BACKGROUND
tbx <dig> is a member of the t-box family of genes.
t-box genes are expressed during embryonic development and have been found to regulate cell specification and organogenesis  <cit> .
they are also well-known for the roles they play in many human developmental syndromes  <cit> .
tbx <dig> is known to function as a transcriptional repressor and is required for embryonic development and for the normal development of the mammary gland  <cit> .
in mice models, homozygous mutations in which the function of tbx <dig> is completely lost are embryonic lethal while haploinsufficiency of tbx <dig> results in significantly reduced branching of ductal trees in adult animals  <cit> .
in humans, mutations that result in the haploinsufficiency and loss of function of tbx <dig> ultimately cause ulnar mammary syndrome   <cit> .
ums is an autosomal dominant disorder characterized by mammary gland hypoplasia and affects limb, apocrine-gland, teeth, hair, and genital development.
besides tbx3's role in early mammary gland development, various studies have also supported a role for tbx <dig> in breast cancer development.
the tbx <dig> gene is located at the 12q <dig> region which is frequently amplified in a variety of malignancies including breast cancer  <cit> .
moreover, tbx <dig> is over-expressed in various breast cancer cell lines as well as primary breast cancer tissues  <cit> .
tbx <dig> is mislocalized to the cytoplasm in primary breast cancer tissues and serum tbx <dig> protein levels were also found to be abnormally high in early stage breast cancer patients  <cit> .
more recently, it has been shown that pma-induced up-regulation of tbx <dig> contributes to breast cancer cell migration  <cit> .
tbx <dig> has been shown to repress the expression of the tumor suppression gene p14arf  <cit>  and the murine homologue p19arf  <cit> .
the p14/19-mdm2-p <dig> pathway plays an important role in regulating cell senescence and protects cells against oncogenic transformation which leads to tumor formation  <cit> .
tbx <dig> over-expression has been shown to immortalize mouse embryonic fibroblast cells by suppressing p19arf  <cit> .
we have previously shown that over-expression of tbx <dig> represses human p14arf by recruiting hdac  <dig>   <dig>   <dig> and  <dig> in the mcf <dig> breast cancer cell line  <cit> .
in order to identify other targets of tbx <dig>  we used chromatin immunoprecipitation-guided ligation and selection  promoter array.
our results showed that  <dig> gene promoters are bound by tbx <dig> in the mcf <dig> breast cancer cell line .
one of the identified genes, nfκbib, is an inhibitor of nf-κb.
studies have shown that nf-κb associated pathways play an important role in cell proliferation, differentiation and apoptosis  <cit> .
specifically, nfκbib inhibits nf-κb by sequestering it in the cytoplasm.
activation of nf-κb occurs upon ubiquitin mediated degradation of nfκbib proteins via serine phosphorylation by iκb kinase .
studies have shown that inhibition of nf-κb activation in mouse mammary glands lead to defective proliferation in lobuloalveolar structures during pregnancy  <cit> , whereas elevated nf-κb activity causes mammary hyperplasia in vivo  <cit> .
furthermore, aberrant activation of nf-κb is related to breast cancer progression, including tumor initiation, proliferation, chemoresistance and tumor metastasis  <cit> .
taken together, these studies suggest that a dysregulation of tbx <dig> expression may contribute to breast cancer development.
further supporting the notion that tbx <dig> plays a role in cancer development, recent studies have shown that increased levels of tbx <dig> enhanced melanoma invasiveness by repressing e-cadherin expression  <cit> .
recent studies have shown that tbx <dig>  a downstream target of wnt/β-catenin in liver cancer, has also been found to be over-expressed in human hepatocellular carcinoma and heptoblastoma  <cit> .
knockdown of tbx <dig> in rat bladder carcinoma cell lines resulted in a lower growth rate and more apoptotic cells than controls, suggesting that tbx <dig> promotes cell proliferation and is a negative regulator of apoptosis  <cit> .
although many studies have shown that a dysregulation of tbx <dig> expression may contribute to cancer progression, no direct evidence shows that tbx <dig> causes breast cancer.
identifying whether tbx <dig> directly promotes breast cancer development and the mechanism by which it does this is important for understanding mammary development as well as the perturbations that may lead to breast cancer.
in the present study, we have demonstrated that over-expression of tbx <dig> in our doxycycline inducible mouse model promotes accelerated mammary gland development and hyperplasia by promoting mammary epithelium cell proliferation.
moreover, we have shown that nfκbib was dramatically down-regulated in the mammary glands of doxycycline induced double transgenic mice.
although over-expression of tbx <dig>  alone, did not cause tumor formation within the mammary gland, our data suggests that the over-expression of tbx <dig> may contribute to breast cancer formation through the inhibition of the nf-κb pathway and stimulation of both mammary epithelial cell and stem-like cell proliferation.
RESULTS
tbx <dig> over-expression is induced in mmtv-rtta; tet-myc-tbx <dig> mammary glands by doxycycline administration
to construct a doxycycline inducible myc-tbx <dig> transgene cassette , myc-tbx <dig> cdna was subcloned downstream of tet operator elements  .
in our transgene expression cassette, the expression of the luciferase reporter gene is regulated by the same promoter as our myc-tbx <dig> transgene.
thus, upon induction with doxycycline, translation of the luciferase reporter gene by its own internal ribosome entry site  can be used as a marker for myc-tbx <dig> overexpression .
in order to express myc-tbx <dig> specifically in the mammary glands of mice, tet-myc-tbx <dig> mice were mated with mmtv-rtta mice.
transgene expression was induced in double transgenic mice by adding 2mg/ml doxycycline to the drinking water.
to verify that the induction of tbx <dig> expression within the mammary glands of mice occurred only upon the addition of doxycycline, luciferase activity was monitored by imaging the mammary glands of both doxycycline induced and un-induced double transgenic mice in vivo, using an iccd camera.
prior to in vivo imaging, mice were sedated by intraperitoneal injection of xylazine and ketamine.
after  <dig> minutes, an aqueous solution of luciferin was injected into the peritoneal cavity to detect luciferase activity and tbx <dig> transgene over-expression.
the in vivo image of the doxycycline induced double transgenic mouse detected a bioluminescent signal 4- <dig> folds above background within all  <dig> pairs of mammary glands.
the bright bioluminescent signal in the cervical midline of the doxycycline induced double transgenic mouse represents the first pair of mammary glands as well as leaky expression of the mmtv promoter within the salivary gland, which is frequently seen in other mmtv models .
no signal was detected in the age-matched un-induced double transgenic littermate control .
to more directly measure the luciferase activity within each mammary gland a luciferase assay was performed using tissue lysates from each mammary gland  of a single doxycycline induced double transgenic mouse .
consistent with the in vivo imaging, all five mammary glands from the doxycycline induced double transgenic mice had high luciferase readings while the un-induced double transgenic littermates showed only baseline readings .
direct tbx <dig> over-expression within the mammary gland was also detected by immunohistochemistry with an anti-tbx <dig> antibody.
tbx <dig> over-expression was detected only in the induced double transgenic mouse mammary gland .
endogenous tbx <dig> expression was not detected .
overall, these results show that tbx <dig> over-expression is specifically induced within all  <dig> mammary glands of our double transgenic mice upon administration of doxycycline.
over-expression of tbx <dig> promotes accelerated mammary gland development by increasing cell proliferation
in mice, the mammary gland development begins shortly after mid-gestation.
five pairs of mammary placodes form at the site of the future nipples  <cit> .
these placodes invaginate and form buds within the mammary fat pad that contain few branches  <cit> .
by birth a simple mammary ductal tree is formed that occupies a small portion of the fat pad  <cit> .
after birth, growth of the mammary gland is relatively quiescent until puberty  <cit> .
at puberty, club-shaped structures called the terminal end buds  form at the tips of the ductal tree.
during this period, cell proliferation in tebs results in ductal elongation through the mammary fat pad.
tebs not only elongate through the fat pad, but also bifurcate to form new primary ducts while secondary side-branches sprout along the extending ducts  <cit> .
the outgrowth of side branches is controlled by several hormones and signaling pathways  <cit> .
at the end of puberty, approximately 10- <dig> weeks of age, tebs reach the edge of the fat pad and disappear  <cit> .
in order to determine the effect of tbx <dig> over-expression on the overall development of the mammary gland, we harvested the 1st and 4th mammary glands from  <dig> doxycycline induced double transgenic mice and from another  <dig> of the un-induced double transgenic littermate controls at four specific time points; 7-weeks, 10-weeks, 12-weeks of age and  <dig>  days postcoitus .
mammary glands harvested at 7-weeks, 10-weeks and 12-weeks were from nulliparous mice, while those harvested at  <dig>  dpc were from uniparous pregnant mice.
whole mount analysis of the 4th mammary gland revealed that at 7-weeks and 10-weeks of age, tebs in the control mice had not reached the edge of the fat pad, whereas the tebs in doxycycline induced double transgenic mice were observed at the edge of the fat pad or had disappeared , suggesting that over-expression of tbx <dig> promotes accelerated ductal elongation.
hematoxylin and eosin staining of both the 1st and 4th mammary glands of the doxycycline induced double transgenic mice displayed increased primary and secondary side branching at all time points when compared to their un-induced double transgenic littermate controls .
we also observed an increase in tertiary side branching although this has been known to occur in response to estrous cycle  <cit> .
in addition, pregnant doxycycline induced double transgenic mice at  <dig>  dpc also displayed more alveoli tissue than the un-induced double transgenic controls .
the samples used for whole mount analysis were from two independent founder lines and the results were consistent between these two lines.
several in vitro studies have suggested that the over-expression of tbx3/tbx <dig> leads to the bypass of senescence and promotes cell proliferation  <cit> .
to determine whether the observed accelerated development of the mammary glands in tbx <dig> over-expressing mice is due to an increase in cell proliferation, we performed an edu cell proliferation assay.
the 4th mammary glands from pregnant doxycycline induced and un-induced double transgenic mice were harvested at  <dig>  dpc and used for the assay.
the proportion of nucleated cells incorporating edu was quantified by fluorescence microscopy  and normalized to the total cell number in each 20× field.
after quantification, we found that the percentage of edu positive cells is significantly  higher in mammary glands over-expressing tbx <dig>  than their un-induced controls .
this result suggests that over-expression of tbx <dig> may promote accelerated mammary gland development by promoting mammary epithelial cell proliferation in vivo.
since highly proliferative tissues are associated with carcinogenesis, we next analysed the histology of the 3rd mammary glands of  <dig> week old mice to identify if any unusual morphological changes have occurred.
hematoxylin and eosin staining of the doxycycline induced double transgenic mouse mammary gland showed mild focal hyperplasia  and discontinued ductal epithelium  when compared to the littermate control.
by the age of  <dig> months, none of the doxycycline induced double transgenic mice had developed tumors.
tbx <dig> represses nfκbib
in our double transgenic mouse model in which tbx <dig> was over-expressed, we observed accelerated development of the mammary gland from  <dig> weeks of age through pregnancy; specifically enhanced branching and ductal elongation.
moreover mice that over-expressed tbx <dig> also had a significantly higher percentage of proliferating mammary epithelial cells than controls.
together these data suggest that tbx <dig> may be regulating genes that play a role in cell proliferation.
identifying the mechanism by which tbx <dig> promotes accelerated mammary gland development will help to further elucidate its possible role in breast cancer development.
dysregulation of the nf-κb associated pathways have been shown to play a role in breast cancer development  <cit> .
moreover, it has been shown that elevated nf-κb activity causes mammary hyperplasia in vivo  <cit> .
due to this observed phenotype and our previous unpublished data in which tbx <dig> binds to the promoter of nfκbib in mcf <dig> cells, we investigated the role tbx <dig> may play in regulating the nfκb pathway.
to verify that tbx <dig> does indeed regulate the nfκbib promoter, we performed a luciferase assay.
briefly, cos- <dig> cells were transfected with either pcdna <dig> -myc  or pcdna <dig> -myc-tbx <dig> expression vector together with the pgl3-nfκbib luciferase reporter construct and a β-galactosidase control plasmid  using lipofectamine  <dig>  forty-eight hours later, cell lysates were harvested and used to perform the luciferase assay.
β-galactosidase enzyme activity was measured and used to normalize luciferase activity.
the luciferase assay revealed that the activity of the nfκbib promoter is significantly repressed  when tbx <dig> is over-expressed in cos- <dig> cells .
to determine whether the nfκbib protein was down-regulated upon over-expression of tbx <dig> within the mammary gland, immunohistochemistry was performed on the mammary glands of doxycycline induced and un-induced double transgenic mice at  <dig> weeks of age.
staining revealed the nfκbib expression was down-regulated in the doxycycline induced double transgenic mouse when compared to its un-induced double transgenic littermate control .
these data suggest that over-expression of tbx <dig> may promote cell proliferation within the mammary gland by repressing the expression of nfκbib.
over-expression of tbx <dig> is associated with an increase in mammary stem-like cells
another mechanism by which tbx <dig> over-expression may promote accelerated mammary gland development is through the proliferation of mammary stem cells.
expression of tbx <dig> has been shown to promote the proliferation of breast cancer stem cells in vitro  <cit> , suggesting that tbx <dig> may also promote mammary stem cell proliferation.
a study showed that a single lin-cd24+cd29high cell is able to generate a functional mammary gland, providing strong evidence that these cells are mammary stem cells  <cit> .
thus, to isolate and analyze the mammary stem-like cell population we first subtracted the mammary lin+  cells.
cd <dig> is considered as an endothelial cell marker, and cd <dig> and ter <dig> are considered as hematopoietic cell markers  <cit> .
therefore, lin+  cells are considered a terminally differentiated cell population.
in contrast, cd <dig> is a skin stem cell marker  <cit>  and cd <dig> is found on neuronal stem cells  <cit> , therefore cd29+ and cd24+ cells are considered mammary stem-like cells  <cit> .
to determine whether over-expression of tbx <dig> promotes the proliferation of mammary stem-like cells, we dissected mammary glands from two mice at  <dig> weeks of age from the doxycycline induced double transgenic group and their un-induced double transgenic littermates and isolated the mammary stem-like cells using the previously mentioned cell markers.
the gating strategy for lin- cells and cd24+ cd29high cells is shown in figure 5a.
facs analysis revealed that over-expression of tbx <dig> did not affect the overall frequency of lin- cells in the mammary glands of doxycycline induced and un-induced mice,  <dig> % and  <dig> %, respectively .
however, within the lin- population, there was a significant increase in the frequency of cd24+cd29high cells in the doxycycline induced double transgenic mice versus un-induced control;  <dig> % and  <dig> % respectively  .
the average and standard deviations from both mice in each group are presented in figure 5b.
these results suggest that over-expression of tbx <dig> may promote proliferation of mammary stem-like cells.
discussion
the tbx <dig> t-box transcription factor plays an important role in early mammary development  <cit> .
mutations that cause haploinsufficiency of tbx <dig> result in mammary gland hypoplasia in both mice and human  <cit> .
on the other hand, tbx <dig> is over-expressed in a variety of cancers, including breast cancer  <cit> .
although tbx <dig> over-expression has been associated with oncogenesis by its known ability to inhibit p14arf expression and bypass senescence or by contributing to breast cancer cell migration  <cit> , no direct evidence has been shown to suggest that over-expression of tbx <dig>  alone, can induce tumor formation within the mammary gland.
in this study, we over-expressed tbx <dig> within the mammary glands of mice, using a tissue-specific, doxycycline inducible transgenic system.
transgenic mouse models using constitutive promoters have provided information about specific genes and breast cancer development, particularly oncogene function  <cit> .
however, there are significant limitations to these systems due to the lack of control of transgene expression.
the ability to control tbx <dig> expression is critical since homozygous tbx <dig> knockout is embryonic lethal and constitutive over-expression is potentially toxic  <cit> .
we implemented a tet-on system in our transgenic mouse model so that tbx <dig> transgene expression is inducible in a time and tissue-specific manner  <cit> , enabling us to test possible tbx <dig> function in tumorigenesis in the mammary glands.
an advantage of our mouse model is the ability to use luciferase expression as an indication of tbx <dig> transgene expression .
in this way, we are able to monitor tbx <dig> expression without sacrificing the animal.
using in vivo imaging as well as a luciferase assay, we were able to show that transgene expression is tightly controlled by doxycycline administration .
our results show that this system is reliable and transgene expression could be induced in all five pairs of mammary glands.
previous studies have shown that the five pairs of mouse mammary glands are differentially regulated by tbx <dig> during early development.
for example, in tbx <dig> knockout studies, homozygous mutations resulted in the absence of mammary placodes, except for an occasional induction of the second pair of mammary placodes  <cit> .
heterozygous mutations of tbx <dig> caused decreased branching morphogenesis in the first three pairs of mammary glands, but had no significant impact on the fourth and fifth pairs of mammary glands  <cit> .
in  <dig>  day old tbx <dig> heterozygous embryos, 75% of the first pair of mammary glands was missing with no nipple or ductal tree formation while the second pair of mammary glands was affected to a lesser extent  <cit> .
although these studies suggest that tbx <dig> regulates murine mammary glands differently, we found that over-expression of tbx <dig> promotes accelerated mammary gland development in both the first and fourth mammary glands  as well as the second, third and fifth mammary glands .
research has solidified a role for tbx <dig> in the early development of the mammary gland.
tbx <dig> homozygous mutant mice results in mammary gland hypoplasia while heterozygous mutations of tbx <dig> caused decreased branching morphogenesis in mammary glands  <cit> .
our research complements these previous studies showing that tbx <dig> over-expression within the mammary glands causes hyperplasia, promoting increased secondary and tertiary branching as well as accelerated ductal elongation.
it is also important to discuss that we have over-expressed human tbx <dig> within the mammary glands of mice.
it has been shown that human tbx <dig> and mouse tbx <dig> are 97% homologous at the protein level.
our group and others have demonstrated that human tbx <dig> is functional in mouse cells  <cit> .
furthermore, atbx <dig> knockout mouse model was able to recapitulate the phenotype seen in humans with ulnar mammary syndrome .
in a study performed by papaioannou et al., a mutation in the mouse tbx <dig> gene that closely corresponds to truncation mutations seen in some individuals with ums resulted in a deficiency in mammary placode induction and the absence or reduction of mammary buds in mutant embryos, corresponding to the mammary gland hypoplasia seen in patients with ums.
moreover, the deficiency in the development of limb elements in individuals with ums was also reflected in limb abnormalities in the tbx <dig> mutant mice.
mutant mice had deformities in the forelimb digits, foot and fibula resulting from a failure in the development of posterior limb elements.
this study exemplifies that the tbx <dig> protein plays a similar role in the development of the mammary glands in both human and mice.
the mechanism by which tbx <dig> over-expression promotes hyperplasia in mammary glands needs to be elucidated.
using an edu cell proliferation assay, we showed that over-expression of tbx <dig> resulted in a dramatic increase in cell proliferation within the mammary glands of pregnant doxycycline induced double transgenic mice at  <dig>  dpc .
although cell proliferation was not directly quantified for the other developmental time points , the similarity in the observed accelerated mammary gland development suggests that the increase in cell proliferation at  <dig>  dpc may also play a role in causing the accelerated branching and elongation of ducts during the other phases of mammary gland growth.
a study has shown that mammary epithelia lacking the gene encoding nfκbia contained increased nfkb activity as well as increased ductal branching and widespread intraductal hyperplasia  <cit> , similar to results seen in our study.
furthermore, aberrant activation of nf-κb increased cell proliferation and breast cancer progression  <cit> .
in this study, we found that tbx <dig> inhibits the promoter activity of nfκbib in vitro .
upon further analysis, in vivo, we observed that nfκbib expression was dramatically reduced in doxycycline induced double transgenic mice as compared to its un-induced double transgenic littermate controls .
taken together, our results suggest a mechanism by which tbx <dig> over-expression represses nfkbib/nfkbib expression to enhance cell proliferation and promote mammary gland hyperplasia.
however, tbx <dig> is a multifunctional transcription factor and the nfkb pathway could be one of many pathways regulated by tbx <dig>  wnt signaling has also been shown to play a major role in regulating mammary gland development  <cit> .
a tbx3-/- mouse model lacked expression of lef <dig> and wnt10b  <cit> , suggesting that wnt signaling is a downstream target of tbx <dig> and that tbx <dig> may regulate mammary gland development via the wnt signaling pathway.
additional experiments can be done to further elucidate other mechanisms by which tbx <dig> over-expression promotes mammary hyperplasia.
studies have suggested a role for tbx3/tbx <dig> in regulating the self-renewal of mouse embryonic stem  cells as well as breast cancer stem-like cells  <cit> .
mouse es cells require leukemia inhibitory factor  to maintain their undifferentiated state  <cit> .
mouse es cells genetically modified to over-express tbx <dig> and grown in culture without lif were able to maintain their undifferentiated state  <cit> .
knockdown of tbx <dig> expression in mouse es cells resulted in a loss of self-renewal, causing these cells to differentiate  <cit> .
these findings suggest that tbx <dig> expression is necessary to maintain mouse es cells in their undifferentiated state and plays a functional role to promote self-renewal.
a recent study has proposed a model in which the expression of tbx <dig> in cancer cells promotes the expansion of cancer stem-like cells through paracrine fibroblast growth factor  signaling  <cit> .
over-expression of tbx <dig> increased the proportion of cancer stem-like cells in mcf <dig> cells by nine-fold as well as lead to an increase in tumorsphere formation and tumor initiation  <cit> , suggesting that tbx <dig> is sufficient to promote normal and cancer stem like cell phenotypes.
due to its role in promoting proliferation of mouse es cells and breast cancer stem-like cells as well as its requirement for early mammary gland development, tbx <dig> may also play a role in regulating mammary stem cell proliferation.
mammary glands consist of two cell lineages: myoepithelial and luminal epithelial cells.
both of them arise from a common progenitor, the mammary stem cell.
research has shown that a single lin-cd24+cd29high cell is able to generate a functional mammary gland, suggesting that these cells are mammary stem cells  <cit> .
to determine whether over-expression of tbx <dig> affects mammary stem cell proliferation, we performed facs analysis of the stem-like cell population, lin-cd24+cd29high, in doxycycline induced double transgenic mice and their un-induced littermate controls.
we found that over-expression of tbx <dig> significantly increased the frequency of lin-cd24+cd29high stem-like cell population , indicating that tbx <dig> expression is associated with an increased number of mammary stem-like cells.
this could explain another mechanism by which tbx <dig> over-expression can cause hyperplasia and accelerated mammary gland development.
further studies of the mechanisms by which tbx <dig> regulates mammary stem-like cells are required to improve our understanding of mammary gland development and tbx <dig> function.
CONCLUSIONS
tbx <dig> over-expression causes mammary gland hyperplasia possibly by inhibiting nfκbib expression and thus promoting cell proliferation.
also, over-expression of tbx <dig> is associated with an increased number of mammary stem-like cells suggesting another mechanism by which tbx <dig> may promote mammary gland hyperplasia and contribute to breast cancer development.

 tbx is a member of the t-box family of genes. t-box genes are expressed during embryonic development and have been found to regulate cell specification and organogenesis. they are also well-known for the roles they play in many human developmental syndromes. tbx is known to function as a transcriptional repressor and is required for embryonic development and for the normal development of the mammary gland. in mice models, homozygous mutations in which the function of tbx is completely lost are embryonic lethal while haploinsufficiency of tbx results in significantly reduced branching of ductal trees in adult animals. in humans, mutations that result in the haploinsufficiency and loss of function of tbx ultimately cause ulnar mammary syndrome. ums is an autosomal dominant disorder characterized by mammary gland hypoplasia and affects limb, apocrine-gland, teeth, hair, and genital development. besides tbx3's role in early mammary gland development, various studies have also supported a role for tbx in breast cancer development. the tbx gene is located at the 12q region which is frequently amplified in a variety of malignancies including breast cancer. moreover, tbx is over-expressed in various breast cancer cell lines as well as primary breast cancer tissues. tbx is mislocalized to the cytoplasm in primary breast cancer tissues and serum tbx protein levels were also found to be abnormally high in early stage breast cancer patients. more recently, it has been shown that pma-induced up-regulation of tbx contributes to breast cancer cell migration. tbx has been shown to repress the expression of the tumor suppression gene p14arf and the murine homologue p19arf. the p14/19-mdm2-p pathway plays an important role in regulating cell senescence and protects cells against oncogenic transformation which leads to tumor formation. tbx over-expression has been shown to immortalize mouse embryonic fibroblast cells by suppressing p19arf. we have previously shown that over-expression of tbx represses human p14arf by recruiting hdac and in the mcf breast cancer cell line. in order to identify other targets of tbx we used chromatin immunoprecipitation-guided ligation and selection promoter array. our results showed that gene promoters are bound by tbx in the mcf breast cancer cell line. one of the identified genes, nfκbib, is an inhibitor of nf-κb. studies have shown that nf-κb associated pathways play an important role in cell proliferation, differentiation and apoptosis. specifically, nfκbib inhibits nf-κb by sequestering it in the cytoplasm. activation of nf-κb occurs upon ubiquitin mediated degradation of nfκbib proteins via serine phosphorylation by iκb kinase. studies have shown that inhibition of nf-κb activation in mouse mammary glands lead to defective proliferation in lobuloalveolar structures during pregnancy, whereas elevated nf-κb activity causes mammary hyperplasia in vivo. furthermore, aberrant activation of nf-κb is related to breast cancer progression, including tumor initiation, proliferation, chemoresistance and tumor metastasis. taken together, these studies suggest that a dysregulation of tbx expression may contribute to breast cancer development. further supporting the notion that tbx plays a role in cancer development, recent studies have shown that increased levels of tbx enhanced melanoma invasiveness by repressing e-cadherin expression. recent studies have shown that tbx a downstream target of wnt/β-catenin in liver cancer, has also been found to be over-expressed in human hepatocellular carcinoma and heptoblastoma. knockdown of tbx in rat bladder carcinoma cell lines resulted in a lower growth rate and more apoptotic cells than controls, suggesting that tbx promotes cell proliferation and is a negative regulator of apoptosis. although many studies have shown that a dysregulation of tbx expression may contribute to cancer progression, no direct evidence shows that tbx causes breast cancer. identifying whether tbx directly promotes breast cancer development and the mechanism by which it does this is important for understanding mammary development as well as the perturbations that may lead to breast cancer. in the present study, we have demonstrated that over-expression of tbx in our doxycycline inducible mouse model promotes accelerated mammary gland development and hyperplasia by promoting mammary epithelium cell proliferation. moreover, we have shown that nfκbib was dramatically down-regulated in the mammary glands of doxycycline induced double transgenic mice. although over-expression of tbx alone, did not cause tumor formation within the mammary gland, our data suggests that the over-expression of tbx may contribute to breast cancer formation through the inhibition of the nf-κb pathway and stimulation of both mammary epithelial cell and stem-like cell proliferation.  tbx over-expression is induced in mmtv-rtta; tet-myc-tbx mammary glands by doxycycline administration to construct a doxycycline inducible myc-tbx transgene cassette, myc-tbx cdna was subcloned downstream of tet operator elements. in our transgene expression cassette, the expression of the luciferase reporter gene is regulated by the same promoter as our myc-tbx transgene. thus, upon induction with doxycycline, translation of the luciferase reporter gene by its own internal ribosome entry site can be used as a marker for myc-tbx overexpression. in order to express myc-tbx specifically in the mammary glands of mice, tet-myc-tbx mice were mated with mmtv-rtta mice. transgene expression was induced in double transgenic mice by adding 2mg/ml doxycycline to the drinking water. to verify that the induction of tbx expression within the mammary glands of mice occurred only upon the addition of doxycycline, luciferase activity was monitored by imaging the mammary glands of both doxycycline induced and un-induced double transgenic mice in vivo, using an iccd camera. prior to in vivo imaging, mice were sedated by intraperitoneal injection of xylazine and ketamine. after minutes, an aqueous solution of luciferin was injected into the peritoneal cavity to detect luciferase activity and tbx transgene over-expression. the in vivo image of the doxycycline induced double transgenic mouse detected a bioluminescent signal 4- folds above background within all pairs of mammary glands. the bright bioluminescent signal in the cervical midline of the doxycycline induced double transgenic mouse represents the first pair of mammary glands as well as leaky expression of the mmtv promoter within the salivary gland, which is frequently seen in other mmtv models. no signal was detected in the age-matched un-induced double transgenic littermate control. to more directly measure the luciferase activity within each mammary gland a luciferase assay was performed using tissue lysates from each mammary gland of a single doxycycline induced double transgenic mouse. consistent with the in vivo imaging, all five mammary glands from the doxycycline induced double transgenic mice had high luciferase readings while the un-induced double transgenic littermates showed only baseline readings. direct tbx over-expression within the mammary gland was also detected by immunohistochemistry with an anti-tbx antibody. tbx over-expression was detected only in the induced double transgenic mouse mammary gland. endogenous tbx expression was not detected. overall, these results show that tbx over-expression is specifically induced within all mammary glands of our double transgenic mice upon administration of doxycycline. over-expression of tbx promotes accelerated mammary gland development by increasing cell proliferation in mice, the mammary gland development begins shortly after mid-gestation. five pairs of mammary placodes form at the site of the future nipples. these placodes invaginate and form buds within the mammary fat pad that contain few branches. by birth a simple mammary ductal tree is formed that occupies a small portion of the fat pad. after birth, growth of the mammary gland is relatively quiescent until puberty. at puberty, club-shaped structures called the terminal end buds form at the tips of the ductal tree. during this period, cell proliferation in tebs results in ductal elongation through the mammary fat pad. tebs not only elongate through the fat pad, but also bifurcate to form new primary ducts while secondary side-branches sprout along the extending ducts. the outgrowth of side branches is controlled by several hormones and signaling pathways. at the end of puberty, approximately 10- weeks of age, tebs reach the edge of the fat pad and disappear. in order to determine the effect of tbx over-expression on the overall development of the mammary gland, we harvested the 1st and 4th mammary glands from doxycycline induced double transgenic mice and from another of the un-induced double transgenic littermate controls at four specific time points; 7-weeks, 10-weeks, 12-weeks of age and days postcoitus. mammary glands harvested at 7-weeks, 10-weeks and 12-weeks were from nulliparous mice, while those harvested at dpc were from uniparous pregnant mice. whole mount analysis of the 4th mammary gland revealed that at 7-weeks and 10-weeks of age, tebs in the control mice had not reached the edge of the fat pad, whereas the tebs in doxycycline induced double transgenic mice were observed at the edge of the fat pad or had disappeared, suggesting that over-expression of tbx promotes accelerated ductal elongation. hematoxylin and eosin staining of both the 1st and 4th mammary glands of the doxycycline induced double transgenic mice displayed increased primary and secondary side branching at all time points when compared to their un-induced double transgenic littermate controls. we also observed an increase in tertiary side branching although this has been known to occur in response to estrous cycle. in addition, pregnant doxycycline induced double transgenic mice at dpc also displayed more alveoli tissue than the un-induced double transgenic controls. the samples used for whole mount analysis were from two independent founder lines and the results were consistent between these two lines. several in vitro studies have suggested that the over-expression of tbx3/tbx leads to the bypass of senescence and promotes cell proliferation. to determine whether the observed accelerated development of the mammary glands in tbx over-expressing mice is due to an increase in cell proliferation, we performed an edu cell proliferation assay. the 4th mammary glands from pregnant doxycycline induced and un-induced double transgenic mice were harvested at dpc and used for the assay. the proportion of nucleated cells incorporating edu was quantified by fluorescence microscopy and normalized to the total cell number in each 20× field. after quantification, we found that the percentage of edu positive cells is significantly higher in mammary glands over-expressing tbx than their un-induced controls. this result suggests that over-expression of tbx may promote accelerated mammary gland development by promoting mammary epithelial cell proliferation in vivo. since highly proliferative tissues are associated with carcinogenesis, we next analysed the histology of the 3rd mammary glands of week old mice to identify if any unusual morphological changes have occurred. hematoxylin and eosin staining of the doxycycline induced double transgenic mouse mammary gland showed mild focal hyperplasia and discontinued ductal epithelium when compared to the littermate control. by the age of months, none of the doxycycline induced double transgenic mice had developed tumors. tbx represses nfκbib in our double transgenic mouse model in which tbx was over-expressed, we observed accelerated development of the mammary gland from weeks of age through pregnancy; specifically enhanced branching and ductal elongation. moreover mice that over-expressed tbx also had a significantly higher percentage of proliferating mammary epithelial cells than controls. together these data suggest that tbx may be regulating genes that play a role in cell proliferation. identifying the mechanism by which tbx promotes accelerated mammary gland development will help to further elucidate its possible role in breast cancer development. dysregulation of the nf-κb associated pathways have been shown to play a role in breast cancer development. moreover, it has been shown that elevated nf-κb activity causes mammary hyperplasia in vivo. due to this observed phenotype and our previous unpublished data in which tbx binds to the promoter of nfκbib in mcf cells, we investigated the role tbx may play in regulating the nfκb pathway. to verify that tbx does indeed regulate the nfκbib promoter, we performed a luciferase assay. briefly, cos- cells were transfected with either pcdna -myc or pcdna -myc-tbx expression vector together with the pgl3-nfκbib luciferase reporter construct and a β-galactosidase control plasmid using lipofectamine forty-eight hours later, cell lysates were harvested and used to perform the luciferase assay. β-galactosidase enzyme activity was measured and used to normalize luciferase activity. the luciferase assay revealed that the activity of the nfκbib promoter is significantly repressed when tbx is over-expressed in cos- cells. to determine whether the nfκbib protein was down-regulated upon over-expression of tbx within the mammary gland, immunohistochemistry was performed on the mammary glands of doxycycline induced and un-induced double transgenic mice at weeks of age. staining revealed the nfκbib expression was down-regulated in the doxycycline induced double transgenic mouse when compared to its un-induced double transgenic littermate control. these data suggest that over-expression of tbx may promote cell proliferation within the mammary gland by repressing the expression of nfκbib. over-expression of tbx is associated with an increase in mammary stem-like cells another mechanism by which tbx over-expression may promote accelerated mammary gland development is through the proliferation of mammary stem cells. expression of tbx has been shown to promote the proliferation of breast cancer stem cells in vitro, suggesting that tbx may also promote mammary stem cell proliferation. a study showed that a single lin-cd24+cd29high cell is able to generate a functional mammary gland, providing strong evidence that these cells are mammary stem cells. thus, to isolate and analyze the mammary stem-like cell population we first subtracted the mammary lin+ cells. cd is considered as an endothelial cell marker, and cd and ter are considered as hematopoietic cell markers. therefore, lin+ cells are considered a terminally differentiated cell population. in contrast, cd is a skin stem cell marker and cd is found on neuronal stem cells, therefore cd29+ and cd24+ cells are considered mammary stem-like cells. to determine whether over-expression of tbx promotes the proliferation of mammary stem-like cells, we dissected mammary glands from two mice at weeks of age from the doxycycline induced double transgenic group and their un-induced double transgenic littermates and isolated the mammary stem-like cells using the previously mentioned cell markers. the gating strategy for lin- cells and cd24+ cd29high cells is shown in figure 5a. facs analysis revealed that over-expression of tbx did not affect the overall frequency of lin- cells in the mammary glands of doxycycline induced and un-induced mice, % and %, respectively. however, within the lin- population, there was a significant increase in the frequency of cd24+cd29high cells in the doxycycline induced double transgenic mice versus un-induced control; % and % respectively. the average and standard deviations from both mice in each group are presented in figure 5b. these results suggest that over-expression of tbx may promote proliferation of mammary stem-like cells. discussion the tbx t-box transcription factor plays an important role in early mammary development. mutations that cause haploinsufficiency of tbx result in mammary gland hypoplasia in both mice and human. on the other hand, tbx is over-expressed in a variety of cancers, including breast cancer. although tbx over-expression has been associated with oncogenesis by its known ability to inhibit p14arf expression and bypass senescence or by contributing to breast cancer cell migration, no direct evidence has been shown to suggest that over-expression of tbx alone, can induce tumor formation within the mammary gland. in this study, we over-expressed tbx within the mammary glands of mice, using a tissue-specific, doxycycline inducible transgenic system. transgenic mouse models using constitutive promoters have provided information about specific genes and breast cancer development, particularly oncogene function. however, there are significant limitations to these systems due to the lack of control of transgene expression. the ability to control tbx expression is critical since homozygous tbx knockout is embryonic lethal and constitutive over-expression is potentially toxic. we implemented a tet-on system in our transgenic mouse model so that tbx transgene expression is inducible in a time and tissue-specific manner, enabling us to test possible tbx function in tumorigenesis in the mammary glands. an advantage of our mouse model is the ability to use luciferase expression as an indication of tbx transgene expression. in this way, we are able to monitor tbx expression without sacrificing the animal. using in vivo imaging as well as a luciferase assay, we were able to show that transgene expression is tightly controlled by doxycycline administration. our results show that this system is reliable and transgene expression could be induced in all five pairs of mammary glands. previous studies have shown that the five pairs of mouse mammary glands are differentially regulated by tbx during early development. for example, in tbx knockout studies, homozygous mutations resulted in the absence of mammary placodes, except for an occasional induction of the second pair of mammary placodes. heterozygous mutations of tbx caused decreased branching morphogenesis in the first three pairs of mammary glands, but had no significant impact on the fourth and fifth pairs of mammary glands. in day old tbx heterozygous embryos, 75% of the first pair of mammary glands was missing with no nipple or ductal tree formation while the second pair of mammary glands was affected to a lesser extent. although these studies suggest that tbx regulates murine mammary glands differently, we found that over-expression of tbx promotes accelerated mammary gland development in both the first and fourth mammary glands as well as the second, third and fifth mammary glands. research has solidified a role for tbx in the early development of the mammary gland. tbx homozygous mutant mice results in mammary gland hypoplasia while heterozygous mutations of tbx caused decreased branching morphogenesis in mammary glands. our research complements these previous studies showing that tbx over-expression within the mammary glands causes hyperplasia, promoting increased secondary and tertiary branching as well as accelerated ductal elongation. it is also important to discuss that we have over-expressed human tbx within the mammary glands of mice. it has been shown that human tbx and mouse tbx are 97% homologous at the protein level. our group and others have demonstrated that human tbx is functional in mouse cells. furthermore, atbx knockout mouse model was able to recapitulate the phenotype seen in humans with ulnar mammary syndrome. in a study performed by papaioannou et al., a mutation in the mouse tbx gene that closely corresponds to truncation mutations seen in some individuals with ums resulted in a deficiency in mammary placode induction and the absence or reduction of mammary buds in mutant embryos, corresponding to the mammary gland hypoplasia seen in patients with ums. moreover, the deficiency in the development of limb elements in individuals with ums was also reflected in limb abnormalities in the tbx mutant mice. mutant mice had deformities in the forelimb digits, foot and fibula resulting from a failure in the development of posterior limb elements. this study exemplifies that the tbx protein plays a similar role in the development of the mammary glands in both human and mice. the mechanism by which tbx over-expression promotes hyperplasia in mammary glands needs to be elucidated. using an edu cell proliferation assay, we showed that over-expression of tbx resulted in a dramatic increase in cell proliferation within the mammary glands of pregnant doxycycline induced double transgenic mice at dpc. although cell proliferation was not directly quantified for the other developmental time points, the similarity in the observed accelerated mammary gland development suggests that the increase in cell proliferation at dpc may also play a role in causing the accelerated branching and elongation of ducts during the other phases of mammary gland growth. a study has shown that mammary epithelia lacking the gene encoding nfκbia contained increased nfkb activity as well as increased ductal branching and widespread intraductal hyperplasia, similar to results seen in our study. furthermore, aberrant activation of nf-κb increased cell proliferation and breast cancer progression. in this study, we found that tbx inhibits the promoter activity of nfκbib in vitro. upon further analysis, in vivo, we observed that nfκbib expression was dramatically reduced in doxycycline induced double transgenic mice as compared to its un-induced double transgenic littermate controls. taken together, our results suggest a mechanism by which tbx over-expression represses nfkbib/nfkbib expression to enhance cell proliferation and promote mammary gland hyperplasia. however, tbx is a multifunctional transcription factor and the nfkb pathway could be one of many pathways regulated by tbx wnt signaling has also been shown to play a major role in regulating mammary gland development. a tbx3-/- mouse model lacked expression of lef and wnt10b, suggesting that wnt signaling is a downstream target of tbx and that tbx may regulate mammary gland development via the wnt signaling pathway. additional experiments can be done to further elucidate other mechanisms by which tbx over-expression promotes mammary hyperplasia. studies have suggested a role for tbx3/tbx in regulating the self-renewal of mouse embryonic stem cells as well as breast cancer stem-like cells. mouse es cells require leukemia inhibitory factor to maintain their undifferentiated state. mouse es cells genetically modified to over-express tbx and grown in culture without lif were able to maintain their undifferentiated state. knockdown of tbx expression in mouse es cells resulted in a loss of self-renewal, causing these cells to differentiate. these findings suggest that tbx expression is necessary to maintain mouse es cells in their undifferentiated state and plays a functional role to promote self-renewal. a recent study has proposed a model in which the expression of tbx in cancer cells promotes the expansion of cancer stem-like cells through paracrine fibroblast growth factor signaling. over-expression of tbx increased the proportion of cancer stem-like cells in mcf cells by nine-fold as well as lead to an increase in tumorsphere formation and tumor initiation, suggesting that tbx is sufficient to promote normal and cancer stem like cell phenotypes. due to its role in promoting proliferation of mouse es cells and breast cancer stem-like cells as well as its requirement for early mammary gland development, tbx may also play a role in regulating mammary stem cell proliferation. mammary glands consist of two cell lineages: myoepithelial and luminal epithelial cells. both of them arise from a common progenitor, the mammary stem cell. research has shown that a single lin-cd24+cd29high cell is able to generate a functional mammary gland, suggesting that these cells are mammary stem cells. to determine whether over-expression of tbx affects mammary stem cell proliferation, we performed facs analysis of the stem-like cell population, lin-cd24+cd29high, in doxycycline induced double transgenic mice and their un-induced littermate controls. we found that over-expression of tbx significantly increased the frequency of lin-cd24+cd29high stem-like cell population, indicating that tbx expression is associated with an increased number of mammary stem-like cells. this could explain another mechanism by which tbx over-expression can cause hyperplasia and accelerated mammary gland development. further studies of the mechanisms by which tbx regulates mammary stem-like cells are required to improve our understanding of mammary gland development and tbx function.  tbx over-expression causes mammary gland hyperplasia possibly by inhibiting nfκbib expression and thus promoting cell proliferation. also, over-expression of tbx is associated with an increased number of mammary stem-like cells suggesting another mechanism by which tbx may promote mammary gland hyperplasia and contribute to breast cancer development. 
in humans, mutations that result in the haploinsufficiency of tbx causes ulnar mammary syndrome characterized by mammary gland hypoplasia as well as other congenital defects. although over-expression of tbx alone, did not induce tumor formation it did promote accelerated mammary gland development by increasing mammary epithelial cell proliferation. overall, our data suggests that over-expression of tbx may contribute to breast cancer development by promoting accelerated mammary gland development through the inhibition of the nf-κb pathway and stimulation of both mammary epithelial cell and stem-like cell proliferation. we also show that tbx directly binds to and represses nfκbib, an inhibitor of the nf-κb pathway known to play a role in regulating cell proliferation. lastly, we also show that the over-expression of tbx is associated with an increase in mammary stem-like cells. in addition to its role in mammary gland development, various studies have also supported a role for tbx in breast cancer development. the t-box transcription factor tbx is necessary for early embryonic development and for the normal development of the mammary gland. tbx is over-expressed in various breast cancer cell lines as well as cancer tissue and has been found to contribute to breast cancer cell migration. homozygous mutations, in mice, are embryonic lethal while heterozygous mutations result in perturbed mammary gland development. although many studies have shown that a dysregulation of tbx expression may contribute to cancer progression, no direct evidence shows tbx causes breast cancer. in this study, we created doxycycline inducible double transgenic mice to test whether tbx over-expression can induce tumor formation within the mammary gland. previous studies have suggested that tbx contributes to cancer development by its ability to bypass senescence by repressing the expression of p14arf-tumor suppressor.
 the t-box transcription factor tbx is necessary for early embryonic development and for the normal development of the mammary gland. homozygous mutations, in mice, are embryonic lethal while heterozygous mutations result in perturbed mammary gland development. in humans, mutations that result in the haploinsufficiency of tbx causes ulnar mammary syndrome characterized by mammary gland hypoplasia as well as other congenital defects. in addition to its role in mammary gland development, various studies have also supported a role for tbx in breast cancer development. tbx is over-expressed in various breast cancer cell lines as well as cancer tissue and has been found to contribute to breast cancer cell migration. previous studies have suggested that tbx contributes to cancer development by its ability to bypass senescence by repressing the expression of p14arf-tumor suppressor. although many studies have shown that a dysregulation of tbx expression may contribute to cancer progression, no direct evidence shows tbx causes breast cancer.  in this study, we created doxycycline inducible double transgenic mice to test whether tbx over-expression can induce tumor formation within the mammary gland. although over-expression of tbx alone, did not induce tumor formation it did promote accelerated mammary gland development by increasing mammary epithelial cell proliferation. we also show that tbx directly binds to and represses nfκbib, an inhibitor of the nf-κb pathway known to play a role in regulating cell proliferation. lastly, we also show that the over-expression of tbx is associated with an increase in mammary stem-like cells.  overall, our data suggests that over-expression of tbx may contribute to breast cancer development by promoting accelerated mammary gland development through the inhibition of the nf-κb pathway and stimulation of both mammary epithelial cell and stem-like cell proliferation. 