![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Vol. 13, No. 23, pp. 3136-3148, December 1, 1999
1 G.W. Hooper Foundation, Department of Microbiology, School of Medicine, University of California at San Francisco (UCSF), San Francisco, California 94143-0552 USA; 2 Nina Ireland Laboratory of Developmental Neurobiology, Department of Psychiatry, School of Medicine, UCSF, San Francisco, California 94143-0984 USA; 3 Department of Anatomy and Program in Developmental Biology, School of Medicine, UCSF, San Francisco, California 94143-0452 USA
 |
Abstract |
|---|
 Top
 Abstract
 Introduction
Results
Discussion
Materials and methods
References
|
|---|
In mammals, the first branchial arch (BA1) develops into a number of craniofacial skeletal elements including the jaws and teeth. Outgrowth and patterning of BA1 during early embryogenesis is thought to be controlled by signals from its covering ectoderm. Here we used Cre/loxP technology to inactivate the mouse Fgf8 gene in this ectoderm and have obtained genetic evidence that FGF8 has a dual function in BA1: it promotes mesenchymal cell survival and induces a developmental program required for BA1 morphogenesis. Newborn mutants lack most BA1-derived structures except those that develop from the distal-most region of BA1, including lower incisors. The data suggest that the BA1 primordium is specified into a large proximal region that is controlled by FGF8, and a small distal region that depends on other signaling molecules for its outgrowth and patterning. Because the mutant mice resemble humans with first arch syndromes that include agnathia, our results raise the possibility that some of these syndromes are caused by mutations that affect FGF8 signaling in BA1 ectoderm.
[Key Words: Agnathia; Barx1; BMP4; endothelin-1; FGF8; first branchial arch]
| |
Introduction |
|---|
|
Top
Abstract
Introduction
Results
Discussion
Materials and methods
References
|
|---|
In vertebrates, many structures including parts
of the face develop from small primordia or "buds" consisting of
undifferentiated mesenchymal cells covered by a layer of epithelium.
One of these primordia is the first branchial arch (BA1), which in
mammals develops into teeth, skeletal elements of the jaws, lateral
skull wall, and middle ear, as well as part of the tongue and other soft tissue derivatives. In the mouse embryo, BA1 first becomes apparent at the six- to eight-somite stage [approximately embryonic day (E) 8.25] as a small swelling on the side of the head. This bud
rapidly increases in size as cranial neural crest cells migrate into
and proliferate within the arch. This neural crest-derived mesenchyme,
which is termed ectomesenchyme and is localized immediately subadjacent
to the covering epithelium, differentiates into cartilagenous (chondrocranial) and osseous (dermatocranial) structures. The central
core of BA1 mesenchyme is derived from somitomeres and forms
craniofacial muscle and vascular tissue. At ~E9.5 the outgrowing BA1
on each side of the head develops into the primordia of the mandibular
and maxillary arches, which grow toward the ventral midline.
Subsequently, multiple fusions involving the paired mandibular arches,
maxillary arches, and the frontonasal process establish the basic form
of the face. Errors in these complex morphogenetic events cause
craniofacial anomalies including defects of the mandible, which are
among the most common malformations in humans. More than 130 human
syndromes appear to involve incorrect development of the BA1. The most
severe of these syndromes are associated with agnathia, a condition in
which the lower jaw and other BA1-derived structures are absent (for
review, see Bixler et al. 1985
; Escobar 1993).
Classic experimental embryological studies have suggested that
development of BA1 mesenchyme is controlled by signals from its
covering ectoderm, which regulate cell proliferation, survival, patterning, and differentiation. In turn, signals from the mesenchyme may influence development of the ectoderm. The molecular basis of these
epithelial-mesenchymal interactions is not yet well understood. Genetic analysis in mice has provided evidence that members of several
homeobox gene families are necessary for normal BA1 development (for
review, see Francis-West et al. 1998). However, relatively little is
known about the precise role of the epithelium and the signaling
molecules it produces in the regulation of outgrowth and patterning at
early stages of BA1 development.
Members of the FGF family, particularly FGF8, have been implicated as
epithelial signals that regulate gene expression during BA1
development. When beads soaked in FGF8 (FGF8-beads) were inserted into
isolated mandibular mesenchymal explants, expression of several genes
could be induced (for review, see Peters and Balling 1999). Although
such bead implantation studies provide valuable clues to the identity
of signaling molecules and potential target genes, the long-term
consequences of altering gene expression on skeletal patterning cannot
be addressed in organ culture. Furthermore, firm conclusions about gene
function, particularly when several members of a multigene family are
coexpressed, require loss-of-function studies. However, standard
genetic approaches cannot be used to analyze FGF8 function in craniofacial
development because Fgf8
/ embryos die during
gastrulation (Sun et al. 1999). Here we have used
Cre/loxP technology to circumvent this problem
and to generate mutant embryos in which the Fgf8 gene is
inactivated in BA1 ectoderm. Our analysis identifies FGF8 as an
epithelial signal essential for the outgrowth and patterning of BA1
from the earliest stages of its development.
| |
Results |
|---|
|
Top
Abstract
Introduction
Results
Discussion
Materials and methods
References
|
|---|
Cre-mediated inactivation of Fgf8 in BA1 ectoderm
Inactivation of a particular gene in a specific tissue can be
achieved by mating mice carrying a mutant allele in which essential regions of the gene are flanked by loxP sites (the recognition sequence for the site-specific DNA recombinase, Cre) with mice that
express the cre gene in the tissue of interest (Gu et al. 1994; Tsien et al. 1996; Kulkarni et al. 1999). To inactivate Fgf8 in BA1, we used mice carrying
Fgf8flox, an allele in which vital coding exons are
flanked by loxP sites. Fgf8flox has
wild-type Fgf8 activity but can be converted to a null allele (Fgf8
2,3) by Cre-mediated
recombination (Meyers et al. 1998). Using the mating scheme outlined in
Figure 1A we produced animals that were compound
heterozygotes for Fgf8flox and a null allele of
Fgf8, and which also carried Nes-cre1, a transgene
containing a modified cre gene (Lewandoski et al. 1997) under
the control of the rat Nestin promoter and intron-2 enhancer (Zimmerman
et al. 1994; A. Trumpp, G.R. Martin, and J.M. Bishop, unpubl.). In
Fgf8flox/Fgf8 null;Nes-cre1
embryos, hereafter referred to as Fgf8;Nes-cre or mutant embryos,
Cre-mediated conversion of Fgf8flox to a null allele occurs
only in cells that express Nes-cre1, resulting in complete loss of
Fgf8 gene function in those cells and their descendants. Their
Fgf8flox/Fgf8 null littermates that did not inherit
Nes-cre1 are phenotypically normal and served as controls.
|
The stage and tissue specificity of Nes-cre1 activity was
determined by crossing males carrying Nes-cre1 to females
carrying the Z/AP reporter gene, which produces human
alkaline phosphatase (hAP) only after it has undergone Cre-mediated
recombination (Lobe et al. 1999). A detailed description of the results
of that analysis will be reported elsewhere. Here we focused on an
assessment of cre activity in the head. In
Nes-cre1;Z/AP embryos assayed at ~E9.0
(16-somite stage), little hAP activity was detected in the brain, but
strong activity was detected in surface ectoderm, particularly that
covering BA1 (Fig. 1B,C). The domain of Nes-cre1 activity in
BA1 ectoderm had a sharp caudal limit at the level of the first pharyngeal groove (red arrowheads in Fig. 1B,C). Analysis at earlier stages suggested that Cre-mediated recombination first occurred in the
cranial surface ectoderm at ~E7.75, and that subsequently, recombination occurred in cells on the rostral side of BA1 before it
occurred in those on the caudal side (data not shown; see below).
During BA1 development, Fgf8 is expressed in a dynamic pattern
in the surface ectoderm (Crossley and Martin 1995; Mahmood et al. 1995;
Kettunen and Thesleff 1998). At E8.5, when the nascent arch is a
discrete bud, Fgf8 expression is detected at low levels throughout the ectoderm, but subsequently becomes more robust in the
rostral- and caudal-most aspects of BA1 (Fig. 1D,E; data not shown). By
E9.5, Fgf8 expression becomes restricted to the ectoderm on
the rostral side. By E10.5, when the maxillary and mandibular primordia
have expanded, Fgf8 is abundant in the so-called "oral
ectoderm," which covers the caudal side of the maxillary and the
rostral side of the mandibular arches (see Fig. 6O).
In Fgf8;Nes-cre embryos, Fgf8 is inactivated in the surface ectoderm of prospective BA1. By E9.0 (14-18 somites), no Fgf8 RNA was detected in BA1, although it was detected in all other normal Fgf8 expression domains (n = 5; Fig. 1G,H; data not shown). Significantly, in two slightly younger embryos a small patch of Fgf8-expressing cells was detected on the caudal side of BA1 (yellow arrow, Fig. 1F). This patch appeared to be a subset of the normal caudal Fgf8 expression domain. The presence of this patch confirms that inactivation does not occur synchronously across the arch, and shows that some cells on the caudoproximal side transiently express Fgf8.
Mutant embryos survive to birth but lack most BA1-derived structures
The swelling that heralds BA1 development is evident in Fgf8;Nes-cre embryos, but by E9.0 it is clearly smaller than normal (Figs. 1E,H and 2A,B). As the embryos mature, there is relatively little expansion of either the mandibular or maxillary primordia, whereas development in neighboring regions, such as the hyoid arch (second branchial arch), appears normal (Fig. 2A-D; data not shown). It is important to note, however, that outgrowth of the mutant BA1 is not completely arrested, as the mandibular primordia extend distally and meet at the midline (white arrowhead in Fig. 2D; see also Fig. 6D,H,L). Furthermore, at E11.25 a distinct hillock (yellow arrow in Fig. 2D) forms in caudoproximal BA1, immediately adjacent to the external acoustic meatus (EAM). This hillock appears to form in the region in which Fgf8 expression was detected transiently at E9.0 (yellow arrow in 1F).
|
Fgf8;Nes-cre mutants die shortly after birth. Newborns appear grossly normal except for severe craniofacial defects and the presence of an ectodermal covering over the prospective mouth (Fig. 2E,F; data not shown). The lungs do not inflate suggesting that lethality is caused by anoxia. Characterization of the craniofacial defects in Fgf8;Nes-cre embryos revealed that cartilagenous elements thought to develop from BA1 by E14.5, such as the ala temporalis and incus (maxillary arch-derived), and body of Meckel's cartilage (mandibular arch-derived) fail to develop. However mutant embryos have a vestigial malleus (Ma*) and rostral process (RP*), the distal symphyseal end of Meckel's cartilage (Fig. 2G-N).
Normally, between E14.5 and birth, a number of bones develop from BA1 neural crest-derived mesenchyme (summarized in Fig. 2O). In Fgf8;Nes-cre newborns, most of these bones are absent. A few mandibular arch-derived elements remain, including alveolar bone associated with RP* (Fig. 2L), Ma*, and some small ossified elements in the general region of the gonial and tympanic bones (asterisk in Fig. 2N). Of the maxillary arch-derived dermal elements, the mutants contain the distal maxilla (Mx*) and portions of the squamosal (Fig. 2L). Molars are absent, but vestigial lower incisors are observed in association with RP* (Figs. 2L and 5H). A summary of skeletal development in Fgf8;Nes-cre mutants is presented in Figure 2O.
Newborn mutants had other abnormalities consistent with a failure of
BA1 development, including a small disorganized tongue (microglossia)
and a truncated and misrouted mandibular division of the trigeminal
nerve (data not shown). There was some variation in the mutant
phenotype. The majority (28 of 42) displayed the phenotype described
above, whereas the remaining mutants showed a slightly less severe
phenotype, with a small increase in the extent of dermal bone
development. In many cases, this less severe phenotype was observed on
only one side of the head, which is reminiscent of asymmetries found in
a variety of human congenital syndromes (for review, see Escobar 1993).
FGF8 is necessary for survival of mesenchymal cells in BA1
Analysis of Fgf8-null mutant homozygotes has shown that
Fgf8 is required for cell migration at primitive streak stages
of development (Sun et al. 1999). Therefore, we speculated that the early failure of BA1 outgrowth in mutant embryos might be due to a lack
of neural crest cell migration into the BA1 primordium. To investigate
this possibility we assayed for Cad6, Crabp1, and Ap2.2 expression, which marks migrating neural crest cells
(Mitchell et al. 1991; Maden et al. 1992; Ruberte et al. 1992; Inoue et al. 1997). Expression of these markers was similar in mutant and normal
embryos (Fig. 3A,B; data not shown), indicating that
despite loss of Fgf8 function in BA1, the neural crest cell
population follows its normal migration path and appears normal in size.
|
To determine why the mutant BA1 is hypoplastic, we then studied cell
proliferation by assaying for BrdU incorporation. BrdU-labeled cells
were detected in the mutant BA1 at E9.5, at least 12 hr after
Cre-mediated inactivation of Fgf8, and the ratio of
BrdU-labeled to unlabeled cells appeared roughly the same as in the
normal arch (data not shown). This result suggests that FGF8 is not
required for cell proliferation in BA1. We then assayed for cell
survival by staining in whole mount for Nile blue sulfate (NBS) uptake, which marks dying and dead cells (Bowen 1981), and by performing TUNEL
assays on tissue sections to detect apoptosis. In the BA1 region of
control embryos at E8.5-E10.0, NBS staining was observed only
proximally (Fig. 3C,E), where trigeminal ganglion cells die (Noden
1983; Davies and Lumsden 1984). In Fgf8;Nes-cre embryos, however, extensive cell death was detected at E8.75-E10.0 (Fig. 3D,F;
data not shown). At E9.5, intense NBS staining was detected throughout
the BA1 primordium (Fig. 3D) except at the extreme distal tip near the
ventral midline (Fig. 3F). The area in which dying cells were detected
stretched proximally to the trigeminal swelling and included the region
forming the maxillary arch (Fig. 3D,H). The results of the TUNEL
analysis indicated that the observed abnormal cell death, which was
detected exclusively in the mesenchyme of BA1, is due to apoptosis
(Fig. 3G,H). Together these data suggest that the lack of BA1
development in Fgf8;Nes-cre embryos is due, at least in part,
to apoptosis of a substantial proportion of the cells that normally
populate the arch, and therefore, that FGF8 produced in the surface
ectoderm is essential for their survival.
Patterned expression of some regulatory genes is maintained in the hypoplastic BA1 of Fgf8;Nes-cre mutants
Because a proportion of mutant BA1 mesenchyme survives and forms a
hypoplastic arch, we performed an analysis to identify genes that
require FGF8 signaling and to determine the extent of mesenchymal
patterning in the absence of FGF8. Pitx1, a bicoid-related homeobox gene (Lanctot et al. 1997; Szeto et al. 1999) is normally coexpressed with Fgf8 in the oral ectoderm of the mandibular
and maxillary primordia, and is also expressed in the underlying
mesenchyme (Fig. 4A). The oral mesenchyme also
expresses Lhx7, a LIM homeodomain encoding gene (Grigoriou et
al. 1998; Tucker et al. 1999) (Fig. 4C,E). Expression of both genes was
clearly detected in the mutant BA1, but the signals were less extensive
than normal (Fig. 4A-F), presumably because BA1 mesenchyme is
hypocellular. These results show that mutant BA1 is sufficiently
healthy to express at least some of the genes that mark the oral side
of BA1, and suggest that some aspects of oral-aboral patterning are
intact in the absence of Fgf8.
|
Dlx1, Dlx2, and Dlx5 are three members of
the Dlx multigene family related to the Drosophila distalless
gene, which are thought to regulate morphogenesis along the P-D axis
of BA1 (Qiu et al. 1995, 1997; Depew et al. 1999). Dlx1 and
Dlx2 are expressed in the ectomesenchyme along most of the
P-D length of BA1, including the maxillary primordium (Fig. 4G; data
not shown). In contrast, Dlx5 expression is not detected in
the maxillary primordium and is restricted within the distal two-thirds
of the mandibular primordium (Qiu et al. 1997; Depew et al. 1999) (Fig.
4I). Expression of all three genes was detected in BA1 of mutant
embryos in roughly their normal domains (Fig. 4H,J). This suggests that
cells in the mutant arch have sufficient P-D positional information to maintain differential expression of these genes.
To further investigate the extent to which distal BA1 is patterned, we
assayed for Msx1 and Bmp4 expression. Between E9.5 and E10.5, Msx1 expression is normally restricted to the
distal (medial) ectomesenchyme and Bmp4 expression is detected
in distal BA1 ectoderm overlying the Msx1 expression domain
(Tucker et al. 1998a) (Fig. 5A,C). Both Msx1
and Bmp4 expression appeared relatively normal in mutant BA1
(Fig. 5B,D). Similar results (not shown) were obtained with probes for
Msx2, dHand, and eHand, other genes that are
normally expressed in distal BA1 (Thomas et al. 1998). Pax9,
which is required for tooth development, is normally detected in the
mandibular arches at E11.5 in four spots, two distal and two proximal,
representing the prospective incisor and molar domains, respectively
(Neubuser et al. 1997; Peters et al. 1998) (Fig. 5E). In the mutant
embryos, Pax9 was detected in only two spots near the ventral
midline (Fig 5F), which presumably mark the regions in which the
incisors will form. These early gene expression patterns are consistent
with the observation that the distal-most portions of BA1 are the least
affected: in newborn Fgf8;Nes-cre mutants the distal part of
the maxilla and the distal mandible, including truncated lower
incisors, are present (Fig. 5G,H; summarized in Fig. 2O). These data
suggest that FGF8 is not required for development of distal BA1.
|
Identification of FGF8-responsive genes
FGF8-bead implantation experiments have identified Lhx7,
and the closely related gene Lhx6, which are coexpressed in
oral mesenchyme of mandibular and maxillary arches at E10.5 (Figs. 4C,E; 6A,C), as potential targets of FGF8 signaling in BA1 (Grigoriou et al. 1998). Interestingly, although we found that
Lhx7, is expressed in its normal domain in
Fgf8;Nes-cre mutants (Fig. 4D,F), Lhx6 expression
was not detected in mutant BA1 (Fig. 6B,D). Thus, our data suggest that
they are independently regulated in vivo.
|
Barx1 is another homeobox gene previously identified as
inducible by FGF8 (Tucker et al. 1998b). Beginning at E9.5 it is
expressed in mesenchyme throughout the proximal but not in the distal
portion of BA1 (Fig. 6E,G; data not shown). In E9.5-E11.5
Fgf8;Nes-cre embryos, Barx1 RNA was not detectable,
except in a small patch of cells on the caudal side of the mandibular
arch (yellow arrows in Fig. 6F,H; data not shown). Significantly, this
patch of Barx1 expression was localized in mesenchyme that
appeared to underlie the region in which Fgf8 expression was
detected transiently at E9.0 (cf. Figs. 1F and 6F). At E9.5, the mutant
ectoderm that appears to overlie the patch of Barx1-expressing
cells expresses Endothelin-1 (Et1; yellow arrow, Fig. 6N).
Et1 RNA was not detected elsewhere in mutant BA1 ectoderm, but
was detected in the second and third branchial arch epithelium (Fig.
6N; data not shown). In contrast, in control embryos, Et1 RNA
was detected at low levels throughout the epithelium of branchial
arches 1, 2, and 3 (Clouthier et al. 1998) (Fig. 6M). By E10.5,
Goosecoid (Gsc) expression, which is normally detected
throughout the caudal half of BA1 (Rivera-Perez et al. 1995; Yamada et
al. 1995) (Fig. 6I,K) is restricted in the mutants to the region in
which Barx1- and Et1-expressing cells are found
(yellow arrow, Fig. 6J,L). These results, summarized in Figure 6O, show
that expression of Lhx6, Barx1, Et1, and
Gsc in BA1 are dependent, directly or indirectly, on FGF8
signaling. Furthermore, they suggest that transient expression of
Fgf8 on the caudal side of BA1 at E9.0 is sufficient to induce
local expression of Barx1, Et1, and Gsc, but
that continued expression of Fgf8 is not required to maintain it.
| |
Discussion |
|---|
|
Top
Abstract
Introduction
Results
Discussion
Materials and methods
References
|
|---|
Inactivation of Fgf8 by Cre-mediated recombination in the ectoderm of the nascent first branchial arch severely impairs development of the BA1 primordium. This appears to be due to both apoptosis of the mesenchyme and failure to express a set of genes essential for BA1 morphogenesis. Newborn mutants lack most BA1-derived structures, except those formed from the distal-most region. These results provide genetic evidence that the ectoderm produces factors essential for BA1 outgrowth and patterning at a very early stage. They further suggest a model in which BA1 is specified into two domains at early stages in its development, a proximal region that is dependent on FGF8 signaling for its outgrowth and patterning, and a distal domain that is controlled by other signaling molecules (Fig. 7).
|
FGF8 is an essential signal for cell survival in proximal BA1
Shortly after Fgf8 is inactivated in the nascent BA1
epithelium, there is a brief period of cell death that peaks at
~E9.5, during which a large proportion of proximal but not distal
BA1 mesenchyme undergoes apoptosis. This cell death is presumably responsible for the small size of BA1 from an early stage of its development in the mutant embryos, and most likely contributes to the
final mutant phenotype. Thus, one important conclusion from our study
is that FGF8 is required, directly or indirectly, for cell survival in
proximal BA1. Preliminary data suggest that reduction in the level of
FGF8 also causes cell death in the developing limb and brain (M. Lewandoski, E. Storm, and G.R. Martin, unpubl.). In contrast, analysis
of Fgf8/ embryos indicates
that during gastrulation FGF8 is not required for cell proliferation or
survival, but instead is necessary for cell migration at the primitive
streak stage (Sun et al. 1999). Thus, it appears that FGF8 performs
different functions in different developmental contexts.
There is a substantial body of evidence from experimental studies in
vitro showing that FGFs, particularly FGF1 and FGF2, can function as
survival factors for a wide variety of cell types including neurons,
glia, endothelial cells, and smooth muscle cells (for review, see
Szebenyi and Fallon 1999). For example, it has been found that addition
of FGF2 to explants of trunk neural crest enhances cell survival
without stimulating mitosis (Kalcheim 1989), and local application of
beads containing FGF4 prevents apoptosis in dental mesenchyme isolated
from the mandibular arch at E13 (Vaahtokari et al. 1996). Despite the
wealth of data from such in vitro studies, previous genetic analysis
has provided little evidence that FGFs are required for cell survival.
Null mutations have been generated in Fgf2 and at least 10 other mouse FGF genes, as well as all four FGF receptor (FGFR) genes
(Goldfarb 1996; Floss et al. 1997; Dono et al. 1998; Min et al. 1998;
Ortega et al. 1998; Weinstein et al. 1998; Zhou et al. 1998; Sekine et al. 1999; Sun et al. 1999; D. Ornitz, pers. comm.; C. Basilico, pers.
comm.), but to our knowledge, effects specifically on cell survival
have been described only in Fgf4/ (Feldman et al.
1995) and Fgfr2/ (Arman et al. 1998)
embryos. These mutants display a similar early postimplantation lethal
phenotype in which the inner cell mass dies.
One interesting question is why a proportion of the cells in proximal
BA1 survive. One possibility is that their survival is dependent on
other FGF family members, such as Fgf9, which is detected at
low levels in both wild-type (Kettunen and Thesleff 1998) and mutant
BA1 epithelium (data not shown), or on other types of signals. Another
intriguing possibility is based on the premise that FGFs and other
survival factors that signal through receptor tyrosine kinases (RTKs)
function to prevent apoptosis by stimulating the antiapoptotic activity
of Ras (for review, see Downward 1998), and the observation that FGF
signaling can induce the expression of Sprouty (Spry) genes,
which encode inhibitors of RTK signaling (Hacohen et al. 1998; Casci et
al. 1999; Kramer et al. 1999; Minowada et al. 1999; Reich et al. 1999).
Thus, the extent of cell survival in a given tissue may be determined
by the balance between factors that stimulate and inhibit Ras activity. We have found that the expression of at least one member of the Sprouty
gene family, Spry2, is greatly diminished in BA1 of
Fgf8;Nes-cre embryos (data not shown), supporting the
hypothesis based on FGF-bead implantation experiments that FGF8
positively regulates Sprouty gene expression in BA1 (Minowada et al.
1999). The reduced level of Spry2 in mutant BA1 mesenchyme
might compensate partially for the reduced Ras activity caused by the
inactivation of Fgf8 by derepressing other RTK pathways
normally inhibited by Sprouty, and thereby promote cell survival.
FGF8 induces gene expression necessary for proximal BA1 morphogenesis
Fgf8;Nes-cre embryos lack most of the chondrocranial and
dermatocranial elements that form the jaws, the lateral skull wall, and
middle ear (Fig. 2O). It is possible that their failure to develop is
due solely to the reduction in cell number that occurs between E8.75
and E10. However, it seems likely that the failure of surviving cells
to express genes downstream of FGF8 also contributes to the final
phenotype. One genetic pathway that is affected in Fgf8;Nes-cre mutants involves Endothelin-1 (ET1) (Levin
1995). Mice in which the genes encoding ET1 or its receptor
(ETA) have been inactivated display a pleiotropic phenotype,
including severe effects on the lower jaw and other BA1-derived
structures (Kurihara et al. 1994; Clouthier et al. 1998). Although ET1
signaling is not required for the early stages of arch outgrowth and
BA1 appears grossly normal until E10.5 in
Et1/ mutants (Kurihara et al.
1994), it is required for expression of Gsc (Clouthier et al.
1998), which is necessary for formation of some craniofacial skeletal
elements (Rivera-Perez et al. 1995; Yamada et al. 1995). Our results
show that Et1 and Gsc expression are dependent on
FGF8 signaling, indicating that FGF8 is upstream of this pathway (Fig.
7). Consistent with this hypothesis, the BA1 structures that are
missing or malformed in Et1/
and EtA/ mice are
also affected in Fgf8;Nes-cre embryos. However, loss of
Fgf8 function in BA1 results in a more severe phenotype. This could be explained either by the hypocellularity of the
Fgf8;Nes-cre BA1 or by the lack of expression of genes that
are downstream of FGF8 but not ET1 signaling.
One good candidate for such a gene is Barx1 (Tissier-Seta et
al. 1995). Previous studies have shown that Barx1 expression, which is normally restricted to the region that gives rise to the same
elements that fail to develop in Fgf8;Nes-cre embryos, can be
induced in explants of mandibular arch mesenchyme by FGF8 (Tucker et
al. 1998b). Our data show that FGF8 is required on both the rostral and
caudal side of BA1 to establish the Barx1 expression domain.
We found that Lhx6 is also downstream of FGF8. Surprisingly,
Lhx7 expression does not require FGF8, despite the fact that
Lhx6 and Lhx7, which appear to be expressed in the
same cells on the oral side of the arch, can both be induced by placing FGF8-beads in isolated mandibular arch mesenchyme (Grigoriou et al.
1998). A possible explanation for these findings is that Lhx7 expression is induced by an FGF family member other than FGF8.
FGF8 as a switch that induces but does not maintain the BA1 developmental program
An important question is whether FGF8 functions solely to induce gene expression or whether it is also necessary to maintain it. We were able to address this question because Fgf8 is expressed transiently in a few epithelial cells on the caudoproximal side before it is inactivated. After Fgf8 RNA is no longer detected, Et1 is expressed in what appears to be the same caudal cell population. Moreover Barx1 and subsequently Gsc are induced in the adjacent underlying ectomesenchyme. The finding that expression of Et1, Barx1, and Gsc in the mutant BA1 is restricted to the region in which Fgf8 is expressed transiently suggests that FGF8 is necessary and sufficient to induce expression of these genes in vivo. Furthermore, because Barx1 and Gsc expression persists in this region through E11.5, at least 60 hr after Fgf8 has been inactivated, we propose that FGF8 can function as a switch to induce a gene expression cascade that rapidly becomes independent of FGF8 signaling. Thus, Barx1 and Gsc expression may be maintained by other signals produced in the epithelium, possibly ET1.
Analysis of the skeletons of Fgf8;Nes-cre newborns indicated
that the only proximal BA1-derived skeletal elements present are a
malleus-like middle ear element and nearby amorphous cartilagenous and
bony fragments (Fig. 2O). It seems likely that they develop from cells
on the caudal side of the mutant BA1 where outgrowth is observed at
early stages in close proximity to a structure (the EAM) that will form
the outer ear canal. Cells in this outgrowth apparently express
Gsc, which is required for malleus development (Rivera-Perez
et al. 1995; Yamada et al. 1995). Because this outgrowth develops in
the region that was exposed transiently to FGF8 signaling, the presence
of a vestigial malleus in the Fgf8;Nes-cre newborns does not
contradict the hypothesis that FGF8 is required for development of the
entire proximal domain of BA1.
Control of distal BA1 development
In contrast to the lack of development of skeletal elements derived
from proximal BA1, structures derived from the distal portion of the
arch are invariably present in Fgf8;Nes-cre newborns (Fig.
2O). There is genetic evidence that development of the distal region of
BA1 is dependent on Msx1, as the distal maxilla and mandible
(including the incisors), the very structures that are present in
Fgf8;Nes-cre pups, fail to form in Msx1 null embryos (Satokata and Maas 1994). As expected, Msx1 expression was
detected in its normal domain in distal BA1 of Fgf8;Nes-cre
embryos. Although it is formally possible that the distal domain
develops because Fgf8 is expressed transiently in distal BA1
ectoderm, we think this unlikely because no such Fgf8
expression was detected in the mutant embryos. Instead, we suggest that
development of distal BA1 is not dependent on FGF8. This leaves open
the question of what controls Msx1 expression? It is possible
that FGF signaling is involved, as several FGFs have been found to
induce Msx1 in dental mesenchyme (Bei and Maas 1998; Kettunen
and Thesleff 1998), at the border of the neural plate (Streit and Stern
1999), and in the limb (Wang and Sassoon 1995). Other than
Fgf8, the only FGF family member presently known to be
expressed in BA1 epithelium before E12.5 is Fgf9 (Kettunen and
Thesleff 1998;
http://honeybee.helsinki.fi/toothexp/index.htm). Thus, FGF9 or some other FGF that has yet to be identified may provide
the signal necessary for outgrowth and patterning of the distal domain
of BA1. The hypothesis that another FGF in addition to FGF8 is required
for BA1 development could also explain the pattern of expression we
observed for Pax9, a marker for the prospective tooth-forming
domains in the oral mesenchyme, which can be induced in isolated
mesenchyme by any of several different FGFs (Neubuser et al. 1997). In
the mutant BA1, Pax9 expression is absent in the proximal,
molar domain because it is dependent on FGF8, whereas Pax9
expression is detected in the distal, incisor domain, perhaps because
it is induced by an FGF signal other than FGF8.
Another factor that may play a role in the development of the distal
domain is BMP4, as it too is capable of inducing Msx1 expression in BA1 (Vainio et al. 1993; Tucker et al. 1998a,b), limb
(Wang and Sassoon 1995), and brain (Furuta et al. 1997), and is
expressed in distal BA1 of Fgf8;Nes-cre mutants. In view of
the evidence that BMP signaling plays a role in determining tooth
identity (Tucker et al. 1998b), and that FGF and BMP signaling pathways
function antagonistically to regulate tooth induction (Neubuser et al.
1997), it seems likely that interactions between the two types of
signals control development of the distal-most BA1 structures.
Concluding remarks
The Fgf8;Nes-cre mutants resemble humans with first arch
syndromes that include agnathia. Agnathia alone occurs very rarely, and
is often associated with holoprosencephaly and sometimes with situs
inversus totalis, or both (Pauli et al. 1983; Bixler et al. 1985; Leech
et al. 1988; Escobar 1993). Significantly, embryos that are compound
heterozygous for a null and a hypomorphic allele of Fgf8 show
forebrain defects including holoprosencephaly (E. Meyers, E. Storm, and
G.R. Martin, unpubl.) and display abnormalities in left-right asymmetry
determination (Meyers and Martin 1999). Therefore, it is tempting to
speculate that mutations in Fgf8 or in genes directly upstream
or downstream of it might cause some of the human syndromes
characterized by agnathia/micrognathia.
| |
Materials and methods |
|---|
|
Top
Abstract
Introduction
Results
Discussion
Materials and methods
References
|
|---|
Production and analysis of mutant embryos
Production and full characterization of the Nes-cre1 transgenic
mouse line will be reported elsewhere. The Fgf8flox and
Fgf82,3n alleles (Meyers et al. 1998)
were maintained on a mixed genetic background. Fgf8;Nes-cre
mutants were produced using the breeding scheme outlined in Figure 1A
and genotyped using previously described primers (Lewandoski et al.
1997; Meyers et al. 1998; Sun et al. 1999). Histological analysis of
embryos, scanning electron microscopy, and skeletal preparations were
carried out as described by Depew et al. (1999).
Nes-cre1;Z/AP double hemizygotes (E7.5-E11.5)
were stained for alkaline phosphatase activity essentially as described by Lobe et al. (1999).
Cell death analysis
For whole mount NBS staining, E8.0-E10.5 embryos were dissected, washed in PBS, and incubated for 30-45 min at 37°C in filtered NBS solution [10 mg/ml NBS (Sigma N-5632) in PBS containing 0.1% Tween 20]. Embryos were then washed several times in PBS at room temperature and photographed immediately. TUNEL analysis was performed on paraffin sections using the In Situ Cell Death Detection kit (Boehringer-Mannheim) following the manufacturer's protocol.
RNA in situ hybridization
Whole mount RNA in situ hybridization analysis was carried out as
previously described (Neubuser et al. 1997) using riboprobes prepared
from plasmids described in references cited for each gene.
Fgf8 RNA was detected using a probe for sequences in exons 2 and 3, which are deleted in the Fgf82,3 and
the Fgf82,3n mutant alleles. The
expression of each gene was analyzed in at least three
Fgf8;Nes-cre embryos at each stage.
| |
Acknowledgments |
|---|
We are very grateful to Andras Nagy for providing the Z/AP reporter line, and thank the following for providing plasmids used to prepare probes used in this study: J.F. Brunet (Barx1); E. deRobertis (Gsc); J. Drouin (Pitx1); B. Hogan (Bmp4); V. Pachnis (Lhx6, Lhx7); P. Sharpe (Msx1); M. Takeichi (Cad6); and M. Yanagisawa (Et1). We are grateful to A. Gannon and D. Trail for excellent technical assistance. We also thank our colleagues in the Martin and Bishop laboratories for helpful discussion and critical readings of the manuscript. A.T. was the recipient of postdoctoral fellowships from the Deutsche Forschungsgemeinschaft, Human Frontiers Science Program, and the California Division of the American Cancer Society. This work was supported by grants from the March of Dimes and Nina Ireland (to J.L.R.R.), the Howard Hughes Medical Institute Research Resources Program grant (76296-549901) to the UCSF School of Medicine, and National Institutes of Health grants KO2 MH01046 (to J.L.R.R.), RO1 CA44338 (to J.M.B.), and RO1 HD34380 (to G.R.M.).
The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.
| |
Footnotes |
|---|
Received September 13, 1999; revised version accepted October 19, 1999.
4 Present address: Swiss Institute for Experimental Cancer Research (ISREG), CH 1066, Epalinges, Lausanne, Switzerland.
5 Corresponding author.
E-MAIL gmartin{at}itsa.ucsf.edu; FAX (415) 476-3493
| |
References |
|---|
|
Top
Abstract
Introduction
Results
Discussion
Materials and methods
References
|
|---|
