branchless
Branching morphogenesis of mouse submandibular glands is regulated by multiple growth factors. Ex vivo branching of intact
submandibular glands decreases when either FGFR2 expression is
downregulated or soluble recombinant FGFR2b competes out the endogenous growth factors. However, a combination of neutralizing antibodies to FGF1, FGF7 and
FGF10 is required to inhibit branching in the intact gland, suggesting that multiple FGF isoforms are required for branching. Exogenous FGFs added to submandibular epithelial rudiments cultured without mesenchyme induce distinct morphologies. FGF7 induces epithelial budding, whereas FGF10 induces duct
elongation, and both are inhibited by FGFR or ERK1/2 signaling inhibitors. However, a PI3-kinase inhibitor also decreases FGF7-mediated epithelial
budding, suggesting that multiple signaling pathways exist. FGF receptors were immunolocalized and changes in FGFR, FGF and MMP gene expression were analyzed to identify the mechanisms of FGF-mediated morphogenesis. FGFR1b and FGFR2b are present throughout the epithelium,
although FGFR1b is more highly expressed around the periphery of the buds and the duct tips. FGF7 signaling increases FGFR1b and FGF1 expression, and MMP2 activity, when compared with FGF10, resulting in increased cell proliferation and expansion of the epithelial bud, whereas
FGF10 stimulates localized proliferation at the tip of the duct. FGF7- and FGF10-mediated morphogenesis is inhibited by an MMP inhibitor and a neutralizing antibody to FGF1, suggesting that both FGF1 and MMPs are essential downstream mediators of epithelial morphogenesis. Taken together, these data suggests that FGFR2b signaling involves a regulatory network of
FGFR1b/FGF1/MMP2 expression that mediates budding
and duct elongation during branching morphogenesis (Steinberg, 2005).
FGF and lung morphogenesis Expression of human keratinocyte growth factor (KGF/FGF-7), directed to epithelial cells of
the developing embryonic lung of transgenic mice, disrupts normal pulmonary morphogenesis
during the pseudoglandular stage of development. By embryonic day 15.5 (E15.5), lungs of
transgenic surfactant protein C (SP-C)-KGF mice resemble those of humans with pulmonary
cystadenoma. Lungs are cystic, filling the thoracic cavity, composed of numerous
dilated saccules lined with glycogen-containing columnar epithelial cells. The normal distribution of
SP-C proprotein in the distal regions of respiratory tubules is disrupted. Columnar epithelial cells
lining the papillary structures stain variably and weakly for this distal respiratory cell marker.
Mesenchymal components are preserved in the transgenic mouse lungs, yet the architectural
relationship of the epithelium to the mesenchyme is altered. SP-C-KGF transgenic mice fail to
survive gestation to term, dying before E17.5. Culturing mouse fetal lung explants in the presence
of recombinant human KGF also disrupts branching morphogenesis and results in similar cystic
malformation of the lung. Thus, it appears that precise temporal and spatial expression of KGF is
likely to play a crucial role in the control of branching morphogenesis (Simonet, 1995).
Development of the seminal vesicle (SV) is elicited by androgens and is dependent on
epithelial-mesenchymal interactions. Androgenic signal transmission from the
androgen-receptor-positive mesenchyme to the epithelium has been postulated to involve paracrine
factors. Keratinocyte growth factor (KGF), a member of the fibroblast growth factor family, is
produced by stromal/mesenchymal cells and acts specifically on epithelial cells. Newborn
SVs placed in organ culture undergo androgen-dependent growth and differentiation. Addition of a
KGF-neutralizing monoclonal antibody to this system caused striking inhibition of both SV growth
and branching morphogenesis. This inhibition is due to a decline in epithelial proliferation and
differentiation, as the mesenchymal layer is not affected by anti-KGF treatment. When KGF
(100 ng/ml) is substituted for testosterone in the culture medium, SV growth is approximately
50% that observed with an optimal dose of testosterone (10[-7] M). These findings suggest
that KGF is present during a time of active SV morphogenesis and functions as an important
mediator of androgen-dependent development (Alarid, 1994).
Mouse lung development begins when two lung buds sprout from the epithelium of the embryonic
gut. Patterning of the airways is then accomplished by the outgrowth and repetitive branching of
the two lung buds, a process called branching morphogenesis. One of the four fibroblast growth
factor (FGF) receptor genes, FGFR2, is expressed in the epithelium of a number of embryonic
organs, including the lung buds. To block the function of FGFR2 during branching morphogenesis of
the lung, without affecting its function in other embryonic tissues, the human surfactant protein C
promoter was used to target expression of a dominant negative FGFR2 exclusively to lung bud
epithelium in transgenic mice. Newborn mice expressing the transgene are completely normal
except that instead of normally developed lungs they have two undifferentiated epithelial tubes that
extended from the bifurcation of the trachea down to the diaphragm, a defect that results in
perinatal death. Thus, the dominant negative FGF receptor completely blocks airway branching
and epithelial differentiation, without prohibiting outgrowth, establishing a specific role for FGFs in
branching morphogenesis of the mammalian lung (Peters, 1994).
Embryonic mouse lung epithelium was separated from its mesenchyme and cultured under
mesenchyme-free conditions. When covered with Matrigel, the cultured epithelium undergoes
branching morphogenesis in medium containing acidic fibroblast growth factor (aFGF), in which
the epithelial cells construct a simple columnar cell layer forming a lumen, as seen in normal
development. The epithelial growth and branching morphogenesis induced by aFGF is
completely inhibited by an antibody against aFGF. Heparin causes extra epithelial growth in
cooperation with aFGF, but its use results in luminal expansion instead of enhanced branching.
Basic FGF induces abnormal morphogenesis of the epithelium, though the lumen formed is lined
by a simple columnar cell layer. Epidermal growth factor can not maintain epithelial cell growth,
and the epithelium becomes a smaller and smoother ball than that at the start of cultivation. When
covered with a collagen gel instead of Matrigel, the epithelium remains in its initial form in the presence of aFGF, neither
newly branching nor becoming a smooth ball. These results show that the
epithelium of lung rudiments is able to branch under mesenchyme-free culture conditions in
which a basement membrane matrix and aFGF are substituted for the mesenchyme (Nogawa, 1995).
During mouse lung morphogenesis, the distal mesenchyme regulates the growth and
branching of adjacent endoderm. Fibroblast growth factor 10
(Fgf10) is expressed dynamically in the mesenchyme adjacent to the distal buds from
the earliest stages of lung development. The temporal and spatial patterns of gene
expression suggest that Fgf10 plays a role in directional outgrowth and possibly
induction of epithelial buds, and that positive and negative regulators of Fgf10 are
produced by the endoderm. In transgenic lungs overexpressing Shh in the endoderm,
Fgf10 transcription is reduced, suggesting that high levels of SHH downregulate
Fgf10. Addition of FGF10 to embryonic day 11.5 lung tissue (endoderm plus
mesenchyme) in Matrigel or collagen gel culture elicits a cyst-like expansion of the
endoderm after 24 hours. In Matrigel, but not collagen, this is followed by extensive
budding after 48-60 hours. This response involves an increase in the rate of
endodermal cell proliferation. The activity of FGF1, FGF7 and FGF10 was also tested
directly on isolated endoderm in Matrigel culture. Under these conditions, FGF1 elicits
immediate endodermal budding, while FGF7 and FGF10 initially induce expansion of
the endoderm. However, within 24 hours, samples treated with FGF10 give rise to
multiple buds, while FGF7-treated endoderm never progress to bud formation, at all
concentrations of factor tested. Although exogenous FGF1, FGF7 and FGF10 have
overlapping activities in vitro, their in vivo expression patterns are quite distinct in
relation to early branching events. It is concluded that during early lung development,
localized sources of FGF10 in the mesoderm regulate endoderm proliferation and bud
outgrowth (Bellusci, 1997).
The interactions between fibroblast growth factors (FGF) and their receptors have important roles in
mediating mesenchymal-epithelial cell interactions during embryogenesis. In particular, Fgf10 is
predicted to function as a regulator of brain, lung and limb development on the basis of its
spatiotemporal expression pattern in the developing embryo. To define the role of Fgf10,
Fgf10-deficient mice were generated. Fgf10-/- mice die at birth due to the lack of lung development. Tracheae are
formed, but subsequent pulmonary branching morphogenesis is disrupted. In addition, mutant mice
have complete truncation of the fore- and hind-limbs. In Fgf10-/- embryos, limb bud formation is
initiated but outgrowth of the limb buds does not occur; however, formation of the clavicles is not
affected. Analysis of the expression of marker genes in the mutant limb buds indicates that the apical
ectodermal ridge and the zone of polarizing activity do not form. Thus, Fgf10 serves as an essential regulator of lung and limb formation (Sekine, 1999).
Morphogenesis of the mouse lung involves reciprocal
interactions between the epithelial endoderm and the
surrounding mesenchyme, leading to an invariant early
pattern of branching that forms the basis of the respiratory
tree. There is evidence that Fgf10 and Bmp4,
expressed in the distal mesenchyme and endoderm,
respectively, play important roles in branching
morphogenesis. To examine these roles in more detail, an in vitro culture system has been exploited in which isolated
endoderm is incubated in Matrigel substratum with Fgf-loaded
beads. In addition, a Bmp4 lacZ line of
mice was used in which lacZ faithfully reports Bmp4 expression.
Analysis of lung endoderm in vivo shows a dynamic pattern
of Bmp4 lacZ expression during bud outgrowth, extension
and branching. In vitro, Fgf10 induces both proliferation
and chemotaxis of isolated endoderm, whether it is derived
from the distal or proximal lung. Moreover, after 48 hours,
Bmp4 lacZ expression is upregulated in the endoderm closest
to the bead. Addition of 30-50 ng/ml of exogenous purified
Bmp4 to the culture medium inhibits Fgf-induced budding
or chemotaxis, and inhibits overall proliferation. By
contrast, the Bmp-binding protein Noggin enhances Fgf-induced
morphogenesis. Based on these and other results,
a model is proposed for the combinatorial roles of Fgf10
and Bmp4 in branching morphogenesis of the lung (Weaver, 2000).
A model is presented for the dynamic interaction of growth factors in lung
bud morphogenesis. Throughout early development Shh is expressed
in the endoderm and patched1 in the adjacent
mesoderm. (1) Shortly after initiation of bud growth, Fgf10 is transcribed
at high levels in distal mesenchyme but only very low levels of
Bmp4 expression are seen in the endoderm. Studies in
transgenic embryos support the hypothesis that one function of Shh
is to promote proliferation of the mesenchyme through Ptc and
possibly Gli-dependent pathway(s). Shh may also downregulate
Fgf10 expression. Present
results suggest that Fgf10 promotes both the proliferation
of the endoderm and its outward movement. (2) As
bud outgrowth continues, endodermal Bmp4 expression increases.
Meanwhile, Fgf10 expression gradually decreases at the tip but is
upregulated laterally, in this case asymmetrically, by unknown
mechanisms. (3) As the Fgf10 expression domain moves laterally, it
overlies proximal endoderm. These results suggest that a lateral bud
can only be induced where the level of Bmp4 falls below a threshold. (4) Before undergoing dichotomous branching, the distal
endoderm expresses such high levels of Bmp4 that forward
movement stops. (5) It is hypothesized that the mechanism that
regulates Fgf10 at the tip now drives expression laterally and
symmetrically, leading to the outgrowth of two new buds. The cycle
of outgrowth, promotion of mesenchymal proliferation and
endoderm movement begins again (Weaver, 2000).
Studies in Drosophila and chick have shown that members of the Sprouty
family are inducible negative regulators of growth factors that act through tyrosine kinase receptors. Fibroblast Growth Factor 10 (FGF10) is a key positive regulator of lung branching morphogenesis. Direct evidence is provided
that mSprouty2 is dynamically expressed in the peripheral endoderm in embryonic lung and is downregulated in the clefts between new
branches at E12.5. mSprouty2 is expressed in a domain restricted in time and space, adjacent to that of Fgf10 in the
peripheral mesenchyme. By E14.5, Fgf10 expression is restricted to a narrow domain of mesenchyme along the extreme edges of the
individual lung lobes, whereas mSprouty2 is most highly expressed in the subjacent epithelial terminal buds. FGF10 beads upregulate the
expression of mSprouty2 in adjacent epithelium in embryonic lung explant culture. Lung cultures treated with exogenous FGF10 show
greater branching and higher levels of mSpry2 mRNA. Conversely, Fgf10 antisense oligonucleotides reduce branching and decrease
mSpry2 mRNA levels. However, treatment with exogenous FGF10 or antisense Fgf10 does not change Shh and FgfR2 mRNA levels in the
lungs. Sprouty2 function during lung development was investigated by using two different but complementary approaches. The targeted over-expression of mSprouty2 in the peripheral lung epithelium in vivo, using the Surfactant Protein C promoter, results in a low level of
branching, lung lobe edges abnormal in appearance and the inhibition of epithelial proliferation. Transient high-level overexpression of
mSpry2 throughout the pulmonary epithelium by intra-tracheal adenovirus microinjection also results in a low level of branching. These
results indicate that mSPROUTY2 functions as a negative regulator of embryonic lung morphogenesis and growth (Mailleux, 2001).
Mammalian lung develops as an evagination of ventral gut endoderm into the underlying mesenchyme. Iterative epithelial branching, regulated by the surrounding mesenchyme, generates an elaborate network of airways from the initial lung bud. Fibroblast growth factors (FGFs) often mediate epithelial-mesenchymal interactions and mesenchymal Fgf10 is essential for epithelial branching in the developing lung. However, no FGF has been shown to regulate lung mesenchyme. In embryonic lung, Fgf9 is detected in airway epithelium and visceral pleura at E10.5, but is restricted to the pleura by E12.5. Mice homozygous for a targeted disruption of Fgf9 exhibit lung hypoplasia and early postnatal death. Fgf9-/- lungs exhibit reduced mesenchyme and decreased branching of airways, but show significant distal airspace formation and pneumocyte differentiation. These results suggest that Fgf9 affects lung size by stimulating mesenchymal proliferation. The reduction in the amount of mesenchyme in Fgf9-/- lungs limits expression of mesenchymal Fgf10. A model whereby FGF9 signaling from the epithelium and reciprocal FGF10 signaling from the mesenchyme coordinately regulate epithelial airway branching and organ size during lung embryogenesis (Colvin, 2001b).
Lineage formation in the lung mesenchyme is poorly understood. Using a transgenic mouse line expressing LacZ under the control of Fgf10 regulatory sequences, the pool of Fgf10-positive cells in the distal lung mesenchyme has been shown to contain progenitors of the parabronchial smooth muscle cells. Fgf10 gene expression is slightly repressed in this transgenic line. This allowed creation of a hypomorphic Fgf10 phenotype by expressing the LacZ transgene in a heterozygous Fgf10 background. Hypomorphic Fgf10 mutant lungs display a decrease in ß-galactosidase-positive cells around the bronchial epithelium associated with an accumulation of ß-galactosidase-expressing cells in the distal mesenchyme. This correlates with a marked reduction of alpha smooth muscle actin (SMA) expression, thereby demonstrating that FGF10 is mostly required for the entry of mesenchymal cells into the parabronchial smooth muscle cell lineage. The failure of exogenous FGF10 to phosphorylate its known downstream targets ERK and AKT in lung mesenchymal cultures strongly suggests that FGF10 acts indirectly on the progenitor population via an epithelial intermediate. This study provides support for a role of epithelial BMP4 in mediating the formation of parabronchial smooth muscle cells (Mailleux, 2005).
The results indicate a decrease in Bmp4 expression in
Fgf10LacZ/– embryos. This reduction in Bmp4
expression seems to primarily occur in the epithelium. These results are
consistent with previous reports showing that FGF10 upregulates epithelial
Bmp4 transcription. Overexpression of Bmp4 in the distal lung
epithelium using the surfactant protein C promoter leads to ectopic expression
of alpha-SMA in the distal mesenchyme. While addition of recombinant SHH
induces alpha-SMA expression on isolated lung mesenchymal explants, overexpression of Shh in the distal lung epithelium in vivo does
not modify alpha-SMA expression. This may be explained by the lack of
upregulation of Bmp4 in the epithelium or the mesenchyme upon
overexpression of Shh in vivo, by
contrast to the induction of Bmp4 expression by SHH in vitro (Mailleux, 2005).
Consistent with a major role for Bmp4 in SMC differentiation,
recombinant BMP4 induces alpha-SMA expression in lung mesenchyme explants in
vitro after 48 hours of culture. These results strongly suggest that BMP4
induces SMC formation by acting directly on the mesenchyme. It is therefore
proposed that FGF10 expressed by the distal mesenchyme may contribute to
parabronchial SMC formation via the upregulation of BMP4 synthesis by the
epithelium. The failure to induce alpha-SMA expression in all cells can be
explained by the presence of other cell types in the mesenchymal explants,
e.g. the endothelial cells. In addition, these explants also contain a layer
of mesothelium, producing FGF9, which has been shown to prevent the
differentiation of the smooth muscle cells (Mailleux, 2005).
FGF and neural crest A study of the molecules noggin and fibroblast growth factor (FGF) and FGF's receptor in the induction of the prospective neural crest in
Xenopus laevis embryos has been carried out, using the expression of the gene Xslug as a marker for the neural crest.
When a truncated FGF receptor (XFD) is expressed ectopically in order to block FGF signaling, Xslu expression is inhibited. The
effect of XFD on Xslu is specific and can be reversed by the coinjection of the wild-type FGF receptor (FGFR). Inhibition of
Xslu expression by XFD is not a consequence of neural plate inhibition, as was shown by analyzing Xsox-2 expression. Xslu induction is inhibited when
ectoderm expressing XFD is transplanted into the prospective neural fold region of embryos. The
neural crest can also be induced by an interaction between neural plate and epidermis. Since this induction is suppressed by the presence
of XFD in the neural plate, and not in the epidermis, it suggests that the neural crest is induced by FGF from the epidermis. However,
treatment of neural plate with FGF is not able to induce Xslug expression, showing that in addition to FGF, other non-FGF factors
are also required. It has been suggested that the ectopic ventral expression of Xslu produced by overexpression of noggin
mRNA results from an interaction of noggin with a ventral signal. Overexpression of XFD inhibits this effect, suggesting that FGF
could be one component involved in this ventral signaling. Overexpression of FGFR produces a remarkable increase in the expression
of Xslu in the posterior neural folds and around the blastopore. Injections in different blastomeres of the embryo suggest that the
target cells of this effect are the ventral cells. A model in which the induction of the neural crests at the border of
the neural plate requires functional FGF signaling, which possibly interacts with a neural inducer such as noggin (Mayor, 1997).
The cranial neural crest gives rise to most of the skeletal tissues of the skull. Matrix-mediated tissue interactions have been implicated in the skeletogenic differentiation of crest cells, but little is known of the role that growth factors might play in this process. The discovery that mutations in fibroblast growth factor receptors (FGFRs) cause the major craniosynostosis syndromes implicates FGF-mediated signaling in the skeletogenic differentiation of the cranial neural crest. In vitro, mesencephalic neural crest cells respond to exogenous FGF2 in a dose-dependent manner, with 0.1 and 1 ng/ml causing enhanced proliferation, and 10 ng/ml inducing cartilage differentiation. In longer-term cultures, both endochondral and membrane bone are formed. FGFR1, FGFR2 and FGFR3 are all detectable by immunohistochemistry in the mesencephalic region, with particularly intense expression at the apices of the neural folds from which the neural crest arises. FGFRs are also expressed by subpopulations of neural crest cells in culture. Collectively, these findings suggest that FGFs are involved in the skeletogenic differentiation of the cranial neural crest (Sarkar, 2001).
The neural crest is a unique cell population induced at the lateral border of
the neural plate. Neural crest is not produced at the anterior border of the
neural plate, which is fated to become forebrain. The roles of BMPs, FGFs,
Wnts, and retinoic acid signaling in neural crest induction were analyzed by
using an assay developed for investigating the posteriorization of the neural
plate. Using specific markers for the anterior neural plate border and the
neural crest, the posterior end of early neurula embryos, was shown to be able to transform the anterior neural plate border into neural crest cells. In addition, tissue expressing anterior neural plate markers, induced by an intermediate level of BMP activity, is transformed into neural crest by posteriorizing signals. This transformation is mimicked by bFGF, Wnt-8, or retinoic acid treatment and is also inhibited by expression of the dominant negative forms of
the FGF receptor, the retinoic acid receptor, and Wnt signaling molecules. The
transformation of the anterior neural plate border into neural crest cells is
also achieved in whole embryos, by retinoic acid treatment or by use of a
constitutively active form of the retinoic acid receptor. By analyzing the
expression of mesodermal markers and various graft experiments, the expression
of the mutant retinoic acid receptor has been shown to directly affect the ectoderm. A two-step model is proposed for neural crest induction. Initially, BMP levels intermediate to those required for neural plate and epidermal
specification induce neural folds with an anterior character along the entire
neural plate border. Subsequently, the most posterior region of this anterior
neural plate border is transformed into the neural crest by the posteriorizing
activity of FGFs, Wnts, and retinoic acid signals. A unifying model is discussed where lateralizing and posteriorizing signals are presented as two stages of the same inductive process required for neural crest induction (Villanueva, 2002).
It is suggested that at the early-gastrula stage, a gradient of BMP
activity is established in the ectoderm, which specifies the
neural plate, the neural plate border, and the epidermis at
progressively higher concentrations of BMP. The
neural plate border, induced at a precise location within the
mediolateral axis of the ectoderm, has an anterior character. Later, between early and midgastrula stage, signals presumably originating from the ventrolateral
mesoderm transform a region of the anterior neural plate
border into prospective neural crest cells. A role for
this mesoderm in neural crest induction has been shown. The spread of
these molecules from the mesoderm into the ectoderm consequently locates them only in large animal caps, explaining why the neural crest was not induced when small animal caps were used. These signals could correspond
to Wnt8 and eFGF, since it is known that they are
expressed in the ventrolateral mesoderm, and could correspond
to lateralizing signals. However, the neural crest is not
specified at this stage; this does not occur until the end of
gastrulation. Thus, additional signals are required for the final induction
of the neural crest. Finally, as gastrulation proceeds, the
ventrolateral mesoderm becomes localized to the posterior
region of the embryo, where it continues to produce Wnt8,
eFGF, and possibly retinoic acid, as well as another, as yet
unknown, posteriorizing agent(s) that generates an anterior-posterior gradient of these morphogenes. This gradient would be required for the final specification of the neural crest in the most posterior region of the neural plate border. Thus, the lateral-posterior regions of the neural plate border receive the lateralizing/posteriorizing signals for an extended period of time, finally specifying them as neural crest. In contrast, the anterior neural plate border does not receive such signals or these are inhibited by other agents produced by the anterior regions of the embryo, such as cerberus or dkk1, two known Wnts inhibitors, and, as a consequence, this border region does not develop as neural crest cells. It is tempting to speculate that the anterior-posterior differences within the neural crest could be controlled by a similar mechanism (Villanueva, 2002).
The vertebrate head skeleton is derived in part from neural crest cells, which physically interact with head ectoderm, mesoderm and endoderm to shape the pharyngeal arches. The cellular and molecular nature of these interactions is poorly understood, and the function of endoderm in this process has been explored. By genetic ablation and reintroduction of endoderm in zebrafish, it has been shown that endoderm is required for the development of chondrogenic neural crest cells, including their identity, survival and differentiation into arch cartilages. Using a genetic interference approach, Fgf3 has been identified as a critical component of endodermal function that allows the development of posterior arch cartilages. Together, these results reveal that the endoderm provides differential cues along the anteroposterior axis to control ventral head skeleton development and demonstrate that this function is mediated in part by Fgf3 (David, 2002).
In vertebrates, endoderm and mesoderm are specified by Nodal-related signals, members of the transforming growth factor ß superfamily. Nodal activities are mediated by type I TGFß receptors ALK4 and ALK7 in mammals and most likely by their relative, Taram-A (tar) in zebrafish. Activation of the Nodal pathway by expression of a constitutively active version of tar (tar*) leads to a respecification of early zebrafish blastomeres to an endodermal fate, consistent with the model that high levels of Nodal signaling are sufficient to direct cells to become endoderm. Downstream of nodal signaling, endoderm formation further involves the homeobox transcription factors Mixer/Bonnie and clyde (bon) and the recently identified Sox-related factor Casanova (cas). Zebrafish embryos in which Nodal signals are inactive develop neither endoderm nor mesoderm while, in contrast, bon or cas mutants form mesoderm but little or no endoderm. In particular, cas mutants lack expression of all endodermal markers and derivatives. Interestingly, cas embryos are not rescued by activation of Nodal-related ligands or of the Tar cascade, consistent with its proposed function downstream. However, they provide a permissive environment for endoderm development since tar*-activated wild-type blastomeres can autonomously restore endoderm formation when grafted into cas embryos (David, 2002 and references therein).
cas and bon mutants were used to test whether or not pharyngeal endoderm is required for formation of the pharyngeal skeleton. Both mutants lack most of the ventral cartilage of the head skeleton and these gene functions are required after cephalic neural crest migration, when crest normally contacts the endoderm. Neural crest has been fate mapped in cas and it has been show that, in the absence of endoderm, cephalic neural crest cells remain as a cluster on the surface of the yolk sac and down regulate the expression of pre-chondrogenic markers. cas neural crest cells still have the ability to develop into cartilage when transplanted into wild-type embryos, showing that the cas gene is not required autonomously in neural crest cells but rather in their environment. Furthermore, endoderm can rescue head cartilage formation when reintroduced by grafting wild-type tar*-injected cells into cas embryos. Finally, FGF has been implicated in signaling from endoderm to neural crest by demonstrating that endodermal expression of FGF3 is specifically required for the formation of the posterior, branchial arches. Altogether, these results demonstrate a requirement for pharyngeal endoderm and FGF signaling in the control of head neural crest fates and cartilage induction, and identify FGF3 as the first endodermal signal with an AP restricted function in the pharyngeal region (David, 2002).
Deletion of chromosome 22q11, the most common microdeletion detected in humans, is associated with a life-threatening array of birth defects. Although 90% of affected individuals share the same three megabase deletion, their phenotypes are highly variable and includes craniofacial and cardiovascular anomalies, hypoplasia or aplasia of the thymus with associated deficiency of T cells, hypocalcemia with hypoplasia or aplasia of the parathyroids, and a variety of central nervous system abnormalities. Because ablation of neural crest in chicks produces many features of the deletion 22q11 syndrome, it has been proposed that haploinsufficiency in this region impacts neural crest function during cardiac and pharyngeal arch development. Few factors required for migration, survival, proliferation and subsequent differentiation of pharyngeal arch neural crest and mesoderm-derived mesenchyme into their respective cardiovascular, musculoskeletal, and glandular derivatives have been identified. However, the importance of epithelial-mesenchymal interactions and pharyngeal endoderm function is becoming increasingly clear. Fibroblast growth factor 8 is a signaling molecule expressed in the ectoderm and endoderm of the developing pharyngeal arches and known to play an important role in survival and patterning of first arch tissues. A dosage-sensitive requirement has been demonstrated for FGF8 during development of pharyngeal arch, pharyngeal pouch and neural crest-derived tissues. FGF8 deficient embryos have lethal malformations of the cardiac outflow tract, great vessels and heart due, at least in part, to failure to form the fourth pharyngeal arch arteries, altered expression of Fgf10 in the pharyngeal mesenchyme, and abnormal apoptosis in pharyngeal and cardiac neural crest. The Fgf8 mutants display the complete array of cardiovascular, glandular and craniofacial phenotypes seen in human deletion 22q11 syndromes. This represents the first single gene disruption outside the typically deleted region of human chromosome 22 to fully recapitulate the deletion 22q11 phenotype. FGF8 may operate directly in molecular pathways affected by deletions in 22q11 or function in parallel pathways required for normal development of pharyngeal arch and neural crest-derived tissues. In either case, Fgf8 may function as a modifier of the 22q11 deletion and contribute to the phenotypic variability of this syndrome (Frank, 2002).
Tbx1 haploinsufficiency causes aortic arch abnormalities in mice because of early growth and remodeling defects of the fourth pharyngeal arch arteries. The function of Tbx1 in the development of these arteries is probably cell non-autonomous, since the gene is not expressed in structural components of the artery but in the surrounding pharyngeal endoderm. It is hypothesized that Tbx1 may trigger signals from the pharyngeal endoderm directed to the underlying mesenchyme. The expression patterns of Fgf8 and Fgf10, which partially overlap with Tbx1 expression pattern, are altered in Tbx1–/– mutants. In particular, Fgf8 expression is abolished in the pharyngeal endoderm. To understand the significance of this finding for the pathogenesis of the mutant Tbx1 phenotype, Tbx1 and Fgf8 mutants were crossed. Double heterozygous Tbx1+/–;Fgf8+/– mutants present with a significantly higher penetrance of aortic arch artery defects than do Tbx1+/–;Fgf8+/+ mutants, while Tbx1+/+;Fgf8+/– animals are normal. Fgf8 mutation increases the severity of the primary defect caused by Tbx1 haploinsufficiency, i.e. early hypoplasia of the fourth pharyngeal arch arteries, consistent with the time and location of the shared expression domain of the two genes. Hence, Tbx1 and Fgf8 interact genetically in the development of the aortic arch. These data provide the first evidence of a genetic link between Tbx1 and FGF signaling, and the first example of a modifier of the Tbx1 haploinsufficiency phenotype. It is speculated that the FGF8 locus might affect the penetrance of cardiovascular defects in individuals with chromosome 22q11 deletions involving TBX1 (Vitelli, 2002).
An analysis is presented of cardiovascular and pharyngeal arch development in mouse embryos hypomorphic for Fgf8. Fgf8 compound heterozygous (Fgf8neo/–) embryos have been generated. Although early analysis has demonstrated that some of these embryos have abnormal left-right (LR) axis specification and cardiac looping reversals, the number and type of cardiac defects present at term suggests an additional role for Fgf8 in cardiovascular development. Most Fgf8neo/– mutant embryos survive to term with abnormal cardiovascular patterning, including outflow tract, arch artery and intracardiac defects. In addition, these mutants have hypoplastic pharyngeal arches, small or absent thymus and abnormal craniofacial development. Neural crest cells (NCCs) populate the pharyngeal arches and contribute to many structures of the face, neck and cardiovascular system, suggesting that Fgf8 may be required for NCC development. Fgf8 is expressed within the developing pharyngeal arch ectoderm and endoderm during NCC migration through the arches. Analysis of NCC development in Fgf8neo/– mutant embryos demonstrates that NCCs are specified and migrate, but undergo cell death in areas both adjacent and distal to where Fgf8 is normally expressed. This study defines the cardiovascular defects present in Fgf8 mutants and supports a role for Fgf8 in development of all the pharyngeal arches and in NCC survival (Abu-Issa, 2002).
At the border of the neural plate, the induction of the neural crest can be achieved by interactions with the epidermis, or with the underlying mesoderm. Wnt signals are required for the inducing activity of the epidermis in chick and amphibian embryos. The molecular mechanisms of neural crest induction by the mesoderm has been analyzed in Xenopus embryos. Using a recombination assay, it has been shown that prospective paraxial mesoderm induces a panel of neural crest markers (Slug, FoxD3, Zic5 and Sox9), whereas the future axial mesoderm only induces a subset of these genes. This induction is blocked by a dominant negative (dn) form of FGFR1. However, neither dnFGFR4a nor inhibition of Wnt signaling prevents neural crest induction in this system. Among the FGFs, FGF8 is strongly expressed by the paraxial mesoderm. FGF8 is sufficient to induce the neural crest markers FoxD3, Sox9 and Zic5 transiently in the animal cap assay. In vivo, FGF8 injections also expand the Slug expression domain. This suggests that FGF8 can initiate neural crest formation and cooperates with other DLMZ-derived factors to maintain and complete neural crest induction. In contrast to Wnts, eFGF or bFGF, FGF8 elicits neural crest induction in the absence of mesoderm induction and without a requirement for BMP antagonists. In vivo, it is difficult to dissociate the roles of FGF and WNT factors in mesoderm induction and neural patterning. In most cases, effects on neural crest formation are parallel to altered mesoderm or neural development. However, neural and neural crest patterning can be dissociated experimentally using different dominant-negative manipulations: while Nfz8 blocks both posterior neural plate formation and neural crest formation, dnFGFR4a blocks neural patterning without blocking neural crest formation. These results suggest that different signal transduction mechanisms may be used in neural crest induction, and anteroposterior neural patterning (Monsoro-Burq, 2003).
The chordin/Bmp system provides one of the best examples of extracellular signaling regulation in animal development. Chordin homozygous mutant mice, generated by targeted mutagenesis, show, at low penetrance, early lethality and a ventralized gastrulation phenotype. The mutant embryos that survive die perinatally, displaying an extensive array of malformations that encompass most features of DiGeorge and Velo-Cardio-Facial syndromes in humans. Chordin secreted by the mesendoderm is required for the correct expression of Tbx1 and other transcription factors involved in the development of the pharyngeal region. The chordin mutation provides a mouse model for head and neck congenital malformations that frequently occur in humans and suggests that chordin/Bmp signaling may participate in their pathogenesis (Bachiller, 2003).
To study the interaction of Chrd with genes known to cause
DiGeorge or DiGeorge-like phenotypes in mice, the expression of
Tbx1 and Fgf8 was analyzed in Chrd mutant embryos. Tbx1 is a member of the T-box family of transcription factors. It maps within the DGS/VCFS 22q11 microdeletion in humans
and has been shown to cause DiGeorge-like phenotype upon inactivation
in mice. Expression of Tbx1 is altered in
Chrd-/- embryos. In wild-type E7.5 animals, Tbx1 is expressed in the foregut (future pharyngeal endoderm) and head mesoderm. At this stage, mutant littermates showed a clear reduction in the levels of Tbx1 expression in the same areas. The reduction in Tbx1 mRNA is equally clear in the pharyngeal region of Chrd homozygous embryos at E8.0, E8.5 and E9.0. Transverse histological sections show that at the cellular level the abundance of Tbx1 transcripts is drastically reduced in endoderm, both in the pharynx and foregut up to the level of the hepatic diverticulum. Diminution in the concentration of Tbx1 mRNA
is also evident in mesoderm, including head, splanchnic and
somatic mesoderm in the peripharyngeal region. In addition, Tbx1
expression at E9 in the mesodermal core of the first pharyngeal arch is
diffuse, extending to most of the arch, and Tbx1 transcripts are
absent from the otic vesicle (Bachiller, 2003).
Fgf8 is a secreted growth factor expressed in a variety of
tissues, including the pharyngeal endoderm and neighboring mesoderm.
During early development, Fgf8 is required for gastrulation and the establishment of the left/right axis of symmetry. At later stages of Fgf8 is required for limb and craniofacial development. Recent experiments have shown that mice with reduced Fgf8 activity present a spectrum of cardiovascular and pharyngeal defects that closely mimic DiGeorge syndrome. In addition, Fgf8 expression is abolished in the pharyngeal endoderm of Tbx1-/- mutants and both genes interact genetically during the differentiation of the pharyngeal arch arteries. At E9, Fgf8 expression in Chrd mutants is normal in the mid-hindbrain isthmus, frontonasal prominence and tail. However, in pharyngeal endoderm, Fgf8 transcript levels are drastically reduced. The reduction of Tbx1 and Fgf8 expression in Chrd-/- embryos suggest that both genes act downstream of Chrd in the same regulatory pathway. These experiments do not determine whether Chrd is required for the maintenance or for the induction of Tbx1 and Fgf8 in the pharynx and neighboring tissues. To test whether Chrd can induce Tbx1 and Fgf8, Chrd mRNA (50 pg) was injected into the ventral region of Xenopus embryos at the four-cell stage. Ventral marginal zone (VMZ) explants were dissected at early gastrula, cultured until sibling embryos reached early neurula stage, and analyzed by RTPCR. Tbx1 and Fgf8 mRNAs are expressed at high levels in whole embryos and dorsal marginal zone (DMZ) explants at this stage, and at low levels in VMZ explants. Upon microinjection, Chrd mRNA increases the levels of Tbx1 and Fgf8 in VMZ. In situ hybridization of microinjected Xenopus embryos confirmed that the Tbx1 transcripts induced by Chrd mRNA are located in pharyngeal endoderm. It is concluded that Chrd, a Bmp antagonist, can induce Tbx1 and Fgf8 expression in Xenopus embryos, and is required for full expression of these genes in the pharyngeal region of the mouse embryo (Bachiller, 2003).
Fibroblast growth factor (Fgf) proteins are important regulators of pharyngeal arch development. Analyses of Fgf8 function in chick and mouse and Fgf3 function in zebrafish have demonstrated a role for Fgfs in the differentiation and survival of postmigratory neural crest cells (NCC) that give rise to the pharyngeal skeleton. An earlier, essential function for Fgf8 and Fgf3 is described in zebrafish in regulating the segmentation of the pharyngeal endoderm into pouches. Using time-lapse microscopy, it has been shown that pharyngeal pouches form by the directed lateral migration of discrete clusters of endodermal cells. In animals doubly reduced for Fgf8 and Fgf3, the migration of pharyngeal endodermal cells is disorganized and pouches fail to form. Transplantation and pharmacological experiments show that Fgf8 and Fgf3 are required in the neural keel and cranial mesoderm during early somite stages to promote first pouch formation. In addition, animals doubly reduced for Fgf8 and Fgf3 have severe reductions in hyoid cartilages and the more posterior branchial cartilages. By examining early pouch and later cartilage phenotypes in individual animals hypomorphic for Fgf function, it was found that alterations in pouch structure correlate with later cartilage defects. A model is presented in which Fgf signaling in the mesoderm and segmented hindbrain organizes the segmentation of the pharyngeal endoderm into pouches. Moreover, it is argued that the Fgf-dependent morphogenesis of the pharyngeal endoderm into pouches is critical for the later patterning of pharyngeal cartilages (Crump, 2004).
Fgfs direct embryogenesis of several organs, including the lung, limb, and anterior pituitary. Male-to-female sex reversal occurs in mice lacking Fibroblast growth factor 9 (Fgf9), demonstrating a novel role for FGF signaling in testicular embryogenesis. Fgf9-/- mice also exhibit lung hypoplasia and die at birth. Reproductive system phenotypes range from testicular hypoplasia to complete sex reversal, with most Fgf9-/- XY reproductive systems appearing grossly female at birth. Fgf9 appears to act downstream of Sry to stimulate mesenchymal proliferation, mesonephric cell migration, and Sertoli cell differentiation in the embryonic testis. While Sry is found only in some mammals, Fgfs are highly conserved. Thus, Fgfs may function in sex determination and reproductive system development in many species (Colvin, 2001a).
Male and female mouse gonads at embryonic day 11.0 (E11.0) are morphologically identical in different gonads medial to each mesonephros. By E13.5, the testis is twice the size of the ovary and exhibits morphologically complex testicular cords. Three male-specific events are known to direct early testiculogenesis: cell proliferation, cell migration, and testicular cord formation. An increase in proliferation at the coelomic lining of the gonad (the coelomic epithelium) occurs between E11.3 and E12.1. This proliferation gives rise to Sertoli cells (a supporting cell lineage) early on and to interstitial cells throughout this period. Cells contributing to the interstitium, including vascular endothelial cells and peritubular myoid cells, migrate into the testis from the mesonephros and are required for testicular cord formation. Testicular cord development begins at about E12.0 with clustering of Sertoli and germ cells, followed by rearrangement so that Sertoli cells surround the germ cells. Testicular cords isolate male germ cells from interstitial cells, and prevent male germ cells from entering meiosis. Ovarian germ cells, which are not enclosed by supporting cells, progress by E13.5 to the first meiotic division (Colvin, 2001a and references therein).
The testis regulates further male reproductive development. Until E13.5, both sexes have Mullerian and Wolffian ducts in each mesonephros. Sertoli cells produce Mullerian inhibiting substance (MIS). MIS causes regression of the Mullerian ducts, which, in the absence of MIS, form the oviducts, uterus, and upper vagina. Interstitial Leydig cells produce testosterone, which induces formation of Wolffian duct derivatives, including the epididymis, vas deferens, and seminal vesicles. In females, the absence of testicular MIS and testosterone results in development of Mullerian structures and regression of the Wolffian ducts. Targeted deletion of Mis or its receptor results in development of Mullerian structures in XY mice (Colvin, 2001a and references therein).
Testicular expression of Sry, a transcription factor gene on the Y chromosome, is essential for increased proliferation in, and mesonephric cell migration into, the mouse testis. Sry is expressed in mouse testis between E10.5 and E12.5 and is necessary and sufficient to induce male development. Deletion of Sry generates XY ovaries and mice with a female phenotype, and addition of an Sry transgene generates XX males. A potential downstream target of Sry is Sox9, an autosomal transcription factor expressed in Sertoli cells. Mutations in SRY and SOX9 have been identified in human XY females with gonadal dysgenesis (Colvin, 2001a and references therein).
Fgf9 appears to act downstream of Sry, but the signaling relationship between Sox9 and Fgf9 is unclear. Sry is essential for each mode of mesenchymal expansion in the early testis: proliferation and mesonephric cell migration. Thus, reduced mesenchyme in Fgf9-/- XY gonads suggests that Sry and Fgf9 act along the same developmental pathway. Testicular Fgf9 expression begins shortly after the onset of Sry expression at E10.5, consistent with Fgf9 acting downstream of Sry. Some Fgf9-/- XY gonads exhibit aberrant Sox9 expression, but Fgf9 is not required to induce Sox9 expression in the testis or to maintain Sox9 expression through E18.5. Analysis of Fgf9 expression in Sox9-deficient gonads would determine if Sox9 is required to induce testicular Fgf9 expression. Unfortunately, Sox9 heterozygous mice die at birth precluding the generation of homozygous mutant embryos, and embryos derived by introducing Sox9 homozygous mutant ES cells into tetraploid blastocysts, die by E11.5. Correlation between testicular cord formation and Sox9 expression in Fgf9-/- XY gonads suggests that Fgf9 may regulate Sox9 expression indirectly by facilitating testicular development (Colvin, 2001a).
Fgf9 affects early steps in testiculogenesis, including Sertoli cell development, gonadal cell proliferation, and mesonephric cell migration. Pre-Sertoli cells originate from multipotential cells in the coelomic epithelium and proliferate at the coelomic epithelium between E11.3-E11.5. Impaired Fgf9-/- Sertoli cell development suggests that Fgf9 could directly induce Sertoli cell specification, proliferation, and/or maintenance of differentiation. Loss of signaling from Sertoli cells could then secondarily impair mesenchymal proliferation and mesonephric cell migration. Full Sertoli cell differentiation probably requires testicular cord formation, and maintenance of Sertoli differentiation may require contact with peritubular myoid cells and the basal lamina. Thus, Fgf9 could also facilitate Sertoli cell differentiation by promoting mesenchymal expansion and testicular cord formation (Colvin, 2001a).
Proliferation at the coelomic epithelium gives rise to Sertoli and interstitial cells during an initial burst of proliferation (E11.3-E11.5), and to interstitial cells after this time. Proliferation below the coelomic epithelium in E12.5 Fgf9-/- XY gonads is reduced relative to controls, indicating that Fgf9 is essential for normal proliferation at this stage. Decreased numbers of Sertoli and interstitial cells are observed in Fgf9-/- gonads by E12.5. This, and the onset of testicular Fgf9 expression between E10.5-E11.5, suggests that Fgf9 may mediate the initial stage of proliferation as well (Colvin, 2001a).
Mesonephric cell migration into the testis at E11.3-E16.5 contributes to interstitial cell populations, including vascular endothelial, myoepithelial, and peritubular myoid cells. Exogenous FGF9 induces mesonephric cell migration into E11.5 XX gonads, suggesting that FGF9 in the early testis could act as a chemotactic factor for mesonephric cells. When mesonephric migration into XX gonads is artificially induced, XX gonads exhibit testicular cord formation and increased Sox9 expression. Conversely, blocking mesonephric cell migration in culture impairs testicular cord formation, indicating that impaired mesonephric cell migration could contribute to Fgf9-/- sex reversal. Analysis of mesonephric cell migration into Fgf9-/- XY gonads will test this hypothesis. Mesonephric cells that migrate into the testis are proliferating, suggesting that one molecular signal could induce both migration and proliferation. In the embryonic lung, FGF10 stimulates both migration and proliferation of epithelial cells (Colvin, 2001a).
Loss of Fgf9 has been shown to result in a block of testis development and a male to female sex-reversed phenotype; however, the function of Fgf9 in sex determination was unknown. Fgf9 is now shown to be necessary for two steps of testis development just downstream of the male sex-determining gene, Sry: (1) for the proliferation of a population of cells that give rise to Sertoli progenitors; and (2) for the nuclear localization of an FGF receptor (FGFR2) in Sertoli cell precursors. The nuclear localization of FGFR2 coincides with the initiation of Sry expression and the nuclear localization of SOX9 during the early differentiation of Sertoli cells and the determination of male fate (Schmahl, 2004).
It has been known for some time that many cell-surface growth factor receptors can accumulate within the nucleus. However, the biological relevance of this event is not known. It has been speculated that nuclear growth factor receptors may act as weak transcription factors, topoisomerases and/or nuclear kinases. Nuclear FGF receptors have been observed in spliceosomes. This is particularly interesting, since several components of the sex-determination pathway (including SRY, SOX9 and the +KTS isoform of WT) have been shown to associate with splicing factors, and have been demonstrated to have splicing activity. Another function of nuclear FGF receptors may be to phosphorylate nuclear substrates; forced nuclear translocation of FGF receptors leads to an increase in the phosphorylation of nuclear proteins, and some activities of the nuclear receptor are abolished by deactivation of the kinase domain. The discovery of the sex-specific subcellular localization of FGFR2 in the nuclei of Sertoli precursors provides a well-characterized biological context in which to study the function of nuclear growth factor receptors. In this context, the transition of FGFR2 from the cell membrane to the nucleus suggests that the nuclear localization of cell-surface receptors is linked to the initiation of cell differentiation. It is not yet clear how proliferation of Sertoli cell precursors in the coelomic epithelium and subsequent commitment to the Sertoli fate are interwoven; however, these findings suggest that FGF signaling may be involved in bridging these two processes essential to testis development (Schmahl, 2004).
Brief exposure of rats to high-dose estrogen during the neonatal period interrupts prostate development in a lobe-specific manner and predisposes the gland to dysplasia with aging, a phenomenon referred to as developmental estrogenization. These effects are initiated through altered steroid receptor expression; however, the immediate downstream targets remain unclear. Developmental expression of Shh-ptc-gli has been shown to be downregulated in the dorsolateral prostate following estrogenization, and this is responsible, in part, for branching deficits observed in that prostatic region specifically. In the present study, the roles were examined of Fgf10 signaling during rat prostate development and as a mediator of the developmental estrogenized phenotype. Fgf10 and Fgf R2iiib localize to the distal signaling center of elongating and branching ducts in separate prostate lobes where they regulate the expression of multiple morphoregulatory genes including Shh, ptc, Bmp7, Bmp4, Hoxb13, and Nkx3.1. Ventral and lateral lobe organ cultures and mesenchyme-free ductal cultures demonstrate a direct role for Fgf10/FgfR2iiib in ductal elongation, branching, epithelial proliferation, and differentiation. Based on these findings, a model is proposed depicting the localized expression and feedback loops between several morphoregulatory factors in the developing prostate that contribute to tightly regulated branching morphogenesis. Similar to Shh-ptc-gli, neonatal estrogen exposure downregulates Fgf10, Fgf R2iiib, and Bmp7 expression in the dorsolateral prostate while ventral lobe expression of these genes is unaffected. Lateral prostate organ culture experiments demonstrate that growth and branching inhibition as well as Fgf10/FgfR2iiib suppression are mediated directly at the prostatic level. Furthermore, exogenous Fgf 10 fully rescues the growth and branching deficits due to estrogen exposure. Together, these studies demonstrate that alterations in Fgf10 signaling are a proximate cause of Shh-ptc-gli and Bmp7 downregulation that together result in branching inhibition of the dorsolateral prostate following neonatal estrogen exposure (Huang, 2005).
In humans and mice, mutations in Hoxa13 cause malformation of limb and genitourinary (GU) regions. In males, one of the most common GU malformations associated with loss of Hoxa13 function is hypospadia, a condition defined by the poor growth and closure of the urethra and glans penis. By examining early signaling in the developing mouse genital tubercle, Hoxa13 has been found to be essential for normal expression of Fgf8 and Bmp7 in the urethral plate epithelium. In Hoxa13GFP-mutant mice, hypospadias occur as a result of the combined loss of Fgf8 and Bmp7 expression in the urethral plate epithelium, as well as the ectopic expression of noggin (Nog) in the flanking mesenchyme. In vitro supplementation with Fgf8 restores proliferation in homozygous mutants to wild-type levels, suggesting that Fgf8 is sufficient to direct early proliferation of the developing genital tubercle. However, the closure defects of the distal urethra and glans can be attributed to a loss of apoptosis in the urethra, which is consistent with reduced Bmp7 expression in this region. Mice mutant for Hoxa13 also exhibit changes in androgen receptor expression, providing a developmental link between Hoxa13-associated hypospadias and those produced by antagonists to androgen signaling. Finally, a novel role for Hoxa13 in the vascularization of the glans penis is also identified (Morgan, 2003).
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