BCL11B is Expressed at all Stages of Incisor Development
BCL11B is expressed in the ectoderm of the first branchial arch at E9.5 and E10.5 and in the molar at all stages of development 
. To determine the function of BCL11B in the developing incisor, we analyzed BCL11B expression on sagittal sections of the mandibular incisor from E11.5 to birth. At initiation (E11.5) and early bud (E12.5) stages, BCL11B was expressed in the thickened epithelium; lower levels of BCL11B were detected in the underlying mesenchyme (). High levels of BCL11B persisted in the dental epithelium at cap stage (E14.5), whereas mesenchymal cells surrounding CLs and the follicle continued to express lower levels (). At early (E16.5) and late (E18.5) bell stages, BCL11B was detected in the lingual epithelium and in the labial OEE, and at lower levels in the papillary mesenchyme surrounding both CLs, dental follicle, SR, and ameloblasts at all stages of differentiation (1). BCL11B was expressed in the tissue surrounding the tip of the incisor and the vestibular lamina, an invagination of the oral epithelium that gives rise to the oral vestibule ().
BCL11B expression during incisor development.
Reduced Epithelial Proliferation between Initiation and Bud Stage in Bcl11b−/− Incisors
The first morphological sign of tooth development is the thickening of the oral epithelium at E11.5. At this stage, wild-type and Bcl11b−/−
incisors were morphologically indistinguishable (; wild-type BrdU index
33.2±4.0%). By E12.5 the mutant epithelium appeared approximately one-half the thickness of the wild-type, and cells at the leading edge of the mutant epithelium were less elongated and poorly polarized (). A 2.7-fold decrease in epithelial proliferation of the Bcl11b−/−
incisor was detected at E12.5 (; wild-type BrdUindex
Epithelial invagination defect in Bcl11b−/− developing incisors between initiation and early bud stage.
Several signaling molecules and transcription factors orchestrate invagination of the epithelium between initiation and early bud stages. For example, BMP4, a critical signaling molecule that regulates tooth initiation and morphogenesis 
, is expressed in the dental epithelium and underlying mesenchyme during the initiation of tooth development at E11.5 (). Bmp4
expression largely shifts to the dental mesenchyme by early bud stage in wild-type mice () 
. Alterations in Bmp4
expression were not detected in Bcl11b−/−
incisors at E11.5 (). However, the Bcl11b−/−
epithelium failed to down-regulate expression of Bmp4
at E12.5 (). The expression patterns of other critical signaling molecules and transcription factors, including Activin
, and Msx1
, were not altered in Bcl11b−/−
incisors at the bud stage (Fig. S2
Altered Development of Bcl11b−/− Incisors at Cap Stage
The wild-type, mandibular incisor is characterized by a cap-like shape of the dental epithelium at E14.5, with an enamel knot in the center and protruding CLs (). The enamel knot is a transitory signaling center that is characterized by minimal proliferation and clearly defined apoptosis 
. In contrast, the CLs are highly proliferative with a low apoptotic index 
. The Bcl11b−/−
incisor exhibited a delay in epithelial invagination and protrusion of both CLs at E14.5 (). BrdU-labeling studies revealed that the incisor epithelium of Bcl11b−/−
mice was hypoproliferative compared to that of wild-type mice (27.6±4.1% and 51.8±2.7% BrdU-positive cells, respectively), whereas mesenchymal proliferation appeared unchanged from controls ().
Alterations in Bcl11b−/− incisor development at cap stage.
Proliferation of the dental epithelium at cap stage is controlled in part by FGF10, which is derived from mesenchymal cells of the dental papilla 
. The Bcl11b−/−
papillary mesenchyme was essentially devoid of Fgf10
transcripts at early E14 and ectopic Fgf10
expression was noted between the dental epithelium and vestibular lamina, the latter of which exhibited impaired invagination (). The delay of initiation of mesenchymal Fgf10
expression may contribute to decreased dental epithelial proliferation, delayed invagination of the dental epithelium, and subsequently decreased size of the mutant incisor.
The expression patterns of Shh
, and Tbx1
, were unaltered in Bcl11b−/−
incisors at cap stage (Fig. S3
Apoptotic cells were predominantly localized in the enamel knot of wild-type incisors at E14.5 (Fig. S4A
). However, very few apoptotic cells were detected in the Bcl11b−/−
enamel knot (Fig. S4B
), consistent with delayed incisor development in Bcl11b−/−
Reduced Size and Disruption of Labial-lingual Asymmetry at Bell Stage in Bcl11b−/− Incisors
Wild-type incisors at E16.5 are characterized by a large labial CL, which contains stem cells that give rise to ameloblasts (). In contrast, the lingual CL of wild-type incisors is relatively smaller, consistent with reduced developmental potential on the lingual side of the incisor (). Bcl11b−/− incisors were reduced in size by approximately half at this stage (compare ) and were characterized by a hypocellular labial CL (compare ), an enlarged lingual CL (compare ), and elongated cells resembling ameloblasts along the length of the lingual epithelium (compare ).
Morphological defects in Bcl11b−/− incisor development at bell stage.
The posterior basal epithelium of the labial CL of Bcl11b−/−
mice was hypoproliferative relative to that of wild-type mice at E16.5 (23.2±4.9% and 42.0±5.0% BrdU-positive cells, respectively; ). However, neither the apoptotic index (Figs. S4C and D
) nor proliferation in the lingual CL () of mutant incisors was significantly different from wild-type.
By E18.5, wild-type incisors developed a large labial (region I, ) and a small lingual () CL. Ameloblast differentiation occurs sequentially in the labial IEE with mitotic pre-ameloblasts (), post-mitotic secretory ameloblasts (), and mature ameloblasts () in regions II, III, and IV, respectively 
. Ameloblasts are not present on the lingual side of the wild-type incisor, but rather a thin layer of non-polarized epithelial cells is found on this aspect of the developing tooth ().
incisors were reduced in size at E18.5 (compare ). However, the mutant lingual CL was enlarged (compare ), and the labial CL was markedly hypoplastic (compare ) to the point of resembling the lingual CL of wild-type mice in both size and morphology (compare ). Mutant ameloblasts were smaller and disorganized at all stages of differentiation along the labial epithelium (compare ), and an abnormal layer of polarized cells resembling ameloblasts was observed in the anterior region of the lingual epithelium (compare ). Finally, Bcl11b−/−
incisors were approximately one-half the length of wild-type incisors at birth and correspondingly narrower across the entire tooth (Fig. S5
These results demonstrate that BCL11B plays an important role in development of the labial CL and differentiation of ameloblasts, while simultaneously suppressing these processes on the lingual side of the incisor.
Delay in Ameloblast Development and Ectopic Formation of Lingual Ameloblast-like Cells in Bcl11b−/− Incisors
To determine if labial ameloblasts and lingual ameloblast-like cells underwent differentiation in Bcl11b−/−
incisors, we examined expression of sonic hedgehog (Shh
) and amelogenin (Amelx
), markers of pre-ameloblasts 
and mature ameloblasts 
, respectively. Shh
expression was observed in a gradient along the length of the labial IEE of wild-type incisors at E16.5 and E18.5, with the most intense staining in the posterior region (). Shh
expression was greatly reduced in the labial epithelium of Bcl11b−/−
mice, and ectopic Shh
transcripts were detected in the lingual epithelium at E16.5 and E18.5 (). The expression pattern of Gli1
, a mediator of SHH signaling 
, reflected changes in Shh
expression in the mutant incisor (Fig. S6
Altered ameloblast development in Bcl11b−/− incisors.
Amelx expression was greatly reduced in the labial epithelium of Bcl11b−/− mice at E16.5 (compare ) but recovered to a level similar to that of wild-type mice by E18.5 (). Ectopic Amelx expression was observed in the anterior lingual IEE of Bcl11b−/− mice at E18.5 (compare ), consistent with the presence of terminally differentiated ameloblasts in the lingual epithelium.
These results demonstrate that BCL11B plays a key role in the establishment and/or enforcement of developmental incisor asymmetry and cellular differentiation within the ameloblast lineage.
Alteration of the FGF Signaling at Bell Stage in Bcl11b−/− Incisors
Asymmetric development of the CLs is controlled by several signaling pathways. FGFs and their intracellular antagonists, the Sprouty proteins, are crucial for proper development of the labial and lingual CLs 
. FGF3 and FGF10 are key mesenchymal instructive signals that cooperatively stimulate proliferation of the incisor epithelium at bell stage 
is expressed exclusively within the posterior labial mesenchyme in wild-type incisors (), whereas Fgf10
transcripts are more widely distributed around the labial CL and to a lesser extent in the lingual mesenchyme ().
Labial to lingual reversal of expression of FGF and Sprouty genes in Bcl11b−/− incisor.
The Fgf3 expression domain, which is located in the mesenchyme just anterior to the labial CL in wild-type mice, was absent in Bcl11b−/− mutants at E16.5 and E18.5. However, Fgf3 was ectopically expressed in the mesenchyme adjacent to the lingual CL in Bcl11b−/− mice (). The expression pattern of Fgf10 was altered in Bcl11b−/− incisors in a manner that was qualitatively similar to that of Fgf3 ().
Epithelial FGF9 forms a positive-feedback signaling loop with mesenchymal FGF3 and FGF10 on the labial side of the wild-type incisor 
RNA was detected anterior to the labial CL of the wild-type incisor (). This Fgf9
-positive domain was reduced in Bcl11b−/−
incisors, and ectopic expression of Fgf9
was detected in the lingual epithelium at E16.5 and E18.5 ().
Sprouty proteins are responsible, in part, for inhibition of ameloblast differentiation in the lingual epithelium 
RNA was detected in the mesenchyme adjacent to the labial CL, and at lower levels in the posterior lingual and labial epithelium in wild-type mice at E16.5 and E18.5 (). Spry2
expression was detected predominantly in the posterior lingual and labial epithelium of the wild-type incisor ().
Spry4 expression was up-regulated in the lingual basal epithelium and underlying mesenchyme of Bcl11b−/− incisors at E16.5 and E18.5, and down-regulated on the labial side of the developing Bcl11b−/− incisor at both developmental stages (). Spry2 expression was up-regulated in the lingual CL and slightly down-regulated in the labial epithelium of Bcl11b−/− mice at E16.5 and E18.5 ().
Mesenchymal FGF10 stimulates expression of Lunatic Fringe (Lfrn
), which encodes a secretory molecule that modulates the Notch pathway 
. To determine if the Notch pathway was altered in Bcl11b−/−
incisors at bell stage, we examined expression patterns of Lfrn
RNA was detected predominantly along the length of IEE and in the posterior OEE of wild-type incisors at E16.5 and E18.5 (Figs. S7A and C
expression was down-regulated at the posterior end of the labial CL of Bcl11b−/−
incisors at both E16.5 and E18.5 (Figs. S7B and D
). Ectopic Lfrn
expression was detected in the posterior part of the mutant lingual epithelium at E18.5 (Fig. S7D
). Loss of BCL11B did not affect the level of expression or localization of Notch1
transcripts. However, Notch1
expression reflected the morphological expansion and contraction of lingual and labial SR, respectively, in Bcl11b−/−
incisors (Figs. S7E
These findings highlight dysregulation of the FGF signaling pathways as being central to the incisor phenotype of Bcl11b−/−
mice. As asymmetric expression of Fgf3
contribute to asymmetric development of labial and lingual EpSC niches 
. Thus, the complete reversal of asymmetric Fgf3
expression, together with that of Fgf9
, likely underlies the enhanced and repressed development of the lingual and labial CLs, respectively, in Bcl11b−/−
Disruption of TGFβ Signaling at Bell Stage in Bcl11b−/− Mice
The TGFβ family members, BMP4 and activin βA, and the antagonist FST play key roles in the generation and maintenance of asymmetric ameloblast localization during incisor development. For example, FST inhibits ameloblast differentiation on the lingual side of the incisor, whereas BMP4 promotes it on the labial side. In contrast, activin enhances development of the labial CL, whereas BMP4 limits CL growth 
Bmp4 expression was detected predominantly in the labial mesenchyme, anterior to the labial CL, in wild-type mice at E16.5. Lower levels of Bmp4 transcripts were present in the mesenchyme underlying the lingual epithelium and in an anterior region of the ameloblast layer (). The boundaries of mesenchymal Bmp4 expression were disrupted in Bcl11b−/− incisors at E16.5, with ectopic expression noted in the mesenchyme posterior to the lingual CL. Expression of Bmp4 in the ameloblast layer appeared reduced in mutants at this stage (). At E18.5, Bmp4 transcripts were detected predominantly in the labial epithelium, in a wide region of labial mesenchyme, and at lower levels on the lingual side of the wild-type incisor (). Bmp4 expression increased uniformly in all of these domains in Bcl11b−/− mutants at E18.5 ().
Altered expression of TGFβ genes and Fst in Bcl11b−/− incisor.
Activin expression was restricted to the labial mesenchyme directly underlying the posterior epithelium, within the tip of the labial CL, and in the posterior part of the dental follicle in wild-type mice at E16.5 and E18.5 (). Activin expression was lost within the labial mesenchyme and epithelium in Bcl11b−/− mice at both developmental stages. However, ectopic mesenchymal expression of activin was observed around the lingual CL and follicular expression of activin appeared to be delocalized in the Bcl11b−/− incisors at E16.5 and E18.5 (asterisks in ).
transcripts were observed in the OEE on the labial and lingual sides at E16.5 (; see also 
expression in the OEE persisted at E18.5, and Fst
transcripts were also detected in highly-defined domains at the anterior epithelial tip of the incisor on both labial and lingual sides (; data not shown). In contrast, Fst
transcripts were diffusely distributed throughout the labial and lingual epithelium of Bcl11b−/−
incisors, particularly at the anterior (incisal) tip of the epithelium, and in the papillary mesenchyme at E16.5 (). Fst
expression within the posterior region of the wild-type incisor at E18.5 was indistinguishable from that of Bcl11b−/−
mice (data not shown). However, we noted a dramatic expansion of the Fst
expression domain within the anterior labial epithelium at E18.5. Additionally, Bcl11b−/−
incisors failed to extinguish Fst
expression along the length of the labial OEE at E18.5 ().
Cell Autonomous Effects of BCL11B in Lingual Epithelium
BCL11B is expressed in both ectodermal-derived epithelium and neural crest-derived mesenchyme (). We created lines conditionally null for Bcl11b
expression in both germinal layers to determine the expression domain responsible for BCL11B-mediated suppression of ameloblast differentiation in the lingual epithelium. Mice harboring an epithelial-specific deletion of Bcl11b
), which were created by crossing floxed Bcl11bL2/L2
mice with the K14-
cre deleter strain 
, clearly lacked BCL11B in the entire dental epithelium (). However, specific BCL11B expression persisted in the dental mesenchyme and other non-epithelium-derived tissues. Bcl11bep−/−
mice expressed the pre-ameloblast marker Shh
with an ectopic gradient along the length of the lingual epithelium at E16.5 (), and this persisted at a lower level at E18.5 (Fig. S8B
) However, Bcl11bep−/−
mice did not express Amelx
in the lingual epithelium at either E16.5 () or E18.5 (Fig. S8E
). Considered together, these results suggest that Bcl11bep−/−
mice initiate but do not complete ameloblast differentiation within the lingual dental epithelium.
Ectopic lingual expression of ameloblast markers and signaling molecules in Bcl11bep−/− incisor.
Next, we examined the expression of several genes encoding signaling molecules to determine the effect of epithelium-specific inactivation of Bcl11b on generation of labial-lingual asymmetry at E16.5. A low level of ectopic expression of Fgf3, Fgf9, and activin was detected on the lingual side of the Bcl11bep−/− incisor, and this was qualitatively similar to Bcl11b−/− incisors (compare , , and ). However, we did not observe alterations in the expression patterns of these signaling molecules on the labial side of the Bcl11bep−/− incisor (), as described previously for Bcl11b−/− mice (see and ).
The size and shape of the Bcl11bep−/− incisors were similar to control incisors (see ), and the slight variations in lingual gene expression in Bcl11bep−/− incisors did not result in altered amelogenesis as determined by X-ray micro-CT radiography performed on P21 mandibles (Fig. S9).
Excision of the Bcl11b
locus in neural crest-derived mesenchyme using the Wnt1
-cre deleter line (Bcl11bmes−/−
; see Figs. S10A and B
) did not result in altered morphology or disrupted gene expression patterns (Shh
, and activin
; Figs. S10C
-J; see also S8C and F).
These data indicate that epithelial, but not mesenchymal Bcl11b expression is required for suppression of ectopic pre-ameloblast formation in the lingual epithelium. However, loss of Bcl11b in the epithelium is not sufficient for the lingual pre-ameloblasts to persist or to undergo further differentiation into mature, Amelx-positive ameloblasts.
FGF Signaling Negatively Regulates BCL11B Expression in the Lingual IEE and SR
The ectopic development of lingual pre-ameloblasts expressing Shh
mice is similar to that reported in Spry4−/−; Spry2+/−
mice. Loss of Sprouty gene expression results in abnormal FGF gene expression and establishment of a FGF positive-feedback signaling loop on the lingual side of the incisor. In addition, Spry4−/−; Spry2+/−
mice were characterized by up-regulated expression of Etv4
(previously known as Pea3
), which are considered to be transcriptional targets of FGF signaling, and indicative of activation of the FGF signaling pathway(s) in mutant incisors 
. We assessed expression of BCL11B in incisors from Spry4−/−; Spry2+/−
embryos in order to determine if the FGF signaling pathway(s) regulates BCL11B expression.
BCL11B was highly expressed in the entirety of the wild-type lingual epithelium at E16.5, including the CL, anterior OEE, and IEE (; see also ). In contrast, BCL11B protein levels were dramatically decreased in the Spry4−/−; Spry2+/− incisor, particularly within the lingual IEE and SR, while BCL11B levels in the lingual OEE and labial epithelium were largely unaffected ().
Inhibition of Bcl11b expression in the lingual IEE of Spry4−/−; Spry2+/− mice at E16.5.
Expression of Tbx1
, which is also important for incisor developmental asymmetry, was increased in the lingual epithelium of Spry4−/−; Spry2+/−
. These findings prompted us to examine Tbx1
expression in Bcl11b−/−
was predominantly expressed in the posterior basal epithelium on the labial side of wild-type incisors at both E16.5 and E18.5, and diffusely at a much lower level in the lingual epithelium (Figs. S11A and C
). We observed striking up-regulation of Tbx1
expression in the lingual IEE of Bcl11b−/−
mice at E16.5 and E18.5 (Figs. S11B and D
), suggesting that BCL11B directly or indirectly represses the Tbx1
expression in the lingual epithelium, and that up-regulation of Tbx1
expression in Spry4−/−; Spry2+/−
may occur through down-regulation of BCL11B protein levels. These findings place BCL11B downstream of FGF signaling and upstream of Tbx1
expression in the lingual epithelium of the developing incisor. Tbx1
expression was severely decreased in the labial epithelium of Bcl11b−/−
mice at E16.5 (Fig. S11B
). Labial expression of Tbx1
in the Bcl11b−/−
incisor recovered by E18.5 (Fig. S11D
), suggesting that another factor(s) may compensate for loss of BCL11B expression in the control of expression of Tbx1
in the labial epithelium.
These above findings suggest that the FGF signaling pathways regulate BCL11B expression in the lingual epithelium, and we hypothesized that inactivation of FGF signaling may lead to up-regulation of BCL11B expression within the labial IEE. In order to test this hypothesis, we assessed BCL11B expression in Fgf3−/−; Fgf10+/−
incisors; however, BCL11B immunostaining was indistinguishable from wild-type incisors (Fig. S12
). It is conceivable that another FGF family member(s) may compensate for loss of Fgf3
expression and partial loss of Fgf10
expression by enforcing the repression of BCL11B expression within the labial IEE 
. Indeed, Fgf3−/−; Fgf10+/−
and wild-type incisors are nearly identical in size (Fig. S12
), suggesting that loss or partial loss of these two signaling molecules did not compromise proliferation during incisor development. Finally, it is possible that regulation of BCL11B expression within the labial epithelium may not involve the FGF signaling pathways, as was clearly evident on the lingual side ().