterozygous females; the morning on which a vaginal plug was detected was designated E0.5. Timed matings between CBA and C3H/He mice provided homozygous wild-type (+/+) control embryos; heterozygous control embryos were littermates of homozygous kreislers, from matings of homozygous kreisler stud males with heterozygous females.

For anatomical analysis, embryos were fixed in half-strength Karnovsky fixative and embedded in Araldite; serial sections were cut at 1 μm and stained with toluidine blue. Measurements of whole unfixed embryos were made using an eyepiece graticule of a stereomicroscope. The embryos to be measured were immersed in phosphate-buffered saline, supplemented with 10% foetal calf serum to preserve embryonic morphology.

For immunocytochemistry, embryos were embedded in wax, serially sectioned at 7 μm, and exposed to an affinity-purified rabbit polyclonal antibody raised against a synthetic peptide corresponding to a sequence from bovine CRABP I. The antibody has been characterised by Eriksson et al. (1987) and shown to be specific for CRABP I by Maden et al. (1992). Bound antibody was detected with the ABC kit (Vector Laboratories), using an HRP-conjugated secondary antibody and diaminobenzidine to localise the reaction product, followed by counterstaining with haematoxylin, as described by Maden et al. (1992) 

For in situ hybridisation on sections, we followed the autoradiographic protocol of Wilkinson and Green (1991), using 35S-labelled antisense RNA probes. The probe for Krox-20 (a 600 bp PstI-ApaI fragment, including the zinc finger region) was a gift from D. Wilkinson. Probes for Hoxa-3 (Gaunt, 1987), Hoxd-4 (Gaunt et al., 1989), Hoxb-2 and Hoxb-1 (Wilkinson et al., 1989b) were as previously reported. In all cases, mutant (kr/kr) embryos were matched with phenotypically normal controls (+/+, or +/kr littermates) at the same stage (as judged by the number of somites) and were embedded and sectioned side by side with them in the same paraffin block. Thus each slide carried adjacent parallel sets of sections of normal and mutant embryos. To maximise opportunities for spatial and quantitative comparisons between probes as well as between mice, the Krox-20 and Hoxa-3 probes were used on sections at opposite ends of the same slide. Slides were exposed for 7-9 days before developing. For photography, the autoradiographs were viewed on a BioRad confocal scanning microscope in transmission mode, and dark-field and bright-field images were superimposed digitally. Whole-mount in situ hybridisations followed the protocol of Wilkinson (1992), using probes for Hoxb-1, Hoxb-2 and Hoxd-4. Stained embryos were cleared in glycerol before being photographed. In some cases, the embryos were embedded in gelatinealbumin after in situ hybridisation and sectioned with a vibratome at a thickness of 50 μm.

Whole-mount nerve staining was performed using the antibody 2H3 (a gift of Jane Dodd), using the protocol described by Lumsden and Keynes (1989). Once stained, embryos were dissected and mounted in 90% glycerol, 10% PBS and 0.02% sodium azide. In some cases, embryos were embedded in gelatine-albumin and sectioned with a vibratome at a thickness of 100 μm.

Cell death was revealed using acridine orange (3,6-bis[dimethylamino]acridine) which is excluded from living cells (Speiji, 1971; Wolf and Ready, 1991) but specifically stains apoptotic cells. Embryos were dissected from extraembryonic tissues and incubated in PBS containing 10% foetal calf serum and 5 μg/ml acridine orange for 15 minutes at room temperature. They were then thoroughly rinsed before being mounted in PBS with 10% serum. Staining was photographed within 1 hour of mounting using the fluorescein filter set of a Zeiss Axiophot fluorescence photomicroscope. In some cases, the image was recorded using the rhodamine channel of a BioRad confocal microscope where the final images are a superimposition of a projected fluorescence image with a transmitted-light phase-contrast image. In some cases, once the pattern of cell death had been recorded the embryos were rinsed from underneath the coverslip and processed for whole-mount in situ hybridisation to reveal the pattern of Krox-20 expression.

Anatomy

Homozygous kreisler embryos are easily identifiable upon external examination at E9.5 by the abnormal shape of the hindbrain and the lateral positioning of the ear rudiments (Deol, 1964). Out of a total of 160 embryos from matings between heterozygotes, 38 embryos with the kreisler phenotype were obtained; and out of 666 embryos from matings of heterozygotes with homozygotes, 332 had the kreisler phenotype. Longitudinal sections of E9.5 homozygous kreisler embryos and of their phenotypically normal littermates confirm Deol’s (1964) account of the differences. As shown in Fig. 1, in the hindbrain of the kreisler homozygote, there is a complete absence of morphological segmentation posterior to the boundary between r3 and r4. The otocysts, however, are still present at roughly the level where r5 would normally be seen, or displaced only slightly anterior to their normal location; the centres of the otocysts lie 215 ± 23 μm posterior to the r3/4 boundary in homozygous kreisler embryos, as against 295 ± 24 μm for stage-matched controls [mean ± s.e.m., n=27]. In addition, the rudiment of the facial ganglion on each side of the hindbrain is extended posteriorly from its normal position, adjacent to r4, so as to form a single ganglionic mass that continues over a distance corresponding to two or three rhombomere widths. The extension of the facial ganglion in homozygous kreisler embryos lies between the otocyst and the hindbrain and is correlated with the lateral displacement of the otic placode away from the hindbrain. A further abnormality of the homozygous kreisler embryo at this stage is the presence of cell death in the region that would normally constitute r4 and perhaps the anterior part of r5: large quantities of pycnotic cell debris can be seen here (Fig. 1D). (Further details of the pattern of cell death will be described No such cell death is seen in the heterozygote (Fig. 1C) or in wild-type controls (data not shown).

Fig. 1.

Longitudinal sections of embryos at E9.5 showing the segmental pattern of the hindbrain and adjacent tissues. (A) Phenotypically normal heterozygous (+/kr) embryo. (B) Homozygous kreisler (kr/kr) littermate. These embryos were obtained by mating a +/kr female with a kr/kr male. (C,D) Details from A and B respectively, showing the hindbrain adjacent to the otocyst. In the homozygous kreisler embryo (D), note the absence of rhombomere boundaries posterior to the r3/4 boundary, the thickening of the neural tube and the necrotic cell debris (arrow) at the levels normally corresponding to r4 and r5, and the wide gap between the hindbrain and the otocyst. Labelling: O, otocyst; fg, rudimentlater.)

Fig. 1.

Longitudinal sections of embryos at E9.5 showing the segmental pattern of the hindbrain and adjacent tissues. (A) Phenotypically normal heterozygous (+/kr) embryo. (B) Homozygous kreisler (kr/kr) littermate. These embryos were obtained by mating a +/kr female with a kr/kr male. (C,D) Details from A and B respectively, showing the hindbrain adjacent to the otocyst. In the homozygous kreisler embryo (D), note the absence of rhombomere boundaries posterior to the r3/4 boundary, the thickening of the neural tube and the necrotic cell debris (arrow) at the levels normally corresponding to r4 and r5, and the wide gap between the hindbrain and the otocyst. Labelling: O, otocyst; fg, rudimentlater.)

Expression pattern of Krox-20

The Krox-20 gene provides a particularly useful marker for analysis of hindbrain segmentation. In wild-type embryos it is expressed in r3 and then in r5, with a period when expression can be detected in both domains (Wilkinson et al., 1989a; Hunt et al., 1991a; Schneider-Maunoury et al., 1993). Fig. 2 illustrates the differences in expression patterns of Krox-20 between wild-type and homozygous kreisler embryos towards the end of this period, at approximately the 22-somite stage (E9.5). In the wild-type embryo, there is strong expression in r5, adjacent to the ear, whilst the expression in r3 is beginning to fade (Fig. 2A). In the homozygous kreisler embryo, by contrast, no expression can be seen in the region adjacent to the ear (Fig. 2B) but there is still expression in r3 (Fig. 2C). This anterior domain of expression, although normal in position, is abnormal in that it persists in homozygous kreisler embryos with as many as 25 somites, a stage by which control (wild-type) embryos have ceased to show any expression in r3. Twelve sets of sections of homozygous kreisler specimens at E9.5 all show Krox-20 expression in r3 with no expression in r5 (Table 1).

Table 1.

Expression of Krox-20 in the hindbrain of wild-type and homozygous kreisler embryos

Expression of Krox-20 in the hindbrain of wild-type and homozygous kreisler embryos
Expression of Krox-20 in the hindbrain of wild-type and homozygous kreisler embryos
Fig. 2.

(A-C) The pattern of Krox-20 expression as seen by in situ hybridization in longitudinal sections of the hindbrain at the stage of approximately 22 somites (E9.5).(A) Normal (+/+) embryo showing strong expression in r5 (adjacent to the otocysts) and weaker expression in r3. (B) Homozygous kreisler embryo showing absence of expression adjacent to the otocysts.(C) More dorsal section of the same embryo as in (B), showing strong expression in r3. Labelling: O, otocyst. Autoradiographic silver grains appear red. (D,E) Whole-mount in situ hybridization, to reveal the pattern of expression of Hoxb-2 (2.8) viewed dorsally.(D) Heterozygous kr/+ embryo control, showing the normal pattern of expression of Hoxb-2, which is expressed in posterior regions up to the boundary between r2 and r3. (E) Homozygous kreisler embryo, showing a pattern of Hoxb-2 expression identical to the control. (F,G) Whole-mount in situ hybridization, to reveal the pattern of expression of Hoxb-1 (2.9). Dorsal views. (F) Heterozygous kreisler embryo control, showing the normal domain of expression restricted to r4.(G) Homozygous kreisler embryo, showing expression again in the r4 region, but extending somewhat more caudally than normal (at least in the ventral neural tube). The expected length of the normal r4 domain is marked. (H,I) The pattern of Hoxa-3 (1.5) expression as seen by in situ hybridization in longitudinal sections of the hindbrain at the stage of approximately 22 somites (E9.5); same embryos as in Fig. 2. (H) Normal (+/+) embryo showing a sharp Hoxa-3 (1.5) expression boundary just anterior to the otocysts and coincident with the boundary between r4 and r5. (I) Homozygous kreisler embryo showing a boundary again just anterior to the otocysts plus an ectopic domain of expression in r3. Labelling: O, otocyst. Autoradiographic silver grains appear red. (J,K) Coronal vibratome sections through the hindbrain of embryos stained by whole-mount in situ hybridization, to reveal the pattern of expression of Hoxd-4 (4.2). (J) Heterozygous kr/+ embryo control, showing the normal pattern of expression of Hoxd-4, which is seen in r7 and regions posterior to this. (K) Homozygous kreisler embryo, in which the anterior limit of expression of Hoxd-4 appears almost level with the caudal edge of the otocyst, about two rhombomere-widths anterior to its normal location. Bars: for A-C,H and I, 100 μm, and for D-G,J and K, 125 μm.

Fig. 2.

(A-C) The pattern of Krox-20 expression as seen by in situ hybridization in longitudinal sections of the hindbrain at the stage of approximately 22 somites (E9.5).(A) Normal (+/+) embryo showing strong expression in r5 (adjacent to the otocysts) and weaker expression in r3. (B) Homozygous kreisler embryo showing absence of expression adjacent to the otocysts.(C) More dorsal section of the same embryo as in (B), showing strong expression in r3. Labelling: O, otocyst. Autoradiographic silver grains appear red. (D,E) Whole-mount in situ hybridization, to reveal the pattern of expression of Hoxb-2 (2.8) viewed dorsally.(D) Heterozygous kr/+ embryo control, showing the normal pattern of expression of Hoxb-2, which is expressed in posterior regions up to the boundary between r2 and r3. (E) Homozygous kreisler embryo, showing a pattern of Hoxb-2 expression identical to the control. (F,G) Whole-mount in situ hybridization, to reveal the pattern of expression of Hoxb-1 (2.9). Dorsal views. (F) Heterozygous kreisler embryo control, showing the normal domain of expression restricted to r4.(G) Homozygous kreisler embryo, showing expression again in the r4 region, but extending somewhat more caudally than normal (at least in the ventral neural tube). The expected length of the normal r4 domain is marked. (H,I) The pattern of Hoxa-3 (1.5) expression as seen by in situ hybridization in longitudinal sections of the hindbrain at the stage of approximately 22 somites (E9.5); same embryos as in Fig. 2. (H) Normal (+/+) embryo showing a sharp Hoxa-3 (1.5) expression boundary just anterior to the otocysts and coincident with the boundary between r4 and r5. (I) Homozygous kreisler embryo showing a boundary again just anterior to the otocysts plus an ectopic domain of expression in r3. Labelling: O, otocyst. Autoradiographic silver grains appear red. (J,K) Coronal vibratome sections through the hindbrain of embryos stained by whole-mount in situ hybridization, to reveal the pattern of expression of Hoxd-4 (4.2). (J) Heterozygous kr/+ embryo control, showing the normal pattern of expression of Hoxd-4, which is seen in r7 and regions posterior to this. (K) Homozygous kreisler embryo, in which the anterior limit of expression of Hoxd-4 appears almost level with the caudal edge of the otocyst, about two rhombomere-widths anterior to its normal location. Bars: for A-C,H and I, 100 μm, and for D-G,J and K, 125 μm.

We examined Krox-20 expression at earlier stages by wholemount in situ hybridisation, using embryos from matings of +/kr females with kr/kr males. Before about the 14-somite stage (E9), we could not distinguish homozygous mutant kreisler embryos from +/kr littermates by their external appearance and, in embryos that we examined at the 3-5 somite stage (E8.0), we could not see any difference with regard to Krox-20 expression either: all these very young embryos had only a single domain of Krox-20 expression in the anterior hindbrain, as is normal, corresponding to the future rhombomere r3 (Hunt et al., 1991a; Schneider-Maunoury et al., 1993). Embryos at the 6-12 somite stage, however, fell into two distinct classes (Fig. 5A, B): some (15 out of 27) showed the wild-type pattern of two stripes, corresponding to the future rhombomeres r3 and r5, while others (12 out of 27) showed only a single domain of expression, corresponding to the future r3. We presume that these latter embryos were homozygous mutants. No intermediate cases, with a partial, weakened or narrowed domain of Krox-20, were seen. This strongly suggests that Krox-20 fails ever to be switched on in its r5 domain in kreisler homozygotes.

Expression pattern of Hoxb-2

In the following sections, we present data on Hox gene expression according to the anteroposterior sequence of their normal expression domains, beginning with Hoxb-2. This gene is normally expressed in the neural tube at E9.5 in a domain extending posteriorly from the boundary between r2 and r3 (Wilkinson et al., 1989b); again, our control (kreisler het-erozygote) specimens confirm this (n=5, all whole mounts) (Fig. 2D). We have five kreisler homozygotes hybridised for Hoxb-2 at this stage, all as whole mounts. They all show that the gene is expressed in its normal domain, bounded by the r2/3 boundary (Fig. 2E). It is possible that there may have been some abnormality in the modulation of Hoxb-2 expression within its domain (Sham et al., 1993), but this was difficult to judge from our specimens.

Expression pattern of Hoxb-1

In agreement with previous accounts of the normal expression pattern (Murphy et al., 1989; Wilkinson et al., 1989b; Frohman et al., 1990), we find that in our E9.5 control (kreisler heterozygote) embryos Hoxb-1 is expressed in a single sharply demarcated domain in the neural tube, coinciding with r4 (Fig. 2F). In the kreisler homozygotes, for which we have five informative sets of Hoxb-1 sections and five whole-mount in situ hybridisation specimens, the gene is likewise expressed in a single domain, whose anterior boundary is located in the normal place, at the interface between r3 and r4. The posterior boundary of the expression in the neural tube appears to extend a little further than normal posteriorly, by perhaps as much as half a rhombomere width into the region that would normally be r5 (Fig. 2G).

Expression pattern of Hoxa-3

The Hoxa-3 gene in a wild-type embryo is expressed in posterior regions up to a single sharp anterior limit that coincides with the boundary between r4 and r5, just anterior to the anterior end of the otocyst (Fig. 2H) (Gaunt 1987, 1988; Hunt et al., 1991b); expression is most intense at the anterior end of the domain, in r5 (Hunt et al., 1991b). In homozygous kreisler embryos the pattern of Hoxa-3 expression is more complex (Fig. 2I). Four sets of serial sections and a similar number of whole-mount Hoxa-3 in situ hybridisation specimens all show the same result. There is still a boundary just anterior to the otocyst, with expression posterior to this, even though this level no longer corresponds to a morphologically identifiable rhombomere boundary. However, this expression boundary appears less sharp than in control embryos and there is no sign of the heightened intensity normally characteristic of r5. We shall argue below that the cells in this region of the mutant that express Hoxa-3 are displaying a misplaced r7 character, not an r5 character. In addition to the main, posterior expression domain of Hoxa-3, the mutant has a weak domain of expression in r3, coinciding with the r3 domain of Krox-20 expression and separated from the posterior Hoxa-3 domain by a gap.

Expression pattern of Hoxd-4

Hoxd-4 is normally expressed in the neural tube in a domain comprising r7 and the regions posterior to this; its anterior limit of expression is diffuse, with a smooth gradient of expression in r7 and no detectable expression anterior to the boundary between r6 and r7 (Gaunt et al., 1989; Frohman et al., 1990; Hunt et al., 1991a). We have confirmed this in our control (kreisler heterozygote) whole-mount in situ hybridisation specimens at E9.5 (n=7) (Fig. 2J). We have 8 homozygous kreisler embryos hybridised for Hoxd-4 at this stage, all as whole mounts. In these embryos, the anterior limit of expression has the same diffuse character but appears to be shifted anteriorly by approximately two rhombomeres, such that the graded expression normally seen in r7 is now seen in the region that would normally be r5 (Fig. 2K).

Expression of CRABP I

At E9.5, cellular retinoic-acid binding protein I (CRABP I) can be detected in the hindbrain of control (kreisler heterozygote) embryos in r2 (weakly) and (strongly) in r4, r5 and r6, with a sharp boundary at the interface between r3 and r4 and a diffuse boundary at the posterior end (Dencker et al., 1990; Maden et al., 1992) (Fig. 3A). The protein is also strongly expressed in the adjacent neural crest cells, including those of the developing facial (VII) and glossopharyngeal (IX) ganglia and those that are destined to populate the second and third branchial arches; in this region the crest cells are divided into two distinct populations, anterior and posterior to the otocyst (Fig. 3C).

Fig. 3.

Sagittal section through the head, showing the distribution of CRABP I protein, as revealed by immunocytochemistry (brown staining), which marks specific developing rhombomeres. Intense staining can be seen also in individual differentiating neurons. Numbering refers to identifiable rhombomeres. (A) Heterozygous kr/+ embryo control, showing the normal pattern of CRABP I protein, which is concentrated in r4, r5 and r6, fading out in r7. (B) Homozygous kreisler embryo, showing staining in the r4 region, fading out in the region that would normally correspond to r5. The generally lower intensity of staining in comparison to the control is probably not significant – it was not a consistent finding (unlike the altered distribution). Coronal sections through the hindbrain, showing migrating neural crest cells, are strongly positive for CRABP I. (C) Heterozygous kr/+ embryo control, in which CRABP I marks neural crest cells which migrate as two discrete streams on either side of the otocyst. (D) Homozygous kreisler embryo, in which CRABP I marks a single mass of neural crest cells, which appears to diverge into two streams as cells migrate past the otocyst. Bar, 100 μm.

Fig. 3.

Sagittal section through the head, showing the distribution of CRABP I protein, as revealed by immunocytochemistry (brown staining), which marks specific developing rhombomeres. Intense staining can be seen also in individual differentiating neurons. Numbering refers to identifiable rhombomeres. (A) Heterozygous kr/+ embryo control, showing the normal pattern of CRABP I protein, which is concentrated in r4, r5 and r6, fading out in r7. (B) Homozygous kreisler embryo, showing staining in the r4 region, fading out in the region that would normally correspond to r5. The generally lower intensity of staining in comparison to the control is probably not significant – it was not a consistent finding (unlike the altered distribution). Coronal sections through the hindbrain, showing migrating neural crest cells, are strongly positive for CRABP I. (C) Heterozygous kr/+ embryo control, in which CRABP I marks neural crest cells which migrate as two discrete streams on either side of the otocyst. (D) Homozygous kreisler embryo, in which CRABP I marks a single mass of neural crest cells, which appears to diverge into two streams as cells migrate past the otocyst. Bar, 100 μm.

In the hindbrain of kreisler homozygotes, strong expression is seen in what would normally be r4 but there is no expression of CRABP I posterior to r4, in the region that would normally correspond to r5 and r6 (Fig. 3B). As for the neural crest of the mutant, CRABP I is detected in the facial ganglion and also in a more caudal set of cells, between the hindbrain and the otocyst, level with what would normally be r5.

There is no crest-free region next to the otocyst; instead, CRABP I marks a single continuous group of neural-crest cells adjacent to the hindbrain that diverge into two streams apparently entering the second and third branchial arches (Fig. 3D). The pattern is similar in all of the 8 sets of sections of homozygous kreisler embryos that we have stained for CRABP I.

Neuroanatomical pattern

To see how the pattern of gene expression is reflected in the neuroanatomy, we have used the antibody 2H3 (Dodd et al., 1988) to examine the distribution of differentiating neurons and the arrangement of cranial nerves. In a normal embryo, the distribution of nerve cell bodies and developing fibre tracts in the hindbrain itself reveals the system of rhombomeres. At E9.5, the 2H3 antibody reveals a subset of developing neurons within the hindbrain. At this stage in a heterozygous kreisler embryo, the staining distinguishes r3 and r5 as regions relatively free of axons, since neuronal differentiation is delayed in these rhombomeres (Fig. 4A). In contrast, in homozygous kreisler embryos of a similar stage, there is only a single region relatively free of axons, which appears to correspond to r3, the delayed differentiation of axons characteristic of r5 appears completely absent (Fig. 4B). At a slightly later stage, the 2H3 antibody reveals the concentration of axons along rhombomere boundaries. Fig. 4C shows the hindbrain of heterozygous kreisler embryos; the neuronal staining reveals clear r1/2, r2/3, r3/4, r4/5 and r5/6 boundaries. In the homozygous mutant (Fig. 4D), no such boundaries are seen posterior to the r3/4 boundary, and the overall pattern is as though r5 and r6 have been excised, so that r4 now abuts r7. In the periphery, the abnormalities are confined to the region posterior to the otocyst. The glossopharyngeal ganglion and nerve, normally associated with r6, and the abducens nerve, which normally derives from r5 and r6 (Lumsden and Keynes, 1989), are both missing (Fig. 4E-H). Although there are nerve fibres following part of the distal pathway normally taken by the glossopharyngeal nerve (Fig. 4F), these fibres appear to derive from the adjacent vagal ganglion. These findings were repeatably seen both in whole mounts and reconstructions from serial sections (n=7 homozygotes and n=8 heterozygotes).

Fig. 4.

(A,B) The early (E9.5) pattern of neuronal differentiation within the hindbrain, revealed with the antibody 2H3. (A) Flat-mounted hindbrain of a heterozygous kreisler embryo. Two relatively axon-free regions can be identified, these reflect the delayed neuronal differentiation characteristic of rhombomeres 3 and 5. (B) Flatmounted hindbrain of a homozygous kreisler embryo. Only a single axonfree region can be identified – corresponding to rhombomere 3. Numbering refers to identifiable rhombomeres. (C,D) The organisation of rhombomere boundaries revealed using 2H3 antibody staining of E10.5 embryos showing alignment of axons along rhombomere boundaries. (C) Flatmounted hindbrain of heterozygous kreisler embryo showing a series of rhombomere boundaries demarcating rhombomeres 1-6. (D) Flat-mounted hindbrain of a homozygous kreisler embryo showing a reduced number of rhombomere boundaries demarcating only rhombomeres 1-4. Numbering refers to identifiable rhombomeres. (E,F) Lateral view of whole E10.5 embryos stained with the antibody 2H3 and cut along the midline to reveal the pattern of peripheral cranial nerves. (E) Heterozygous kreisler embryo showing the normal pattern of mouse cranial nerves and ganglia. (F) Homozygous kreisler embryo showing normal patterns for the V and VII nerves, but an apparent fusion of nerves IX and X. Note that while in dorsal regions the IX ganglion appears absent the pattern of the IX nerve more ventrally appears normal. Labelling: V-trigeminal, VII-facial, IX-glossopharyngeal and X-vagus. (G,H) Parasagittal vibratome sections, 100 μm thick, of E11.5 embryos stained as whole mounts before sectioning. The position of the field of view within the section is illustrated in the accompanying schematic drawings. Note for both sections anterior is to the left. (G) Heterozygous kreisler embryo showing the characteristic dorsal exit of the abducens nerve from the hindbrain and its rostral trajectory. Homozygous kreisler embryo showing an equivalent section, but with a complete absence of any neuronal structure resembling (F) the abducens. A branch of the facial nerve and the hypoglossal nerve, provide good markers of position within the embryo. LabellingVI, abducens; VII, branch of the facial nerve; XII, hypoglossal nerve. Bar, 100 μm.

Fig. 4.

(A,B) The early (E9.5) pattern of neuronal differentiation within the hindbrain, revealed with the antibody 2H3. (A) Flat-mounted hindbrain of a heterozygous kreisler embryo. Two relatively axon-free regions can be identified, these reflect the delayed neuronal differentiation characteristic of rhombomeres 3 and 5. (B) Flatmounted hindbrain of a homozygous kreisler embryo. Only a single axonfree region can be identified – corresponding to rhombomere 3. Numbering refers to identifiable rhombomeres. (C,D) The organisation of rhombomere boundaries revealed using 2H3 antibody staining of E10.5 embryos showing alignment of axons along rhombomere boundaries. (C) Flatmounted hindbrain of heterozygous kreisler embryo showing a series of rhombomere boundaries demarcating rhombomeres 1-6. (D) Flat-mounted hindbrain of a homozygous kreisler embryo showing a reduced number of rhombomere boundaries demarcating only rhombomeres 1-4. Numbering refers to identifiable rhombomeres. (E,F) Lateral view of whole E10.5 embryos stained with the antibody 2H3 and cut along the midline to reveal the pattern of peripheral cranial nerves. (E) Heterozygous kreisler embryo showing the normal pattern of mouse cranial nerves and ganglia. (F) Homozygous kreisler embryo showing normal patterns for the V and VII nerves, but an apparent fusion of nerves IX and X. Note that while in dorsal regions the IX ganglion appears absent the pattern of the IX nerve more ventrally appears normal. Labelling: V-trigeminal, VII-facial, IX-glossopharyngeal and X-vagus. (G,H) Parasagittal vibratome sections, 100 μm thick, of E11.5 embryos stained as whole mounts before sectioning. The position of the field of view within the section is illustrated in the accompanying schematic drawings. Note for both sections anterior is to the left. (G) Heterozygous kreisler embryo showing the characteristic dorsal exit of the abducens nerve from the hindbrain and its rostral trajectory. Homozygous kreisler embryo showing an equivalent section, but with a complete absence of any neuronal structure resembling (F) the abducens. A branch of the facial nerve and the hypoglossal nerve, provide good markers of position within the embryo. LabellingVI, abducens; VII, branch of the facial nerve; XII, hypoglossal nerve. Bar, 100 μm.

Patterns of cell death

We used acridine orange to compare the distributions of dead and dying cells (Wolf and Ready, 1991) in heterozygous and homozygous kreisler embryos. The stages of greatest interest are those preceding the appearance of the pattern of rhombomeres and of the visible defect in the homozygous mutants. As described above, there is a period before overt segmentation when it is possible to use the expression pattern of Krox-20 to distinguish phenotypically normal embryos (which express Krox-20 in two domains, corresponding to future r3 and r5) from homozygous kreisler embryos (which express Krox-20 in only one domain, corresponding to future r3). This difference begins to be apparent at about the 6-somite stage. Embryos up to and including the 7-somite stage do not show any obvious difference in the amount or distribution of cell death: it appears uniformly low. A total of 44 embryos at stages between 2 and 7 somites have been examined for the pattern of cell death. Of these 12 embryos, all at the 7-somite stage, have also been stained for Krox-20 expression, revealing 3 as homozygous kreisler mutants and 9 as heterozygous (Fig. 5A-D). By the 14-somite stage, however, at which homozygous kreisler embryos can be distinguished by their morphology as well as their pattern of gene expression, dramatic differences are evident in the pattern of cell death. In homozygous kreisler embryos, it is greatly enhanced within the neuroepithelium of r2, r3 and r4 in comparison with heterozygous kreisler embryos (Fig. 5E,F). In confirmation of the pattern seen in Fig. 1D, levels of cell death remain elevated in r4 of homozygous kreisler embryos but have declined in r3 and r2 by the latest stage examined, about the 23-somite stage (Fig. 5G,H).

In the homozygous kreisler mutant, rhombomeres 5 and 6 are lost

Our results confirm that, in the homozygous kreisler mutant, there is a fundamental defect in the organisation of the hindbrain. In this respect, we agree with the previous observations of Deol (1964) and Frohman et al. (1993). We have analysed the mutant at E9.5 in terms of the expression patterns of Krox-20 and a number of different Hox genes, as did Frohman et al. (1993). In addition, we have examined the cranial nerves, the distribution of CRABP-I and the pattern of cell death. To investigate the origin of the abnormalities seen at E9.5, we have also examined cell death and Krox-20 expression at earlier stages.

The abnormalities in the homozygous kreisler hindbrain are most obvious morphologically at the level where r4 would normally meet r5. The underlying pattern of gene expression shows only minor abnormalities anterior to the level of the r4/5 boundary, but major abnormalities posterior to this. Thus we see no expression of Krox-20 in what should be r5; CRABP I fades out posterior to the region of r4, instead of fading out posterior to r6; and the Hoxd-4 domain is shifted approximately two rhombomere widths anteriorly, so that its most anterior expression is in the region of r5 instead of r7. The full pattern of gene expression is summarised in Fig. 6. It can be seen that a simple interpretation of the phenotype is possible: the tissue that would normally form r5 and r6 has been deleted or converted in character so that it is absorbed into a neighbouring rhombomere. This explains not only the anterior shift of Hoxd-4 expression but also the loss of the r5 Krox-20 domain and the restricted distribution of CRABP I. The interpretation is supported by the anatomy of the cranial nerves in homozygous kreisler embryos: the glossopharyngeal (IX) nerve and ganglion, normally associated with r6, and the abducens nerve, which normally originates from r5 and r6, are both missing. Further support comes from our findings on the expression of the FGF-3 (int-2) gene. These observations will be presented in detail elsewhere, (McKay, Lumsden and Lewis, unpublished data), in connection with the interpretation of the abnormalities of the inner ear, but in brief we find that FGF-3, which is normally expressed in r5 and r6, is not expressed in this region of the homozygous kreisler mutant. The question of how the disturbances seen at E9.5 arise will be discussed below, but we should first consider how our results compare with those of Frohman et al. (1993).

Fig. 5.

Changing patterns of cell death in the kreisler mutant revealed by acridine orange staining.(A) An embryo with approximately 7 somites showing the wild-type expression pattern of Krox-20 typical of heterozygous kreisler embryos. (B) An embryo with approximately 7 somites showing a failure of Krox-20 expression in r5 which is typical of homozygous kreisler embryos. (C) A confocal image of the pattern of cell death in the embryo same embryo as in A, this embryo is presumably a heterozygous kreisler embryo as judged from the pattern of Krox-20. It is little different from the pattern of cell death seen in D. (D) A confocal image of the pattern of cell death in the same embryo as shown in B, this embryo is presumably a homozygous kreisler embryo as judged from the pattern of Krox-20. For both C and D, dying cells appear pale pink and are superimposed on a phase-contrast image. (E) Fluorescent image of a heterozygous kreisler embryo with about 14 somites. Arrows mark intense areas of cell death around the closing otic vesicle. Cell death in the hindbrain is restricted to the roof plate few dying cells are seen within the neuroepithelium. (F) Fluorescent image of a homozygous kreisler embryo with about 14 somites. Arrows mark cell death (out of focus) around the closing otic vesicle. Cell death is evident with the neuroepithelium of rhombomeres 2, 3 and 4. (G) Fluorescent image of the pattern of cell death in a heterozygous kreisler embryo with about 22 somites. Scattered cell death is visible, mostly within the roof plate of the hindbrain. (H) Fluorescent image of the pattern of cell death in a homozygous kreisler embryo with about 22 somites. Extensive cell death can be seen within the neuroepithelium of rhombomere 4. For all images anterior is to the left. Bar, 100 μm.

Fig. 5.

Changing patterns of cell death in the kreisler mutant revealed by acridine orange staining.(A) An embryo with approximately 7 somites showing the wild-type expression pattern of Krox-20 typical of heterozygous kreisler embryos. (B) An embryo with approximately 7 somites showing a failure of Krox-20 expression in r5 which is typical of homozygous kreisler embryos. (C) A confocal image of the pattern of cell death in the embryo same embryo as in A, this embryo is presumably a heterozygous kreisler embryo as judged from the pattern of Krox-20. It is little different from the pattern of cell death seen in D. (D) A confocal image of the pattern of cell death in the same embryo as shown in B, this embryo is presumably a homozygous kreisler embryo as judged from the pattern of Krox-20. For both C and D, dying cells appear pale pink and are superimposed on a phase-contrast image. (E) Fluorescent image of a heterozygous kreisler embryo with about 14 somites. Arrows mark intense areas of cell death around the closing otic vesicle. Cell death in the hindbrain is restricted to the roof plate few dying cells are seen within the neuroepithelium. (F) Fluorescent image of a homozygous kreisler embryo with about 14 somites. Arrows mark cell death (out of focus) around the closing otic vesicle. Cell death is evident with the neuroepithelium of rhombomeres 2, 3 and 4. (G) Fluorescent image of the pattern of cell death in a heterozygous kreisler embryo with about 22 somites. Scattered cell death is visible, mostly within the roof plate of the hindbrain. (H) Fluorescent image of the pattern of cell death in a homozygous kreisler embryo with about 22 somites. Extensive cell death can be seen within the neuroepithelium of rhombomere 4. For all images anterior is to the left. Bar, 100 μm.

Fig. 6.

A schematic summary of the patterns of gene expression in the hindbrain of heterozygous kreisler or wild-type embryos and homozygous kreisler embryos.

Fig. 6.

A schematic summary of the patterns of gene expression in the hindbrain of heterozygous kreisler or wild-type embryos and homozygous kreisler embryos.

Frohman et al. (1993) analysed kreisler in terms of the expression of Krox-20, FGF-3, Hoxb-1, Hoxb-3 and Hoxb-4 and also examined the skeletal anatomy of the branchial arches in the later embryo. A striking finding was that there were abnormalities in part of the hyoid bone, indicating that struc-tures with a second arch character had developed in the third arch. Normally, the skeleton of each arch is formed by neural crest cells that migrate from the adjacent rhombomeres, carrying their positional specification with them (Noden, 1983; Lumsden et al., 1991; Hunt et al., 1991a,b). The hyoid bone abnormalities in homozygous kreisler embryos, therefore, are suggestive of a homeotic conversion conferring an abnormally anterior character on the rhombomeres supplying crest cells to the third arch. This is indeed the interpretation that we favour. It does not, however, tally easily with the pattern of gene expression as detailed by Frohman et al. (1993) in homozygous kreisler embryos. Although their findings agree with ours in many respects, there are differences between their account and ours in the relative locations of the boundaries of the Hox gene expression domains, and these led Frohman et al. (1993) to a different view of the basic abnormality.

In particular, our account implies that, in homozygous kreisler mice, the expression patterns of Hoxa-3 and Hoxb-1 do not overlap, whilst the account of Frohman et al. (1993) implies that there are two rhombomeres in which Hoxb-1 and Hoxb-3 overlap. In other words, Frohman et al. (1993) report that Hox genes are expressed in abnormal combinations in homozygous kreisler embryos, whereas we find only that they are expressed in abnormal locations (with minor qualifications).

We believe that differences of subjective judgement may explain most of the discrepancies. The expression boundaries in question are not sharp and several anatomical landmarks are shifted in the mutant, making precise interpretation difficult. For this reason we have, for most of the genes examined, used a combination of sections and whole mounts, instead of simply relying on sections. It is, however, possible that the expression pattern of Hoxb-3 may be slightly different from that of its paralog Hoxa-3, and that of Hoxb-4 from that of its paralog Hoxd-4. But our CRABP I and neuroanatomical data support the view that the primary defect is a loss of r5 and r6, and that the combinations of genes and anatomical features expressed in any given region of the mutant hindbrain are combinations encountered also in a normal hindbrain, even if at a different site.

A further possible reason for differences between our account given here and that of Frohman et al. (1993) may lie in small differences in the staging of the embryos. As we shall see below, changes in the pattern of gene expression with age would be expected according to our interpretation.

Secondary features of the kreisler phenotype may reflect misregulation of Krox-20

The simple summary of the kreisler phenotype in terms of loss of r5 and r6 has to be qualified in two respects. First, expression of Krox-20 in its r3 domain persists slightly longer than normal and this persistence is correlated with weak ectopic expression of Hoxa-3 in r3. It is known that the product of Krox-20 is able to modulate the level of expression of Hoxb-2 (Sham et al., 1993), and it may have a similar effect on Hoxa-3; that is, the misregulation of Hoxa-3 in r3 might be secondary to misregulation of Krox-20 in r3. Another possible interpretation, as Frohman et al. (1993) suggest, is that in homozygous kreisler embryos r3 may be partially converted to an r5 character. But this is not reflected in the neuroanatomy, and other features of this region fail to show a corresponding transformation.

A second, minor way in which the kreisler phenotype differs from a simple loss of r5 and r6 concerns the appearance of the Hox gene expression boundaries: although Hoxb-1 and Hoxa-3 both show expression boundaries in the absence of visible segment boundaries, these expression boundaries appear blurred. This could be interpreted in two ways. As Swiatek and Gridley (1993) and Schneider-Maunoury et al. (1993) have shown, the Krox-20 gene product is critical for the maintenance of morphologically distinct rhombomeres in the region from r2 to r6. In the absence of boundaries defined by the confrontation of cells that are dissimilar with regard to expression of Krox-20, there may be a loss of the usual prohibition on cell movement between segmental compartments (Fraser et al., 1990; Guthrie et al., 1993). Hence, a mixing of cells that do express Hoxa-3 with cells that do not could account for the fuzziness of the Hoxa-3 boundary, for example. Alternatively, it could be that the Krox-20 gene product is required not only for the maintenance of segments but also to help sharpen the spatial regulation of Hox gene expression and co-ordinate it precisely with the segment boundaries, as the products of the pair rule genes do in Drosophila (Ingham and Martinez-Arias, 1986).

The kreisler mutation alters the distribution of neural crest cells

In normal (wild-type or heterozygous kreisler) embryos, r5 produces few if any surviving neural crest cells (Adelmann, 1925; Tan and Morriss-Kay, 1985; Lumsden et al., 1991; Graham et al., 1993); it thus serves to separate the neural crest into two distinct streams of cells, which stain with CRABP I, and which enter branchial arches two and three. However, in homozygous kreisler embryos, the corresponding region does appear to produce neural crest (Hertwig, 1944; Deol, 1964; and our own observations): the absence of r5 means that no crestfree region exists adjacent to the otocyst. Indeed the CRABP I staining pattern suggests that crest cells from r4 migrate laterally not only into the second branchial arch (as they normally would) but also posteriorly and laterally into the third arch. This behaviour of the neural crest in kreisler homozygotes matches Frohman et al. (1993) observations of ectopic second arch structures developing within the third arch, as discussed earlier. An explanation for the seemingly abnormal migration pathways of the crest cells from the r4 region is suggested below.

The kreisler gene acts upstream of Krox-20

What deductions do our data allow as to the place of the kreisler gene in the hierarchy of genetic control? A key finding is that in kreisler homozygotes Krox-20 does not even begin to be expressed in its r5 domain. This implies that the kreisler gene function lies upstream from Krox-20.

Schneider-Maunoury et al. (1993) and Swiatek and Gridley (1993) have raised transgenic mice in which Krox-20 has been mutated by homologous recombination so as to produce an inactive product. In contrast with our kreisler homozygotes, the homozygous Krox-20 mutant embryos initially switch on expression of the (defective) Krox-20 gene in two hindbrain domains corresponding to the future r3 and r5; but, through lack of functional Krox-20 protein, these domains then disappear. The result is an embryo in which r3 and r5 appear to have been deleted and in which there is a loss of morphological segmentation, with no sign of the normal r2/r3, r3/r4, r4/r5 or r5/r6 rhombomere boundaries. Formation of these morphological boundaries seems to require a confrontation between cells expressing functional Krox-20 and cells not expressing this gene (Lumsden, 1990; Guthrie and Lumsden, 1991). The loss of a subset of morphological rhombomere boundaries in kreisler can therefore be explained as a consequence of the loss of the r5 domain of Krox-20 expression. Misregulation of Krox-20 cannot be the whole explanation of the kreisler phenotype, however, since the kreisler defects extend beyond the region affected in the Krox-20 mutants: not only r5, but also r6 is missing in kreisler.

The kreisler defect overlaps with but does not coincide with the defect due to knock-out of Hoxa-1

Transgenic mice in which the Hoxa-1 gene has been knocked out by homologous recombination also have some features in common with kreisler (Lufkin et al., 1991; Chisaka et al., 1992; Dollé et al., 1993; Mark et al., 1993). Like kreisler and Krox-20 mutants, Hoxa-1 mutants show a deletion of a region that includes r5 and a corresponding localised loss of segmentation in the posterior hindbrain; and, in the case of kreisler and the Hoxa-1 mutants, this is coupled with characteristic abnormalities in the positioning and development of the otocysts. But there are also discordances, which can be summed up by saying that, whereas kreisler lacks r5 and r6, the defect in Hoxa-1 mutants is in r4 and r5 (Mark et al., 1993). Neither gene, therefore, is likely to be acting purely as a regulator of the other. The known timing of expression of the two genes gives no direct evidence as to whether kreisler acts upstream or downstream of Hoxa-1: Hox genes, like kreisler, are already activated early, before E8.5 (Gaunt, 1987). It should be noted that the location of the kreisler gene on chromosome 2 does not match that of any other gene known to be involved in hindbrain patterning and, in particular, it cannot belong to any of the Hox complexes.

Cell death in the r4 region of the kreisler hindbrain may be a late consequence of a homeotic conversion of prospective r5 and r6 cells to an r4 character

The pattern of cell death in kreisler presents a puzzle: although it is rhombomeres r5 and r6 that are lost, it is predominantly in the neighbourhood of r4 that we see cell death (although there is also some cell death in r2 and r3). It is possible that the prospective r5 and r6 territories are lost by cell death at a very early stage, before the onset of Krox-20 expression, but this is not supported by our findings. A more attractive interpretation is suggested by the observation that, in the kreisler homozygote at E9.5, the neural tube at the level of r4 not only contains more dying cells than normal, but also has much thicker walls. The cell death appears to be disposing of a cell surplus. Where might this surplus have come from? A very plausible answer is that, in the kreisler homozygote, cells that would normally have received r5 or r6 positional specifications have instead received an r4 specification and, in consequence, have combined with the other, normally located, r4 cells to form a single giant r4 domain, but one in which the extra cells are accommodated through an increase of thickness rather than an increase of length. The cell death seen at E9.5 would then be a late regulative phenomenon, occurring in reaction to a much earlier aberration of positional specification. In other words, the primary effect of the mutation may be a homeotic conversion of prospective r5 and r6 cells to the character of r4, with the consequence that those cells are absorbed into r4 and eventually eliminated by cell death. Such down-regulation of excessive cell numbers through cell death has been observed in many systems (Raff, 1992) and in particular appears to be the mechanism for regulation of over-large segments in Drosophila (Magrassi and Lawrence, 1988; Ish-Horowicz and Gyurkovics, 1988).

If the re-packing of the homeotically transformed r5 and r6 cells into a giant, thick-walled r4 domain takes some time to occur, some of the discrepancies between Frohman et al. (1993) account and ours may be simply the result of analysing the pattern of Hox gene expression at a slightly different stage in the process. The abnormality that Frohman et al. (1993) see in the hyoid bone also has a very straightforward interpretation along these lines: neural crest cells migrating at an early stage into the third branchial arch from the level of r5 and r6 would carry with them an r4 specification instead of an r5 or r6 specification, and so would form ectopic second arch structures. As the regions of hindbrain at the r4, r5 and r6 levels become telescoped into a single thickened r4, this stream of neural crest cells would take on the appearance that we see at E9.5, diverging and extending backwards from the r4 region into both the second and third branchial arches.

Our study of Krox-20 expression in kreisler mutants gives direct evidence that the kreisler gene acts early, before E8.5, and regulates expression of Krox-20. If the above interpretation of the pattern of cell death is accepted, it would seem that the kreisler gene product must also regulate expression of other early patterning genes at the level of r5 and r6, and in particular Hox genes, on which positional specification is thought to depend. Whether or not this speculation is right, it seems clear that the kreisler gene is required, and required early, for the construction of a specific subset of hindbrain segments. Thus, in mutant phenotype at least, it is reminiscent of a gap gene in Drosophila. To discover whether this is more than a superficial analogy, it will be necessary to clone the kreisler gene.

We thank David Wilkinson for the gift of Krox-20 probe and for generous help and advice; Anthony Graham for his substantial help too. We are grateful to Gail Martin, Greg Barsh and their colleagues for communicating their results before publication. We also thank members of the ICRF Developmental Biology Unit for many useful discussions and comments, and most especially the animal unit at Clare Hall for taking such expert care of our kreisler mouse colony.

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