BACKGROUND
the mammalian runx <dig> belongs to the runt domain family of transcription factors.
the members of this gene family, runx <dig>  runx <dig> and runx <dig> are key regulators of lineage-specific gene expression in major developmental pathways  <cit> .
the three runx proteins recognize the same dna-motif and regulate their target genes through interaction with a common group of transcriptional co-activators or co-repressors  <cit> .
interestingly, however, the functional overlaps are minor and each runx protein has a distinct subset of biological functions.
accordingly, each of the corresponding runx knockout  mice displays a unique subset of phenotypic abnormalities  <cit> .
this lack of functional redundancy results from a tightly regulated spatio/temporal expression mediated by an intricate transcriptional control  <cit> .
for example, both runx <dig> and runx <dig> genes are expressed in developing dorsal root ganglia, but in different classes of sensory neurons  <cit>  and both are expressed in mature t cells, but at different stages during t cell development  <cit> .
in the mouse embryo, expression of runx <dig> is first detected in definitive hematopoietic stem cells  and in endothelial cells at hsc emergence sites  <cit> .
these occurrences correlate well with the earlier findings that homozygous disruption of runx <dig> results in a complete absence of fetal liver hematopoiesis  <cit> .
postnatally, runx <dig> is highly expressed in several hematopoietic lineages including myeloid, b- and t-lymphoid cells  <cit>  and is required for megakaryocytic maturation  <cit> .
in contrast to the critical role of runx <dig> during embryogenesis, it is less essential for adult hematopoiesis  <cit> , albeit conditional inactivation is associated with a number of hematopoietic abnormalities including myeloproliferation  <cit> .
runx <dig> is also expressed in a number of other tissues at specific time windows during embryogenesis  <cit> .
however, due to the early  lethality of homozygous null runx <dig> mice  <cit> , relatively little is known about the function of runx <dig> in these tissues.
in developing thymus runx <dig> is more abundantly expressed in the cortex  <cit>  during early stages of thymopoiesis  <cit> .
transgenic mice that express a dominant-negative form of runx <dig> display defects in maturation of single positive  cd <dig> and sp cd <dig> thymocytes  <cit> .
additionally, naive t cells from these mice exhibited increased production of il <dig>  lack of gata <dig> expression and enhanced th <dig> differentiation of cd4+ helper-t cells  <cit> .
selective inactivation of runx <dig> in t-cell progenitors demonstrated that runx <dig> acts in double negative  thymocytes to repress cd <dig> expression and to up-regulate cd <dig> as cells differentiate into the double positive  thymocytes  <cit> .
es cells bearing a mutant runx <dig> lacking the c-terminal repression sub-domain, failed to adequately contribute to the thymus in chimera mice  <cit> .
in the knock-in model of this mutant, the overall number of thymocytes was significantly reduced and the thymus contained a higher proportion of sp cd <dig> cells  <cit> .
we have previously shown that transcription of runx <dig> is regulated by two distantly located promoter regions designated p <dig> and p <dig> for the distal and proximal, respectively  <cit> .
the p <dig> is nested within a particularly large and evolutionary conserved cpg island  <cit> , while no such cpg rich region is found in p <dig>  the two promoters are regulated in a cell type-specific manner and respond to mitogenic stimulation  <cit> .
the p1- and p2-derived primary transcripts are processed into a diverse repertoire of alternatively spliced mrnas that are differentially expressed in various cell types and at different developmental stages  <cit> .
the p <dig> and p <dig> mrnas differ at their 5'-coding regions  <cit> , and the resulting protein isoforms differ in their biological functions  <cit> .
similarly, a number of p2-derived splice-variants bear shorter reading frames that lack the transcriptional activation domain and can act in a dominant negative manner  <cit> .
forced expression of such isoforms results in transcriptional repression of runx <dig> target genes  <cit> , and in blocking of myeloid differentiation  <cit> .
hence, differential usage of p <dig> versus p <dig> has a profound effect on the repertoire and nature of runx <dig> isoforms in the cell, which likely impacts on the regulation of runx <dig> target genes  <cit> .
significantly, disruption of p <dig> by the 12; <dig> chromosomal translocation results in the most common subtype of childhood acute lymphoblastic leukemia  <cit> .
p1- and p2-transcripts also differ in the structure and function of their 5' untranslated region .
the p1-5'utr is relatively short and mediates cap-dependent translation, whereas the p2-5'utr is particularly long, contains an internal ribosome entry site  and mediates cap-independent translation  <cit> .
these structurally and functionally different 5'utrs could have a profound effect on translation efficiency of runx <dig> mrnas.
indeed, in cell lines where p <dig> transcription predominated, p2-5' ires-dependent translation was more efficient than p1-5' cap-dependent translation  <cit> .
ires-dependent translation is particularly important during development, in times when cap-dependent translation in proliferating or differentiating cells is down-regulated  <cit> .
while it is clear that the regulation of runx <dig> expression by alternative p1/p <dig> usage is a potentially key paradigm in the in vivo regulation of runx <dig>  very little is known about the role of the p1/p <dig> switch in mediating tissue and stage specific expression of runx <dig> during development.
to investigate the biological function of the p1/p <dig> switch during embryogenesis, we generated mice bearing a hypomorphic runx <dig> allele by inserting a neomycin  gene into the p <dig> region .
mice homozygous for the p2neo allele  displayed a markedly attenuated p <dig> activity.
nevertheless, these mutant mice developed to term, but newborns died within a few days after birth.
we show that the p1/p <dig> switch is developmentally regulated and that the two promoters are alternatively used during embryogenesis.
p <dig> mediated transcription is required during early thymopoiesis for the proper development of the thymus and for differentiation of dn and dp cd4/cd <dig> t cells.
attenuation of p <dig> activity results in impaired development of fetal liver hematopoietic precursors and in enhanced apoptosis of thymocytes due to altered expression of t-cell receptors .
the data describe previously unknown functions of runx <dig>  delineate the activity of p <dig> and p <dig> in embryogenesis, show unequivocally that their function during development is non redundant and underscore the significance of the two promoters in runx <dig> biology.
RESULTS
generation of runx <dig> p2neo/neo mice and phenotypic analysis of mutant newborns
null mutation in runx <dig> results in embryonal lethality  <cit> .
as p <dig> activity starts early in embryogenesis , we envisaged a scenario in which germline inactivation of p <dig> will also result in embryonal death.
to avoid this occurrence we explored means to selectively attenuate the activity of runx <dig> p <dig>  we have previously demonstrated that the genomic region upstream the transcription start sites of runx <dig> p <dig> was important for regulated transcription of runx <dig> in various cell lines  <cit> .
we thus assessed whether insertion of a neo cassette into this region affects p <dig> activity in transfected cells .
transfection experiments indicated that insertion of a neo cassette into the p <dig> region specifically attenuated p2-mediated transcription in several cell lines and encouraged us to utilize this approach for evaluating the biological activity of runx <dig> p <dig> in vivo.
runx <dig> p <dig> was targeted in es cells using a construct in which the neo cassette was placed at the same site as in the p2neo-ren construct , but in a transcriptional orientation opposite to that of runx <dig>  and was flanked by lox p sites .
homologous recombination yielded es clones , which were used to generate several runx1p2neo/+ chimeric males that passed on the runx <dig> mutation through the germ line.
mating f <dig> heterozygotes with 129/sv, icr or mf <dig> mice generated runx1p2neo/neo mice with an inbred or a mixed background.
heterozygous runx1p2neo/+  mice appeared phenotypically indistinguishable from their wt littermates.
however, homozygous runx1p2neo/neo  neonates were reduced in size, gained little weight and most of them died  <dig> to  <dig> days post natally .
to further characterize the phenotype/genotype relationships of the mutant mice, heterozygotes p2neo/+ mice were mated with two different strains of transgenic mice expressing cre driven by either pgk or eiia promoters  <cit>  .
the resulting heterozygotes bearing the remaining loxp sequence within the p2-xbai site were mated and offspring homozygous to p2-loxp were obtained at the expected mendelian proportion .
significantly, runx1p2loxp/loxp  mice were indistinguishable form wt littermates indicating that the early lethality phenotype of p2neo/neo newborns was due to the presence of the neo cassette in the p <dig> region.
attenuated runx <dig> expression in p2neo/neo mice
the above-described phenotypic manifestation of the p2neo/neo allele posed a question as to how the expression level of runx <dig> was affected.
to address this issue, runx <dig> expression in various tissues was examined using rt-pcr, immunohistochemistry  and rna in situ hybridization  .
in tissues of p2neo/neo mice, such as kidney and tongue, where runx <dig> expression is normally mediated by both p <dig> and p <dig>  only p1-transcripts were detected .
in contrast, no runx <dig> expression was detected by either rt-pcr or ihc in developing gastric epithelium where only p <dig> is normally active .
p2neo is therefore a hypomorphic allele of runx <dig> in which p <dig> activity is largely diminished.
interestingly, however, no developmental abnormalities were detected by macroscopic and histological examination of kidney, stomach and tongue of mutant embryos or newborns.
the most obvious and consistent macroscopic abnormality was a profound reduction in size of the thymus of newborn p2neo/neo mice as compared to wt .
western blot analysis, rish using a p <dig> specific probe and rt-pcr showed that during early thymogenesis   <dig> – <dig> ) transcription of runx <dig> in wt thymus was predominately mediated by p <dig> , and that at this stage almost no runx <dig> was detected in p2neo/neo thymus .
the p <dig> activity in wt thymus was switched-on between e <dig> –e <dig>  and persisted thereafter, whereas the activity of p <dig>  which predominated during embryogenesis, gradually decreased after birth .
importantly, once switched-on the p <dig> activity was apparently unaffected in p2neo/neo mice, as evidenced by northern blot analysis of runx <dig> mrnas in newborns .
together, the complementary results of runx <dig> expression studies and morphological analyses showed that expression of runx <dig> in p2neo/neo mice was compromised both temporally and spatially, resulting in adverse effect on thymus development.
impaired thymus development in runx <dig> p2neo/neo mice
thymus development in mouse embryo begins at e <dig>  ensued by immigration of lymphocyte precursors into the thymic rudiment at e <dig> .
development proceeds through stage specific interactions between thymocytes and thymic epithelium that are essential for the formation of thymic architecture  <cit> .
runx <dig> is highly expressed in developing thymocytes, but not in thymic epithelium  <cit> .
macroscopic examinations revealed that thymi of p2neo/neo neonates were about one-third of the normal size .
this diminished size was caused by a profound reduction in the number of thymocytes during mutant thymus development .
histological examinations revealed no gross morphological differences between p2neo/neo and wt at e <dig>  and e <dig>  , except for scattered cysts in p2neo/neo thymi .
these cysts are thought to result from accelerated epithelial turnover within centers of epithelial islands  <cit> .
at e <dig>  however, extensive histological differences between wt and p2neo/neo emerged .
wt thymus had a clear demarcation of cortex and medulla.
wt cortex was densely cellular with small hyperchromatic thymocytes, while the medulla was paler, contained a lower number of thymocytes admixed with macrophages, epithelial cells, and antigen presenting cells .
mutant thymus, on the other hand, was much smaller, hypocellular, had a scalloped capsular surface and consisted predominantly of epithelial cells, with no evidence of an organized cortex and medulla.
at e <dig>  the histological defects were even more pronounced; the p2neo/neo thymus still lacked corticomedullary organization and contained occasional cysts .
ihc using anti runx <dig> antibodies revealed a lower number of runx <dig> positive cells in e <dig>  and e <dig>  p2neo/neo thymi compared to wt .
runx <dig> is mostly expressed in dn cd4-/cd8- immature thymocytes, where it regulates cd <dig> expression  <cit> .
ihc of e <dig> –e <dig>  wt thymus revealed that these runx <dig> positive immature thymocytes were predominantly in the cortex and less in the medulla , as also shown by the rna in-situ hybridization data .
in p2neo/neo thymus on the other hand, the fewer runx <dig> positive cells were scattered randomly within the organ .
at e <dig>  most of runx <dig> expressing dn cd4-/cd8- thymocytes were found in the sub-cortical band , whereas the cortical, dp cd4+/cd8+ thymocytes did not express runx <dig> .
in e <dig>  p2neo/neo thymus, many runx <dig> positive cells were still in the cortex and only after birth, when the mutant thymus gradually gained wt morphology, were runx <dig> expressing hyperchromatic cells seen in the scb .
but even then, the overall size of mutant thymus remained much smaller compared to wt .
the data indicate that normal embryonal thymopoiesis depends on a proper p2-mediated transcription of runx <dig> which when attenuated during the e <dig> –e <dig>  time period results in impaired thymus development.
increased apoptosis in embryonal p2neo/neo thymocytes
to directly assess whether the thymic hypocellularity of p2neo/neo embryos was a consequence of enhanced cell death, the proportion of apoptotic thymocytes was determined by flow cytometry.
at e <dig>  a much higher proportion of p2neo/neo thymocytes  compared to wt  bound annexin v, indicating increased apoptosis among p2neo/neo thymocytes .
additionally, increased proportion of necrotic cells positive to both annexin v and propidium iodid , was observed in p2neo/neo thymus .
the enhanced cell death of e <dig>  p2neo/neo thymocytes led to a three-fold reduction in the proportion of viable thymocytes in mutant thymus .
at e <dig> – <dig>  the p <dig> promoter is switched on , expression of runx <dig> in p2neo/neo thymocytes increased  and recovery of thymopoiesis commenced.
the end result was an increased proportion of viable p2neo/neo thymocytes at e <dig>  almost to wt level .
the enhanced apoptosis in p2neo/neo e <dig>  and e <dig>  thymus was also demonstrated by in situ tunel labeling and ihc of activated caspase- <dig> .
the proportion of non-viable thymocytes at various stages of thymus development is shown in fig.
3b.
at early stages, apoptosis was significantly higher in p2neo/neo fetuses compared to wt, but gradually declined concomitant with the increase in p1-mediated expression of runx <dig>
we next asked whether the proliferative capacity of p2neo/neo thymocytes was also affected.
e <dig>  thymocytes were isolated, analyzed by facs and similar numbers of viable cells cultured in-vitro under proliferation stimulating conditions .
purified p2neo/neo and wt thymocytes displayed a similar thymidine incorporation rate, indicating that the ability of thymocytes to proliferate was hardly affected by the attenuated expression of runx <dig> during early thymopoiesis.
this conclusion was supported by immunostaining of thymocytes with the cell proliferation-associated antigen ki- <dig>  in e <dig>  p2neo/neo thymus the number of ki- <dig> positive cells was lower than in wt , reflecting the hypocellularity of mutant thymus .
nevertheless, significant numbers of p2neo/neo thymocytes did express ki- <dig>  indicating normal progression through the cell cycle.
together, these results show that p2neo/neo thymocytes possess proliferation and cell cycle progression capabilities similar to wt, but attenuating runx <dig> expression during early developmental stages rendered thymocytes more susceptible to apoptosis, leading to thymic hypocellularity.
to address whether the increased apoptosis of p2neo/neo thymocytes was due to loss of a cell-autonomous function of runx <dig>  we examined the capacity of p2neo/neo thymocytes to populate fetal thymic organ culture .
e <dig>  wt thymic lobes were reconstituted, at a saturating cell number, with either wt or p2neo/neo e <dig>  fetal liver  cells that contain hematopoietic progenitor cells .
at ftoc day  <dig> and  <dig>  cells were extracted out of the lobes and analyzed by facs for annexin v binding.
p2neo/neo hpc gave rise to a significantly lower number of ftoc cells compared to wt and most p2neo/neo derived cells were annexin vhigh .
these data further demonstrated the intrinsic requirement for p2-mediated expression of runx <dig> during early embryonal thymopoiesis.
the enhanced apoptosis of p2neo/neo thymocytes is associated with increased expression of tcrβ/tcrγδ
the above results raised the question of how does attenuated expression of runx <dig> cause the increased apoptosis of p2neo/neo thymocytes.
the majority of immature thymocytes with a non-productive rearrangement of tcrβ gene die through fas/fas ligand-mediated apoptosis  <cit> .
only a small subset of immature thymocytes with productive tcrβ or γδ rearrangement are selected for further maturation associated with rapid proliferation  <cit> .
interestingly, the percentage of tcrβ/tcrγδ expressing thymocytes was significantly higher in e <dig>  to e <dig>  p2neo/neo fetuses compared to wt , as was the mean-fluorescence-intensity  level of tcrβ+ thymocytes .
these data indicate an enhanced tcrβ/γδ expression on p2neo/neo thymocytes compared to wt during the early stages of embryonal thymopoiesis.
runx <dig> acts in transfected cells as a negative regulator of tcrβ transcription  <cit> .
it is thus possible that the diminished runx <dig> expression between e <dig>  to e <dig>  caused de-repression of tcr transcription in p2neo/neo thymocytes, leading to the increased level of tcrβ/γδ.
supporting this assumption are data demonstrating that upregulation of tcrβ/γδ in p2neo/neo thymocytes occurred at the transcriptional level , and that down regulation to wt levels occurred at e <dig>  , upon turning-on of p <dig> .
we next addressed whether increased expression of tcrβ/γδ in p2neo/neo thymocytes was associated with the increased susceptibility to apoptosis of the mutant cells.
annexin v staining was monitored in fractionated tcrβ+ or tcrγδ+ thymocytes .
in e <dig>  and e <dig>  embryos 50–65% of wt and 80–90% of p2neo/neo tcrβ+ thymocytes were apoptotic as were ~95% of both wt and p2neo/neo tcrγδ+ thymocytes .
additionally, at these developmental stages a significantly higher proportion of p2neo/neo thymocytes express the tcrs .
thus, the enhanced apoptosis in p2neo/neo compared to wt thymus , was closely associated with an increase in tcrβ/γδ expression.
of note, mfi of annexin v+ apoptotic p2neo/neo thymocytes, was lower compared to the wt , presumably due to a lower level of cell surface phosphatidyl-serine in p2neo/neo cells.
together, the complementary outcome of these experiments indicates that runx <dig> functions in vivo as a negative regulator of tcr expression.
when the temporal expression of runx <dig> is attenuated, levels of tcrβ+/tcrγδ+ thymocytes increase, which may lead to enhanced cell death.
when p <dig> is turned-on, restoring runx <dig> expression in p2neo/neo cells, apoptosis in mutant thymocytes assumes normal levels.
impaired thymocyte differentiation in p2neo/neo embryos
we next employed flow cytometry to study thymocyte differentiation in p2neo/neo embryos and neonates.
first we examined the cd4/cd <dig> double negative  population at e <dig>  when nearly 100% of thymocytes are dn.
the dn population is divided into four sub-groups, dn <dig> to dn <dig>  according to the expression of cd <dig> and cd <dig> surface markers.
compared to wt, p2neo/neo embryos displayed an increase in the proportion of dn <dig>  thymocytes and a corresponding reduction in dn <dig> cells  , resulting in a significant increase in the ratio dn3/dn <dig> among p2neo/neo thymocytes .
to examine p1/p <dig> usage in the dn sub-groups we used flow cytometry to sort e <dig>  thymocytes based on surface expression of cd <dig> and cd <dig> , and levels of p <dig> and p <dig> transcripts was determined.
the levels of p <dig> and p <dig> expression did not differ significantly between dn <dig> and dn <dig> subgroups and was similar to the level shown in fig.
1d.
next we examined the cd8/cd <dig> double positive population.
at e <dig> , p2neo/neo embryos displayed a  <dig> -fold reduction in the proportion of double positive  cd4+/cd8+ thymocytes as compared to wt .
at e <dig>  the decrease in proportion of dp thymocytes in p2neo/neo embryos was more pronounced and accompanied by a marked increase in an abnormal population of cd4+/cd8low cells .
a similar subpopulation of immature cd4+/cd8low was previously observed in mice with thymocyte specific targeting of runx <dig>  <cit>  and in mice bearing a hypomorphic cbfβ allele  <cit> .
it was postulated that this abnormal cd4+/cd8low population represents immature dp thymocytes in which cd <dig> expression was diminished and cd <dig> expression was partially de-repressed  <cit> .
delayed differentiation of p2neo/neo thymocytes was still apparent at post-natal day   <dig>  , but recovery to wt proportions was noted at p <dig> .
taken together, the change in the proportion of dn3/dn <dig> thymocytes at early thymopoiesis, the decrease in dp cd4/cd <dig> thymocytes and the increase in the abnormal sp cd4+/cd8low population, indicate that during embryonal thymopoiesis p <dig> activity is also required for regulation of cd4/cd <dig> expression and thereby for the proper differentiation of dn and dp thymocytes.
reduction in committed t-cell precursors  in p2neo/neo fetuses
runx <dig> is essential for embryonal hematopoiesis and runx1-/- mice lack definitive hematopoiesis as well as hemapoietic colony forming activity in vitro  <cit> .
in developing fl, runx <dig> is expressed in hsc and in early progenitors  <cit> , and in wt fl both promoters are active , whereas p <dig> activity was largely attenuated .
to assess hematopoietic activity, individual fls from wt and p2neo/neo embryos were isolated and analyzed for in vitro colony formation.
as shown in fig.
6b, the number of colonies grown in p2neo/neo cultures was significantly lower compared to wt.
however, because the size of the colonies in p2neo/neo and wt cultures was similar , the results indicate that p2neo/neo fl cells contained fewer progenitors compared to wt.
this conclusion was supported by the finding that e <dig>  and e <dig>  p2neo/neo embryos showed marked reduction  in the proportion of gr-1+/cd18+ and cd11a+/cd11b+ cells compared to wt littermates .
we next assessed the effect of the p2neo/neo hypomorph on the development of fl hpc.
runx <dig> expressing fl hpc also express cd <dig>  c-kit and cd <dig>  <cit> .
in e <dig>  and e <dig>  p2neo/neo embryos, a 2-fold reduction in the proportion of cd44+/c-kit+ hpc compared to wt littermates was observed , and even greater reduction was observed in cd <dig> positive cells at e <dig> –e <dig>  .
fl hpc that are b220low/c-kit+/cd19- represent an hpc population restricted to the t-cell and natural killer cell lineages  <cit> .
these fl progenitors, termed t-cell precursors , are thought to represent the immediate developmental step before thymic immigration.
because the hypocellularity of p2neo/neo thymus was noted at the earliest developmental stages , the proportion of tcps in wt and p2neo/neo was compared.
facs analysis of b220low/c-kit+/cd19- e <dig>  fl cells, revealed a fourfold reduction in the proportion of p2neo/neo tcps compared to wt .
these results indicated that the proportion of runx <dig> expressing fl hpc, particularly the tcp subset, was markedly reduced in p2neo/neo compared to wt.
this conclusion was supported by in vitro ftoc progenitor assay, which monitors the capacity of fl tcps to reconstitute thymic lobes  <cit>  .
when e <dig>  wt and p2neo/neo fl cells were cultured under limiting dilution conditions in ftocs, the frequency of successfully populated thymic lobes was lower for p2neo/neo tcps as compared to wt  .
additionally, when day  <dig> ftocs cells were obtained and cd4/cd <dig> positive cells analyzed by facs, an abnormal differentiation of p2neo/neo thymocytes was observed with increased proportion of the abnormal population dp cd4+/cd8low and a twofold reduction in the proportion of sp cd4+ or cd8+ compared to wt .
this ftoc result corresponds well with the in vivo delayed maturation of p2neo/neo fetal thymocytes described in fig.
<dig>  collectively, these data indicate that in p2neo/neo embryos the development of fl hpc of different lineages was impaired.
runx <dig> p2neo allele failed to rescue the embryonal lethality phenotype of runx1-/- mice
homozygous disruption of runx <dig> results in a complete absence of fl hematopoiesis and the mice die between e <dig>  and e <dig>  of hemorrhages in the central nervous system  <cit> .
p2neo/neo mice were born and showed no sign of hemorrhages, indicating that expression levels of runx <dig> in homozygous p2neo/neo during early embryogenesis were sufficient to rescue the runx1-/- phenotype.
this occurrence raised the question of whether runx <dig> levels provided by activity of a single p <dig> will also be sufficient to rescue the null phenotype.
to address this issue we generated a compound mutant strain runx1lz; runx1p2neo by mating runx1lz/+ mice  <cit>  with runx1p2neo/+.
these mice had one null allele expressing a fused runx1-lacz protein and one p2neo allele expressing wt p <dig> .
compound mutant runx1lz;runx1p2neo embryos died between e <dig>  and e <dig>  and suffered from hemorrhages in the nervous system .
the most extensive bleedings was seen in the 4th ventricle, the ventral metencephalon and spinal cord.
moreover, the liver of e <dig>  compound mutant embryos was depleted of hematopoietic cells , indicating lack of definitive hematopoiesis, similar to the previously reported runx1-/- phenotype  <cit> .
these results indicated, that during midgestation, angiogenesis and definitive hematopoiesis are highly sensitive to the dosage of runx <dig> as activity of a single p <dig> was not sufficient to rescue the runx1-/- phenotype.
this conclusion is consistent with the previously reported data which was based on transplantation assays  <cit> .
this observation may also pertain to the unique role of p <dig> activity during midgestation angiogenesis and/or early hematopoiesis.
early lethality phenotype of p2neo/neo neonates is rescued by excision of the neo cassette in early t cell progenitors
as noted earlier, p2neo/neo neonates die within a few days after birth.
the reasons for this early lethality are not clear.
while p2-mediated expression diminished in several p2neo/neo tissues, including stomach, intestine and kidney , the most striking abnormalities were noted in thymic development and thymopoiesis.
we thus addressed whether these abnormalities could be rescued by specific removal of the neo cassette in early thymopoiesis and whether this occurrence will extend the life span of p2neo/neo neonates.
runx1-p2neo/neo mice were mated with a lck-cre transgenic line  <cit>  to excise the neo and regain p <dig> activity in thymocytes .
contrary to p2neo/neo mice, the p2/lck-cre mice developed normally and outwardly were indistinguishable from wt littermates.
nevertheless, embryonal thymopoiesis of p2/lck-cre was not completely normal and embryos exhibited a lower number of runx <dig> expressing thymocytes .
this may be due to an incomplete excision of the neo cassette in all t cell progenitors.
significantly, however, runx <dig> expression level in p2/lck-cre was sufficient to allow for normal thymic development.
of note, p2-mediated runx <dig> expression in other runx <dig> expressing cells, such as epithelial cells, was not rescued in p2/lck-cre mice .
discussion
runx <dig> is an important cell-linage specific regulator of definitive hematopoiesis and thymopoiesis.
the unique spatio/temporal expression pattern of runx <dig> is attained through alternative usage of its two promoters, p <dig> and p <dig>  <cit>  that also impacts on translation efficacy and repertoire of protein isoforms  <cit> .
while it was clear that transcriptional regulation of runx <dig> expression is an important element of its biological function, little was known about the role of the two promoters during development, and about how and when they are differentially regulated.
is the alternative promoter usage of human and mouse runx1/runx <dig> a reflection of vertebrates' higher complexity?
differential usage of alternative promoters enables diversified transcriptional regulation within a single locus and may thus serve as a molecular basis for the higher complexity of certain vertebrates' systems, such as the immune system.
indeed, while the runx genes are highly conserved throughout the animal kingdom, more primitive animals such as the nematode c. elegance and the sea urchin contain only one gene regulated by a single promoter  <cit> .
this single promoter is the equivalent of vertebrates' p <dig>
differential p1/p <dig> usage during embryogenesis
the use of hypomorphic alleles for studying essential genes at the organismal level is a well-established approach.
we employed mice homozygous for a hypomorphic runx <dig> allele, which caused a profound attenuation of p <dig> activity, to investigate the biological role of p <dig> during embryogenesis.
specifically, we analyzed the impact of the resulting hypomorphic runx <dig> expression on fl hpc development and early thymopoiesis.
we found that during embryogenesis runx <dig> is developmentally regulated through alternative usage of p <dig> and p <dig>  in certain tissues, including kidney, tongue and liver both p <dig> and p <dig> are active, whereas in others such as intestinal epithelium and early thymocytes p <dig> activity predominates.
contrary to runx <dig> null mice, in which embryonic lethality occurs at ~e <dig>   <cit> , p2neo/neo mice develop to term, implying that during early development the activity of p <dig> prevails.
this conclusion is supported by the finding that both p1- and p2-transcripts are found in fl of e <dig>  embryos  <cit> .
the notion that both p <dig> and p <dig> are active during early hematopoiesis correlates well with the finding that p2neo/neo embryos displayed moderate and severe hematopoietic defects in the liver and thymus, respectively.
p <dig> activity is essential for embryonal thymopoiesis and thymus development
thymic development in the mouse begins at ~e <dig>   <cit> .
runx <dig> is highly expressed in thymocytes, but not in thymus epithelium  <cit> .
using in-situ hybridization, rt-pcr and ihc we demonstrate that during early thymopoiesis activity of p <dig> predominates.
the p <dig> is upregulated at e <dig>  and its activity persists thereafter, while the activity of p <dig> declines after birth.
at e <dig> the thymic rudiment is colonized by the first wave of committed t-cell precursors   <cit>  which endure in the thymus until e <dig>  <cit> .
it is tempting to speculate that p <dig> activity is confined to descendants of the first wave tcp, whereas p <dig> is active in thymocytes originating in the second wave.
consistent with this hypothesis, the first wave p2neo/neo thymocytes contained a markedly reduced level of runx <dig> compared to wt, whereas in second wave thymocytes the level was similar to wt due to switch-on of p <dig> at e <dig> .
these second wave thymocytes grow exponentially after birth, when p <dig> is more active, and become the major cell population in the thymus.
during early stages of thymus development  a marked reduction in thymic cellularity was observed in p2neo/neo embryos.
we show that this occurrence was due to enhanced apoptosis of p2neo/neo thymocytes.
but why were e <dig>  to e <dig>  p2neo/neo thymocytes more susceptible to apoptosis than wt cells?
selection processes in the thymus ensure that peripheral t cells are responsive to foreign antigens but tolerant to self-antigens.
this occurs through positive and negative β-selections.
thymocytes, whose tcr engaged peptide/mhc molecules productively, escape their inherent programmed cell death and subsequently are induced to proliferate.
on the other hand, thymocytes that react strongly with self-peptide-mhc molecules are actively eliminated  <cit> .
according to the avidity model, selection occurs only when signals received through the tcr fall between two thresholds: signals below the one are incompatible with survival , while signals above the other result in deletion  <cit> .
we hypothesize that the elevated tcr levels in p2neo/neo thymocytes disturbed this delicate balance and disposed a larger proportion of p2neo/neo t cells to apoptosis.
several studies in transfected cells have demonstrated the involvement of runx <dig> in transcription regulation of tcrs expression  <cit> .
specifically, we have shown that runx <dig> can act as a transcriptional repressor in tcr regulation  <cit> .
thus, it is conceivable that the increased levels of tcrs in embryonic p2neo/neo thymocytes resulted from the reduced runx <dig> expression in these cells, and that the higher tcrs levels lead to a higher avidity of tcr-mhc interactions, which programmed the cells to apoptosis.
this hypothesis is supported by runx1f/f/lck-cre mice, which show enhanced negative selection of runx1-/- thymocyte  <cit> , and by the finding that upon recovery of runx <dig> expression in p2neo/neo thymocytes, tcr expression and apoptosis resumed wt levels.
potentially related, mice homozygous for a knock-in runx <dig> cdna that are lacking the c-terminal vwrpy, exhibit thymic hypocellularity  <cit> .
the vwrpy motif, which is highly conserved among all runx proteins, is essential for recruitment of the transcriptional co-repressor gro/tle  <cit>  and subsequent repression of target genes  <cit> .
thymic hypocellularity was also noted in mice bearing a hypomorphic allele of cbfβ, the non-dna binding partner of runx <dig>  which is essential for the function of runx proteins  <cit> .
in addition to the marked hypocellularity, thymi of p2neo/neo embryos contained an increased proportion of dn <dig>  thymocytes, a reduced proportion of dp cd4+/cd8+ thymocytes and a pronounced increase in an abnormal population of immature cd4+/cd8low cells.
the increase in dn <dig> and cd4+/cd8low thymocyte populations was previously observed in adult runx1f/f/lck-cre mice due to diminished runx <dig> in dn thymocytes and the consequent derepression of cd <dig>  <cit> .
more recently a similar increase in cd4+/cd8low thymocytes was also observed in fetuses of cbfβrss/- mice bearing the hypomorphic cbfβ allele  <cit> , indicating that cbfβ is required for cd <dig> silencing by runx <dig>  abnormally high proportion of cd4+ thymocytes was also noted in embryos of the runx <dig> knock-in mutant lacking the vwrpy motif  <cit> .
this finding suggests that runx1-mediated repression of cd <dig> in dn thymocytes involves the recruitment of gro/tle, as in case of runx3-mediated silencing of cd <dig> in dp thymocytes  <cit> .
attenuation of p <dig> activity affects development of fl hpc
runx <dig> is essential for the emergence of embryonal hsc  <cit> .
analysis of fl hematopoiesis in p2neo/neo embryos revealed that the emergence and commitment of hpc to either myeloid or lymphoid lineages was not affected by attenuation of p <dig>  however, the noted delay in differentiation and expansion of various p2neo/neo progenitor populations indicated that development of fl hpc was sensitive to diminution in p <dig> activity.
interestingly, the overall phenotype of fl hpc in p2neo/neo embryos resembled the phenotype observed by cai et al  <cit>  in heterozygous runx1+/- fetuses, suggesting a gene dosage effect.
in developing fl activity of p <dig> emerged early and persisted throughout gestation  <cit> .
as p <dig> activity was largely unaffected in p2neo/neo hpc, it is conceivable that the hypomorphic expression in p2neo/neo fl creates conditions resembling haploinsufficiency  <cit> , in line with the large body of literature documenting hematopoietic defects due to hemizygous dosage of runx <dig>  <cit> .
however, it is worth noting that the effect of p2neo/neo hypomorph is not merely through gene dosage, because contrary to runx1+/- the p2neo/neo neonates die within a few days after birth.
rescue of the early lethality of p2neo/neo neonates
despite the diminished p2-activity and reduced runx <dig> dosage, p2neo/neo embryos developed to term.
thus the residual p <dig> activity plus the apparent wt expression of p <dig> were sufficient to support normal development of p2neo/neo embryos through gestation.
however, inactivation in p2neo/neo embryos of one p <dig> allele, as in the compound mutant runx1lz;runx1p2neo, which further reduced runx <dig> dosage, caused hemorrhages in the nervous system and mid-gestation  lethality.
this result defined the threshold level requirement of runx <dig> for rescue of the nervous system hemorrhages.
while p2neo/neo embryos developed to term, neonates die within a few days after birth.
specific removal of the floxed p2-neo cassette by crossing p2neo/+ mice with lck-cre transgenics resumed p <dig> expression in early thymopoiesis, allowed for normal thymic development and rescued the neonatal lethality.
of note, the neonatal lethality was rescued even though expression of p <dig> in the gastrointestinal tract of p2neo/neo/lck-cre mice was not resumed.
this occurrence indicated that lethality of p2neo/neo neonates was not due to lack of runx <dig> expression in gastrointestinal tract epithelium, despite the striking reduction of nutrients in the gastrointestinal tract of p2neo/neo neonates .
as athymic nude mice live through adulthood, it is conceivable that impaired thymopoiesis in p2neo/neo embryos, especially at the negative selection step, resulted in emergence of pathogenic t cells that cause the neonatal lethality.
finally, as the n-terminus of runx <dig> proteins encoded by p2-mrna differs from that of p <dig>  <cit> , it is tempting to speculate that the hematopoietic phenotypes of p2neo/neo embryos were caused not only by gene dosage, but are due to the lack of p <dig> isoforms.
elucidating the specific role of the p1- and p2-protein isoforms in runx <dig> biology, particularly early thymopoiesis, awaits further investigations.
CONCLUSIONS
differential usage of alternative promoters enables diversification of transcriptional regulation within a single locus and thereby plays a significant role in the control of gene expression.
during mouse embryogenesis the spatio/temporal expression of runx <dig> is developmentally regulated by alternative usage of two mutually distinct promoters, p <dig> and p <dig>  these promoters are not only differentially regulated, but also give rise to mrnas with variant 5'-utrs and to structurally different protein isoforms.
our studies support a model whereby attenuation of p <dig> affects mrna levels and protein isoforms repertoire within the cell and thereby creates the hematopoietic phenotype of p2neo/neo embryos.
the findings provide in vivo evidence for the non-redundant function of p <dig> and p <dig> and underscore the significance of alternative promoter usage in runx <dig> biology.

 the mammalian runx belongs to the runt domain family of transcription factors. the members of this gene family, runx runx and runx are key regulators of lineage-specific gene expression in major developmental pathways. the three runx proteins recognize the same dna-motif and regulate their target genes through interaction with a common group of transcriptional co-activators or co-repressors. interestingly, however, the functional overlaps are minor and each runx protein has a distinct subset of biological functions. accordingly, each of the corresponding runx knockout mice displays a unique subset of phenotypic abnormalities. this lack of functional redundancy results from a tightly regulated spatio/temporal expression mediated by an intricate transcriptional control. for example, both runx and runx genes are expressed in developing dorsal root ganglia, but in different classes of sensory neurons and both are expressed in mature t cells, but at different stages during t cell development. in the mouse embryo, expression of runx is first detected in definitive hematopoietic stem cells and in endothelial cells at hsc emergence sites. these occurrences correlate well with the earlier findings that homozygous disruption of runx results in a complete absence of fetal liver hematopoiesis. postnatally, runx is highly expressed in several hematopoietic lineages including myeloid, b- and t-lymphoid cells and is required for megakaryocytic maturation. in contrast to the critical role of runx during embryogenesis, it is less essential for adult hematopoiesis, albeit conditional inactivation is associated with a number of hematopoietic abnormalities including myeloproliferation. runx is also expressed in a number of other tissues at specific time windows during embryogenesis. however, due to the early lethality of homozygous null runx mice, relatively little is known about the function of runx in these tissues. in developing thymus runx is more abundantly expressed in the cortex during early stages of thymopoiesis. transgenic mice that express a dominant-negative form of runx display defects in maturation of single positive cd and sp cd thymocytes. additionally, naive t cells from these mice exhibited increased production of il lack of gata expression and enhanced th differentiation of cd4+ helper-t cells. selective inactivation of runx in t-cell progenitors demonstrated that runx acts in double negative thymocytes to repress cd expression and to up-regulate cd as cells differentiate into the double positive thymocytes. es cells bearing a mutant runx lacking the c-terminal repression sub-domain, failed to adequately contribute to the thymus in chimera mice. in the knock-in model of this mutant, the overall number of thymocytes was significantly reduced and the thymus contained a higher proportion of sp cd cells. we have previously shown that transcription of runx is regulated by two distantly located promoter regions designated p and p for the distal and proximal, respectively. the p is nested within a particularly large and evolutionary conserved cpg island, while no such cpg rich region is found in p the two promoters are regulated in a cell type-specific manner and respond to mitogenic stimulation. the p1- and p2-derived primary transcripts are processed into a diverse repertoire of alternatively spliced mrnas that are differentially expressed in various cell types and at different developmental stages. the p and p mrnas differ at their 5'-coding regions, and the resulting protein isoforms differ in their biological functions. similarly, a number of p2-derived splice-variants bear shorter reading frames that lack the transcriptional activation domain and can act in a dominant negative manner. forced expression of such isoforms results in transcriptional repression of runx target genes, and in blocking of myeloid differentiation. hence, differential usage of p versus p has a profound effect on the repertoire and nature of runx isoforms in the cell, which likely impacts on the regulation of runx target genes. significantly, disruption of p by the 12; chromosomal translocation results in the most common subtype of childhood acute lymphoblastic leukemia. p1- and p2-transcripts also differ in the structure and function of their 5' untranslated region. the p1-5'utr is relatively short and mediates cap-dependent translation, whereas the p2-5'utr is particularly long, contains an internal ribosome entry site and mediates cap-independent translation. these structurally and functionally different 5'utrs could have a profound effect on translation efficiency of runx mrnas. indeed, in cell lines where p transcription predominated, p2-5' ires-dependent translation was more efficient than p1-5' cap-dependent translation. ires-dependent translation is particularly important during development, in times when cap-dependent translation in proliferating or differentiating cells is down-regulated. while it is clear that the regulation of runx expression by alternative p1/p usage is a potentially key paradigm in the in vivo regulation of runx very little is known about the role of the p1/p switch in mediating tissue and stage specific expression of runx during development. to investigate the biological function of the p1/p switch during embryogenesis, we generated mice bearing a hypomorphic runx allele by inserting a neomycin gene into the p region. mice homozygous for the p2neo allele displayed a markedly attenuated p activity. nevertheless, these mutant mice developed to term, but newborns died within a few days after birth. we show that the p1/p switch is developmentally regulated and that the two promoters are alternatively used during embryogenesis. p mediated transcription is required during early thymopoiesis for the proper development of the thymus and for differentiation of dn and dp cd4/cd t cells. attenuation of p activity results in impaired development of fetal liver hematopoietic precursors and in enhanced apoptosis of thymocytes due to altered expression of t-cell receptors. the data describe previously unknown functions of runx delineate the activity of p and p in embryogenesis, show unequivocally that their function during development is non redundant and underscore the significance of the two promoters in runx biology.  generation of runx p2neo/neo mice and phenotypic analysis of mutant newborns null mutation in runx results in embryonal lethality. as p activity starts early in embryogenesis, we envisaged a scenario in which germline inactivation of p will also result in embryonal death. to avoid this occurrence we explored means to selectively attenuate the activity of runx p we have previously demonstrated that the genomic region upstream the transcription start sites of runx p was important for regulated transcription of runx in various cell lines. we thus assessed whether insertion of a neo cassette into this region affects p activity in transfected cells. transfection experiments indicated that insertion of a neo cassette into the p region specifically attenuated p2-mediated transcription in several cell lines and encouraged us to utilize this approach for evaluating the biological activity of runx p in vivo. runx p was targeted in es cells using a construct in which the neo cassette was placed at the same site as in the p2neo-ren construct, but in a transcriptional orientation opposite to that of runx and was flanked by lox p sites. homologous recombination yielded es clones, which were used to generate several runx1p2neo/+ chimeric males that passed on the runx mutation through the germ line. mating f heterozygotes with 129/sv, icr or mf mice generated runx1p2neo/neo mice with an inbred or a mixed background. heterozygous runx1p2neo/+ mice appeared phenotypically indistinguishable from their wt littermates. however, homozygous runx1p2neo/neo neonates were reduced in size, gained little weight and most of them died to days post natally. to further characterize the phenotype/genotype relationships of the mutant mice, heterozygotes p2neo/+ mice were mated with two different strains of transgenic mice expressing cre driven by either pgk or eiia promoters. the resulting heterozygotes bearing the remaining loxp sequence within the p2-xbai site were mated and offspring homozygous to p2-loxp were obtained at the expected mendelian proportion. significantly, runx1p2loxp/loxp mice were indistinguishable form wt littermates indicating that the early lethality phenotype of p2neo/neo newborns was due to the presence of the neo cassette in the p region. attenuated runx expression in p2neo/neo mice the above-described phenotypic manifestation of the p2neo/neo allele posed a question as to how the expression level of runx was affected. to address this issue, runx expression in various tissues was examined using rt-pcr, immunohistochemistry and rna in situ hybridization. in tissues of p2neo/neo mice, such as kidney and tongue, where runx expression is normally mediated by both p and p only p1-transcripts were detected. in contrast, no runx expression was detected by either rt-pcr or ihc in developing gastric epithelium where only p is normally active. p2neo is therefore a hypomorphic allele of runx in which p activity is largely diminished. interestingly, however, no developmental abnormalities were detected by macroscopic and histological examination of kidney, stomach and tongue of mutant embryos or newborns. the most obvious and consistent macroscopic abnormality was a profound reduction in size of the thymus of newborn p2neo/neo mice as compared to wt. western blot analysis, rish using a p specific probe and rt-pcr showed that during early thymogenesis – ) transcription of runx in wt thymus was predominately mediated by p, and that at this stage almost no runx was detected in p2neo/neo thymus. the p activity in wt thymus was switched-on between e –e and persisted thereafter, whereas the activity of p which predominated during embryogenesis, gradually decreased after birth. importantly, once switched-on the p activity was apparently unaffected in p2neo/neo mice, as evidenced by northern blot analysis of runx mrnas in newborns. together, the complementary results of runx expression studies and morphological analyses showed that expression of runx in p2neo/neo mice was compromised both temporally and spatially, resulting in adverse effect on thymus development. impaired thymus development in runx p2neo/neo mice thymus development in mouse embryo begins at e ensued by immigration of lymphocyte precursors into the thymic rudiment at e. development proceeds through stage specific interactions between thymocytes and thymic epithelium that are essential for the formation of thymic architecture. runx is highly expressed in developing thymocytes, but not in thymic epithelium. macroscopic examinations revealed that thymi of p2neo/neo neonates were about one-third of the normal size. this diminished size was caused by a profound reduction in the number of thymocytes during mutant thymus development. histological examinations revealed no gross morphological differences between p2neo/neo and wt at e and e, except for scattered cysts in p2neo/neo thymi. these cysts are thought to result from accelerated epithelial turnover within centers of epithelial islands. at e however, extensive histological differences between wt and p2neo/neo emerged. wt thymus had a clear demarcation of cortex and medulla. wt cortex was densely cellular with small hyperchromatic thymocytes, while the medulla was paler, contained a lower number of thymocytes admixed with macrophages, epithelial cells, and antigen presenting cells. mutant thymus, on the other hand, was much smaller, hypocellular, had a scalloped capsular surface and consisted predominantly of epithelial cells, with no evidence of an organized cortex and medulla. at e the histological defects were even more pronounced; the p2neo/neo thymus still lacked corticomedullary organization and contained occasional cysts. ihc using anti runx antibodies revealed a lower number of runx positive cells in e and e p2neo/neo thymi compared to wt. runx is mostly expressed in dn cd4-/cd8- immature thymocytes, where it regulates cd expression. ihc of e –e wt thymus revealed that these runx positive immature thymocytes were predominantly in the cortex and less in the medulla, as also shown by the rna in-situ hybridization data. in p2neo/neo thymus on the other hand, the fewer runx positive cells were scattered randomly within the organ. at e most of runx expressing dn cd4-/cd8- thymocytes were found in the sub-cortical band, whereas the cortical, dp cd4+/cd8+ thymocytes did not express runx. in e p2neo/neo thymus, many runx positive cells were still in the cortex and only after birth, when the mutant thymus gradually gained wt morphology, were runx expressing hyperchromatic cells seen in the scb. but even then, the overall size of mutant thymus remained much smaller compared to wt. the data indicate that normal embryonal thymopoiesis depends on a proper p2-mediated transcription of runx which when attenuated during the e –e time period results in impaired thymus development. increased apoptosis in embryonal p2neo/neo thymocytes to directly assess whether the thymic hypocellularity of p2neo/neo embryos was a consequence of enhanced cell death, the proportion of apoptotic thymocytes was determined by flow cytometry. at e a much higher proportion of p2neo/neo thymocytes compared to wt bound annexin v, indicating increased apoptosis among p2neo/neo thymocytes. additionally, increased proportion of necrotic cells positive to both annexin v and propidium iodid, was observed in p2neo/neo thymus. the enhanced cell death of e p2neo/neo thymocytes led to a three-fold reduction in the proportion of viable thymocytes in mutant thymus. at e – the p promoter is switched on, expression of runx in p2neo/neo thymocytes increased and recovery of thymopoiesis commenced. the end result was an increased proportion of viable p2neo/neo thymocytes at e almost to wt level. the enhanced apoptosis in p2neo/neo e and e thymus was also demonstrated by in situ tunel labeling and ihc of activated caspase-. the proportion of non-viable thymocytes at various stages of thymus development is shown in fig. 3b. at early stages, apoptosis was significantly higher in p2neo/neo fetuses compared to wt, but gradually declined concomitant with the increase in p1-mediated expression of runx we next asked whether the proliferative capacity of p2neo/neo thymocytes was also affected. e thymocytes were isolated, analyzed by facs and similar numbers of viable cells cultured in-vitro under proliferation stimulating conditions. purified p2neo/neo and wt thymocytes displayed a similar thymidine incorporation rate, indicating that the ability of thymocytes to proliferate was hardly affected by the attenuated expression of runx during early thymopoiesis. this conclusion was supported by immunostaining of thymocytes with the cell proliferation-associated antigen ki- in e p2neo/neo thymus the number of ki- positive cells was lower than in wt, reflecting the hypocellularity of mutant thymus. nevertheless, significant numbers of p2neo/neo thymocytes did express ki- indicating normal progression through the cell cycle. together, these results show that p2neo/neo thymocytes possess proliferation and cell cycle progression capabilities similar to wt, but attenuating runx expression during early developmental stages rendered thymocytes more susceptible to apoptosis, leading to thymic hypocellularity. to address whether the increased apoptosis of p2neo/neo thymocytes was due to loss of a cell-autonomous function of runx we examined the capacity of p2neo/neo thymocytes to populate fetal thymic organ culture. e wt thymic lobes were reconstituted, at a saturating cell number, with either wt or p2neo/neo e fetal liver cells that contain hematopoietic progenitor cells. at ftoc day and cells were extracted out of the lobes and analyzed by facs for annexin v binding. p2neo/neo hpc gave rise to a significantly lower number of ftoc cells compared to wt and most p2neo/neo derived cells were annexin vhigh. these data further demonstrated the intrinsic requirement for p2-mediated expression of runx during early embryonal thymopoiesis. the enhanced apoptosis of p2neo/neo thymocytes is associated with increased expression of tcrβ/tcrγδ the above results raised the question of how does attenuated expression of runx cause the increased apoptosis of p2neo/neo thymocytes. the majority of immature thymocytes with a non-productive rearrangement of tcrβ gene die through fas/fas ligand-mediated apoptosis. only a small subset of immature thymocytes with productive tcrβ or γδ rearrangement are selected for further maturation associated with rapid proliferation. interestingly, the percentage of tcrβ/tcrγδ expressing thymocytes was significantly higher in e to e p2neo/neo fetuses compared to wt, as was the mean-fluorescence-intensity level of tcrβ+ thymocytes. these data indicate an enhanced tcrβ/γδ expression on p2neo/neo thymocytes compared to wt during the early stages of embryonal thymopoiesis. runx acts in transfected cells as a negative regulator of tcrβ transcription. it is thus possible that the diminished runx expression between e to e caused de-repression of tcr transcription in p2neo/neo thymocytes, leading to the increased level of tcrβ/γδ. supporting this assumption are data demonstrating that upregulation of tcrβ/γδ in p2neo/neo thymocytes occurred at the transcriptional level, and that down regulation to wt levels occurred at e, upon turning-on of p. we next addressed whether increased expression of tcrβ/γδ in p2neo/neo thymocytes was associated with the increased susceptibility to apoptosis of the mutant cells. annexin v staining was monitored in fractionated tcrβ+ or tcrγδ+ thymocytes. in e and e embryos 50–65% of wt and 80–90% of p2neo/neo tcrβ+ thymocytes were apoptotic as were ~95% of both wt and p2neo/neo tcrγδ+ thymocytes. additionally, at these developmental stages a significantly higher proportion of p2neo/neo thymocytes express the tcrs. thus, the enhanced apoptosis in p2neo/neo compared to wt thymus, was closely associated with an increase in tcrβ/γδ expression. of note, mfi of annexin v+ apoptotic p2neo/neo thymocytes, was lower compared to the wt, presumably due to a lower level of cell surface phosphatidyl-serine in p2neo/neo cells. together, the complementary outcome of these experiments indicates that runx functions in vivo as a negative regulator of tcr expression. when the temporal expression of runx is attenuated, levels of tcrβ+/tcrγδ+ thymocytes increase, which may lead to enhanced cell death. when p is turned-on, restoring runx expression in p2neo/neo cells, apoptosis in mutant thymocytes assumes normal levels. impaired thymocyte differentiation in p2neo/neo embryos we next employed flow cytometry to study thymocyte differentiation in p2neo/neo embryos and neonates. first we examined the cd4/cd double negative population at e when nearly 100% of thymocytes are dn. the dn population is divided into four sub-groups, dn to dn according to the expression of cd and cd surface markers. compared to wt, p2neo/neo embryos displayed an increase in the proportion of dn thymocytes and a corresponding reduction in dn cells, resulting in a significant increase in the ratio dn3/dn among p2neo/neo thymocytes. to examine p1/p usage in the dn sub-groups we used flow cytometry to sort e thymocytes based on surface expression of cd and cd, and levels of p and p transcripts was determined. the levels of p and p expression did not differ significantly between dn and dn subgroups and was similar to the level shown in fig. 1d. next we examined the cd8/cd double positive population. at e, p2neo/neo embryos displayed a -fold reduction in the proportion of double positive cd4+/cd8+ thymocytes as compared to wt. at e the decrease in proportion of dp thymocytes in p2neo/neo embryos was more pronounced and accompanied by a marked increase in an abnormal population of cd4+/cd8low cells. a similar subpopulation of immature cd4+/cd8low was previously observed in mice with thymocyte specific targeting of runx and in mice bearing a hypomorphic cbfβ allele. it was postulated that this abnormal cd4+/cd8low population represents immature dp thymocytes in which cd expression was diminished and cd expression was partially de-repressed. delayed differentiation of p2neo/neo thymocytes was still apparent at post-natal day, but recovery to wt proportions was noted at p. taken together, the change in the proportion of dn3/dn thymocytes at early thymopoiesis, the decrease in dp cd4/cd thymocytes and the increase in the abnormal sp cd4+/cd8low population, indicate that during embryonal thymopoiesis p activity is also required for regulation of cd4/cd expression and thereby for the proper differentiation of dn and dp thymocytes. reduction in committed t-cell precursors in p2neo/neo fetuses runx is essential for embryonal hematopoiesis and runx1-/- mice lack definitive hematopoiesis as well as hemapoietic colony forming activity in vitro. in developing fl, runx is expressed in hsc and in early progenitors, and in wt fl both promoters are active, whereas p activity was largely attenuated. to assess hematopoietic activity, individual fls from wt and p2neo/neo embryos were isolated and analyzed for in vitro colony formation. as shown in fig. 6b, the number of colonies grown in p2neo/neo cultures was significantly lower compared to wt. however, because the size of the colonies in p2neo/neo and wt cultures was similar, the results indicate that p2neo/neo fl cells contained fewer progenitors compared to wt. this conclusion was supported by the finding that e and e p2neo/neo embryos showed marked reduction in the proportion of gr-1+/cd18+ and cd11a+/cd11b+ cells compared to wt littermates. we next assessed the effect of the p2neo/neo hypomorph on the development of fl hpc. runx expressing fl hpc also express cd c-kit and cd. in e and e p2neo/neo embryos, a 2-fold reduction in the proportion of cd44+/c-kit+ hpc compared to wt littermates was observed, and even greater reduction was observed in cd positive cells at e –e. fl hpc that are b220low/c-kit+/cd19- represent an hpc population restricted to the t-cell and natural killer cell lineages. these fl progenitors, termed t-cell precursors, are thought to represent the immediate developmental step before thymic immigration. because the hypocellularity of p2neo/neo thymus was noted at the earliest developmental stages, the proportion of tcps in wt and p2neo/neo was compared. facs analysis of b220low/c-kit+/cd19- e fl cells, revealed a fourfold reduction in the proportion of p2neo/neo tcps compared to wt. these results indicated that the proportion of runx expressing fl hpc, particularly the tcp subset, was markedly reduced in p2neo/neo compared to wt. this conclusion was supported by in vitro ftoc progenitor assay, which monitors the capacity of fl tcps to reconstitute thymic lobes. when e wt and p2neo/neo fl cells were cultured under limiting dilution conditions in ftocs, the frequency of successfully populated thymic lobes was lower for p2neo/neo tcps as compared to wt. additionally, when day ftocs cells were obtained and cd4/cd positive cells analyzed by facs, an abnormal differentiation of p2neo/neo thymocytes was observed with increased proportion of the abnormal population dp cd4+/cd8low and a twofold reduction in the proportion of sp cd4+ or cd8+ compared to wt. this ftoc result corresponds well with the in vivo delayed maturation of p2neo/neo fetal thymocytes described in fig. collectively, these data indicate that in p2neo/neo embryos the development of fl hpc of different lineages was impaired. runx p2neo allele failed to rescue the embryonal lethality phenotype of runx1-/- mice homozygous disruption of runx results in a complete absence of fl hematopoiesis and the mice die between e and e of hemorrhages in the central nervous system. p2neo/neo mice were born and showed no sign of hemorrhages, indicating that expression levels of runx in homozygous p2neo/neo during early embryogenesis were sufficient to rescue the runx1-/- phenotype. this occurrence raised the question of whether runx levels provided by activity of a single p will also be sufficient to rescue the null phenotype. to address this issue we generated a compound mutant strain runx1lz; runx1p2neo by mating runx1lz/+ mice with runx1p2neo/+. these mice had one null allele expressing a fused runx1-lacz protein and one p2neo allele expressing wt p. compound mutant runx1lz;runx1p2neo embryos died between e and e and suffered from hemorrhages in the nervous system. the most extensive bleedings was seen in the 4th ventricle, the ventral metencephalon and spinal cord. moreover, the liver of e compound mutant embryos was depleted of hematopoietic cells, indicating lack of definitive hematopoiesis, similar to the previously reported runx1-/- phenotype. these results indicated, that during midgestation, angiogenesis and definitive hematopoiesis are highly sensitive to the dosage of runx as activity of a single p was not sufficient to rescue the runx1-/- phenotype. this conclusion is consistent with the previously reported data which was based on transplantation assays. this observation may also pertain to the unique role of p activity during midgestation angiogenesis and/or early hematopoiesis. early lethality phenotype of p2neo/neo neonates is rescued by excision of the neo cassette in early t cell progenitors as noted earlier, p2neo/neo neonates die within a few days after birth. the reasons for this early lethality are not clear. while p2-mediated expression diminished in several p2neo/neo tissues, including stomach, intestine and kidney, the most striking abnormalities were noted in thymic development and thymopoiesis. we thus addressed whether these abnormalities could be rescued by specific removal of the neo cassette in early thymopoiesis and whether this occurrence will extend the life span of p2neo/neo neonates. runx1-p2neo/neo mice were mated with a lck-cre transgenic line to excise the neo and regain p activity in thymocytes. contrary to p2neo/neo mice, the p2/lck-cre mice developed normally and outwardly were indistinguishable from wt littermates. nevertheless, embryonal thymopoiesis of p2/lck-cre was not completely normal and embryos exhibited a lower number of runx expressing thymocytes. this may be due to an incomplete excision of the neo cassette in all t cell progenitors. significantly, however, runx expression level in p2/lck-cre was sufficient to allow for normal thymic development. of note, p2-mediated runx expression in other runx expressing cells, such as epithelial cells, was not rescued in p2/lck-cre mice. discussion runx is an important cell-linage specific regulator of definitive hematopoiesis and thymopoiesis. the unique spatio/temporal expression pattern of runx is attained through alternative usage of its two promoters, p and p that also impacts on translation efficacy and repertoire of protein isoforms. while it was clear that transcriptional regulation of runx expression is an important element of its biological function, little was known about the role of the two promoters during development, and about how and when they are differentially regulated. is the alternative promoter usage of human and mouse runx1/runx a reflection of vertebrates' higher complexity? differential usage of alternative promoters enables diversified transcriptional regulation within a single locus and may thus serve as a molecular basis for the higher complexity of certain vertebrates' systems, such as the immune system. indeed, while the runx genes are highly conserved throughout the animal kingdom, more primitive animals such as the nematode c. elegance and the sea urchin contain only one gene regulated by a single promoter. this single promoter is the equivalent of vertebrates' p differential p1/p usage during embryogenesis the use of hypomorphic alleles for studying essential genes at the organismal level is a well-established approach. we employed mice homozygous for a hypomorphic runx allele, which caused a profound attenuation of p activity, to investigate the biological role of p during embryogenesis. specifically, we analyzed the impact of the resulting hypomorphic runx expression on fl hpc development and early thymopoiesis. we found that during embryogenesis runx is developmentally regulated through alternative usage of p and p in certain tissues, including kidney, tongue and liver both p and p are active, whereas in others such as intestinal epithelium and early thymocytes p activity predominates. contrary to runx null mice, in which embryonic lethality occurs at ~e, p2neo/neo mice develop to term, implying that during early development the activity of p prevails. this conclusion is supported by the finding that both p1- and p2-transcripts are found in fl of e embryos. the notion that both p and p are active during early hematopoiesis correlates well with the finding that p2neo/neo embryos displayed moderate and severe hematopoietic defects in the liver and thymus, respectively. p activity is essential for embryonal thymopoiesis and thymus development thymic development in the mouse begins at ~e. runx is highly expressed in thymocytes, but not in thymus epithelium. using in-situ hybridization, rt-pcr and ihc we demonstrate that during early thymopoiesis activity of p predominates. the p is upregulated at e and its activity persists thereafter, while the activity of p declines after birth. at e the thymic rudiment is colonized by the first wave of committed t-cell precursors which endure in the thymus until e. it is tempting to speculate that p activity is confined to descendants of the first wave tcp, whereas p is active in thymocytes originating in the second wave. consistent with this hypothesis, the first wave p2neo/neo thymocytes contained a markedly reduced level of runx compared to wt, whereas in second wave thymocytes the level was similar to wt due to switch-on of p at e. these second wave thymocytes grow exponentially after birth, when p is more active, and become the major cell population in the thymus. during early stages of thymus development a marked reduction in thymic cellularity was observed in p2neo/neo embryos. we show that this occurrence was due to enhanced apoptosis of p2neo/neo thymocytes. but why were e to e p2neo/neo thymocytes more susceptible to apoptosis than wt cells? selection processes in the thymus ensure that peripheral t cells are responsive to foreign antigens but tolerant to self-antigens. this occurs through positive and negative β-selections. thymocytes, whose tcr engaged peptide/mhc molecules productively, escape their inherent programmed cell death and subsequently are induced to proliferate. on the other hand, thymocytes that react strongly with self-peptide-mhc molecules are actively eliminated. according to the avidity model, selection occurs only when signals received through the tcr fall between two thresholds: signals below the one are incompatible with survival, while signals above the other result in deletion. we hypothesize that the elevated tcr levels in p2neo/neo thymocytes disturbed this delicate balance and disposed a larger proportion of p2neo/neo t cells to apoptosis. several studies in transfected cells have demonstrated the involvement of runx in transcription regulation of tcrs expression. specifically, we have shown that runx can act as a transcriptional repressor in tcr regulation. thus, it is conceivable that the increased levels of tcrs in embryonic p2neo/neo thymocytes resulted from the reduced runx expression in these cells, and that the higher tcrs levels lead to a higher avidity of tcr-mhc interactions, which programmed the cells to apoptosis. this hypothesis is supported by runx1f/f/lck-cre mice, which show enhanced negative selection of runx1-/- thymocyte, and by the finding that upon recovery of runx expression in p2neo/neo thymocytes, tcr expression and apoptosis resumed wt levels. potentially related, mice homozygous for a knock-in runx cdna that are lacking the c-terminal vwrpy, exhibit thymic hypocellularity. the vwrpy motif, which is highly conserved among all runx proteins, is essential for recruitment of the transcriptional co-repressor gro/tle and subsequent repression of target genes. thymic hypocellularity was also noted in mice bearing a hypomorphic allele of cbfβ, the non-dna binding partner of runx which is essential for the function of runx proteins. in addition to the marked hypocellularity, thymi of p2neo/neo embryos contained an increased proportion of dn thymocytes, a reduced proportion of dp cd4+/cd8+ thymocytes and a pronounced increase in an abnormal population of immature cd4+/cd8low cells. the increase in dn and cd4+/cd8low thymocyte populations was previously observed in adult runx1f/f/lck-cre mice due to diminished runx in dn thymocytes and the consequent derepression of cd. more recently a similar increase in cd4+/cd8low thymocytes was also observed in fetuses of cbfβrss/- mice bearing the hypomorphic cbfβ allele, indicating that cbfβ is required for cd silencing by runx abnormally high proportion of cd4+ thymocytes was also noted in embryos of the runx knock-in mutant lacking the vwrpy motif. this finding suggests that runx1-mediated repression of cd in dn thymocytes involves the recruitment of gro/tle, as in case of runx3-mediated silencing of cd in dp thymocytes. attenuation of p activity affects development of fl hpc runx is essential for the emergence of embryonal hsc. analysis of fl hematopoiesis in p2neo/neo embryos revealed that the emergence and commitment of hpc to either myeloid or lymphoid lineages was not affected by attenuation of p however, the noted delay in differentiation and expansion of various p2neo/neo progenitor populations indicated that development of fl hpc was sensitive to diminution in p activity. interestingly, the overall phenotype of fl hpc in p2neo/neo embryos resembled the phenotype observed by cai et al in heterozygous runx1+/- fetuses, suggesting a gene dosage effect. in developing fl activity of p emerged early and persisted throughout gestation. as p activity was largely unaffected in p2neo/neo hpc, it is conceivable that the hypomorphic expression in p2neo/neo fl creates conditions resembling haploinsufficiency, in line with the large body of literature documenting hematopoietic defects due to hemizygous dosage of runx. however, it is worth noting that the effect of p2neo/neo hypomorph is not merely through gene dosage, because contrary to runx1+/- the p2neo/neo neonates die within a few days after birth. rescue of the early lethality of p2neo/neo neonates despite the diminished p2-activity and reduced runx dosage, p2neo/neo embryos developed to term. thus the residual p activity plus the apparent wt expression of p were sufficient to support normal development of p2neo/neo embryos through gestation. however, inactivation in p2neo/neo embryos of one p allele, as in the compound mutant runx1lz;runx1p2neo, which further reduced runx dosage, caused hemorrhages in the nervous system and mid-gestation lethality. this result defined the threshold level requirement of runx for rescue of the nervous system hemorrhages. while p2neo/neo embryos developed to term, neonates die within a few days after birth. specific removal of the floxed p2-neo cassette by crossing p2neo/+ mice with lck-cre transgenics resumed p expression in early thymopoiesis, allowed for normal thymic development and rescued the neonatal lethality. of note, the neonatal lethality was rescued even though expression of p in the gastrointestinal tract of p2neo/neo/lck-cre mice was not resumed. this occurrence indicated that lethality of p2neo/neo neonates was not due to lack of runx expression in gastrointestinal tract epithelium, despite the striking reduction of nutrients in the gastrointestinal tract of p2neo/neo neonates. as athymic nude mice live through adulthood, it is conceivable that impaired thymopoiesis in p2neo/neo embryos, especially at the negative selection step, resulted in emergence of pathogenic t cells that cause the neonatal lethality. finally, as the n-terminus of runx proteins encoded by p2-mrna differs from that of p, it is tempting to speculate that the hematopoietic phenotypes of p2neo/neo embryos were caused not only by gene dosage, but are due to the lack of p isoforms. elucidating the specific role of the p1- and p2-protein isoforms in runx biology, particularly early thymopoiesis, awaits further investigations.  differential usage of alternative promoters enables diversification of transcriptional regulation within a single locus and thereby plays a significant role in the control of gene expression. during mouse embryogenesis the spatio/temporal expression of runx is developmentally regulated by alternative usage of two mutually distinct promoters, p and p these promoters are not only differentially regulated, but also give rise to mrnas with variant 5'-utrs and to structurally different protein isoforms. our studies support a model whereby attenuation of p affects mrna levels and protein isoforms repertoire within the cell and thereby creates the hematopoietic phenotype of p2neo/neo embryos. the findings provide in vivo evidence for the non-redundant function of p and p and underscore the significance of alternative promoter usage in runx biology. 
the data delineate the activity of p and p in embryogenesis and describe previously unknown functions of runx the findings show unequivocally that the role of p1/p during development is non redundant and underscore the significance of alternative promoter usage in runx biology. early thymus development and thymopoiesis were also abrogated as reflected by thymic hypocellularity and loss of corticomedullary demarcation. mice homozygous for the hypomorphic allele developed to term, but died within a few days after birth. mice deficient in runx lack definitive hematopoiesis and die in mid-gestation. we employed mice bearing a hypomorphic runx allele, with a largely diminished p activity, to investigate the biological role of alternative p1/p usage. differentiation of cd4/cd thymocytes was impaired and their apoptosis was enhanced due to altered expression of t-cell receptors. while the alternative usage of p and p likely plays an important role in runx biology, very little is known about the function of the p1/p switch in mediating tissue and stage specific expression of runx during development. alternative promoters usage is an important paradigm in transcriptional control of mammalian gene expression. however, despite the growing interest in alternative promoters and their role in genome diversification, very little is known about how and on what occasions those promoters are differentially regulated. expression of runx is regulated by two functionally distinct promoters designated p and p differential usage of these two promoters creates diversity in distribution and protein-coding potential of the mrna transcripts.
 alternative promoters usage is an important paradigm in transcriptional control of mammalian gene expression. however, despite the growing interest in alternative promoters and their role in genome diversification, very little is known about how and on what occasions those promoters are differentially regulated. runx transcription factor is a key regulator of early hematopoiesis and a frequent target of chromosomal translocations in acute leukemias. mice deficient in runx lack definitive hematopoiesis and die in mid-gestation. expression of runx is regulated by two functionally distinct promoters designated p and p differential usage of these two promoters creates diversity in distribution and protein-coding potential of the mrna transcripts. while the alternative usage of p and p likely plays an important role in runx biology, very little is known about the function of the p1/p switch in mediating tissue and stage specific expression of runx during development.  we employed mice bearing a hypomorphic runx allele, with a largely diminished p activity, to investigate the biological role of alternative p1/p usage. mice homozygous for the hypomorphic allele developed to term, but died within a few days after birth. during embryogenesis the p1/p activity is spatially and temporally modulated. p activity is required in early hematopoiesis and when attenuated, development of liver hematopoietic progenitor cells was impaired. early thymus development and thymopoiesis were also abrogated as reflected by thymic hypocellularity and loss of corticomedullary demarcation. differentiation of cd4/cd thymocytes was impaired and their apoptosis was enhanced due to altered expression of t-cell receptors.  the data delineate the activity of p and p in embryogenesis and describe previously unknown functions of runx the findings show unequivocally that the role of p1/p during development is non redundant and underscore the significance of alternative promoter usage in runx biology. 