Hox-7 expression during murine craniofacial development

Analysis of developmental mutations in the fruit fly Drosophila melanogaster has demonstrated the importance of a family of genes in controlling pattern formation and segment identity during insect development (Nüsslein-Volhard and Wieschaus, 1980). Many of these genes have been isolated and found to contain a nucleotide sequence of 180 bp called the homeobox, which codes for a helix-turn-helix protein structure very similar to that previously found in proteins determining mating types in yeast. These have been shown to bind DNA and act as transcription factors (Scott et al. 1989). Mutational analysis of Drosophila embryos has shown that homeobox-containing genes play a key role in determining the anterior-posterior axis, segment formation and polarity, and finally the identities of the segments themselves (reveiwed by Akam, 1987 and Ingham, 1988).

Since their discovery in Drosophila (McGinnis et al. 1984; Regulski et al. 1985), homeobox genes have also been found in vertebrates including Xenopus (Carrasco et al. 1984), mice (Holland and Hogan, 1988) and man (Bohcinelli et al. 1985). Extensive analysis in the mouse has yielded the sequences and chromosome locations of many murine homeobox containing genes (Hox). The majority of Hox genes in the mouse are located in 4 clusters on 4 different chromosomes (Graham et al. 1989; Duboule and Dolle, 1989).

A number of other homeobox containing genes have also been isolated both in Drosophila and in vertebrates, which have more diverged sequences than the homeobox sequences of the class 1 homeobox genes (Akam, 1989). These homeobox genes do not appear to be involved in segmentation but seem to play a more important role in organogenesis and cell differentiation. One such gene is the muscle segment homeobox (msh) gene in Drosophila (Gehring, 1987). This gene is expressed in mesoderm, the forming CNS and muscle anlage of the Drosophila embryo but as yet no mutation has been ascribed to it. Since the discovery of this gene several other msh-type genes have been found and described in ascidians, zebrafish, mice (Holland, 1991) and in the quail (Takahashi and Douarin, 1990). One of these msh-like genes isolated in the mouse (Hox-7) does not form part of the four Class I Hox clusters but was located on mouse chromosome 5 (Hill et al. 1989; Robert et al. 1989). Expression of this gene has previously been reported in the facial processes of E10 to E13 mouse embryos and its detailed expression patterns during mouse tooth development has been described (MacKenzie et al. 1991).

In this study, we describe in detail the spatial and temporal expression of Hox-7 during craniofacial formation in the mouse from embryonic day 9.5 (E9.5) to E15.5 by in situ hybridisation. The discrete spatial and temporal expression patterns of Hox-7 in the developing head and face suggest a possible involvement in cellular differentiation leading to the formation of a number of craniofacial structures, including the anterior pituitary, choroid plexus, external ear, brain meninges and skull bones, nasal pits and forming Jacobson’s organs.

Manchester strain mice (MFI) were mated overnight and the day of finding the vaginal plug designated day zero. At E9.5 to E15.5, mothers were killed by ether overdose and embryos were removed aseptically. Following decapitation, embryo heads were fixed overnight in 4% paraformaldehyde/PBS, dehydrated through ascending gradients of alcohol, cleared in chloroform and embedded in fibrowax (Raymond A. Lamb, London). Histological sections were cut at 10 micron thickness in the coronal and frontal planes and mounted on aminoalkylsilane-coated slides under RNAase-free conditions.

Hox-7 cDNAs were isolated from a mouse E8.5 embryonic cDNA library following a screen at reduced stringency with the Drosophila bicoid homeobox (MacKenzie et al. 1991). Sequencing confirmed that two clones isolated were derived from Hox-7 transcripts by comparing the sequences to those already published (Robert et al. 1989; Hill et al. 1989). For in situ hybridisation, a 600 base pair (bp) EcoR1 fragment from one cDNA clone, containing 375 bp of protein coding sequence, was subcloned into the transcription vector pSP72 (MacKenzie et al. 1991). Sense and antisense ³⁵S-labelled riboprobes were generated by standard SP6 and T7 polymerase reactions. The in situ hybridisation protocol was as previously described (Sharpe et al. 1988).

Slides were examined and photographed under light-field and dark-field optics.

In previous studies, transcripts of Hox-7 have been detected in the facial processes of several ages of mouse embryos (Hill et al. 1989; Robert et al. 1989), the developing eye (Monaghan et al. 1991) and a more detailed analysis showed expression in a number of craniofacial structures in particular the mesenchymal condensations around the developing teeth (MacKenzie et al. 1991). This study represents a survey of the expression patterns of Hox-7 in the developing brain and surrounding cranial region and considers their possible functional significance.

At E9.5, Hox-7 is expressed in the mesenchyme of the branchial arches, components of the forming heart e.g. endocardiac cushions, and in the mesenchyme and epithelium of the progress zones of the developing forelimb and hindlimb buds as previously described (Hill et al. 1989; Robert et al. 1989). A band of intense expression is also seen in a narrow streak running from the most rostral areas of the forebrain to the tail neuropore along the line of neural tube closure (Fig. 1). No Hox-7 expression is evident in the forming Rathke’s pouch at this time.

By Ell.5 this expression pattern is still localised to the dorsal midline of the neural tube. A frontal section shows that an extensive area of Hox-7 expression can also be detected in the mesenchyme cells separating the head epidermis from the neuroectoderm (Figs 2, 3). This pattern of expression persists at least until E15.5 and delineates the future meninges, the fields of later ossification that form the skull bones. At E11.5, Hox-7 expression is intense in the anlage of the posterior choroid plexus of the 4th ventricle of the hindbrain, caused by the execution of the pontine flexure where it is continuous with the persistant band of Hox-7 expression in the dorsal midline of the neuroepithelium (Fig. 2), and in the thinning neuroepithelium destined to become the lateral choroid plexus of the lateral ventricles of the developing cerebral cortex prior to the development of the choroid plexus phenotype (Fig. 3). Strong Hox-7 expression is also detectable in the epithelium of Rathke’s pouch (Fig. 2).

By E12.5 and E13.5 Hox-7 expression is obvious not only in the thinning and convoluting areas of neuroepithelium differentiating into the choroid plexus (Fig. 4) but is also present in the immediately adjacent mesenchyme cells (Fig. 5). Hox-7 expression is also evident along the ventral aspect of the forming forebrain in the region of the tuberculum posterius which constitutes a thickening of the floor of the forming brain and represents the posterior margin of the diencephalon and the area of the brain adjacent to the rostral end of the notochord (Fig. 6). The small area of Hox-7 expression shown in the dorsal neuroepithelium of the mid brain (Fig. 6) is due to the slightly oblique plane of section through the area corresponding to the dorsal midline of the neural tube. Hox-7 is expressed in the epithelium of the nasal pits and Rathke’s pouch (Fig. 6). At E12.5 several regions of Hox-7 expression can be discerned within Rathke’s pouch (Fig. 8). Low Hox-7 expression is detected in the portion of Rathke’s pouch closest to the infundibular evagination. Expression is higher in the areas of Rathke’s pouch most distal from the myencephalon (Fig. 8B) with the highest levels of Hox-7 expression detected in the cellular outgrowth of proliferating cells that forms the pars distalis; the area of Rathke’s pouch destined to form the anterior lobe proper (Fig. 8C). At E13.5 Hox-7 expression is detectable in the auricular hillocks; areas of mesenchymal aggregation around the forming auditory meutus, these eventually become the auricle or pinna (Fig. 9). These cells, and their associated epithelium form the cartilage of the pinna, which becomes externally visible at E13.5. From E13.5 to E15.5, Hox-7expression consistently localised in the choroid plexii. This is most obvious at E14.5 in the lateral choroid plexus. (Fig. 10A –G) where Hox-7 is expressed in the plexus extending from the rostral aspect of the telencephalon –forebrain junction and into the ventricles of the cerebral hemispheres.

At E15 Hox-7 expression becomes detectable in mesenchyme derived from the second and third branchial arches (Fig. 11), which later forms components of the middle ear and the styloid process. Hox-7 is now also found in mesenchyme adjacent to the thinning epithelium of Jacobsons organ.

In this study, all the mice were decapitated at the junction of the head and neck so we could not determine if Hox-7 was expressed in derivatives of the third, fourth or fifth branchial arches, which at E9.5 and E10.5 all express Hox-7 in common with the maxillary and mandibular processes and the second branchial arches.

We have independently isolated and cloned a mouse homologue of the Drosophila msh gene, Hox-7, by a low-stringency screening of a cDNA library produced from E8.5 mouse embryo mRNA (MacKenzie et al. 1991). Using in situ hybridisation we have investigated the temporal and spatial expression patterns of Hox-7 in the developing central nervous system and cranium of the mouse embryo between E9.5 and E15.5. Expression of Hox-7 was present in the neuroepithelium surrounding the fusion sites of the neural tube and in Rathke’s pouch. Later in development Hox-7 expression was detected in areas destined to form choroid plexus. Expression of Hox-7 in the telencephalon preceded and accompanied the formation of the lateral choroid plexii. Expression was also seen in the external ear, the eye, the teeth primordia, nasal epithelium, Jacobson’s organ and second arch derivatives.

Between E11 and E15, Hox-7 expression occurs in the areas of the brain known as the choroid plexii. Two of these exist; the lateral choroid plexus (CP), which resides in the lateral ventricles of the cerebral cortex and the posterior CP in the 4th ventricle of the hind brain. The CP has several functions, of which the most important is secretion of the cerebrospinal fluid (CSF) (Crosby et al. 1962; Cserr et al. 1971), and in this capacity is important in sustaining the shape and volume of the ventricles during growth and development of the brain. Thus the CP has to act as the blood-brain barrier to the CSF (Barr and Kiernan, 1988). The mature CP is composed of a layer of microvillous ependymal cells separated from a stromal layer by a basement membrane. This stromal layer contains capillaries uncharacteristic of the brain blood supply in that they contain pores large enough to allow the passage of large molecules. The only barrier preventing the crossing of blood components is afforded by tight junctions between the ependymal cells (Barr and Kiernan, 1988), any breach of which could be fatal (Van Rybroek and Moore, 1990). The localisation of IGFII transcripts (Stylianopouou et al. 1988) and the mRN A of its binding protein; BP-3 A (Tseng et al. 1989) in the CP of adult rat brain combined with the presence of their translation products in the CSF (Haselbacher and Humbel, 1982; Hossenlopp et al. 1986) is indicative of the possible role of the CP as a vehicle of parasynaptic communication at distant sites in the brain (Herkenham, 1987). The CP also acts as a major supplier of iron to neurons via the CSF by producing large amounts of transferrin (Tsutsumi et al. 1989). Interestingly, the areas of dental mesenchyme that expresses Hox-7 (MacKenzie et al. 1991) also produce large amounts of transferrin (Partanen et al. 1984; Dickson et al. 1985).

Hox-7 expression is seen in the neuroepithelium of the forming telencephalon at E11 before any signs of plexus formation are visible (Fig. 3). This would suggest that Hox-7 might act not only as a molecular marker for the plexus anlage but may also be the gene involved in its spatial specification and differentiation. The first morphological signs of plexus formation occur around E11.5 when the neuroepithelium of the telencephalon facing the prosencephalon atrophies and increases its surface area either by rapid cell division or a movement of extant cells over their neighbours. However, this process on its own does not result in the concentrated intraventricular movement of the resulting neuroepithelial corrugations. This force is most probably supplied by the phalanx of ventrally directed dorsal mesenchyme expressing high levels of Hox-7 mRNA whose concerted movement may be responsible for the internalisation of the plexus. The mesenchyme invading and pushing into the choroid plexus is then incorporated into its structure producing an organ with both neuroepithelial and mesenchymal components. This scenario contrasts sharply with that previously observed for tooth formation where non-Hox-7 expressing dental epithelium invades Hox-7 expressing neural crest mesenchyme (MacKenzie et al. 1991). A communication exists between the mesenchyme at the very tip of the involuting choroid plexus and the external Hox-7 expressing mesenchyme up to and including E15.5. In the case of the formation of the posterior choroid plexus, Hox-7 expressing mesenchyme may have merely forced the involution of the tela choroida, already atrophied by the pontine flexure and expressing Hox-7. Alternatively, Hox-7 expressing mesenchyme cells surrounding the brain may exert a constant positive pressure inside the developing cranium; their invasion being allowed by any atrophication of the neuroepithelium.

At the same time as Hox-7 is being expressed in the thinning choroid plexus epithelium, the growth factor gene Vgr-1 is also expressed in the same tissues, including the areas destined to form choroid plexus indicating a possible molecular link between their expression patterns (Jones et al. 1991).

It has been shown that the steroid drug hydrocortisone stimulates the rate of growth of the CP in the developing chick brain (Rychter and Stanstny, 1979). It might be interesting to study the effects of this drug on the development of other organ systems such as the tooth and anterior pituitary to determine if its effects are mediated via Hox-7.

Although a great deal is known about the morphological changes occurring during the development of Rathke’s pouch and the cellular morphological and molecular processes occurring during its differentiation into the cell types of the pituitary, very little is known about the molecular mechanisms of its formation and determination. Rathke’s pouch is formed by an out-pocketing of the stomatodeal epithelium at about E8.5 in the mouse, but the corresponding outgrowth from the developing hypothalamus to form the neurohypophysis or infundibular stalk does not occur until Ell.5 (Rugh, 1990). Hox-7 expression is not seen in Rathke’s pouch until E10.5 to Ell. During the formation of Rathke’s pouch, between E9.5 and E 11.5, the TGF-β- like growth factor BMP-4 is expressed in the floor plate of the area of diencephalon destined to associate with Rathke’s pouch. BMP-4 is also expressed in limb buds, facial anlage and developing heart components; areas where Hox-7 is also known to be expressed (Jones et al. 1991; Hill et al. 1989).

All the cell types that differentiate from Rathke’s pouch involved in endocrine secretion differentiate from an outgrowth of Rathke’s pouch known as the pars distalis. The cell types that differentiate from the pars distalis are mammotrophs (secrete PRL), gonadotrophs (secrete LH and FSH), thyrotrophs (secrete TSH), corticotrophs (secrete ACTH from POMC) and somatotrophs whose secretion of growth hormone is known to be initiated in the E14 embryo by the GHF-1 growth factor expressed in the anlage of the pars distalis (Dolle et al. 1990). Immunological and in situ hybridisation studies in the rat have shown that these cell types can be detected by their products as early as E13 (Lugo, 1989).

The expression of Hox-7 in Rathke’s pouch during its development is concentrated primarily in epithelium determined to form the pars distalis and is almost absent from epithelium determined to form the pars intermedia. Therefore, the expression of Hox-7 in this area may be a factor in determining the differentiation of the above mentioned cell types of the pars distalis. Conversely, the lack of expression in epithelium destined to form the pars intermedia may also determine the differentiation of its cell lineages. Studies on the expression of POMC in the developing pituitary of the rat have shown that expression of POMC mRNA and its translation products in the epithelium of the pars distalis seem to occur in areas of epithelium adjacent to groups of mesenchyme cells rich in glycosaminoglycans (Pintar and Lugo, 1987). Since glycosaminoglycans are known to influence cell differentiation (Gallagher, 1989), the presence of these mesenchyme cells may be influencing the differentiation of the corticotrophic cell type by a mechanism that involves the expression of the Hox-7 gene.

Expression of Hox-7 in the neural plate and lateral ectoderm is detectable prior to neural tube closure at the primitive streak stage in the mouse embryo (R. Hill and D. Davidson, personal communication). Following neural tube closure, Hox-7 shows a persistent expression pattern closely localised to the dorsal midline running from the rostral end of the tube to the caudal end. Hox-7 can be detected in this site in the forming brain even after considerable modifications have occurred up to E15.5 of gestation. A proto-oncogene gene that appears to share this expression pattern is int-2 (Wilkinson et al. 1987). This sharing of a domain of expression at the dorsal midline may be suggestive of the initial involvement of Hox-7 and int-2 in lateral-medial axis formation in the primitive streak embryo and its subsequent involvement in the dorsalisation of the neural tube.

The expression patterns of Hox-7 have been shown to have a certain degree of persistence in mesenchyme derived from the neural crest in formation of the face and teeth or the mesenchymal derivatives of the 2nd, 3rd and 4th branchial arches (MacKenzie et al. 1991). This aside, expression of the Hox-7 gene is largely independent of cell lineage and is more dependent on the spatial situations and interactions of the tissues in which it is expressed. Hox-7 tends to be expressed in areas of epithelial –mesenchymal interaction. It is expressed with equal frequency in both epithelial structures and mesenchymal structures and in the majority of cases is exclusively in either one or the other of the germ layers of the organ system being formed, the choroid plexus being the most obvious exception. The morphological changes seen in the two germ layers expressing the gene seem to be stereotyped for each layer. Where Hox-7 is expressed in mesenchyme, the cells usually aggregate/migrate to differentiate into either chondrogenic tissue, as seen in the formation of the auricle and chondrocranium, or act as a focus in the invagination of epithelium as seen in tooth formation where the cells subsequently make dentine (MacKenzie et al. 1991). Expression in epithelium usually precedes or occurs during epithelial invaginations or atrophy as seen in choroid plexus and Rathke’s pouch. Despite these possible relationships the expression patterns displayed by Hox-7 are very complex. The expression of Hox-7 in the epithelium of the nasal pits and later in the thinning epithelium of the forming Jacobson’s organs can be likened to the expression patterns and morphological rearrangements seen in the formation of the choroid plexus.

Malformations induced by the vitamin A analogue, Isotretinoin, in human children of mothers exposed to the drug during pregnancy include craniofacial abnormalities and heart defects but more consistently involve brain abnormalities such as the cystic dilation of the 4th ventricle roof and hydrocephalus induced by abnormal secretion of cerebrospinal fluid (secreted by the choroid plexus), and malformations or absence of the external ear (Lammer, 1988). All these structures express the Hox-7 gene at some time during their development. Therefore, the expression of Hox-7 may be directly or indirectly affected by this drug following its metabolism to retinoic acid and its metabolites.

Wolf-Hirschhorn syndrome (WHS) is a human genetic disease which manifests itself in the form of severe congenital malformations and has been shown to be associated with deletions in the p arm of human chromosome 4. HOX7 is the human homologue of Hox-7 and has been mapped to the 4pl6.1 band on human chromosome 4 where the deletions associated with most WHS occur (Ivens et al. 1990). WHS is a human midline fusion syndrome resulting in ear malformations, cleft jaws, cases of double rows of teeth (Fujimoto and Wilson, 1990), heart malformations, decreased stature and profound mental retardation as a result of microcephaly (Lurie et al. 1989). All these malformations are associated with organ systems in which Hox-7 is now known to be expressed during their development in the mouse embryo.