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Human Molecular Genetics Pages 945-957  

The variable expressivity and incomplete penetrance of the twist-null heterozygous mouse phenotype resemble those of human Saethre-Chotzen syndrome
Introduction
Results
   M-twist transcripts in the head prior to 16.5 days post-coitum (d.p.c.)
   M-twist expression during mouse odontogenesis
   M-twist transcripts in forming sensory organs from 10.5 to 19.5 d.p.c.
   twist-null heterozygous additional toe phenotype
   Inheritance of the duplicated hallux phenotype
   Neurocranium and chondrocranium malformations in twist-null heterozygotes
   Viscerocranium malformations in twist-null heterozygous animals
   Additional twist-null heterozygous malformations
   M-twistDbHLH heterozygous mice analyses
Discussion
   Defects are observed in tissues derived from cells expressing M-twist
   Hindlimb twist-null heterozygous mouse phenotype and limb polarity
   Craniofacial abnormalities and skull bone formation
   Variability in penetrance and expressivity
   Relevance to human patients with craniosynostosis malformations
   Mutant mice strains as models to study the etiology of human syndromes
Materials And Methods
   In situ hybridization
   Mice analyses
   Skeletal preparations
Acknowledgements
References

The variable expressivity and incomplete penetrance of the twist-null heterozygous mouse phenotype resemble those of human Saethre-Chotzen syndrome

The variable expressivity and incomplete penetrance of the twist-null heterozygous mouse phenotype resemble those of human Saethre-Chotzen syndrome

Patrice Bourgeois1,+, Anne-Laure Bolcato-Bellemin1,+, Jean-Marc Danse1, Agnes Bloch-Zupan2, Kunihiko Yoshiba1,§, Corinne Stoetzel1, Fabienne Perrin-Schmitt1,*

1Laboratoire de Génétique Moléculaire des Eucaryotes du CNRS/INSERM U184, Institut de Chimie Biologique and 2INSERM U424, Institut de Biologie Médicale, Faculté de Médecine, 11 rue Humann, 67085 Strasbourg Cedex, France

Received January 20, 1998; Revised and Accepted March 3, 1998

Most targeted gene mutations are recessive and analyses of gene function often focus on homozygous mutant phenotypes. Here we describe parts of the expression pattern of M-twist in the head of developing wild-type mice and present our analysis of the phenotype of heterozygous twist-null animals at around birth and in adults. A number of twist-null heterozygous mice present skull and limb defects and, in addition, we observed other malformations, such as defects in middle ear formation and the xyphoïd process. Our study is of interest to understand bone formation and the role of M-twist during this process, as within the same animal growth of some bones can be accelerated while for others it can be delayed. Moreover, we show here that expressivity of the mouse mutant heterozygous phenotype is dependent on the genetic background. This information might also be helpful for clinicians, since molecular defects affecting one allele of the human H-twist (TWIST) gene were identified in patients affected with Saethre-Chotzen syndrome (SCS). Expressivity of this syndrome is variable, although most patients present craniofacial and limb malformations resembling those seen in mutant mice. Thus the mutant mouse twist-null strain might be a useful animal model for SCS. The twist-null mutant mouse model, combined with other mutant mouse strains, might also help in an understanding of the etiology of morphological abnormalities that appear in human patients affected by other syndromes.

INTRODUCTION

In mice M-twist is a vital gene, since the two different mutant alleles that have been produced are both homozygous lethal during early embryonic development (1; A.-L. Bolcato-Bellemin et al., in preparation). The entire coding region lies in the first exon of the gene (2), as is the case for Xenopus X-twi (3), human H-twist (4) and avian G-twist (A.-L. Bolcato-Bellemin et al., in preparation). M-twist codes for a protein which belongs to the basic helix-loop-helix (bHLH) transcription factor family (5). The twist-null recessive mutation deletes the entire coding region (first exon of the gene) and the intron and homozygous mutant embryos die at 11.5 days of development with a failure of fusion of the cranial neural folds and defects in head mesenchyme, branchial arch formation and somite organization; in addition, the relative size of the forelimb and hindlimb buds is reversed when compared with the wild-type (1). At the molecular level the twist-null mutation is a real null mutation, as no RNA is synthesized from the mutant allele (A.-L. Bolcato-Bellemin, unpublished results). The M-twistDbHLH recessive mutation deletes the bHLH coding region of the gene, but produces a partial transcript which contains the sequences of the second exon and homozygous mutant conceptuses display disorganized extraembryonic and embryonic germ layers and die at the end of gastrulation (A.-L. Bolcato-Bellemin, unpublished results). Compound heterozygotes die during embryogenesis.

Transcripts of M-twist, G-twist and X-twi are already weakly detected in unfertilized and unincubated eggs and from then onwards, then massively at about gastrulation and at later developmental stages; these RNAs are mainly located in the presumptive endomesodermal germ layer prior to gastrulation, then in mesodermal derivatives such as the branchial arches, the somites and the limb buds (6-8; A.-L. Bolcato-Bellemin et al., in preparation; C. Stoetzel et al., in preparation).

These studies suggest that the homologous vertebrate twist genes are true orthologous genes of Drosophila twist. In Drosophila, twist was identified on the basis of its lethal embryonic phenotype: embryos lacking twist function fail to gastrulate normally, produce no mesoderm and die at the end of embryogenesis with a `twisted' appearance (9,10). Molecular analyses showed that Drosophila twist encodes a protein containing a bHLH motif (5,11) and is a zygotic mesodermal determinant gene, activated in response to a maternal gene cascade (12-15). Later in development twist is implicated in mesodermal cell patterning (15-18).

Human H-twist (TWIST) is located at 7p21 (4,19) and the clinical description of different human diseases mapping to this region (MIM Databank) suggested that H-twist may be involved in one of them. Human patients presenting with Saethre-Chotzen syndrome (SCS; MIM 101400) or acrocephalo-syndactyly type III syndrome (ACS III) were shown to be heterozygous for mutations affecting either the H-twist (TWIST) coding sequence (20-22) or its vicinity (22,23). This developmental disorder is `of autosomal dominant inheritance with a high degree of penetrance' and variable expressivity; `incomplete penetrance' was also documented (ref. 24 and references therein). It is characterized by a broad and variable pattern of malformations, including craniosynostoses, facial asymmetry with a deviated nasal septum, distal limb abnormalities, eyelid ptosis, abnormally small ears with prominent crura and possibly, in addition, brachydactyly, maxillary hypoplasia with a narrow palate, partial cutaneous syndactyly and broad halluces in a valgus position (24). Mild to moderate mental retardation is also reported (25,26). Some of these human abnormalities resemble those observed in mutant M-twist heterozygous mice (20; see also below).

We present here our detailed analyses of the mouse heterozygous mutant phenotype. In particular we show in some strains the presence of an additional toe on either or both hindlegs and either an accelerated or delayed ossification of some bones in the head. Other mild skeletal defects, such as nasal septum deviation and facial asymmetry, are also found in many but not all animals in other genetic backgrounds.

These results are of special interest in the context of comparative molecular embryology: if human patients also present these defects, this will complete the clinical description of SCS. Furthermore, the variable expressivity of the phenotype could be due to different human genealogies. Moreover, the fact that the craniofacial and hindlimb phenotype is not fully penetrant and depends on the genetic background suggests that another gene(s) is implied in the appearance of morphological disorders. Work is possible in mice to try to elucidate this point. In any case, mutant heterozygous M-twist mice appear as animal models that will help in understanding the etiology of human SCS abnormalities and of some other craniosynostosis syndromes.

RESULTS

M-twisttranscripts in the head prior to 16.5 days post-coitum (d.p.c.)

In situ hybridizations have been performed on whole mount embryos up to stage 11.5 d.p.c. and on head tissue sections for later stages to study the spatial localization of M-twist transcripts during late embryogenesis.

M-twist transcripts were previously reported from 9 d.p.c. onward in the entire head mesenchyme and more abundantly in the mesenchymal cells of the mandibular arch and, as soon as they appear, in cells of the second and third branchial arches at 9.5 d.p.c. and in the maxillary process of developing embryos at 10 d.p.c. (2,6,7).

We show here only in situ hybridization to head tissue sections at 12.5, 14.5 and 16.5 d.p.c. (Fig. 1), to briefly illustrate the locations of M-twist transcripts in forming head chondrification centers of the future chondrocranium and neurocranium and in diverse derivatives of the branchial arches, such as the mandible.


Figure 1. M-twist transcripts in 12.5, 14.5 and 16.5 d.p.c. embyo heads. At 12.5 d.p.c. M-twist transcripts are almost uniformly distributed in the entire head mesenchyme and in particular around the oral cavity, in the forming tongue, the mandible and around the pharynx and the nasal chamber. At 14.5 d.p.c. M-twist transcripts are located in the precartilaginous and cartilaginous centres, in the nasal capsule, in the cartilaginous optic capsule and in primordia of the otic ossicles, and in the mesenchyme around the mouth and in the forming skull bones. At 16.5 d.p.c. the M-twist transcript distribution is similar to that of previous stage, but its levels are lower. The bars indicate 1 mm.


Figure 2. M-twist transcript location during odontogenesis. Incisor formation is shown on the left, molar formation on the right, in sections of embryos at 12.5, 14.5 and 16.5 d.p.c. and in newborn mice (19.5 d.p.c.). M-twist transcripts are detected at all stages in neural crest-derived mesenchyme of molar and incisor anlagen. The bars indicate 300 µm.


M-twistexpression during mouse odontogenesis

M-twist transcript location during mouse odontogenesis is illustrated in Figure 2, left, for development of the mandibular incisors and Figure 2, right, for development of the molars. Briefly, M-twist transcripts were detected in the neural crest-derived mesenchyme of the molar and incisor anlagen at 12.5 d.p.c. They were then observed in the mesenchymal structures of incisors and molars at 14.5 d.p.c. and in the posterior part of the pulp as well as in the peridental mesenchyme at 16.5 d.p.c. Finally, they were restricted to the peridental mesenchyme of the first molars and incisors and confined to the mesenchyme facing the inner dental epithelium in specific cusp tips of the second molars of newborn animals.

M-twisttranscripts in forming sensory organs from 10.5 to 19.5 d.p.c.

During eye formation M-twist RNA was detected in the optic cup at 10.5 d.p.c., then in particular in the eyelids and mesothelial layer of the cornea at 12.5 and 14.5 d.p.c.; at 19.5 d.p.c. sparse traces of M-twist RNA were still detected in the fused eyelids (Fig. 3A).


Figure 3. M-twist transcripts in forming sensorial organs. (A) Optic apparatus formation. At 12.5 d.p.c. M-twist transcripts are in the mesodermal cells lying between the corneal ectoderm and the anterior wall of the lens vesicle. At 14.5 d.p.c. they are abundant in the eyelid, but are not present in the neural layer of the retina nor in the future conjunctival sac. Note that the mesodermal condensate that will become the future extrinsic ocular muscle contains high amounts of M-twist transcripts. At 16.5 d.p.c. there are few M-twist transcripts at the line of fusion of upper and lower eyelids. (B) During otic apparatus development from stages 10.5 to 16.5 d.p.c. M-twist transcripts accumulate in particular in the precartilaginous mesenchyme forming the otic capsule (stages 10.5 and 12.5 d.p.c.), along the external acoustic meatus and in the primordium of the pinna (at 12.5 d.p.c.) and along the tubo-tympanic recess (at 14.5 and 16.5 d.p.c.). (C) M-twist RNA accumulates in mesenchymal cells surrounding the olfactory epithelium (C1, stage 14.5 d.p.c.), within the mesenchymal cells of the palate and of the oral surface of the tongue, where there are numerous fungiform papillae (C2, stage 16.5 d.p.c.) and in connective tissue sheath and sinuses of forming vibrissae (C3 and C4, stages 14.5 and 19.5 d.p.c.). During skin differentiation the stratum germinativum contains high levels of M-twist RNA, but not the stratum granulosum (C5 and C6, stages 16.5 and 19.5 d.p.c. respectively). The bars indicate 100 µm.

During development of the otic apparatus M-twist transcript accumulated around the otocyst at 10.5 d.p.c., then in the forming external acoustic meatus and in the primordium of the pinna at 12.5 d.p.c. At 14.5 d.p.c. they were abundant in particular around the tubo-tympanic recess in the condensing mesenchymal cells and as traces in the same tissues at 16.5 d.p.c. (Fig. 3B).

In addition, M-twist transcripts were observed within mesenchymal cells surrounding the olfactory epithelium at 16.5 d.p.c. (Fig. 3C1), along the oral epithelium of the tongue but not in the papillae (Fig. 3C2), in the connective tissues of forming vibrissae (Fig. 3C3 and C4) and within the stratum germinativum of the skin (Fig. 3C5 and C6).

twist-null heterozygous additional toe phenotype

Two heterozygous C57/Bl6 twist-null mice from the Jackson Laboratory were intercrossed and we observed that 59% of their heterozygous progeny present an additional toe close to the hallux (`duplicated hallux'), on either one or both hindfeet (Fig. 4A for 1-week-old mice hindlegs).


Figure 4. Hindleg heterozygous mutant morphology studies. (A) Mutant hindlegs of two newborn mice. Notice that the extra digit (I*) is seen near the hallux. It appears longer than the hallux and lies outside it. The plantar surface of the hindlimb is shown and one additional digit pad beneath the extra digit is visible. (B) 14.5 d.p.c. embryo footplates of two littermates, cleared at 37°C in alkali and glycerol after alcian blue staining. Additional chondrification centres forming the additional digit (I*) are seen at the outer side of the hallux in the heterozygous mutant embryo hindlimb. (C) Skeletal preparation of newborn hindlegs after alcian blue and alizarin red staining. Compare the wild-type hindfoot with one metatarsal (MT) and one cuneiform 1 bone (C1) bearing the hallux, with that of the heterozygous mutant littermate. The latter presents a whole additional digit (I*) formed with three phalanges at the outer part of the foot, near the hallux, and duplicated or broader metatarsal (MT*) and cuneiform 1 bones (C1*). (D) In adults skeletal hindleg preparations, after alizarin red staining, the cuneiform 1 bone (C1) and the metatarsal (MT) on the heterozygous mutant hindleg is of the same size as its counterpart in the wild-type animal. The additional digit is labeled I*. The bars indicate 1 µm.


Figure 5. Neurocranium and chondrocranium abnormalities in twist-null heterozygotes. (A) The cartilage of 14.5 d.p.c. embryos has been stained with alcian blue. The lateral view shows that the head cartilage of heterozygous animals is more developed than that of wild-type littermates (arrows). (B) In 1 week heterozygous mice skeletal preparations, after staining with alcian blue and alizarin red, the definitive suture of the parietal bones is almost formed, while in littermate wild-type embryos the bones are not joined. The arrows mark the distance between the parietal bones. (C) The interparietal bones in the heterozygous animals appear more developed than in the wild-type animals at 17.5 d.p.c. Note the growth delay for the supraoccipital bone in the heterozygous mutant animals. (D) The lower ridge of the interparietal bones in heterozygous mutant newborn mice presents an `arrow shape' and is broader than that of wild-type littermates. The bars indicate 1 mm.

Our observations were made at different developmental stages and show that a supernumerary chondrification center is already detectable in the footplates of 13.5 d.p.c. heterozygous mutant embryos and more obviously at 14.5 d.p.c. (Fig. 4B). Skeletal preparations of newborn and adult mice showed that the supernumerary toe has three phalanges and originates on a metatarsal linked to cuneiform bone 1, as does the hallux, which is composed of two phalanges (Fig. 4C). Heterozygous embryos often present duplicated or broadened metatarsal and cuneiform 1 bones, while these bones are similar in size and width in wild-type and heterozygous adults (Fig. 4D). We observed at least one additional digital pad on the palmar side of the foot that presents the additional toe. More generally, the extremes of this phenotype range from a simple broadening of the hallux to the formation of a complete supernumerary toe with three phalanges, attached to the metatarsal 1 bone. Two isolated cases of duplication of the last phalange of the supernumerary toe were also recovered.

Table 1. Duplicated hallux phenotype inheritance in C57/B16 strains
  No. litters No. animals +/+ twist-null/+ Dead
        (R) (L) (L+R) (Ø)  
A twist-null1/+ × twist-null1/+ 7 55 22 7 10 (59%) 2 13 (41%) 1
B twist-null1/+ × C57/Bl6 11 54 33 3 3 (57%) 2 6 (43%) 7
C twist-null2(L/R)/+ × twist-null2(L/R)/+ 5 30 6 4 3 (75%) 5 4 (25%) 8
D twist-null2(Ø)/+ × twist-null2(Ø)/+ 4 22 10 1 1 (50%) 0 4 (50%) 6
E twist-null2(L/R)/+ × C57/Bl6 27 199 115 23 18 (77%) 24 19 (23%) 1
F twist-null2(Ø)/+ × C57/Bl6 28 193 108 18 10 (75%) 25 18 (25%) 13
G Total   553 294 56 45 58 64 36
        159 (71%)    
      (53%)   223 (40.5%)   (6.5%)
(R), additional toe right hindfoot; (L), additional toe left hindfoot; (Ø), no additional toe; (L+R), additional toe left and right hindfeet; (L/R), additional toe either hindfoot.
twist-null1/+, first generation twist-null/+ animals.
twist-null2/+, second generation twist-null/+ animals.

Table 2. Duplicated hallux phenotype inheritance in CD1 mixed background
  No. litters No. animals +/+ twist-null/+
        (L/R) (NOSE) (Ø)
A twist-null1(L+R)/+ × CD1 5 68 33 6 0 29
B twist-null1(Ø)/+ × CD1 4 31 17 1 0 15
C twist-null2(L/R)/+ × CD1 1 12 5 0 1 6
D twist-null2(Ø)/+ × CD1 5 52 35 0 0 16
E twist-null3/+ × CD1 6 66 43 0 2 21
F twist-null4/+ × CD1 7 78 44 2* 4* 30
G Total   307 177 9 (7%) 7 (5%) 117
        16 (12%)   (88%)
      58% 5% 37%
(L/R), additional toe either hindfoot; (Ø), no additional toe; (NOSE), facial asymmetry.
twist-null1/+, first generation twist-null/+ animals.
twist-null2/+, second generation twist-null/+ animals.
twist-null3/+, third generation twist-null/+ animals.
twist-null4/+, fourth generation twist-null/+ animals.
2* and 4*, two animals with both additional toe and facial asymmetry.

Inheritance of the duplicated hallux phenotype

Inheritance of the duplicated hallux has been followed for several generations and the results obtained are presented in Table 1 for analyses in the C57/Bl6 strain and in Table 2 for those in a mixed CD1 genetic background.

In the intercrosses of the two heterozygous C57/Bl6 twist-null mice from the Jackson Laboratory (twist-null1/+, first generation) 59% (19/32) of the heterozygous progeny presented a duplicated hallux phenotype (Table 1A). There are sporadic additional fingers or toes only in ~1-1.5% of our wild-type animals (not shown). In addition, there is some lethality prior to birth among heterozygous animals, as we recovered 40% (22/55) wild-type animals at birth instead of the expected 33%, and also 1.8% lethality (1/55) within the first 2 weeks after birth.

We followed the progeny of the two heterozygous C57/Bl6 twist-null1males, both of which generated the phenotype. One heterozygous twist-null1 male was backcrossed to C57/Bl6 wild-type partners and 57% (8/14) of the heterozygous animals in his progeny presented the duplicated hallux phenotype (Table 1B). In these crosses we also observed some lethality among heterozygotes prior to birth, as only 25% heterozygotes were recovered at birth instead of the expected 50%, and also 13% (7/54) lethality within the first 2 weeks after birth, probably among heterozygous animals.

Intercrosses of two heterozygous animals of the second generation (twist-null2/+) were then performed. Those animals were obtained from crosses of the C57/Bl6 twist-null1/+male with C57/Bl6 wild-type partners. The intercrosses of two animals with the duplicated hallux phenotype [twist-null2(L/R)/+] generate 75% (12/16) heterozygotes displaying the phenotype (Table 1C), while those of two animals with no phenotype [twist-null2(Ø)/+] generate 50% (2/4) heterozygotes with an additional toe(s) (Table 1D). The twist-null2 heterozygous animals crossed with C57/Bl6 wild-type partners gave the following results: twist-null2 animals with phenotype [twist-null2(L/R)/+] generated 77% (65/84) heterozygoytes with additional toe(s) (Table 1E); twist-null2animals with no phenotype [twist-null2(Ø)/+] generated 75% (55/71) heterozygotes with phenotype. Thus there are no significant differences in phenotype transmission whether parents are with or without phenotype. Again, there is also a noticeable lethality among the heterozygous animals.

Overall, out of 553 animals analyzed 71% (159/223) of the heterozygotes present the duplicated hallux phenotype. Thus this phenotype is linked to the twist-null mutation, but is not fully penetrant and can appear in the progeny of parents without the phenotype. We did not observe any significant differences in the sex ratio nor any obvious effect on general animal behavior.

The twist-null mutation has been outcrossed with animals of the wild-type CD1 strain and the duplicated hallux phenotype was followed for four generations (Table 2). The limb abnormalities rapidly disappeared, as in the first generation in a mixed CD1 background there were only 14% heterozygotes with the phenotype and 2.5% in the fourth generation, while other phenotypes arose (see below).

Neurocranium and chondrocranium malformations intwist-null heterozygotes

The progeny of heterozygous twist-null animals crossed with C57/Bl6 wild-types were collected, analyzed and skeletal preparations were made from animals aged 13.5 d.p.c. to 7 days after birth. After staining with alizarin red S and alcian blue, which stain mineralized bone and cartilage respectively (27), the evolution of cartilage formation and ossification was observed. The sizes and degrees of ossification of different structures in heterozygous animals were compared with those of wild-type littermates (Table 3).

At 13.5 d.p.c. the head cartilage of heterozygous embryos was more developed than in wild-type embryos (Fig. 5A). In 7 day mouse skulls the space between cranial plates was broader in the wild-type than in heterozygotes (Fig. 5B). In addition, the parietal and frontal bones appeared to have grown more rapidly in heterozygous animals. The heterozygous interparietal bone was 26% larger in 17.5 d.p.c. heterozygous embryos (Fig. 5C), but only 19% larger at 7 days post-partum than the corresponding wild-type (Fig. 5D); note also in Figure 5C its thickness and shape in the heterozygous animals.

The supraoccipital bone, which belongs to the chondrocranium, was first seen in alizarin stained material at ~16.5 d.p.c. as two ossification centers, one located on either side of the dorsal midline. These two areas of ossification amalgamated at ~17.5 d.p.c. to form the definitive supraoccipital bone. We observed that at 17.5 d.p.c. it was still not formed in the heterozygous mutant, while it was already ossified and formed in the wild-type (Fig. 5C). However, this size difference was almost undetectable at 7 days post-partum, a stage at which the supraoccipital bone was only 5% smaller in the heterozygous mutant (not shown).

Viscerocranium malformations intwist-null heterozygous animals

Some defects appeared at very low penetrance in the viscerocranium of the heterozygous C57/Bl6 twist-null mutant animals, but the number of animals presenting these defects increased in a CD1 background. In the fourth generation in a CD1 background 5% of the heterozygotes presented the phenotype of an `elephant tusk' appearance, with incisors growing over the nose or below the lower jaw (see Table 2 and Fig. 6A for an adult specimen), due to the fact that the lower and upper incisors were not in register. Skeletal preparations of such animals showed that this obvious asymmetry of the face was due to deviation of both the nasal septum and of the palate bones; the nasal bones were slightly shorter or more curved, resulting in a shortening of the anterior facial regions of the heterozygotes; the entire mutant skull was shorter than that of the littermate wild-type mouse (Fig. 6B); however, the three molars were present on each side of the upper jaw and the lower jaws were symmetrical, without any visible phenotype (not shown).


Figure 6. Other skull abnormalities in twist-null heterozygotes. (A) A wild-type animal (upper picture) is shown for comparison with one littermate twist-null heterozygote (lower picture), where the upper and lower incisors are not facing each other. (B) Skeletal preparations of two littermate adults viewed from the top of the skull. The upper picture presents the wild-type, the lower picture presents the heterozygous skull. Observe the size difference in the two animals and the deviation of the whole nasal septum (curved arrows), with asymmetry of the orbits. (C) Ventral views of the palate bones from newborn mice after staining with alcian blue and alizarin red. There is a growth delay in the ossification process of the bones in the heterozygous mutant mice when compared with wild-type littermates (stars, arrows and double arrows point to the respective bones to be compared). (D) At 18.5 d.p.c. the retrotympanic otic process of the squamosal bone in heterozygous mutant animals is growth delayed when compared with that of wild-type littermates. (E) Ossification of the whole tympanic ring (TR) and its extremities is delayed in the heterozygous mutant animal when compared with wild-type littermates. Observe also at that stage the absence of the manubrium of the malleus (MM), of the malleus (M) and of the processus braevisof the malleus (PB) in mutant heterozygous animals. The stars (*) point to ossification differences at the extremities of the tympanic ring. The bars represent 1 mm.

Other bones which belong to the viscerocranium were obviously growth delayed in C57/Bl6 mice bearing the twist-null mutation. Ossification of the palate bones in newborn heterozygous mice was slightly delayed when compared with that of wild-type littermates (Fig. 6C). In addition, the retrotympanic otic process of the squamosal bone in 18.5 d.p.c. heterozygotes was 30% shorter than in the wild-type (Fig. 6D) and small ossification centers were observed at each end of the tympanic ring in the wild-type but not in the mutant (Fig. 6E). However, these size differences tended to disappear as development of the embryos proceeded: in 7 days post-partum mutants the retrotympanic otic process of the squamosal bone was only 17% shorter than that of wild-type littermates (Fig. 6D).

All the heterozygotes with one or two supernumerary digits presented these skull abnormalities.

Table 3. Size variations of some bones in twist-null(L/R)/+ animals
Stage No. animals Interparietal Parietal Supraoccipital Squamosal
    Width Width Width Length Width
17.5 d.p.c. 14 +26% +6% ND -29% -6%
18.5 d.p.c. 9 +19% +6% -24% -30% -5%
Birth 15 +20% +6% -31% -27% -5%
5 days 9 +11% +8% -7% -24% -
7 days 14 +19% +6% -5% -17% -7%
ND, not determined; - , not measured.

Table 4. Additional bone abnormalities in twist-null/+ animals
  No. animals Sternum or xyphoïd defects Interfrontal bone present
C57/Bl6 wild-type 25 0 10 (40%)
twist-null/+ heterozygotes 36 11 (31%) 33 (92%)

Additionaltwist-null heterozygous malformations

We observed that ~8.5% of the C57/Bl6 heterozygotes were significantly smaller than wild-type littermates. The presence of an interfrontal bone was observed in 92% of the heterozygotes, while usually it was only observed in 50% of the wild-type mice (28). In addition, slight defects in the plexus and xyphoïd process region, such as asymmetry and little holes in the ossified region, were visible in 31% of the heterozygotes and are always associated with cranial abnormalities and polydactyly (Table 4). Cases of slight asymmetric vertebrae were also found and some cases of mice with an asymmetric larynx were observed (not shown), with a weak penetrance.

M-twistDbHLH heterozygous mice analyses

The initial M-twistDbHLH allele in a C57/Bl6 × SJL hybrid background was outcrossed to CD1 and C57/Bl6 genetic backgrounds. In the fifth and sixth generations 73 mice in increasing CD1 and 15 mice in mixed C57/Bl6 backgrounds respectively were analyzed. No animals, either wild-type or heterozygous, presented any obvious phenotype in the hindlimbs or skull. However, rare cases of partial syndactyly linking the second and the third toes, either on one or both hindlimbs, have been recovered in mixed CD1 strains.

Measurements of different skull bones were done on 37 newborn animals in a CD1 background (20 wild-type and 17 heterozygotes) and only seven newborn animals (four wild-type, three heterozygotes) in a C57/Bl6 background. These preliminary results showed that the mean values of the measures on the heterozygotes were ~4-9% higher than their counterparts in wild-type animals (depending on the strain), but that these differences were always within the standard error of the values (not shown); 19% of heterozygotes had a large interfrontal bone, while it was rather small and only observed in ~11% of the wild-types (not shown).

However, rib cage abnormalities were recorded in a total of 36 heterozygous M-twistDbHLH newborn mice, which were compared with 37 wild-type littermates with a CD1 mixed background: typically 70% of the wild-types had 13 pairs of ribs (either seven pairs attached to the sternum and six non-attached or eight attached and five non-attached); this is only observed in 44% of the heterozygotes. In contrast, 42% heterozygous animals, but only 24% of the wild-types, had 14 pairs of ribs (either eight attached and six non-attached or seven attached and seven non-attached). In addition, unilateral on either side (Fig. 7B and B[prime]) or bilateral additional ribs (Fig. 7C and C[prime]) were detected in 22% of heterozygous animals but only in 10% of wild-types.


Figure 7. Thoracic skeletal abnormalities of M-twistDbHLH heterozygous animals. The entire rib cages from wild-type and M-twistDbHLH heterozygous newborn animals are shown from the right side (A, B and C) and only the ribs attached to the sternum as flat mounts from the ventral side (A[prime], B[prime] and C[prime]). (A and A[prime]) Wild-type mice have seven pairs of attached and six pairs of non-attached ribs. (B and B[prime]) M-twistDbHLH/+ rib cage with one bilateral attached additional rib pair (* at the left and at the right side of the rib cage). (C and C[prime]) M-twistDbHLH/+ rib cage with one unilateral attached additional rib (* at the left side of the rib cage). The bars represent 1 mm.

Thus, even in a small number of animals, skeletal morphological abnormalities were observed that were linked to the heterozygous M-twistDbHLH genetic state.

DISCUSSION

Defects are observed in tissues derived from cells expressing M-twist

Among cells that differentiate from the mesodermal sheet the sclerotome derivatives are the easiest to detect and to measure. We report a skeleton phenotype which is not fully penetrant, although significantly linked to the heterozygous state at the M-twist locus, mainly in C57/BL6 strains. In all the crosses performed we observed that a number of heterozygous animals in the progeny appeared with rounder heads than wild-types and with smaller bodies. The smaller size of some heterozygotes might mainly originate from nutritional problems, linked to their facial asymmetry. We focused on studies of the inheritance of the limb and linked cranial dismorphologies. No obvious defects in dermatome or myotome derivatives were observed, although these systems were not studied in detail.

The phenotypic abnormalities observed in structures that belong to the chondrocranium, the neurocranium, the viscerocranium and in the hindfoot are consistent with the fact that M-twist is normally expressed at a high rate in the primordia of these structures. Mesenchymal components join the mesodermal layer to form bones, cartilage and connective tissue in the head. Craniofacial development and cell fates are widely documented (23,29-31). Developmental and molecular studies of limb formation have also been reported (refs 32-34 and references therein). Other structures expressing M-twist at rather high levels are not obviously modified in the mutants: no gross defects or delays in tooth formation nor any eyelid problems nor any forelimb abnormalities were observed in mice. However, a specific study of these structures would be required, possibly in different genetic backgrounds, to confirm this: we observed some unusual molar morphology, but no real studies have been done on these structures. As tooth formation is a well-studied model, it was of interest to notice that during odontogenesis M-twist transcripts were located within mesenchymal cells that originate from the neural cephalic crests and that they are restricted to specific regions of the pulp prior to differentiation of the odontoblasts: this specific localization illustrates regionalization of the peridental mesenchyme from which odontoblasts originate.

Hindlimbtwist-null heterozygous mouse phenotype and limb polarity

A number of genes (i.e. FGF, FGFR and Hox genes) have been shown to be implicated in limb formation and patterning (for reviews see refs 34,35).

The duplicated hallux of heterozygous twist-null mice (resulting in the formula 1*12345, where 1* is attached to the metatarsal 1 bone, as the hallux) might be interpreted as a partial reversed polarity in the hindlimb bud. Diverse cases of progressive addition of digits in the limb buds have been reported, either as a consequence of ZPA or retinoic acid (RA) bead grafting experiments (36). In the chick low dose RA leads to an additional digit two and/or three, while a high dose leads to patterns with additional digits (37,38). The phenotype that we observe in twist-null heterozygous mice might resemble that of the low dose RA-induced phenotype.

The higher value of the M-twist transcript gradient in the limb bud mesenchyme at 10.5 d.p.c. is located in the anterior region, which will give rise to the thumb (or the hallux) (not shown). The limb field mesoderm is reported to determine the initial anteroposterior asymmetry (38), but dorsal signals regulate anteroposterior patterning (39). One possible hypothesis would be that in twist-null heterozygous mice the gradient of M-twist transcripts is not as sharp as in wild-type animals and, as a consequence, the regional anteroposterior pattern in the limb bud is slightly modified through mesodermal downstream gene expression, which results in a partial duplication of digits.

We did not observe any significant forelimb abnormalities, although the forelimb bud expresses M-twist in similar fashion to the hindlimb bud. Further progression in the CD1 background might let such a defect appear. In addition, at least two genes are expressed in forelimb buds and not in hindlimb bud mesoderm at a developmental stage equivalent to that of the forelimb, XlHbox (40,41) and Ghox2 (42). One hypothesis could be that their expression might induce a molecular difference between arms and legs, which could explain why the extra toe phenotype of the twist-null heterozygotes does not appear in forelimbs.

Craniofacial abnormalities and skull bone formation

Bones of the skull derive from cells of different origins: the frontal and parietal bones derive from occipital somites and somitomeres (43), while branchial arch cells, which originate from cephalic neural crest cells, contribute to craniofacial bone, cartilage and teeth. Their ossification occurs either through a membranous model (i.e. for the interparietal bone) or via a chondrification step (i.e. for the lower part of the parietal bone and the upper medial part of the nasal bones and the malleus and incus). The vertebrae originate from the sclerotome.

The craniofacial abnormalities observed in the twist-null heterozygous mice might be classified as: those that arise by precocious chondrification/ossification of bones (the interparietal and parietal bones); those that arise by delayed growth of some bones (tympanic ring and other otic bones, supraoccipital bone, nasal septum and palate bones).

In summary, in twist-null heterozygotes the neurocranium develops more rapidly, the chondrocranium is delayed and both situations are seen for different bones of the viscerocranium. These variations might reflect the dual origin, membranous and cartilaginous, of different bones forming the skull. At a molecular level it might be possible that different amounts of TWIST protein would be required to allow development of the different types of bones. This is the first report of such an opposing effect of a single gene product on bone formation.

Variability in penetrance and expressivity

Variability in the penetrance and expressivity of a mutation has been classically attributed to variability in genetic background. In twist-null mice the duplicated hallux/cranial phenotype was progressively lost, while facial asymmetry was more frequently observed as we progressed from a C57/Bl6 towards a CD1 genetic background. Furthermore, within animals of similar genetic backgrounds there was variability in the morphological defects. Thus we suggest that this variability in the penetrance and expressivity of the phenotype of heterozygous mice is really due to the genetic background. One explanation could be that M-twist is expressed at the same levels in different strains.

Another possibility is that a mutant allele (or variants) of an unknown gene might segregate with the original twist-null mutation. In traditional genetics on yeast or Drosophila it is common to `clean' the chromosome bearing the studied mutation by selection of recombinants with wild-type animals to get rid of such possible co-segregation. This is almost what is reported here. This other gene might present allelic variants within the different wild-type mice strains and be either located distal to the 12 B-C chromosomal location of M-twist (44) or on another chromosome. The heterozygous state at the M-twist locus and different levels of expression of variants of this other gene would have as a consequence the appearance of skeletal abnormalitiesviagenetic interactions. Such an event has been described in the etiology of a spina bifida-like phenotype: unexpectedly, undulated-Patch double mutant mice (un/un and Ph/+) exhibit a phenotype similar to an extreme form of spina bifida in humans, a phenotype which is not found in either homozygous undulated or heterozygous Patch mice (45).

In the case of the Hox4-2 gene the variability between individuals is suggested to arise from genetic background (46). Also, for the heterozygous Cbp mutant mouse phenotype (47), which results in craniofacial and limb abnormalities that resemble those of human patients affected by Rubinstein-Taybi syndrome (48,49), the penetrance of some abnormalities was shown to depend on the genetic background of the animals.

In the case of goosecoid 100% of the homozygous gsc-nullmutants (which have a deletion of the entire GSC protein encoding region), present craniofacial defects, while the rib cage defect phenotype is not fully penetrant and is different in the 129SvEv and C57/Bl6 genetic backgrounds (50). However, in gsc-/- homozygous mice (which have a disrupted homeobox encoding sequence) no rib cage phenotype has been reported in 129 strains (51).

The fact that different structures might be affected in the heterozygotes probably reflects the fact that M-twist is active in a wide variety of cells during early embryogenesis.

Relevance to human patients with craniosynostosis malformations

Craniosynostosis is a premature fusion of calvarial sutures that causes abnormal skull shape. Many genetic craniosynostoses are highly penetrant; their variable expression has suggested that several may represent allelic, rather than genetically distinct, loci. They may involve deficiency of the mesenchymal blastema along the approaching osteogenic fronts of the skull bones, leading to premature fusion, accelerated bone maturation or secondary changes in the sutures due to forces mediated by abnormal development of the base of the skull (reviewed in refs 24,52).

The twist-null heterozygous cranial phenotype depends on genetic background: these observations resemble those reported for human SCS, which has a variable expressivity (24). The coding sequence of the human TWIST gene (20-22), but also sequences in its vicinity (22,23), have been reported to be linked to SCS. However, at present there is no real correlation between the extent of the observed malformations of the patients and the type of identified molecular defect they bear. In human beings no genetic background studies can be performed, but the large number of different morphological abnormalities reported to belong to SCS and which appear in unlinked patients might be a consequence of the different pedigrees of the patients.

Mutant mice strains as models to study the etiology of human syndromes

In the case of the myotonic dystrophy kinase gene the Dmpk mutant mice phenotypes are not identical to the human genetic disease (53,54). Also, Fgfr1- and Fgfr3-deficient heterozygous mice exhibit a phenotype which is opposite to that seen in achondroplastic patients, who seem to bear gain-of-function mutations (52,55,56). In our case, in contrast to the suggested types of alterations that would generate a faithful disease model (57), it is the heterozygous loss-of-function state that mimics the human autosomal dominant phenotype.

Mice mutant for the M-twist gene may serve as animal models to understand the genesis of some morphological human abnormalities, as has been reported for several other mutant mice (58). However, work has still to be performed to elucidate in mice some characteristics found in humans. For instance, would the defects that were observed in formation of the auditory apparatus of twist-null heterozygous mice cause any hearing deficit? In human SCS patients mild hearing deficits are common (24). However, in human patients eyelid ptosis is frequent, but this defect is difficult to observe in mice.

Different genes have been identified as being mutated in patients presenting craniosynostosis and limb phenotypes (MSX2 and FGFRs) and some of their murine counterparts (Msx1, Msx2 and FGFR-1) have been studied (references in ref. 52). A recent report has shown that some patients diagnosed with SCS were not mutated at the TWIST locus, but have a Pro250->Arg mutation in FGFR-3 (22). The similarities in all these human syndromes and developmental biology studies in Drosophila and Mus (52) might suggest that some of these gene products would interact or at least participate in the same developmental process. Molecular probes and mutant mice will possibly help to clarify this situation.

MATERIALS AND METHODS

In situhybridization

Head tissue sections and hybridizations were as described (2) using the XbaI-EcoRI 420 bp M-twist exon 2 fragment as probe.

Mice analyses

The two different twist-null heterozygous males derived at the Jackson Laboratory and the M-twistDbHLH heterozygotes from our laboratory were bred to establish timed matings with C57/Black6 and CD1 partners respectively. Genotyping was carried out either on extra-embryonic tissues, for early embryos, on viscera (heart + lung), for later ones, or on tail biopsies, after birth. Samples were digested overnight with 0.5 mg/ml proteinase K. When extracted from tails or soft tissues the DNA was ethanol precipitated prior to PCR analysis. For extra-embryonic tissues PCR was performed directly on samples that were heated to 95°C for 5 min after proteinase K treatment. PCR reactions were carried out with 100 ng each primer in a thermal cycler (Perkin Elmer Cetus). The first pairs of primers used were either 5[prime]-ACCTTTGGAAGCACAGTCCTCTGAGAGCAG-3[prime] (forward primer, M-twist sequence) with 5[prime]-CAGGACATAGCGTTGGCTACCCGTG-3[prime] (backward primer, Neomycin sequence) or 5[prime]-TGCCCAGTCATAGCCGAATAGCCTC-3[prime] (forward primer, Neomycin sequence) with 5[prime]-GGTGGAATTTGGAAACAGTTGTTTTAAGAA-3[prime] (backward primer, M-twist sequence). Each pair of primers generates a fragment of ~900 bp on the twist-null allele.

The third pair of primers used was 5[prime]-CAGGACATAGCGTTGGCTACCCGTG-3[prime] (forward primer, Neomycin sequence) with 5[prime]-GGCTGTTTTCTATGACCGCT-3[prime] (backward primer, M-twist sequence), which amplifies a 1100 bp fragment on the M-twistDbHLH allele.

All other molecular biology techniques were according to Sambrook et al. (59).

Skeletal preparations

Neonates up to 1 week old were killed, skinned, eviscerated and skeletal preparations were performed according to Inouye (27). Embryonic cartilage preparations of 13.5-16.5 d.p.c. embryos and adult leg preparations were carried out according to Jegalian and De Robertis (60). Specific staining of the cartilage was obtained by immersion in 0.05% alcian blue ethanol/acetic acid 80/20 solution for 24 h and that of mineralized bone by immersion in an aqueous 1% KOH solution containing 37 mg/500 ml alizarin red.

ACKNOWLEDGEMENTS

We are grateful to Prof. P. Chambon for working facilities. We thank Prof. J.V. Ruch for discussions and Dr P. Simpson for reading the manuscript. We also thank P. Sourine, F. Mackay and M. Le Meur for great care of the mice, the oligonucleotide team for all their syntheses, B. Boulay and J.M. Lafontaine for help with illustrations and the secretariat staff for general support. A.-L.B.-B. and P.B. were recipients of thesis grants from le Ministère de l'Enseignement Supérieur et de la Recherche. This work was supported by a grant from l'Association de la Recherche sur le Cancer and by l'Hôpital Universitaire de Strasbourg, the Centre National de la Recherche Scientifique and the Institut National de la Santé et de la Recherche Médicale.

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*To whom correspondence should be addressed. Tel: +33 3 88 24 34 45; Fax: +33 3 88 24 34 78; Email: fabienne@igbmc.u-strasbg.fr
+The first two authors contributed equally to this work
§Present address: Department of Operative Dentistry and Endodontics, Niigata University School of Dentistry, 5274 Gakkocho-dori 2-brancho, Niigata 951, Japan

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Y.-F. Dong, D. Y. Soung, Y. Chang, M. Enomoto-Iwamoto, M. Paris, R. J. O'Keefe, E. M. Schwarz, and H. Drissi
Transforming Growth Factor-{beta} and Wnt Signals Regulate Chondrocyte Differentiation through Twist1 in a Stage-Specific Manner
Mol. Endocrinol., November 1, 2007; 21(11): 2805 - 2820.
[Abstract] [Full Text] [PDF]

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 J. Dent. Res.Home page
K.M. Galler, A. Yasue, A.C. Cavender, P. Bialek, G. Karsenty, and R.N. D'Souza
A Novel Role for Twist-1 in Pulp Homeostasis
J. Dent. Res., October 1, 2007; 86(10): 951 - 955.
[Abstract] [Full Text] [PDF]

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 J. Cell Sci.Home page
M. Hayashi, K. Nimura, K. Kashiwagi, T. Harada, K. Takaoka, H. Kato, K. Tamai, and Y. Kaneda
Comparative roles of Twist-1 and Id1 in transcriptional regulation by BMP signaling
J. Cell Sci., April 15, 2007; 120(8): 1350 - 1357.
[Abstract] [Full Text] [PDF]

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 Cancer Res.Home page
T.-K. Man, M. Chintagumpala, J. Visvanathan, J. Shen, L. Perlaky, J. Hicks, M. Johnson, N. Davino, J. Murray, L. Helman, et al.
Expression Profiles of Osteosarcoma That Can Predict Response to Chemotherapy
Cancer Res., September 15, 2005; 65(18): 8142 - 8150.
[Abstract] [Full Text] [PDF]

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 J. Dent. Res.Home page
P. Pungchanchaikul, M. Gelbier, P. Ferretti, and A. Bloch-Zupan
Gene Expression during Palate Fusion in vivo and in vitro
J. Dent. Res., June 1, 2005; 84(6): 526 - 531.
[Abstract] [Full Text] [PDF]

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 GeneticsHome page
K. F. Oram and T. Gridley
Mutations in Snail Family Genes Enhance Craniosynostosis of Twist1 Haplo-insufficient Mice: Implications for Saethre-Chotzen Syndrome
Genetics, June 1, 2005; 170(2): 971 - 974.
[Abstract] [Full Text] [PDF]