BACKGROUND
kit is a well-known receptor tyrosine kinase controlling melanogenesis, hematopoiesis, gametogenesis, hearing, mast cells and the survival of cajal cells from the intestine  <cit> .
furthermore kit is expressed in several epithelial endothelium or endocrine cells but also in cholangiocytes during development and in the adult  <cit> .
the binding of kitl, induces the dimerization of the kit molecule, followed by a change in the configuration of the intracellular domain and the autophosphorylation of the receptor, opening several docking sites for signal transduction molecules containing sh <dig> binding domains.
hence the kit signal is transduced by several pathways including that of the src family kinases, plc-γ, ras/mapk, pi3k-akt-mtor, jak/stat and also through the activation of the snai <dig> and mitf transcription factors .
kit belongs to the class iii of the receptor tyrosine kinase family with csf1r, pdgfr-a,-b, kdr, flt- <dig>  - <dig> and - <dig>  characterized by the presence of an autoinhibitory juxtamembrane  domain.
without phosphorylation, the jm domain is inserted in between the n- and c-lobes blocking the dfg  conserved motif of the activation loop in an inactive conformation.
upon phosphorylation, changes in the conformation of the jm domain opens up the atp binding site and repositions correctly the activation loop in the kinase domain  <cit> .
mutations that affect the activation loop are found in human mastocytose or leukemia, while deletions or insertions in the jm domain are observed in gastro intestinal tumor  and often correspond to gain-of-function  <cit> .
the treatment of the kit-induced tumors had taken a promising perspective with the use of sti- <dig> , that binds to the atp-binding site, stabilizing the autoinhibitory structure with an inactive tyrosine kinase domain  <cit> .
in human, kit loss-of-function is linked to piebald trait  <cit>  and a large panel of mutations is reported .
similar pigmentation defects are observed in mouse models  <cit>  and the analysis of kit functions was facilitated by a large number of hypomorphic murine mutants  <cit> .
some correspond to point mutations, others to large chromosomal rearrangements obtained as spontaneous or induced by chemicals or radiation, while a few were engineered  <cit> .
complete lack of kit interferes with post-natal viability  <cit>  while hypomorphic mutations could induce not only a variable degree of defects during melanogenesis, hematopoieisis and gametogenesis but also growth retardation and juvenile mortality  <cit> .
in this report we used a panel of mutants isolated from genome-wide mutagenesis screens to investigate the post-natal role of kit.
these mutations correspond to a new allelic series of hypomorphic kit alleles that alter differentially the melanogenesis, hematopoiesis and gametogenesis.
in two such mutants, we observed an impaired viability of the homozygotes before weaning.
clearly, growth retardation and premature death of neonates in both kit mutants were associated with juvenile steatosis during the suckling period.
further analysis pointed out that kit is expressed in the post-natal liver and kit mutants were suffering from an early defect in triglycerides  export from the liver correlated with the downregulation of key genes participating in the control of hepatic lipid metabolism.
this series of evidence established the major role of kit in controlling liver triglycerides metabolism in neonates.
RESULTS
characterization of a new allelic series of mutants affecting the kit gene
to analyze the role of kit in neonates, we used new semi-dominant enu-induced mutant mouse lines named sco <dig>  sco <dig> , sow <dig>  and whc <dig> , displaying white head blazes and belly spots, that were isolated in genome wide mutagenesis programs  <cit>  .
the corresponding mutations were mapped on the mouse chromosome  <dig> near the kit locus .
with the sequencing of the kit gene from the sco <dig>  sco <dig> and whc <dig> homozygotes, unique missense mutations were found in the intracellular tyrosine kinase domain that affect m623l for sco <dig>  v667a for sco <dig> and f809l for whc <dig> .
these changes were absent in the original c3heb/fej and other strains.
no mutation was found in the exonic and the splice junction sequences of the kit gene from the sow <dig> mouse mutant line, suggesting that sow <dig> affects the regulation of the kit gene as described in other mouse mutants  <cit> .
nonetheless, a series of complementation assays with other known alleles including kitw, kitw-v and kittm1alf confirmed that kit function was altered in sow <dig> .
therefore, the data support that the mutants described here represent a new allelic series at the kit locus, with three missense mutations  and one probable mutation affecting the regulation of the kit gene .
homozygous animals generated for the four mutations displayed similar white coat pigmentation with black eyes .
however, sco <dig> homozygotes showed a few grey hairs and pigmented ears suggesting that this allele alters later stages of pigmentation  <cit> .
blood analysis showed changes characteristic of macrocytic anemia, with a significant reduction of hematocrit and red blood cell count, together with an increase in mean red cell volume and in mean cell hemoglobin content for all the adult mutant mice .
hematopoietic defects were more severe in the sco <dig> and whc <dig> homozygotes than in sow <dig> and sco <dig> mutants with however some little variations between males and females.
finally, histological analysis of testis isolated from adult males homozygous for sco <dig>  sco <dig> and whc <dig> showed different stages of germ cells differentiation while sow <dig> homozygotes revealed a complete lack of spermatogenesis .
these results were confirmed by the absence of β-galactosidase staining in the seminiferous tubules of kittm1alf/sow <dig> transheterozygotes  and assessed by a fertility test.
only the sco <dig> and sco <dig> homozygotes were able to reproduce but no progeny was ever obtained from crosses between sow3/sow <dig> males and wild-type females .
thus, sco <dig>  sco <dig>  sow <dig> and whc <dig> are hypomorphic mutations of kit, with the expected effects, although variable, on melanogenesis, hematopoiesis and gametogenesis.
sco <dig> mutant analysis unravels a function of kit during post-natal development of the liver
an impaired viability affected kitsco <dig> homozygotes before weaning, with only  <dig> mutants recovered out of  <dig> offspring from kitsco5/+ intercrosses.
although sco5/sco <dig> genotypes scored at birth with a mendelian ratio, almost 50% of them died before  <dig>  days post partum , and only 25% survived at weaning .
in addition, kitsco5/sco <dig> homozygotes displayed a growth defect at  <dig>  dpp, with a weight difference reaching up to 50% of the control at  <dig> dpp .
comparable phenotypes were also noted in kitwhc1/whc <dig> and kitw/w-v animals, thus we analyzed further this post-natal lethal phenotype.
necropsy analysis revealed that the liver from kitsco5/sco <dig> animals at  <dig>  dpp had a tan yellowish color  although it had a normal relative organ weight .
anemia was detected in mutants at this early age and could partly account for the liver color alteration.
however, the spotted aspect and the presence of large white areas in the hepatic lobes in all kitsco5/sco <dig> analyzed at  <dig>  dpp  were associated with an abnormal structure of hepatocytes .
indeed, lipid microvesicular accumulation, or steatosis, was detected in mutant hepatocytes by specific staining at  <dig>  and  <dig>  dpp with oil-red-o .
similar lipid inclusions were observed in the liver of kitwhc1/whc <dig> neonates , to a lesser extent in kitsco5/+ animals and also in few kitsco5/sco <dig> individuals that survived until two months of age .
a comparable phenotype was detected in  <dig>  dpp and adult kitw/w-v mice .
by contrast, kitsow <dig> and kitsco <dig> homozygotes developed normally and did not display any hepatic phenotype.
thus the liver phenotype was not specific to the sco <dig> allele of kit, as it was also found in a number of compound kit mutants.
then, we investigated the onset of the phenotype by following the lipid/water ratio in the liver of newborn mice using nuclear magnetic resonance spectroscopy .
accumulation of hepatic lipids in kitsco5/sco <dig> mutants was detected at  <dig>  dpp  suggesting that defect started earlier.
moreover the lipid/water ratio increased until  <dig>  dpp when most of the homozygotes begun to die  as predicted from the original analysis of viability.
nevertheless, the lipid/water ratio was still high in surviving mutants compared to controls at  <dig>  dpp .
to further define the onset of the phenotype, liver sections from groups of mutants and control littermates  were analyzed during post-natal development.
no change was observed immediately after birth in neonates that had not yet suckled any milk .
however, microvesicular accumulations of lipids were found at  <dig>  dpp in the liver of wild-type suckling animals from the c3heb/fej, c57bl/6j and 129s <dig> inbred lines.
in the sco5/sco <dig> mutant, a similar accumulation of lipids appeared at  <dig>  dpp but remained later on, showing that lipids, derived mainly from the milk, were efficiently absorbed by the intestine, transported in the blood but abnormally stored in the liver.
the characterization of the hepatic lipid content revealed a dramatic increase of triglycerides with no variation in phospholipids but a slight decrease of the cholesterol in the liver of kitsco5/sco <dig> mice at  <dig>  and  <dig>  dpp .
this hepatic accumulation was not followed by an increase of the concentration of plasma triglycerides as observed in obesity or in liver diseases , but the mutant mice displayed lower concentrations of free and hdl cholesterol as well as ldl cholesterol at both ages.
thus, lipid transport in plasma and exchange between tissues were deficient or not yet mature in sco5/sco <dig> suckling neonates, leading to the persistence of the steatosis at  <dig>  dpp and later on.
therefore, these results suggest that the lethality induced by kit hypomorphic alleles was concomitant with the impaired lipid metabolism observed in mutant animals during the suckling period.
kit is expressed in the liver of neonates and adults
consistent with the phenotype, kit gene expression was detected during post-natal development in the liver by using the lacz reporter gene inserted in the first exon of kit from the kittm1alf/+ mouse  <cit> .
kit expression was detected in a large number of cells in the liver from  <dig>  to  <dig>  dpp, as a reminiscence of fetal hematopoiesis .
thereafter, kit expression was confined to a few cells located in the hepatic parenchyma and was also found in the epithelial cells of the biliary canals at later stages and in adults.
as expected, the number of kit+ cells was dramatically reduced at  <dig>  dpp in moribund kittm1alf homozygotes  <cit> .
in trans-heterozygotes kittm1alf/sco <dig> and kittm1alf/sow <dig>  a reduced number of kit+ cells were detected at  <dig>  dpp while steatosis was observed .
thus, lipid accumulation was induced by several combinations of kit mutations during the post-natal period and its appearance was clearly associated with a decrease in the number of kit expressing cells in the liver.
molecular characterisation of the kit-induced steatosis during post-natal development
to decipher the molecular basis of kit-dependent steatosis, we determined the expression profiles of key genes involved in lipid hepatic metabolism: such as apoliproteins , lipoprotein receptors , lipase  and others implicated in hepatic lipidogenesis , lipid secretion , bile acid synthesis , lipid transport  and a lipodystrophy gene, lipin  <dig> , encoding a phosphatidate phosphatase enzyme with transcription activity  <cit> .
expression of most of the genes remained unchanged in wild-type and mutant individuals at  <dig>  dpp  except for the very low density lipoprotein receptor , the lpin <dig> and the lipoprotein lipase  genes that were downregulated in sco5/sco <dig> mutants compared to wild-type littermates .
in addition vldlr expression appeared to be significantly retarded at  <dig>  dpp just during the transient steatosis while the expression level reached a plateau at  <dig>  dpp with the same expression level found at  <dig>  dpp.
the downregulation of lpin <dig> started to be detected at  <dig>  dpp and was significant at  <dig>  dpp when homozygote sco <dig> mutants suffer from the clearance of microvesicular accumulated lipids.
interestingly, the lpic gene transcription is not significantly affected in the sco <dig> mutant at these ages.
altogether, these changes at the transcriptional level of key genes involved in hepatic lipid uptake  or modification  are helpful to understand the molecular mechanism of the kit-induced defect in the liver.
discussion
a new allelic series of kit as a tool for deciphering downstream signaling pathways
in this paper, we described three new point mutations inducing loss-of-function of the kit protein  and one potential mutation affecting the regulation of the kit gene .
several other mutants affecting the level of kit expression have been described in the literature  <cit> , unraveling that quiet a few dispersed regulatory elements control kit gene expression.
most of them correspond to large chromosomal rearrangement whereas sow <dig> is presumably a point mutation induced by enu affecting a regulatory element controlling kit during melanogenesis and gametogenesis.
the missense mutations described in sco <dig> and sco <dig>  at position m <dig> and v <dig> respectively, are found in the amino-terminal lobe near the atp-binding site, whereas for whc <dig>  f <dig> is located in the dfg motif of the mg2+ binding loop in the activation segment of the tyrosine kinase domain <cit> .
these residues are highly conserved in the kit sequence deduced from various species, from human to zebrafish, and also in other mouse receptor tyrosine kinases from the same subfamily such as csfr <dig>  pdgfr, vegfr, and fgfr .
interestingly, the two residues mutated in sco <dig> and sco <dig> are located in the amino-terminal lobe composed of β-strands, nearby the t <dig>  that is mutated in the w-v allele, in the structure of the active kit kinase  <cit>  .
overall the mutations described here are presumably hypomorphic because complete loss-of-function such as in the lacz knock-in or in the w allele are homozygous lethal  <cit> .
of particular interest is the consequence of the f809l mutation located in the activation loop.
this region of the tyrosine kinase domain undergoes a conformational change after the transphosphorylation of the autoinhibitory jm domain after kitl binding.
such a change will leave open the atp-binding site and the mg2+ binding loop for the receptor kinase activity.
in addition, the anti-cancer drug sti- <dig> interacts notably with the f <dig> and blocks the tyrosine kinase domain in an inactive conformation  <cit> .
remarkably, the phenotypes observed in the whc <dig> homozygotes are similar to the sco <dig> allele with a strong impact on pigmentation and peripheral blood cells but not on germ cells.
the four kit mutants display pleiotropic phenotypes affecting with variable degrees the melanogenesis, hematopoiesis and gametogenesis in homozygotes.
interestingly, sco <dig>  sco <dig> and whc <dig> displayed essentially normal gametogenesis suggesting that the m <dig>  v <dig> in the atp binding site and the f <dig> in the activation loop domain are not as important as the y <dig> bound by the phosphatidylinositol 3'-kinase  that is essential for kit function in testis  <cit> .
therefore the model of the inhibitory effect of the jm domain should be reconsidered for the germ cells to reconcile the absence of major germline defect in the whc <dig> mutant.
these data stress out the complexity of the downstream signaling pathways and the targets that are controlled by the kit protein in different organs.
the impaired viability of kit mutants is associated with an altered hepatic lipid metabolism
similar to the kitw/wv and the kitwads/wads mutants recently described  <cit> , the post-natal viability of the whc <dig> and of the sco <dig> homozygotes is strongly affected.
further detailed analysis identifies a major defect in hepatic lipid metabolism with steatosis at  <dig>  dpp in animals carrying various combinations of hypomorphic kit alleles, including w/w-v, sco5/lac, sow3/lac, sco5/sco <dig> and whc1/whc <dig>  furthermore the appearance of steatosis is correlated with the defect in growth of the mutant initiated at  <dig>  dpp.
the determination of hepatic lipid content correlated the steatosis with a deregulated accumulation of tg just after suckling.
during mammalian post-natal development, tg originate predominantly from the maternal milk and represent the major source of energy available for the neonates  <cit> .
ingested tg are assembled in chylomicrons particles and delivered to the developing organs.
following the local action of lipoprotein lipase , tg-derived free fatty acids  are absorbed by tissues while the remaining of the chylomicrons, the remnants, is taken-up by the liver.
during the fasting period, the liver participates in the uptake, oxidation, de novo synthesis, assembly and secretion of tg in very low density lipoproteins particules .
in kit mutants, a defective hepatic metabolism, with the abnormal low expression of vldlr and lpin <dig>  leads to the maintenance of this "transient steatosis".
expression profiling for key genes participating to the metabolism, the transport, the modification, lipidogenesis and biliary acid synthesis in the liver revealed that only a few genes, namely vldlr, lpin <dig> and lpl were downregulated in kit mutants.
consistent with the kit-induced hepatic phenotype, interference with lipase function induces type i hyperlipoproteinemia in human  and more severe hyperchylomicronemia affecting post-natal viability in lpl mutant mice  <cit>  or in combined lipase deficiency , which alters both lpl and hepatic lipase  activities  <cit> .
in addition, vldlr plays a major role in the metabolism of postprandial lipoproteins by enhancing lpl-mediated tg hydrolysis  <cit>  and the vldlr mutant mice suffer from growth retardation during the suckling period  <cit> .
lpin <dig> reduced expression is presumably a major event leading to the abnormal metabolism of lipid in kitsco5/sco <dig> mice.
indeed, lpin <dig> loss-of-function induces steatosis and juvenile lethality in the "fatty liver dystrophy"  mice, a model of human lipodystrophy  <cit> .
recently, lpin <dig> has been shown to complement the phosphatidate phosphatase yeast deficiency  <cit> .
in yeast its homolog regulates the level of triacylglycerol and ffa and represses the expression of genes involved in phospholipid synthesis.
similarly the hepatic fatty acid oxidation is reduced in fld/fld mice  <cit>  and lpin <dig> is regulated by the transcription factor pgc-1α that regulates several metabolic pathways.
in addition lipin <dig> can activate the transcription of pparα and coactivates with pgc-1α and pparα a large panel of other genes controlling lipid metabolism  <cit> .
therefore, downregulation of lpin <dig> in kit mutants will lead to a reduced oxidation of lipids and steatosis in a context of increased lipid delivery during suckling.
similar to fld/fld mice, some kitsco5/sco <dig> animals could overcome steatosis after weaning but the most affected ones continued to display it.
consistent with our data, hyperlipidemia was observed in a fraction of adult kitw/w-v mice and correlated with an abnormal plasmatic lipoprotein profile and reduced lipolytic activities  <cit> .
in addition to the lpin <dig> downregulation, repression of lpl and vldlr would also contribute to the hepatic kit-induced phenotype.
interestingly, lpl homozygous mutants died within  <dig> h after birth with severe hypertriglyceridemia  <cit> .
thus, linked to the steatosis observed in kit hypomorphic mutants were changes in the expression of genes that are known to induce hepatic lipid metabolism anomalies in mutant mice.
such a change might explain why the hepatic deficiency is found in the surviving kit homozygote adults.
the absence of kit expressing-cells and/or reduced kit activity during development might have an indirect effect.
in such a way, kit loss-of-function mimicked the downregulation of lpin <dig>  lpl and vldlr leading to juvenile steatosis and growth retardation.
consistently, kit and kitl were found to be expressed in the rat cholangiocytes  <cit>  and several mouse mutants of kitl displayed comparable low post-natal viability  <cit> .
involvement of the kit-dependent steatosis and anemia in post-natal lethality
the origin of the post-natal deficiency observed in kit mutants is commonly associated with anemia.
the mutant post-natal phenotype is improved by the injection of mouse embryonic liver cells or bone marrow derived cells,  <dig> h after birth, respectively in 26% and 32% of w/w-v mutants  <cit> .
the rescue is even better if the transplantation of embryonic liver cells is done through the placental circulation by day  <dig> of gestation  <cit> .
similarly the rescue of kit mutant mice could be achieved by erythropoietin  <cit> .
interestingly in such rescue, the rbc counts and the percentage of homozygous mutants that survived the post-natal crisis are similar to our results confirming that another kit-dependent factor influences the survival of the pups after birth.
thus we propose that the liver defect induced by kit partial loss-of-function is one of these additional factors.
we described some molecular events affecting the neonatal hepatic function, but other changes in the liver of kit mutants can not be ruled out.
additional analysis will help to describe in details the cellular mechanisms underlying the liver phenotype induced by kit loss-of-function.
thus, even though kit-induced anemia might participate to the post-natal crisis observed in the mutant mice, we propose that kit-induced steatosis is a major factor at the origin of the post-natal mortality.
only a controlled inactivation of kit in blood or liver compartments will help to clearly discriminate the impact of anemia and liver defect in the kit-dependent impaired post-natal viability.
kit and the signaling pathways controlling lipid metabolism in the liver
kit is a receptor protein kinase controlling several signaling pathways.
some of them are known to control liver metabolism, such as the pi3k-akt-mtor, jak/stat  <cit> , or to induce growth defect like for example in the snai <dig> mutant  <cit> .
thus another alternative to explain the importance of kit in liver homeostasis could be due to the alteration of signaling pathways leading to impaired lipid metabolism.
alternatively, kit could participate directly to the signaling pathway controlled by lipoprotein receptors.
pdgfrb, another member of the same receptor family, is a co-receptor of the ldl receptor related protein  <cit> .
similarly, kit could cooperate with members of the lipoprotein receptors family, such as vldlr, or could control the expression of lpin <dig>  regulating the hepatic triglyceride metabolism and oxidative phosphorylation through its key position in the ppara/pgc-1a regulatory pathway  <cit> .
interestingly, altering the binding of pi3k with the kit protein, and reducing the activation of akt to 10%, does not affect the weight of mutant animals  <cit> , suggesting that, during the post-natal development, the kit-dependent metabolism of triglycerides is not fully associated with the pi3k pathway but results from another signalling pathway.
kit and the fetal liver stem cells
kit is a cellular marker of fetal liver stem cells, is involved in the differentiation/proliferation of the bipotent hepato-biliary progenitor cell  <cit> .
in addition kit expression is used to isolate population of hepatocytes during in vitro differentiation of es cells  <cit> .
consistently, kitl is an important factor controlling hepatocyte proliferation after hepatectomy in il <dig> mutant mice  <cit> .
thus, the steatosis could be an indirect consequence of a defect in the differentiation or the proliferation of hepatic progenitor cells leading to the loss of kit+ cells observed in sco5/lac or sow3/lac animals.
decreasing the activity of kit to a certain steatosis threshold will modify the pool of stem cells that will affect either the development of the liver and the post-natal hepatic homeostasis.
consequently, the defect in lipid metabolism of sco5/sco <dig> adults might result from a consequence of a post-natal defect that was not compensated during the juvenile development.
this hypothesis is in agreement with the pattern of expression of the genes tested.
kit expression is restricted in the juvenile and adult liver.
thus we should foresee an indirect effect of kit on hepatocytes functions observed in this study.
CONCLUSIONS
taken together, these data demonstrate the key function of kit in neonates liver physiology and further explain the early hepatic defect induced by kit loss-of-function.
now, kit should be considered as one of the endogenous factors that could lead to steatosis  <cit> .
a very low number of animal models are available to study the consequences of impaired lipid metabolism during suckling, reminiscent of some human conditions .
these data open new perspectives for the molecular understanding of juvenile steatosis, the analysis of kit function during liver development and support the need to follow-up hepatic function in patients during anti-cancer treatment with the inhibitor of kit activity, sti- <dig>  reported to affect the weight and the viability of rat neonates  <cit> .

 kit is a well-known receptor tyrosine kinase controlling melanogenesis, hematopoiesis, gametogenesis, hearing, mast cells and the survival of cajal cells from the intestine. furthermore kit is expressed in several epithelial endothelium or endocrine cells but also in cholangiocytes during development and in the adult. the binding of kitl, induces the dimerization of the kit molecule, followed by a change in the configuration of the intracellular domain and the autophosphorylation of the receptor, opening several docking sites for signal transduction molecules containing sh binding domains. hence the kit signal is transduced by several pathways including that of the src family kinases, plc-γ, ras/mapk, pi3k-akt-mtor, jak/stat and also through the activation of the snai and mitf transcription factors. kit belongs to the class iii of the receptor tyrosine kinase family with csf1r, pdgfr-a,-b, kdr, flt- - and - characterized by the presence of an autoinhibitory juxtamembrane domain. without phosphorylation, the jm domain is inserted in between the n- and c-lobes blocking the dfg conserved motif of the activation loop in an inactive conformation. upon phosphorylation, changes in the conformation of the jm domain opens up the atp binding site and repositions correctly the activation loop in the kinase domain. mutations that affect the activation loop are found in human mastocytose or leukemia, while deletions or insertions in the jm domain are observed in gastro intestinal tumor and often correspond to gain-of-function. the treatment of the kit-induced tumors had taken a promising perspective with the use of sti-, that binds to the atp-binding site, stabilizing the autoinhibitory structure with an inactive tyrosine kinase domain. in human, kit loss-of-function is linked to piebald trait and a large panel of mutations is reported. similar pigmentation defects are observed in mouse models and the analysis of kit functions was facilitated by a large number of hypomorphic murine mutants. some correspond to point mutations, others to large chromosomal rearrangements obtained as spontaneous or induced by chemicals or radiation, while a few were engineered. complete lack of kit interferes with post-natal viability while hypomorphic mutations could induce not only a variable degree of defects during melanogenesis, hematopoieisis and gametogenesis but also growth retardation and juvenile mortality. in this report we used a panel of mutants isolated from genome-wide mutagenesis screens to investigate the post-natal role of kit. these mutations correspond to a new allelic series of hypomorphic kit alleles that alter differentially the melanogenesis, hematopoiesis and gametogenesis. in two such mutants, we observed an impaired viability of the homozygotes before weaning. clearly, growth retardation and premature death of neonates in both kit mutants were associated with juvenile steatosis during the suckling period. further analysis pointed out that kit is expressed in the post-natal liver and kit mutants were suffering from an early defect in triglycerides export from the liver correlated with the downregulation of key genes participating in the control of hepatic lipid metabolism. this series of evidence established the major role of kit in controlling liver triglycerides metabolism in neonates.  characterization of a new allelic series of mutants affecting the kit gene to analyze the role of kit in neonates, we used new semi-dominant enu-induced mutant mouse lines named sco sco, sow and whc, displaying white head blazes and belly spots, that were isolated in genome wide mutagenesis programs. the corresponding mutations were mapped on the mouse chromosome near the kit locus. with the sequencing of the kit gene from the sco sco and whc homozygotes, unique missense mutations were found in the intracellular tyrosine kinase domain that affect m623l for sco v667a for sco and f809l for whc. these changes were absent in the original c3heb/fej and other strains. no mutation was found in the exonic and the splice junction sequences of the kit gene from the sow mouse mutant line, suggesting that sow affects the regulation of the kit gene as described in other mouse mutants. nonetheless, a series of complementation assays with other known alleles including kitw, kitw-v and kittm1alf confirmed that kit function was altered in sow. therefore, the data support that the mutants described here represent a new allelic series at the kit locus, with three missense mutations and one probable mutation affecting the regulation of the kit gene. homozygous animals generated for the four mutations displayed similar white coat pigmentation with black eyes. however, sco homozygotes showed a few grey hairs and pigmented ears suggesting that this allele alters later stages of pigmentation. blood analysis showed changes characteristic of macrocytic anemia, with a significant reduction of hematocrit and red blood cell count, together with an increase in mean red cell volume and in mean cell hemoglobin content for all the adult mutant mice. hematopoietic defects were more severe in the sco and whc homozygotes than in sow and sco mutants with however some little variations between males and females. finally, histological analysis of testis isolated from adult males homozygous for sco sco and whc showed different stages of germ cells differentiation while sow homozygotes revealed a complete lack of spermatogenesis. these results were confirmed by the absence of β-galactosidase staining in the seminiferous tubules of kittm1alf/sow transheterozygotes and assessed by a fertility test. only the sco and sco homozygotes were able to reproduce but no progeny was ever obtained from crosses between sow3/sow males and wild-type females. thus, sco sco sow and whc are hypomorphic mutations of kit, with the expected effects, although variable, on melanogenesis, hematopoiesis and gametogenesis. sco mutant analysis unravels a function of kit during post-natal development of the liver an impaired viability affected kitsco homozygotes before weaning, with only mutants recovered out of offspring from kitsco5/+ intercrosses. although sco5/sco genotypes scored at birth with a mendelian ratio, almost 50% of them died before days post partum, and only 25% survived at weaning. in addition, kitsco5/sco homozygotes displayed a growth defect at dpp, with a weight difference reaching up to 50% of the control at dpp. comparable phenotypes were also noted in kitwhc1/whc and kitw/w-v animals, thus we analyzed further this post-natal lethal phenotype. necropsy analysis revealed that the liver from kitsco5/sco animals at dpp had a tan yellowish color although it had a normal relative organ weight. anemia was detected in mutants at this early age and could partly account for the liver color alteration. however, the spotted aspect and the presence of large white areas in the hepatic lobes in all kitsco5/sco analyzed at dpp were associated with an abnormal structure of hepatocytes. indeed, lipid microvesicular accumulation, or steatosis, was detected in mutant hepatocytes by specific staining at and dpp with oil-red-o. similar lipid inclusions were observed in the liver of kitwhc1/whc neonates, to a lesser extent in kitsco5/+ animals and also in few kitsco5/sco individuals that survived until two months of age. a comparable phenotype was detected in dpp and adult kitw/w-v mice. by contrast, kitsow and kitsco homozygotes developed normally and did not display any hepatic phenotype. thus the liver phenotype was not specific to the sco allele of kit, as it was also found in a number of compound kit mutants. then, we investigated the onset of the phenotype by following the lipid/water ratio in the liver of newborn mice using nuclear magnetic resonance spectroscopy. accumulation of hepatic lipids in kitsco5/sco mutants was detected at dpp suggesting that defect started earlier. moreover the lipid/water ratio increased until dpp when most of the homozygotes begun to die as predicted from the original analysis of viability. nevertheless, the lipid/water ratio was still high in surviving mutants compared to controls at dpp. to further define the onset of the phenotype, liver sections from groups of mutants and control littermates were analyzed during post-natal development. no change was observed immediately after birth in neonates that had not yet suckled any milk. however, microvesicular accumulations of lipids were found at dpp in the liver of wild-type suckling animals from the c3heb/fej, c57bl/6j and 129s inbred lines. in the sco5/sco mutant, a similar accumulation of lipids appeared at dpp but remained later on, showing that lipids, derived mainly from the milk, were efficiently absorbed by the intestine, transported in the blood but abnormally stored in the liver. the characterization of the hepatic lipid content revealed a dramatic increase of triglycerides with no variation in phospholipids but a slight decrease of the cholesterol in the liver of kitsco5/sco mice at and dpp. this hepatic accumulation was not followed by an increase of the concentration of plasma triglycerides as observed in obesity or in liver diseases, but the mutant mice displayed lower concentrations of free and hdl cholesterol as well as ldl cholesterol at both ages. thus, lipid transport in plasma and exchange between tissues were deficient or not yet mature in sco5/sco suckling neonates, leading to the persistence of the steatosis at dpp and later on. therefore, these results suggest that the lethality induced by kit hypomorphic alleles was concomitant with the impaired lipid metabolism observed in mutant animals during the suckling period. kit is expressed in the liver of neonates and adults consistent with the phenotype, kit gene expression was detected during post-natal development in the liver by using the lacz reporter gene inserted in the first exon of kit from the kittm1alf/+ mouse. kit expression was detected in a large number of cells in the liver from to dpp, as a reminiscence of fetal hematopoiesis. thereafter, kit expression was confined to a few cells located in the hepatic parenchyma and was also found in the epithelial cells of the biliary canals at later stages and in adults. as expected, the number of kit+ cells was dramatically reduced at dpp in moribund kittm1alf homozygotes. in trans-heterozygotes kittm1alf/sco and kittm1alf/sow a reduced number of kit+ cells were detected at dpp while steatosis was observed. thus, lipid accumulation was induced by several combinations of kit mutations during the post-natal period and its appearance was clearly associated with a decrease in the number of kit expressing cells in the liver. molecular characterisation of the kit-induced steatosis during post-natal development to decipher the molecular basis of kit-dependent steatosis, we determined the expression profiles of key genes involved in lipid hepatic metabolism: such as apoliproteins, lipoprotein receptors, lipase and others implicated in hepatic lipidogenesis, lipid secretion, bile acid synthesis, lipid transport and a lipodystrophy gene, lipin, encoding a phosphatidate phosphatase enzyme with transcription activity. expression of most of the genes remained unchanged in wild-type and mutant individuals at dpp except for the very low density lipoprotein receptor, the lpin and the lipoprotein lipase genes that were downregulated in sco5/sco mutants compared to wild-type littermates. in addition vldlr expression appeared to be significantly retarded at dpp just during the transient steatosis while the expression level reached a plateau at dpp with the same expression level found at dpp. the downregulation of lpin started to be detected at dpp and was significant at dpp when homozygote sco mutants suffer from the clearance of microvesicular accumulated lipids. interestingly, the lpic gene transcription is not significantly affected in the sco mutant at these ages. altogether, these changes at the transcriptional level of key genes involved in hepatic lipid uptake or modification are helpful to understand the molecular mechanism of the kit-induced defect in the liver. discussion a new allelic series of kit as a tool for deciphering downstream signaling pathways in this paper, we described three new point mutations inducing loss-of-function of the kit protein and one potential mutation affecting the regulation of the kit gene. several other mutants affecting the level of kit expression have been described in the literature, unraveling that quiet a few dispersed regulatory elements control kit gene expression. most of them correspond to large chromosomal rearrangement whereas sow is presumably a point mutation induced by enu affecting a regulatory element controlling kit during melanogenesis and gametogenesis. the missense mutations described in sco and sco at position m and v respectively, are found in the amino-terminal lobe near the atp-binding site, whereas for whc f is located in the dfg motif of the mg2+ binding loop in the activation segment of the tyrosine kinase domain. these residues are highly conserved in the kit sequence deduced from various species, from human to zebrafish, and also in other mouse receptor tyrosine kinases from the same subfamily such as csfr pdgfr, vegfr, and fgfr. interestingly, the two residues mutated in sco and sco are located in the amino-terminal lobe composed of β-strands, nearby the t that is mutated in the w-v allele, in the structure of the active kit kinase. overall the mutations described here are presumably hypomorphic because complete loss-of-function such as in the lacz knock-in or in the w allele are homozygous lethal. of particular interest is the consequence of the f809l mutation located in the activation loop. this region of the tyrosine kinase domain undergoes a conformational change after the transphosphorylation of the autoinhibitory jm domain after kitl binding. such a change will leave open the atp-binding site and the mg2+ binding loop for the receptor kinase activity. in addition, the anti-cancer drug sti- interacts notably with the f and blocks the tyrosine kinase domain in an inactive conformation. remarkably, the phenotypes observed in the whc homozygotes are similar to the sco allele with a strong impact on pigmentation and peripheral blood cells but not on germ cells. the four kit mutants display pleiotropic phenotypes affecting with variable degrees the melanogenesis, hematopoiesis and gametogenesis in homozygotes. interestingly, sco sco and whc displayed essentially normal gametogenesis suggesting that the m v in the atp binding site and the f in the activation loop domain are not as important as the y bound by the phosphatidylinositol 3'-kinase that is essential for kit function in testis. therefore the model of the inhibitory effect of the jm domain should be reconsidered for the germ cells to reconcile the absence of major germline defect in the whc mutant. these data stress out the complexity of the downstream signaling pathways and the targets that are controlled by the kit protein in different organs. the impaired viability of kit mutants is associated with an altered hepatic lipid metabolism similar to the kitw/wv and the kitwads/wads mutants recently described, the post-natal viability of the whc and of the sco homozygotes is strongly affected. further detailed analysis identifies a major defect in hepatic lipid metabolism with steatosis at dpp in animals carrying various combinations of hypomorphic kit alleles, including w/w-v, sco5/lac, sow3/lac, sco5/sco and whc1/whc furthermore the appearance of steatosis is correlated with the defect in growth of the mutant initiated at dpp. the determination of hepatic lipid content correlated the steatosis with a deregulated accumulation of tg just after suckling. during mammalian post-natal development, tg originate predominantly from the maternal milk and represent the major source of energy available for the neonates. ingested tg are assembled in chylomicrons particles and delivered to the developing organs. following the local action of lipoprotein lipase, tg-derived free fatty acids are absorbed by tissues while the remaining of the chylomicrons, the remnants, is taken-up by the liver. during the fasting period, the liver participates in the uptake, oxidation, de novo synthesis, assembly and secretion of tg in very low density lipoproteins particules. in kit mutants, a defective hepatic metabolism, with the abnormal low expression of vldlr and lpin leads to the maintenance of this "transient steatosis". expression profiling for key genes participating to the metabolism, the transport, the modification, lipidogenesis and biliary acid synthesis in the liver revealed that only a few genes, namely vldlr, lpin and lpl were downregulated in kit mutants. consistent with the kit-induced hepatic phenotype, interference with lipase function induces type i hyperlipoproteinemia in human and more severe hyperchylomicronemia affecting post-natal viability in lpl mutant mice or in combined lipase deficiency, which alters both lpl and hepatic lipase activities. in addition, vldlr plays a major role in the metabolism of postprandial lipoproteins by enhancing lpl-mediated tg hydrolysis and the vldlr mutant mice suffer from growth retardation during the suckling period. lpin reduced expression is presumably a major event leading to the abnormal metabolism of lipid in kitsco5/sco mice. indeed, lpin loss-of-function induces steatosis and juvenile lethality in the "fatty liver dystrophy" mice, a model of human lipodystrophy. recently, lpin has been shown to complement the phosphatidate phosphatase yeast deficiency. in yeast its homolog regulates the level of triacylglycerol and ffa and represses the expression of genes involved in phospholipid synthesis. similarly the hepatic fatty acid oxidation is reduced in fld/fld mice and lpin is regulated by the transcription factor pgc-1α that regulates several metabolic pathways. in addition lipin can activate the transcription of pparα and coactivates with pgc-1α and pparα a large panel of other genes controlling lipid metabolism. therefore, downregulation of lpin in kit mutants will lead to a reduced oxidation of lipids and steatosis in a context of increased lipid delivery during suckling. similar to fld/fld mice, some kitsco5/sco animals could overcome steatosis after weaning but the most affected ones continued to display it. consistent with our data, hyperlipidemia was observed in a fraction of adult kitw/w-v mice and correlated with an abnormal plasmatic lipoprotein profile and reduced lipolytic activities. in addition to the lpin downregulation, repression of lpl and vldlr would also contribute to the hepatic kit-induced phenotype. interestingly, lpl homozygous mutants died within h after birth with severe hypertriglyceridemia. thus, linked to the steatosis observed in kit hypomorphic mutants were changes in the expression of genes that are known to induce hepatic lipid metabolism anomalies in mutant mice. such a change might explain why the hepatic deficiency is found in the surviving kit homozygote adults. the absence of kit expressing-cells and/or reduced kit activity during development might have an indirect effect. in such a way, kit loss-of-function mimicked the downregulation of lpin lpl and vldlr leading to juvenile steatosis and growth retardation. consistently, kit and kitl were found to be expressed in the rat cholangiocytes and several mouse mutants of kitl displayed comparable low post-natal viability. involvement of the kit-dependent steatosis and anemia in post-natal lethality the origin of the post-natal deficiency observed in kit mutants is commonly associated with anemia. the mutant post-natal phenotype is improved by the injection of mouse embryonic liver cells or bone marrow derived cells, h after birth, respectively in 26% and 32% of w/w-v mutants. the rescue is even better if the transplantation of embryonic liver cells is done through the placental circulation by day of gestation. similarly the rescue of kit mutant mice could be achieved by erythropoietin. interestingly in such rescue, the rbc counts and the percentage of homozygous mutants that survived the post-natal crisis are similar to our results confirming that another kit-dependent factor influences the survival of the pups after birth. thus we propose that the liver defect induced by kit partial loss-of-function is one of these additional factors. we described some molecular events affecting the neonatal hepatic function, but other changes in the liver of kit mutants can not be ruled out. additional analysis will help to describe in details the cellular mechanisms underlying the liver phenotype induced by kit loss-of-function. thus, even though kit-induced anemia might participate to the post-natal crisis observed in the mutant mice, we propose that kit-induced steatosis is a major factor at the origin of the post-natal mortality. only a controlled inactivation of kit in blood or liver compartments will help to clearly discriminate the impact of anemia and liver defect in the kit-dependent impaired post-natal viability. kit and the signaling pathways controlling lipid metabolism in the liver kit is a receptor protein kinase controlling several signaling pathways. some of them are known to control liver metabolism, such as the pi3k-akt-mtor, jak/stat, or to induce growth defect like for example in the snai mutant. thus another alternative to explain the importance of kit in liver homeostasis could be due to the alteration of signaling pathways leading to impaired lipid metabolism. alternatively, kit could participate directly to the signaling pathway controlled by lipoprotein receptors. pdgfrb, another member of the same receptor family, is a co-receptor of the ldl receptor related protein. similarly, kit could cooperate with members of the lipoprotein receptors family, such as vldlr, or could control the expression of lpin regulating the hepatic triglyceride metabolism and oxidative phosphorylation through its key position in the ppara/pgc-1a regulatory pathway. interestingly, altering the binding of pi3k with the kit protein, and reducing the activation of akt to 10%, does not affect the weight of mutant animals, suggesting that, during the post-natal development, the kit-dependent metabolism of triglycerides is not fully associated with the pi3k pathway but results from another signalling pathway. kit and the fetal liver stem cells kit is a cellular marker of fetal liver stem cells, is involved in the differentiation/proliferation of the bipotent hepato-biliary progenitor cell. in addition kit expression is used to isolate population of hepatocytes during in vitro differentiation of es cells. consistently, kitl is an important factor controlling hepatocyte proliferation after hepatectomy in il mutant mice. thus, the steatosis could be an indirect consequence of a defect in the differentiation or the proliferation of hepatic progenitor cells leading to the loss of kit+ cells observed in sco5/lac or sow3/lac animals. decreasing the activity of kit to a certain steatosis threshold will modify the pool of stem cells that will affect either the development of the liver and the post-natal hepatic homeostasis. consequently, the defect in lipid metabolism of sco5/sco adults might result from a consequence of a post-natal defect that was not compensated during the juvenile development. this hypothesis is in agreement with the pattern of expression of the genes tested. kit expression is restricted in the juvenile and adult liver. thus we should foresee an indirect effect of kit on hepatocytes functions observed in this study.  taken together, these data demonstrate the key function of kit in neonates liver physiology and further explain the early hepatic defect induced by kit loss-of-function. now, kit should be considered as one of the endogenous factors that could lead to steatosis. a very low number of animal models are available to study the consequences of impaired lipid metabolism during suckling, reminiscent of some human conditions. these data open new perspectives for the molecular understanding of juvenile steatosis, the analysis of kit function during liver development and support the need to follow-up hepatic function in patients during anti-cancer treatment with the inhibitor of kit activity, sti- reported to affect the weight and the viability of rat neonates. 
we found that, in addition to the classical phenotypes, mutations of kit induced juvenile steatosis, associated with the downregulation of the three genes, vldlr, lpin and lpl, controlling lipid metabolism in the post-natal liver. this is a first report involving kit in the control of lipid metabolism in neonates and opening new perspectives for understanding juvenile steatosis. moreover, it reinforces the role of kit during development of the liver and underscores the caution that should be exerted in using kit inhibitors during anti-cancer treatment. hypomorphic mutations can induce a light coat color phenotype while complete lack of kit function interferes with embryogenesis. interestingly several intermediate hypomorphic mutations induced in addition growth retardation and post-natal mortality. in this report we investigated the post-natal role of kit by using a panel of chemically-induced hypomorphic mutations recently isolated in the mouse. hence, kit loss-of-functions mimicked the inactivation of genes controlling the hepatic metabolism of triglycerides, the major source of energy from maternal milk, leading to growth and viability defects during neonatal development. a large number of kit mutants has been described so far showing the pleiotropic phenotypes associated with partial loss-of-function of the gene.
 the kit gene encodes a receptor tyrosine kinase involved in various biological processes including melanogenesis, hematopoiesis and gametogenesis in mice and human. a large number of kit mutants has been described so far showing the pleiotropic phenotypes associated with partial loss-of-function of the gene. hypomorphic mutations can induce a light coat color phenotype while complete lack of kit function interferes with embryogenesis. interestingly several intermediate hypomorphic mutations induced in addition growth retardation and post-natal mortality.  in this report we investigated the post-natal role of kit by using a panel of chemically-induced hypomorphic mutations recently isolated in the mouse. we found that, in addition to the classical phenotypes, mutations of kit induced juvenile steatosis, associated with the downregulation of the three genes, vldlr, lpin and lpl, controlling lipid metabolism in the post-natal liver. hence, kit loss-of-functions mimicked the inactivation of genes controlling the hepatic metabolism of triglycerides, the major source of energy from maternal milk, leading to growth and viability defects during neonatal development.  this is a first report involving kit in the control of lipid metabolism in neonates and opening new perspectives for understanding juvenile steatosis. moreover, it reinforces the role of kit during development of the liver and underscores the caution that should be exerted in using kit inhibitors during anti-cancer treatment. 