BACKGROUND
when and where genes are expressed provides useful information about their function.
this view forms the basis of several genome-scale projects that attempt the systematic localization of mrnas and/or proteins expressed in tissues and cells  <cit> .
when the products of genes remain within the cells that transcribe and translate them, localizing mrnas is especially informative.
cell-autonomously acting proteins include transcription factors, transmembrane proteins and also the majority of metabolic enzymes.
although the expression patterns of many genes that encode enzymes have been determined on a gene-by-gene basis, the systematic effort used in the present study stands out because it not only provides a significant coverage of enzymes encoded in the genome but is conducted in a standardized fashion.
in addition, expression sites are annotated and images are made accessible on a public website  <cit> .
the biochemistry of glycolytic and tca cycle enzymes has been extensively studied, but the expression patterns of the genes encoding these enzymes in a multicellular organism have not yet been systematically examined.
genes encoding glycolytic and tca cycle enzymes are a particularly attractive target for a systematic analysis of expression patterns because their products form pathways.
the glycolytic pathway is located in the cytosol and consists of a sequence of ten steps catalyzed by  <dig> different gene products, some of which are isozymes.
the product of glycolysis  serves as starting material for the mitochondrial tca cycle.
the tca cycle comprises eight distinct steps involving  <dig> gene products, several of which form multisubunit complexes.
while glycolysis and the tca cycle illustrate pathways characterized by sequences of catalytic reactions, mammalian carbonic anhydrases  exemplify a single protein family, all the members of which catalyze the interconversion of carbon dioxide and bicarbonate  <cit> .
notably,  <dig> of the  <dig> cas are catalytically active while the remaining three are ca-related polypeptides .
these have high sequence homology to cas but seem to lack enzymatic activity  <cit> .
the three ca-rps  owe their putative lack of ca activity to substitutions of one or more active site residues  <cit> .
cas are located in different subcellular compartments: ca i, ii, iii, vii  <cit>  and xiii are cytosolic enzymes  <cit> .
ca iv  <cit>  and xv  <cit>  are anchored to the plasma membrane, while ca ix, xii  <cit>  and xiv  <cit>  are transmembrane proteins.
both ca va and ca vb encode mitochondrial enzymes  <cit> , while ca vi is a secreted protein  <cit> .
cas participate in a variety of physiological processes including ph regulation, co <dig> and hco3- transport, ion transport, water and electrolyte balance, ureagenesis, gluconeogenesis and lipogenesis .
we previously developed methods for determining the spatial expression patterns of genes by in situ hybridization  on a transcriptome-wide scale .
here we report the expression patterns of  <dig> genes that encode either catalytic or regulatory subunits of enzymes, representing a quarter of the  <dig> gene products with catalysis-associated activity.
gene expression was analyzed on serially sectioned, stage e <dig>  mouse embryos.
the mid-gestation embryo provides an overview of the entire organism showing well-differentiated organs and tissues resembling those of the adult in their basic design.
one can readily capture all organs in a set of  <dig> serial sections through such an embryo.
the genetic programs underlying e.g.
growth, terminal differentiation, cell migration and apoptosis are still developing at e <dig> , so it is possible to study a wide range of biological processes  <cit> .
this report has three aims.
it provides a listing and a first-pass analysis of the expression of all  <dig> genes; it compares the expression patterns of glycolytic and tca cycle genes; and it compares the expression patterns of  <dig> of the  <dig> known cas in order to illustrate the astonishing diversity of expression patterns exhibited by members of a single multigene family.
RESULTS
expression patterns of mouse genes encoding proteins with catalytic activity
under its "molecular function" category, gene ontology lists approximately  <dig> gene products that exhibit activity associated with catalysis  <cit> .
we have determined the expression patterns of  <dig> of the  <dig> mrnas in e <dig>  mouse embryos using robotic ish.
the expression pattern of each gene was based on the analysis of ~  <dig> sagittal sections,  <dig> μm thick and  <dig> μm apart.
the products of the  <dig> genes included transferases, oxidoreductases, ligases and other enzymes .
genes were selected on the basis of three not mutually exclusive criteria:  the genes scored as 'present' in a duplicate affymetrix microarray chip  hybridized with mrna from e <dig>  mouse embryos ;  all gene products encoding glycolytic and tca cycle enzymes were selected;  for kinases, the intensity of the ma signal was ranked by the raw numerical value, and the  <dig> most strongly-expressed transcripts were selected.
all but three of the  <dig> genes analyzed can be classified into a particular gene ontology  subcategory of enzymatic activity .
on the basis of their domain structures, dip2a, echdc <dig> and echdc <dig> have inferred catalytic activities.
the ish data set used here represents between  <dig> and 77% of the go subcategories.
also, one third of the murine kinome was represented by  <dig> kinases, a subcategory of transferases  <cit> .
of the  <dig> genes,  <dig>  were expressed in one or more embryonic tissues , while only  <dig>  were not detected.
one hundred and six  were expressed ubiquitously ;  <dig>  were expressed regionally .
one hundred and ninety-two  transcripts fell into the "ubiquitous with pattern"  category .
a similar distribution was observed when kinases only were scored; 45% of the kinases were regionally expressed and ~ 30% showed uwp expression.
thus, metabolic enzymes, often viewed as housekeeping proteins that should be expressed ubiquitously, and kinases, involved in signal transduction and hence expected to exhibit tissue-specific expression, showed similar degrees of regionalization of expression.
additional file  <dig> lists all  <dig> genes analyzed and provides information about their overall expression type using the nomenclature of table  <dig> and complemented by a numerical score for expression level .
all ish data generated for this study are fully accessible at the genepaint database  <cit> .
data can be retrieved using gene name, gene symbol , accession number, entrez gene id or genepaint set id   <cit> .
many of the expression patterns of the  <dig> genes are annotated in approximately  <dig> different tissues.
as a result, patterns can be queried by site of expression.
for example, genes expressed in liver and heart can be identified using the structure selection tool of the genepaint database  <cit> .
images of individual sections of each set retrieved can be inspected using image viewers that are integrated into the database.
for additional instructions on how to access genepaint data see references  <cit> .
through our detailed analysis of the expression patterns, we identified numerous genes with tissue-specific expression, which can be used as molecular markers at e <dig>  .
lysyl oxidase-like  <dig>  is expressed in cartilage.
this is consistent with earlier reports that implicated this enzyme in the biogenesis of connective tissues  <cit> .
acetyl-coenzyme a synthetase  <dig> -like  is strongly expressed in the ventricular zone of the developing nervous system  and in liver .
this enzyme ligates acetate to coenzyme a to produce acetyl-coa, a molecule essential in various metabolic pathways.
accordingly, acas2l is reported to be strongly expressed in metabolically active tissues in adult mice  <cit> .
interestingly, however, it is not seen in adult liver  <cit> ; apart from the cns, liver is the only site at which acas2l is expressed in the embryo.
expression in the strongly proliferating cns ventricular zone may reflect the energy requirements of dividing cells.
carboxypeptidase n polypeptide  <dig>  exclusively marks the digestive system with strong expression in liver, intestine, pancreas and stomach.
a previous study reports cpn <dig> expression in embryos as early as e <dig>   <cit> .
the ish data in  <cit>  also show expression at e <dig>  and e <dig>  in erythroid progenitor cells in the embryonic liver and at e <dig>  in hepatocytes.
the subsequent sections of this report focus on glycolysis and tca cycle genes.
the salient features of the expression pattern are briefly described for each gene and, where warranted, patterns are related to those of other genes in the same pathway.
individual images of expression patterns are not provided in this paper since there are more than  <dig> images for the tca cycle genes alone.
instead, the section below provides the genepaint set id and the section number leading to the correct image on the genepaint database  <cit> .
genepaint set ids  can be entered in the search field of the homepage  <cit> .
clicking on the "go" button produces the "results" page on which the "set viewer" link is available.
the appropriate section link  can be selected in the image directory, generating a thumbnail of the section.
this thumbnail can be magnified by clicking any one of three viewer buttons labelled "html", "applet" or "plugin".
expression patterns of glycolytic pathway enzymes
we found that many of the glycolytic and tca cycle genes exhibited a recurrent pattern of expression, which we denoted the "stereotypical energy metabolism pattern" .
the semp, an example of which is shown in fig.
<dig>  is characterized by the following features.
first, expression is seen in a set of  <dig> "signature tissues"  and expression strength is usually not constant across the signature tissues but varies among them.
second, expression is not detected in axial cartilage and long bones.
the matrix shown in fig.
3a is a schematic diagram of the expression of all glycolytic pathway components assessed in signature tissues, axial and limb cartilage.
it can be seen that with the exception of aldolases , at least one gene exhibits a semp  for each step in glycolysis.
<dig> of the  <dig> genes involved in glycolysis show a semp.
hexokinase  <dig>  hk <dig>
this gene is broadly expressed throughout the embryo but signal strength is elevated in the superior and inferior colliculi, spinal chord, dorsal root- and cranial ganglia, and in the telencephalon.
hexokinase  <dig>  hk <dig>
the expression pattern in the nervous system strongly resembles that of hk <dig>  but in contrast to hexokinase  <dig>  hk <dig> is only weakly expressed outside the nervous system except in the epithelial component of the lung.
hexokinase  <dig>  hk <dig>
low and ubiquitous expression characterizes this gene, with some notable exceptions such as the cranial- and dorsal root ganglia , which exhibit strong expression in scattered neurons.
expression is markedly elevated in the perichondrium of the axial skeleton .
hexokinase  <dig>  gck
expression is observed in the anterior pituitary, in the ventromedial nucleus of the hypothalamus and in the preoptic area .
it is also seen the locus coeruleus , the major site of noradrenalin production in the mammalian brain.
outside the cns, gck mrna is seen in the primordium of the pancreas .
among the four genes encoding the allosterically regulated hexokinases, gck stands out because of its spatially restricted expression pattern.
glucose phosphate isomerase  <dig>  gpi <dig>
this gene is expressed in almost every part of the embryo and very strongly in some tissues.
gpi <dig> expression is particularly strong in the ventricular zone of the developing neocortex and spinal cord, in the motor column along the spinal cord, in dorsal root and cranial ganglia and in the cranial nerve nuclei.
the retina , olfactory epithelium, heart, muscles, liver and whisker follicles express gpi <dig>  like gck, gpi <dig> is expressed in the locus coeruleus and in many midbrain neurons .
phosphofructokinase muscle, pfkm
generally, this gene is weakly expressed in many regions of the nervous system.
the most striking expression is in skeletal muscles such as those of the hip  and handplate .
phosphofructokinase liver, pfkl
this gene is strongly expressed.
transcripts are detected in liver, muscles, nervous system and the skeletal anlage.
pfkl transcripts are very abundant in the marginal zone of the cortex, where they form a patch-like pattern .
other obvious sites of expression include midbrain, medulla, spinal cord, dorsal root- and cranial ganglia, thalamus and hypothalamus.
thymus, pancreas, kidney, adrenal gland and lung also express pfkl.
phosphofructokinase platelet, pfkp
the defining feature of pfkp expression is its restriction to the central and peripheral nervous systems.
in the telencephalon, highly regional expression is seen in the piriform cortex  and in scattered neurons of the vetromedial nucleus of the hypothalamus .
in thalamus, midbrain, pons and medulla, numerous single neurons express this gene with very prominent expression in the cranial nerve nuclei .
dorsal root ganglia, motor neurons in the spinal cord, retina and olfactory bulb also express pfkp.
aldolase  <dig>  a isoform, aldoa
aldoa shows strong expression in all muscles, the cardiovascular system, kidney, and the axial and appendicular skeletal elements.
in liver, numerous scattered cells also express aldoa strongly.
transcripts are seen in ganglia, in the motor-column of the spinal cord and in the brain, where elevated expression is detected in the piriform cortex, pons and cranial nerve nuclei.
aldolase  <dig> b isoform, aldob
aldob's expression is highly regional; transcripts are found only in liver and the kidney tubules.
there may be weak aldob expression in the midgut .
aldolase  <dig> c isoform, zebrin ii, aldoc
expression of this isoform is strong in the ventricular zone of neocortex , hippocampus and spinal cord .
strong expression is also seen in neuroepithelium lining the floor of the aqueduct and of the fourth ventricle .
in the case of the fourth ventricle, cells exiting the neuroepithelium express aldoc .
weaker aldoc is seen in cranial and dorsal root ganglia as well as throughout the spinal cord.
notably, aldolase a, b and c collectively constitute a semp when superimposed but none of them alone is a semp.
triosephosphateisomerase, tpi
tpi is expressed throughout the embryo but signal strength varies.
it is elevated in the ventricular zone of the cortex, in the pons, medulla, midbrain, lens, liver, heart, cranial ganglia and choroid plexi.
a particular feature of the expression pattern is the scattered tpi-positive cells located at the striatal-cortical interface .
in addition, this gene is expressed in the spinal cord, most strongly in the motor column .
tpi transcripts are also detected in muscles, the pns, piriform cortex, retina, epidermis, cerebellum, pituitary, lung, facial epithelium, intervertebral discs and joints of limbs.
glyceraldehyde 3-phosphate dehydrogenase spermatogenic, gapdhs
no expression of gapdhs is detected at e <dig> .
glyceraldehyde 3-phosphate dehydrogenase, gapdh
the expression pattern of gapdh is broad with transcripts seen throughout the embryo except in the axial skeletal elements, where no expression is detected.
phosphoglycerate kinase  <dig>  pgk <dig>
pgk <dig> is strongly expressed throughout the integuments, but pgk <dig> transcripts are found in all organs with the exception of the axial skeleton .
phosphoglycerate mutase  <dig>  pgam <dig>
several tissues show strong pgam <dig> expression including the ventricular zone, piriform cortex, cranial nerve nuclei, pituitary, olfactory epithelium, retina, dorsal root ganglia, cranial ganglia, whisker follicles, thymus, salivary glands, kidney and liver.
phosphoglycerate mutase  <dig>  pgam <dig>
the strongest pgam <dig> expression is seen in the integuments, but expression is ubiquitous with the exception of the prevertebrae .
enolase  <dig>  alpha non-neuron enolase  <dig>  eno <dig>
eno <dig> is expressed strongly in the integuments.
with the exception of the prevertebrae, all tissues express eno <dig> weakly.
enolase  <dig>  gamma neuronal enolase <dig>  eno <dig>
eno <dig> is characterized by strong expression in the cns, cranial and dorsal root ganglia.
eno <dig> expression in the cns, however, is highly regionalized with positive and negative tissues intermingling.
for example, there is high expression in the piriform cortex but none in the surrounding cortical cells .
in the neocortex, eno <dig> transcripts are detected in the marginal zone but not elsewhere .
in the pons, transcripts are abundant in the nuclei of the motor trigeminal nucleus , but the rest of the pons shows much less expression.
in the spinal cord, eno <dig> expression is strongest in ventral-most neurons while very little signal is seen in the roof .
outside the nervous system, expression of eno <dig> is seen in salivary glands, and weakly in intestine and lung .
enolase  <dig>  beta muscle enolase  <dig>  eno <dig>
eno <dig> is ubiquitously expressed except in the axial skeleton .
pyruvate kinase, muscle, pkm <dig>
expression of pkm <dig> is ubiquitous in the cns but is elevated in certain structures such as the ventricular zone that lines the left ventricles and the inferior colliculus of the midbrain.
many tissues outside the cns express this gene with the exception of the prevertebrae.
in the liver, most cells do not express pkm <dig> with the exception of blood vessels.
pyruvate kinase liver and red blood cell, pklr
pklr mrna is abundant in liver, lung, salivary gland, teeth primordia and gut .
transcripts of this gene are also readily detected in the motor column of the spinal cord, in the olfactory epithelium and in intervertebral discs.
numerous other sites of expression are observed, but the signal strength is significantly lower than in the aforementioned tissues.
pkm <dig> and pklr superimposed constitute a semp.
expression patterns of tricarboxylic acid cycle enzymes
some of the  <dig> polypeptide chains catalyzing the  <dig> tca cycle steps form multienzyme complexes .
this adds a new dimension to the analysis of expression because the components of multisubunit proteins should be coexpressed.
consequently, they are expected to exhibit similar expression patterns, which should be readily apparent in an ish analysis.
as discussed above,  <dig> of the  <dig> tca cycle genes may also exhibit a semp.
although the variability within the semp is greater in the tca cycle genes than in glycolysis, the stereotypic pattern is still observed.
using the same criteria as for glycolysis, matrix b in fig.
<dig> provides a synopsis of expression and highlights semp results in bold.
for each step of the tca cycle, at least one gene exhibits a semp.
citrate synthase, cs
this gene is most strongly expressed in whisker follicles, cranial ganglia, dorsal root ganglia, salivary gland, differentiating neurons of the olfactory bulb and a subset of cranial nerve nuclei.
expression is also detected in liver, lung, kidney, heart and thymus.
citrate synthase like, csl
this gene is broadly expressed throughout the embryo but signal strength is elevated in the cranial ganglia, liver, muscles, kidney, medulla, pons, heart, salivary gland, dorsal root ganglia, olfactory bulb, thymus, spinal cord, intestine, neocortex, midbrain and diencephalon.
csl is not expressed in the axial skeleton or the long bones of the limbs.
aconitase  <dig>  aco <dig>
the aco <dig> gene is broadly expressed but stronger expression is seen in liver, kidney, lung, adrenal gland and salivary gland.
within the nervous systems, aco <dig> transcripts are elevated in forebrain, brain stem, spinal cord, cranial and dorsal root ganglia.
aconitase  <dig>  aco <dig>
like aco <dig>  aco <dig> is expressed in nearly all tissues.
however, several sites are characterized by regional expression.
for example, elevated expression is seen in the marginal zone of the neocortex ; in the hindbrain, localized expression is seen in several pontine  and medullary nuclei , and in the spinal cord, transcript levels are highest in the motor columns .
dorsal root ganglia are characterized by intermingling of aco2-positive and negative neurons .
isocitrate dehydrogenases complex
the conversion of isocitrate to alpha-ketoglutarate represents an allosterically regulated, rate-limiting step of the krebs cycle.
five genes encoding isocitrate dehydrogenases have been identified in mammals .
idh <dig>  encodes a cytosolic enzyme that uses nadp+ as cofactor and thus is not a krebs cycle enzyme proper but has a major role in lipid metabolism  <cit> .
idh <dig>  also encodes an enzyme that uses nadp+ as cofactor and is located in the mitochondrial matrix.
idh3a, idh3b and idh3g encode enzymes that utilize nad+ as cofactor and reside in the mitochondrial matrix.
these three polypeptides form a heterotetramer comprising two idh3α subunits, one idh3β and one idh3γ subunit  <cit> .
idh3α has been proposed as the catalytic subunit while idh3β and idh3γ have supporting and regulatory roles  <cit> .
it would be thus expected that these genes have similar expression patterns.
studies with recombinant polypeptides also suggest that isocitrate dehydrogenase heteromers are catalytically active only if they contain the idh3a subunit  <cit> .
isocitrate dehydrogenase  <dig>  idh <dig>
idh <dig> encodes a cytosolic enzyme that is strongly expressed in kidney, adrenal gland, intestine, thymus and cartilage.
this gene is weakly expressed in stomach, facial soft tissue, muscle, lung and liver.
idh <dig> stands out because of its strong expression in cns and pns.
it should be noted that idh <dig> synthesizes alpha-ketoglutarate and nadph, which can be utilized by glutamate synthase to generate the neurotransmitter glutamate, perhaps explaining the strong expression in the developing nervous system.
isocitrate dehydrogenase  <dig>  idh <dig>
mitochondrial enzyme-encoding idh <dig> is expressed nearly ubiquitously but the strongest signal is seen in skeletal muscles, the developing heart, choroid plexi  and lens .
there is a notable absence of idh <dig> expression in the developing axial and appendicular cartilage .
isocitrate dehydrogenase  <dig>  alpha, idh3a
idh3a is broadly expressed in the cns with transcripts being observed in nearly all tissues.
nonetheless, this gene stands out among the isocitrate dehydrogenases because of its strong expression in muscles, heart, thymus, intestinal epithelium, liver, cns, pns, and developing whisker follicles.
there is a notable absence of expression in the developing axial and appendicular cartilage, as is typical for semp.
isocitrate dehydrogenase  <dig>  beta, idh3b
isocitrate dehydrogenase  <dig>  gamma, idh3g
the expression patterns of idh3b and idh3g resemble that of idh3a, although the intensity is much weaker.
thus, while idh3a exhibits a definitive semp, this pattern is less pronounced for the β and γ subunits.
the elevated expression of the α subunit can presumably be rationalized if we consider the stoichiometric ratios 2α:1β:1γ in the active enzyme and the implication of the α subunit as the catalytic enzyme, which could potentially drive the complex.
oxoglutarate dehydrogenase complex
oxoglutarate dehydrogenase is a mitochondrial enzyme complex comprising three different subunits  that converts 2-oxoglutatate into succinyl-coa and carbon dioxide.
like pyruvate dehydrogenase, oxoglutarate dehydrogenase forms a large multienzyme complex and the conversion of 2-oxoglutatate into succinyl-coa requires the presence of all three subunits.
consequently, one would predict that ogdh, dld and dlst genes have similar expression patterns.
oxoglutarate dehydrogenase lipoamide, ogdh
dihydrolipoamide s-succinyltransferase, dlst
dihydrolipoamide dehydrogenase, dld
all three genes encoding oxoglutarate dehydrogenase subunits show very similar expression patterns, but only ogdh has strong semp features.
succinate-coenzyme a ligase complex
succinate-coenzyme a ligase is a mitochondrial matrix enzyme that converts succinyl-coa to succinate and coa.
it consists of an invariant alpha subunit  and a beta subunit that is either adp  or gdp-specific .
succinate-coa ligase, alpha subunit, suclg <dig>
succinate-coenzyme a ligase, adp-forming beta subunit, sucla <dig>
both subunits exhibit the typical energy metabolism expression pattern with expression in the cns, pns, muscles, liver, kidney, gastrointestinal tract, adrenal and salivary glands.
there is no expression in axial or limb cartilage.
this stereotypical pattern is somewhat more pronounced for the adp-forming beta subunit than for the alpha subunit.
succinate-coenzyme a ligase, gdp-forming beta subunit, suclg <dig>
although suclg <dig> expression resembles that of suclg and sucla <dig> in some ways, there are striking differences.
generally, cns and pns show very low suclg <dig> expression.
the only exceptions are in the ventricular zone lining the lateral ventricles of the forebrain, the lamina terminalis, the anterior boundary of the thalamus  and the neuronal component of the pituitary , where somewhat elevated expression can be seen.
other notable exceptions to the low expression of suclg <dig> in the cns are the scattered cells of the amygdala, which express the gene strongly .
succinate dehydrogenase complex
this complex consists of four subunits and is located in the inner mitochondrial membrane.
subunit a  is a flavoprotein while subunit b is an iron-sulfur subunit .
subunits c and d are integral membrane proteins .
succinate dehydrogenase complex, subunit a, flavoprotein, sdha
succinate dehydrogenase complex, subunit b, iron sulfur, sdhb
succinate dehydrogenase complex, subunit c, integral membrane protein, sdhc
succinate dehydrogenase complex, subunit d, integral membrane protein, sdhd
since all four polypeptide chains need to be coexpressed to form an active enzyme, it is not unexpected that the expression patterns of the four genes are related.
there are, however, quantitative differences in expression level.
for example, sdhd expression in liver is much more pronounced than in other tissues , while the expression of e.g.
sdhb is fairly similar everywhere .
irrespective of such subtle quantitative differences, all four genes display the stereotypical energy metabolic enzyme pattern of expression.
fumarate hydratase  <dig>  fh <dig>
this gene is broadly expressed and its pattern of expression resembles those of other glycolytic/tca enzymes, although the differences in expression levels are less pronounced than those seen for idh3a .
malate dehydrogenases
malate dehydrogenase  <dig>  nad+ , mdh <dig>
mdh <dig> is essential for the malate-aspartate shuttle, but like idh <dig> it is not a component of the tca cycle.mdh <dig> is expressed in a semp-like manner with additional expression in the facial epithelia .
malate dehydrogenase  <dig>  nad+ , mdh <dig>
mdh <dig>  a tca cycle component, shows a semp expression pattern.
the similarity in expression between mdh <dig> and mdh <dig> is critical since these enzymes play a pivotal role in the malate-aspartate shuttle.
since oxaloacetate cannot enter the mitochondrion directly, it is first converted by mdh <dig> to malate, which readily enters the mitochondrion and is then reconverted to oxaloactetate by mdh <dig>
our study covers the expression of glycolysis and tca cycle genes in full.
at e <dig> , at least one isoform of each step in these pathways shows a semp pattern.
an exception to this rule is aldoa, aldob and aldoc, none of which has a semp alone, though together they constitute a semp.
a finding that emerges from our analysis of tca cycle components is that in cases where the biochemistry depends on a multisubunit complex, the transcripts of at least one subunit show a semp while the other subunits may have uniform expression .
some transcripts of glycolytic pathway genes are expressed highly regionally, such as hexokinase isoform  <dig> .
this indicates a regional function more specialized than energy production.
northern blot data and ish analysis of postnatal and adult tissues detect gck mrna in the lateral hypothalamic area and in the arcuate, ventromedial, dorsomedial and paraventricular nuclei  <cit> .
it has been suggested that gck-positive neurons are "glucose-sensing neurons" that respond to high glucose concentration  <cit> .
the expression pattern of gck at e <dig>  suggests that a glucose-sensing mechanism has become established by this stage in development, since our data demonstrate gck mrna in the precursors of the above-mentioned hypothalamic nuclei.
miki et al.
<cit>  analyzed expression patterns in  <dig> different mouse embryonic and adult tissues using microarrays containing  <dig>  mouse cdnas.
these authors found that genes encoding enzymes catalyzing glycolytic reactions that are closely linked have similar expression patterns.
they also found that transcripts from key regulatory genes exhibited tissue specificity.
to a large extent our data point in the same direction, although the data are obviously different in nature.
carbonic anhydrases
in mouse,  <dig> ca family members have been identified,  <dig> of which are cas proper .
fifteen of the  <dig> members of this gene family were analyzed for expression at e <dig> .
the  <dig> rna probes hybridized ranged between  <dig> and  <dig>  nucleotides in length.
for most of the cas we were able to design templates, and hence probes, that had no sequence homology to any other family member.
however, there was homology of ~ 80% in a few cases, ranging from  <dig> to  <dig> nucleotides .
the highest homology over the longest stretch of dna was seen between the car <dig> probe and car <dig> mrna .
despite this homology, no cross-hybridization occurred.
for example, car <dig> was strongly expressed in blood vessels of the brain and in the lung , but no such expression was seen in these tissues in sections hybridized with authentic car <dig> riboprobe .
# genbank accession number of reference sequence
* ensembl transcript id
§homology of aligned fragments using bl2seq  and percent homology
expression patterns of carbonic anhydrases
carbonic anhydrase  <dig>  car <dig>
strongly expressing cells are scattered throughout the liver  and weak expression approaching background level is seen in various embryonic tissues.
carbonic anhydrase  <dig>  car <dig>
car <dig> expression is regional.
major sites of expression are lung mesenchyme surrounding the bronchi, liver, capillaries distributed throughout the embryo, choroid plexi and cochlea .
in contrast to car <dig>  expression of car <dig> is widespread.
carbonic anhydrase  <dig>  car <dig>
car <dig>  which encodes a cytosolic isozyme, is expressed with a high degree of tissue specificity.
striated muscles, facial cartilage , ureter  and scattered neurons in dorsal root ganglia express car <dig> strongly.
in liver, car <dig> expression is widespread, resembling that of car <dig>
carbonic anhydrase  <dig>  car <dig>
expression of this gene, which encodes a protein anchored to the plasma membrane, is confined to developing glomeruli  and weakly-expressing cells scattered throughout the cns, presumably representing blood vessels.
car <dig> may be an excellent marker for embryonic kidney.
carbonic anhydrase 5a, mitochondrial, car5a
carbonic anhydrase 5b, mitochondria, car5b
no expression of these two mitochondrial enzymes is detected in the mouse embryo at e <dig> .
carbonic anhydrase  <dig>  car <dig>
no expression is detected for the car <dig> gene in the e <dig>  mouse embryo.
carbonic anhydrase  <dig>  car <dig>
this cytoplasmic enzyme shows weak to medium expression throughout the embryo.
carbonic anhydrase  <dig>  car <dig>
expression of this ca-rp is highly regional.
transcripts are abundant in cerebellum , thyroid , clavicle primordium, intestine and stomach .
car <dig> is also detected in heart, dorsal aorta , bronchi , salivary glands and the cortical region of the kidneys .
carbonic anhydrase  <dig>  car <dig>
weak to medium expression can be seen scattered throughout the embryo, although there are also car10-negative tissues such as the axial skeleton .
somewhat elevated expression can be seen in scattered neurons of the cns  and pns  and in the heart .
carbonic anhydrase  <dig>  car <dig>
car <dig> is expressed in the cranial and dorsal root ganglia, olfactory bulb, striatum, amygdala and the midbrain.
particularly striking is the enrichment of these ca-rp transcripts in the boundary cap of the cranial  and dorsal root ganglia .
carbonic anhydrase  <dig>  car <dig>
this is another example of a regionally-expressed ca gene.
choroid plexi, salivary glands and facial as well as ventral integument, including the whisker follicles, express this transmembrane protein strongly .
a medium level of expression is detected in kidney, lung, cerebral cortex, hindbrain, spinal cord and the pns.
carbonic anhydrase  <dig>  car <dig>
car <dig> is strongly expressed in the inner ear and cochlea .
however, the most striking expression site of this gene is at a muscle fiber bundle located posterior to the submandibular gland .
meninges covering the ventral delineation of the hypothalamus and pons exhibit attenuated expression .
carbonic anhydrase  <dig>  car <dig>
this gene is expressed exclusively in the pigment layer of the retina  and choroid plexi .
carbonic anhydrase  <dig>  car <dig>
strong expression is localized in cells scattered throughout the dorsal root ganglia , dorsal spinal cord, pons, tegmentum and inferior colliculus .
a few other structures  also express the gene, but only weakly.
because carbonic anhydrases catalyze a ubiquitous reaction, one might have predicted them to be widely expressed.
however, this was not the case, since half the ca genes showed a considerable degree of regionalization.
although there have been few reports about expression in the developing mouse embryo, we found our results to be generally consistent with published ca data.
at comparable embryonic stages, transcripts of car <dig> were reported in skeletal muscle and ureter  <cit> , and car <dig> mrna was seen in cerebellum, gut, kidney, lung and heart  <cit> .
the expression of car <dig> in choroid plexus and kidney was also consistent with previous reports  <cit> .
the specific expression sites reported here for car <dig>  car <dig>  car <dig> and car <dig> had not been described previously, although expression had been reported in whole embryos and in adult tissues using northern blots and rt-pcr  <cit> .
ma data for car <dig>   <dig> and  <dig> showed them to be absent, but we were able to reveal definite and regionalized expression of these genes using ish.
ma analysis was apparently not sufficiently sensitive to detect mrnas that are confined to a few cells representing a minute fraction of the total tissue from which the rna was extracted.
the developmental expression we report here is also consistent with the expression described in adult tissues.
expression of embryonic car <dig>  <cit> ,  <dig>  <cit> ,  <dig>   <dig>  <cit> ,  <dig>  <cit>  and  <dig>  <cit>  presumably presages that in the adult.
for example, adult car  <dig> protein is abundant in erythrocytes  <cit> .
the expression we describe for car <dig> in the embryonic liver presumably marks sites of hematopoiesis because car <dig> expression resembles that of hematopoietic maturation-associated c-myb  and the hepatocyte differentiation molecular marker hnf4α   <cit> .
furthermore, car <dig> was recently suggested as a target of the transcription factor c-myb  <cit> .
embryonic expression of car <dig> in muscles and car <dig> in glomeruli and blood vessels of the cns presumably presages adult expression in these tissues  <cit> .
in adult rat, car <dig> has been proposed as an immunohistochemical marker for the blood-brain barrier and is thought to play an important role in co2/hco3- homeostasis in the brain  <cit> .
expression in blood vessels of the cns is already apparent in the e <dig>  embryo, suggesting that co2/hco <dig> - conversion is established early in development.
car <dig> has been reported as responsible for supplying hco <dig> - in gluconeogenesis and ureagenesis, pathways restricted to liver mitochondria  <cit> .
consistent with this, car <dig> protein is only detected in adult liver  <cit> .
despite the substantial activities of all gluconeogenesis enzymes, gluconeogenesis itself is reported to proceed at very low rates in fetal hepatocytes and to be triggered by birth itself  <cit> .
this is consistent with our inability to detect car <dig> in the embryo.
finally, car <dig> is normally secreted into saliva and milk  <cit> ; there is no embryonic expression of car <dig>  perhaps because these fluids are not yet produced at e <dig> , although salivary and mammary gland primordia are present at this stage of development.
CONCLUSIONS
the results presented here constitute the first comprehensive description of the expression of all enzymes involved in glycolysis and the tca cycle.
global studies such as this are especially useful for revealing higher degree-associations.
we provide evidence for a stereotypical energy metabolism pattern  of expression .
of the total of  <dig> genes encoding either a glycolysis or a tca cycle enzyme,  <dig> exhibited a semp.
the defining future of a semp is expression in  <dig> "signature tissue".
for the majority of genes classified as semp, expression in all of the  <dig> signature tissue is observed.
examples of exceptions are fh <dig>  sucla and hk <dig>  the existence of a semp raises the possibility that many glycolytic and tca cycle genes may be regulated in a coordinated fashion.
whether this is also true for other metabolic pathways, such as amino acid or protein syntheses, remains to be seen.
our study also makes the point that even so-called housekeeping genes, although quite broadly expressed, may vary significantly among tissues in levels of expression.
kinases have received much attention during recent decades owing to their physiological importance and their high potential as drug targets.
initial efforts in the study of the kinome have concentrated on three specific aspects:  the identification of novel kinases,  the identification of ligands to known kinases, and  the complete characterization of signal transduction to map the biochemical pathways of the kinome fully  <cit> .
as more data become available, so systematic and interactive databases arise, similar to that presented here for managing gene expression data  <cit> .
thus, our study complements the information gathered by the kinome-research community.
at the same time, current trends in kinome research are switching towards structure prediction, structure analysis  and gene expression profiling for eventual comparison in mutant or disease conditions  <cit> .
the expression analysis of  <dig> kinases reported here sets the ground and illustrates the feasibility of a high-throughput expression analysis of the kinome, and provides the expression patterns for over one third of the mouse kinome.
with this study we also corroborate the practicality of high-throughput ish.
the usefulness of comprehensive data sets is even more apparent when all results are accessible through the genepaint database  <cit> .
contrary to the common expectation that enzymes encoded by "housekeeping" genes will be widely expressed, almost half the carbonic anhydrase gene family shows a regionalized expression pattern and a number of them are so highly regionalized that they are suitable as tissue-specific markers.
hence these enzymes show regional diversity in expression, as reported for transcription factors  <cit>  and growth factors  <cit> , both of which are recognized key components of biological regulation.
in summary, this study greatly increases our general knowledge of the expression patterns of many genes that encode enzymes catalyzing various types of metabolic reactions.
although the data are from e <dig>  embryos we believe that information collected at this stage serves as a suitable predictor and starting point for the elucidation of expression patterns in adult mice and presumably also in humans.

 when and where genes are expressed provides useful information about their function. this view forms the basis of several genome-scale projects that attempt the systematic localization of mrnas and/or proteins expressed in tissues and cells. when the products of genes remain within the cells that transcribe and translate them, localizing mrnas is especially informative. cell-autonomously acting proteins include transcription factors, transmembrane proteins and also the majority of metabolic enzymes. although the expression patterns of many genes that encode enzymes have been determined on a gene-by-gene basis, the systematic effort used in the present study stands out because it not only provides a significant coverage of enzymes encoded in the genome but is conducted in a standardized fashion. in addition, expression sites are annotated and images are made accessible on a public website. the biochemistry of glycolytic and tca cycle enzymes has been extensively studied, but the expression patterns of the genes encoding these enzymes in a multicellular organism have not yet been systematically examined. genes encoding glycolytic and tca cycle enzymes are a particularly attractive target for a systematic analysis of expression patterns because their products form pathways. the glycolytic pathway is located in the cytosol and consists of a sequence of ten steps catalyzed by different gene products, some of which are isozymes. the product of glycolysis serves as starting material for the mitochondrial tca cycle. the tca cycle comprises eight distinct steps involving gene products, several of which form multisubunit complexes. while glycolysis and the tca cycle illustrate pathways characterized by sequences of catalytic reactions, mammalian carbonic anhydrases exemplify a single protein family, all the members of which catalyze the interconversion of carbon dioxide and bicarbonate. notably, of the cas are catalytically active while the remaining three are ca-related polypeptides. these have high sequence homology to cas but seem to lack enzymatic activity. the three ca-rps owe their putative lack of ca activity to substitutions of one or more active site residues. cas are located in different subcellular compartments: ca i, ii, iii, vii and xiii are cytosolic enzymes. ca iv and xv are anchored to the plasma membrane, while ca ix, xii and xiv are transmembrane proteins. both ca va and ca vb encode mitochondrial enzymes, while ca vi is a secreted protein. cas participate in a variety of physiological processes including ph regulation, co and hco3- transport, ion transport, water and electrolyte balance, ureagenesis, gluconeogenesis and lipogenesis. we previously developed methods for determining the spatial expression patterns of genes by in situ hybridization on a transcriptome-wide scale. here we report the expression patterns of genes that encode either catalytic or regulatory subunits of enzymes, representing a quarter of the gene products with catalysis-associated activity. gene expression was analyzed on serially sectioned, stage e mouse embryos. the mid-gestation embryo provides an overview of the entire organism showing well-differentiated organs and tissues resembling those of the adult in their basic design. one can readily capture all organs in a set of serial sections through such an embryo. the genetic programs underlying e.g. growth, terminal differentiation, cell migration and apoptosis are still developing at e, so it is possible to study a wide range of biological processes. this report has three aims. it provides a listing and a first-pass analysis of the expression of all genes; it compares the expression patterns of glycolytic and tca cycle genes; and it compares the expression patterns of of the known cas in order to illustrate the astonishing diversity of expression patterns exhibited by members of a single multigene family.  expression patterns of mouse genes encoding proteins with catalytic activity under its "molecular function" category, gene ontology lists approximately gene products that exhibit activity associated with catalysis. we have determined the expression patterns of of the mrnas in e mouse embryos using robotic ish. the expression pattern of each gene was based on the analysis of ~ sagittal sections, μm thick and μm apart. the products of the genes included transferases, oxidoreductases, ligases and other enzymes. genes were selected on the basis of three not mutually exclusive criteria: the genes scored as'present' in a duplicate affymetrix microarray chip hybridized with mrna from e mouse embryos; all gene products encoding glycolytic and tca cycle enzymes were selected; for kinases, the intensity of the ma signal was ranked by the raw numerical value, and the most strongly-expressed transcripts were selected. all but three of the genes analyzed can be classified into a particular gene ontology subcategory of enzymatic activity. on the basis of their domain structures, dip2a, echdc and echdc have inferred catalytic activities. the ish data set used here represents between and 77% of the go subcategories. also, one third of the murine kinome was represented by kinases, a subcategory of transferases. of the genes, were expressed in one or more embryonic tissues, while only were not detected. one hundred and six were expressed ubiquitously; were expressed regionally. one hundred and ninety-two transcripts fell into the "ubiquitous with pattern" category. a similar distribution was observed when kinases only were scored; 45% of the kinases were regionally expressed and ~ 30% showed uwp expression. thus, metabolic enzymes, often viewed as housekeeping proteins that should be expressed ubiquitously, and kinases, involved in signal transduction and hence expected to exhibit tissue-specific expression, showed similar degrees of regionalization of expression. additional file lists all genes analyzed and provides information about their overall expression type using the nomenclature of table and complemented by a numerical score for expression level. all ish data generated for this study are fully accessible at the genepaint database. data can be retrieved using gene name, gene symbol, accession number, entrez gene id or genepaint set id. many of the expression patterns of the genes are annotated in approximately different tissues. as a result, patterns can be queried by site of expression. for example, genes expressed in liver and heart can be identified using the structure selection tool of the genepaint database. images of individual sections of each set retrieved can be inspected using image viewers that are integrated into the database. for additional instructions on how to access genepaint data see references. through our detailed analysis of the expression patterns, we identified numerous genes with tissue-specific expression, which can be used as molecular markers at e. lysyl oxidase-like is expressed in cartilage. this is consistent with earlier reports that implicated this enzyme in the biogenesis of connective tissues. acetyl-coenzyme a synthetase -like is strongly expressed in the ventricular zone of the developing nervous system and in liver. this enzyme ligates acetate to coenzyme a to produce acetyl-coa, a molecule essential in various metabolic pathways. accordingly, acas2l is reported to be strongly expressed in metabolically active tissues in adult mice. interestingly, however, it is not seen in adult liver; apart from the cns, liver is the only site at which acas2l is expressed in the embryo. expression in the strongly proliferating cns ventricular zone may reflect the energy requirements of dividing cells. carboxypeptidase n polypeptide exclusively marks the digestive system with strong expression in liver, intestine, pancreas and stomach. a previous study reports cpn expression in embryos as early as e. the ish data in also show expression at e and e in erythroid progenitor cells in the embryonic liver and at e in hepatocytes. the subsequent sections of this report focus on glycolysis and tca cycle genes. the salient features of the expression pattern are briefly described for each gene and, where warranted, patterns are related to those of other genes in the same pathway. individual images of expression patterns are not provided in this paper since there are more than images for the tca cycle genes alone. instead, the section below provides the genepaint set id and the section number leading to the correct image on the genepaint database. genepaint set ids can be entered in the search field of the homepage. clicking on the "go" button produces the "results" page on which the "set viewer" link is available. the appropriate section link can be selected in the image directory, generating a thumbnail of the section. this thumbnail can be magnified by clicking any one of three viewer buttons labelled "html", "applet" or "plugin". expression patterns of glycolytic pathway enzymes we found that many of the glycolytic and tca cycle genes exhibited a recurrent pattern of expression, which we denoted the "stereotypical energy metabolism pattern". the semp, an example of which is shown in fig. is characterized by the following features. first, expression is seen in a set of "signature tissues" and expression strength is usually not constant across the signature tissues but varies among them. second, expression is not detected in axial cartilage and long bones. the matrix shown in fig. 3a is a schematic diagram of the expression of all glycolytic pathway components assessed in signature tissues, axial and limb cartilage. it can be seen that with the exception of aldolases, at least one gene exhibits a semp for each step in glycolysis. of the genes involved in glycolysis show a semp. hexokinase hk this gene is broadly expressed throughout the embryo but signal strength is elevated in the superior and inferior colliculi, spinal chord, dorsal root- and cranial ganglia, and in the telencephalon. hexokinase hk the expression pattern in the nervous system strongly resembles that of hk but in contrast to hexokinase hk is only weakly expressed outside the nervous system except in the epithelial component of the lung. hexokinase hk low and ubiquitous expression characterizes this gene, with some notable exceptions such as the cranial- and dorsal root ganglia, which exhibit strong expression in scattered neurons. expression is markedly elevated in the perichondrium of the axial skeleton. hexokinase gck expression is observed in the anterior pituitary, in the ventromedial nucleus of the hypothalamus and in the preoptic area. it is also seen the locus coeruleus, the major site of noradrenalin production in the mammalian brain. outside the cns, gck mrna is seen in the primordium of the pancreas. among the four genes encoding the allosterically regulated hexokinases, gck stands out because of its spatially restricted expression pattern. glucose phosphate isomerase gpi this gene is expressed in almost every part of the embryo and very strongly in some tissues. gpi expression is particularly strong in the ventricular zone of the developing neocortex and spinal cord, in the motor column along the spinal cord, in dorsal root and cranial ganglia and in the cranial nerve nuclei. the retina, olfactory epithelium, heart, muscles, liver and whisker follicles express gpi like gck, gpi is expressed in the locus coeruleus and in many midbrain neurons. phosphofructokinase muscle, pfkm generally, this gene is weakly expressed in many regions of the nervous system. the most striking expression is in skeletal muscles such as those of the hip and handplate. phosphofructokinase liver, pfkl this gene is strongly expressed. transcripts are detected in liver, muscles, nervous system and the skeletal anlage. pfkl transcripts are very abundant in the marginal zone of the cortex, where they form a patch-like pattern. other obvious sites of expression include midbrain, medulla, spinal cord, dorsal root- and cranial ganglia, thalamus and hypothalamus. thymus, pancreas, kidney, adrenal gland and lung also express pfkl. phosphofructokinase platelet, pfkp the defining feature of pfkp expression is its restriction to the central and peripheral nervous systems. in the telencephalon, highly regional expression is seen in the piriform cortex and in scattered neurons of the vetromedial nucleus of the hypothalamus. in thalamus, midbrain, pons and medulla, numerous single neurons express this gene with very prominent expression in the cranial nerve nuclei. dorsal root ganglia, motor neurons in the spinal cord, retina and olfactory bulb also express pfkp. aldolase a isoform, aldoa aldoa shows strong expression in all muscles, the cardiovascular system, kidney, and the axial and appendicular skeletal elements. in liver, numerous scattered cells also express aldoa strongly. transcripts are seen in ganglia, in the motor-column of the spinal cord and in the brain, where elevated expression is detected in the piriform cortex, pons and cranial nerve nuclei. aldolase b isoform, aldob aldob's expression is highly regional; transcripts are found only in liver and the kidney tubules. there may be weak aldob expression in the midgut. aldolase c isoform, zebrin ii, aldoc expression of this isoform is strong in the ventricular zone of neocortex, hippocampus and spinal cord. strong expression is also seen in neuroepithelium lining the floor of the aqueduct and of the fourth ventricle. in the case of the fourth ventricle, cells exiting the neuroepithelium express aldoc. weaker aldoc is seen in cranial and dorsal root ganglia as well as throughout the spinal cord. notably, aldolase a, b and c collectively constitute a semp when superimposed but none of them alone is a semp. triosephosphateisomerase, tpi tpi is expressed throughout the embryo but signal strength varies. it is elevated in the ventricular zone of the cortex, in the pons, medulla, midbrain, lens, liver, heart, cranial ganglia and choroid plexi. a particular feature of the expression pattern is the scattered tpi-positive cells located at the striatal-cortical interface. in addition, this gene is expressed in the spinal cord, most strongly in the motor column. tpi transcripts are also detected in muscles, the pns, piriform cortex, retina, epidermis, cerebellum, pituitary, lung, facial epithelium, intervertebral discs and joints of limbs. glyceraldehyde 3-phosphate dehydrogenase spermatogenic, gapdhs no expression of gapdhs is detected at e. glyceraldehyde 3-phosphate dehydrogenase, gapdh the expression pattern of gapdh is broad with transcripts seen throughout the embryo except in the axial skeletal elements, where no expression is detected. phosphoglycerate kinase pgk pgk is strongly expressed throughout the integuments, but pgk transcripts are found in all organs with the exception of the axial skeleton. phosphoglycerate mutase pgam several tissues show strong pgam expression including the ventricular zone, piriform cortex, cranial nerve nuclei, pituitary, olfactory epithelium, retina, dorsal root ganglia, cranial ganglia, whisker follicles, thymus, salivary glands, kidney and liver. phosphoglycerate mutase pgam the strongest pgam expression is seen in the integuments, but expression is ubiquitous with the exception of the prevertebrae. enolase alpha non-neuron enolase eno eno is expressed strongly in the integuments. with the exception of the prevertebrae, all tissues express eno weakly. enolase gamma neuronal enolase eno eno is characterized by strong expression in the cns, cranial and dorsal root ganglia. eno expression in the cns, however, is highly regionalized with positive and negative tissues intermingling. for example, there is high expression in the piriform cortex but none in the surrounding cortical cells. in the neocortex, eno transcripts are detected in the marginal zone but not elsewhere. in the pons, transcripts are abundant in the nuclei of the motor trigeminal nucleus, but the rest of the pons shows much less expression. in the spinal cord, eno expression is strongest in ventral-most neurons while very little signal is seen in the roof. outside the nervous system, expression of eno is seen in salivary glands, and weakly in intestine and lung. enolase beta muscle enolase eno eno is ubiquitously expressed except in the axial skeleton. pyruvate kinase, muscle, pkm expression of pkm is ubiquitous in the cns but is elevated in certain structures such as the ventricular zone that lines the left ventricles and the inferior colliculus of the midbrain. many tissues outside the cns express this gene with the exception of the prevertebrae. in the liver, most cells do not express pkm with the exception of blood vessels. pyruvate kinase liver and red blood cell, pklr pklr mrna is abundant in liver, lung, salivary gland, teeth primordia and gut. transcripts of this gene are also readily detected in the motor column of the spinal cord, in the olfactory epithelium and in intervertebral discs. numerous other sites of expression are observed, but the signal strength is significantly lower than in the aforementioned tissues. pkm and pklr superimposed constitute a semp. expression patterns of tricarboxylic acid cycle enzymes some of the polypeptide chains catalyzing the tca cycle steps form multienzyme complexes. this adds a new dimension to the analysis of expression because the components of multisubunit proteins should be coexpressed. consequently, they are expected to exhibit similar expression patterns, which should be readily apparent in an ish analysis. as discussed above, of the tca cycle genes may also exhibit a semp. although the variability within the semp is greater in the tca cycle genes than in glycolysis, the stereotypic pattern is still observed. using the same criteria as for glycolysis, matrix b in fig. provides a synopsis of expression and highlights semp results in bold. for each step of the tca cycle, at least one gene exhibits a semp. citrate synthase, cs this gene is most strongly expressed in whisker follicles, cranial ganglia, dorsal root ganglia, salivary gland, differentiating neurons of the olfactory bulb and a subset of cranial nerve nuclei. expression is also detected in liver, lung, kidney, heart and thymus. citrate synthase like, csl this gene is broadly expressed throughout the embryo but signal strength is elevated in the cranial ganglia, liver, muscles, kidney, medulla, pons, heart, salivary gland, dorsal root ganglia, olfactory bulb, thymus, spinal cord, intestine, neocortex, midbrain and diencephalon. csl is not expressed in the axial skeleton or the long bones of the limbs. aconitase aco the aco gene is broadly expressed but stronger expression is seen in liver, kidney, lung, adrenal gland and salivary gland. within the nervous systems, aco transcripts are elevated in forebrain, brain stem, spinal cord, cranial and dorsal root ganglia. aconitase aco like aco aco is expressed in nearly all tissues. however, several sites are characterized by regional expression. for example, elevated expression is seen in the marginal zone of the neocortex; in the hindbrain, localized expression is seen in several pontine and medullary nuclei, and in the spinal cord, transcript levels are highest in the motor columns. dorsal root ganglia are characterized by intermingling of aco2-positive and negative neurons. isocitrate dehydrogenases complex the conversion of isocitrate to alpha-ketoglutarate represents an allosterically regulated, rate-limiting step of the krebs cycle. five genes encoding isocitrate dehydrogenases have been identified in mammals. idh encodes a cytosolic enzyme that uses nadp+ as cofactor and thus is not a krebs cycle enzyme proper but has a major role in lipid metabolism. idh also encodes an enzyme that uses nadp+ as cofactor and is located in the mitochondrial matrix. idh3a, idh3b and idh3g encode enzymes that utilize nad+ as cofactor and reside in the mitochondrial matrix. these three polypeptides form a heterotetramer comprising two idh3α subunits, one idh3β and one idh3γ subunit. idh3α has been proposed as the catalytic subunit while idh3β and idh3γ have supporting and regulatory roles. it would be thus expected that these genes have similar expression patterns. studies with recombinant polypeptides also suggest that isocitrate dehydrogenase heteromers are catalytically active only if they contain the idh3a subunit. isocitrate dehydrogenase idh idh encodes a cytosolic enzyme that is strongly expressed in kidney, adrenal gland, intestine, thymus and cartilage. this gene is weakly expressed in stomach, facial soft tissue, muscle, lung and liver. idh stands out because of its strong expression in cns and pns. it should be noted that idh synthesizes alpha-ketoglutarate and nadph, which can be utilized by glutamate synthase to generate the neurotransmitter glutamate, perhaps explaining the strong expression in the developing nervous system. isocitrate dehydrogenase idh mitochondrial enzyme-encoding idh is expressed nearly ubiquitously but the strongest signal is seen in skeletal muscles, the developing heart, choroid plexi and lens. there is a notable absence of idh expression in the developing axial and appendicular cartilage. isocitrate dehydrogenase alpha, idh3a idh3a is broadly expressed in the cns with transcripts being observed in nearly all tissues. nonetheless, this gene stands out among the isocitrate dehydrogenases because of its strong expression in muscles, heart, thymus, intestinal epithelium, liver, cns, pns, and developing whisker follicles. there is a notable absence of expression in the developing axial and appendicular cartilage, as is typical for semp. isocitrate dehydrogenase beta, idh3b isocitrate dehydrogenase gamma, idh3g the expression patterns of idh3b and idh3g resemble that of idh3a, although the intensity is much weaker. thus, while idh3a exhibits a definitive semp, this pattern is less pronounced for the β and γ subunits. the elevated expression of the α subunit can presumably be rationalized if we consider the stoichiometric ratios 2α:1β:1γ in the active enzyme and the implication of the α subunit as the catalytic enzyme, which could potentially drive the complex. oxoglutarate dehydrogenase complex oxoglutarate dehydrogenase is a mitochondrial enzyme complex comprising three different subunits that converts 2-oxoglutatate into succinyl-coa and carbon dioxide. like pyruvate dehydrogenase, oxoglutarate dehydrogenase forms a large multienzyme complex and the conversion of 2-oxoglutatate into succinyl-coa requires the presence of all three subunits. consequently, one would predict that ogdh, dld and dlst genes have similar expression patterns. oxoglutarate dehydrogenase lipoamide, ogdh dihydrolipoamide s-succinyltransferase, dlst dihydrolipoamide dehydrogenase, dld all three genes encoding oxoglutarate dehydrogenase subunits show very similar expression patterns, but only ogdh has strong semp features. succinate-coenzyme a ligase complex succinate-coenzyme a ligase is a mitochondrial matrix enzyme that converts succinyl-coa to succinate and coa. it consists of an invariant alpha subunit and a beta subunit that is either adp or gdp-specific. succinate-coa ligase, alpha subunit, suclg succinate-coenzyme a ligase, adp-forming beta subunit, sucla both subunits exhibit the typical energy metabolism expression pattern with expression in the cns, pns, muscles, liver, kidney, gastrointestinal tract, adrenal and salivary glands. there is no expression in axial or limb cartilage. this stereotypical pattern is somewhat more pronounced for the adp-forming beta subunit than for the alpha subunit. succinate-coenzyme a ligase, gdp-forming beta subunit, suclg although suclg expression resembles that of suclg and sucla in some ways, there are striking differences. generally, cns and pns show very low suclg expression. the only exceptions are in the ventricular zone lining the lateral ventricles of the forebrain, the lamina terminalis, the anterior boundary of the thalamus and the neuronal component of the pituitary, where somewhat elevated expression can be seen. other notable exceptions to the low expression of suclg in the cns are the scattered cells of the amygdala, which express the gene strongly. succinate dehydrogenase complex this complex consists of four subunits and is located in the inner mitochondrial membrane. subunit a is a flavoprotein while subunit b is an iron-sulfur subunit. subunits c and d are integral membrane proteins. succinate dehydrogenase complex, subunit a, flavoprotein, sdha succinate dehydrogenase complex, subunit b, iron sulfur, sdhb succinate dehydrogenase complex, subunit c, integral membrane protein, sdhc succinate dehydrogenase complex, subunit d, integral membrane protein, sdhd since all four polypeptide chains need to be coexpressed to form an active enzyme, it is not unexpected that the expression patterns of the four genes are related. there are, however, quantitative differences in expression level. for example, sdhd expression in liver is much more pronounced than in other tissues, while the expression of e.g. sdhb is fairly similar everywhere. irrespective of such subtle quantitative differences, all four genes display the stereotypical energy metabolic enzyme pattern of expression. fumarate hydratase fh this gene is broadly expressed and its pattern of expression resembles those of other glycolytic/tca enzymes, although the differences in expression levels are less pronounced than those seen for idh3a. malate dehydrogenases malate dehydrogenase nad+, mdh mdh is essential for the malate-aspartate shuttle, but like idh it is not a component of the tca cycle.mdh is expressed in a semp-like manner with additional expression in the facial epithelia. malate dehydrogenase nad+, mdh mdh a tca cycle component, shows a semp expression pattern. the similarity in expression between mdh and mdh is critical since these enzymes play a pivotal role in the malate-aspartate shuttle. since oxaloacetate cannot enter the mitochondrion directly, it is first converted by mdh to malate, which readily enters the mitochondrion and is then reconverted to oxaloactetate by mdh our study covers the expression of glycolysis and tca cycle genes in full. at e, at least one isoform of each step in these pathways shows a semp pattern. an exception to this rule is aldoa, aldob and aldoc, none of which has a semp alone, though together they constitute a semp. a finding that emerges from our analysis of tca cycle components is that in cases where the biochemistry depends on a multisubunit complex, the transcripts of at least one subunit show a semp while the other subunits may have uniform expression. some transcripts of glycolytic pathway genes are expressed highly regionally, such as hexokinase isoform. this indicates a regional function more specialized than energy production. northern blot data and ish analysis of postnatal and adult tissues detect gck mrna in the lateral hypothalamic area and in the arcuate, ventromedial, dorsomedial and paraventricular nuclei. it has been suggested that gck-positive neurons are "glucose-sensing neurons" that respond to high glucose concentration. the expression pattern of gck at e suggests that a glucose-sensing mechanism has become established by this stage in development, since our data demonstrate gck mrna in the precursors of the above-mentioned hypothalamic nuclei. miki et al. analyzed expression patterns in different mouse embryonic and adult tissues using microarrays containing mouse cdnas. these authors found that genes encoding enzymes catalyzing glycolytic reactions that are closely linked have similar expression patterns. they also found that transcripts from key regulatory genes exhibited tissue specificity. to a large extent our data point in the same direction, although the data are obviously different in nature. carbonic anhydrases in mouse, ca family members have been identified, of which are cas proper. fifteen of the members of this gene family were analyzed for expression at e. the rna probes hybridized ranged between and nucleotides in length. for most of the cas we were able to design templates, and hence probes, that had no sequence homology to any other family member. however, there was homology of ~ 80% in a few cases, ranging from to nucleotides. the highest homology over the longest stretch of dna was seen between the car probe and car mrna. despite this homology, no cross-hybridization occurred. for example, car was strongly expressed in blood vessels of the brain and in the lung, but no such expression was seen in these tissues in sections hybridized with authentic car riboprobe. # genbank accession number of reference sequence * ensembl transcript id §homology of aligned fragments using bl2seq and percent homology expression patterns of carbonic anhydrases carbonic anhydrase car strongly expressing cells are scattered throughout the liver and weak expression approaching background level is seen in various embryonic tissues. carbonic anhydrase car car expression is regional. major sites of expression are lung mesenchyme surrounding the bronchi, liver, capillaries distributed throughout the embryo, choroid plexi and cochlea. in contrast to car expression of car is widespread. carbonic anhydrase car car which encodes a cytosolic isozyme, is expressed with a high degree of tissue specificity. striated muscles, facial cartilage, ureter and scattered neurons in dorsal root ganglia express car strongly. in liver, car expression is widespread, resembling that of car carbonic anhydrase car expression of this gene, which encodes a protein anchored to the plasma membrane, is confined to developing glomeruli and weakly-expressing cells scattered throughout the cns, presumably representing blood vessels. car may be an excellent marker for embryonic kidney. carbonic anhydrase 5a, mitochondrial, car5a carbonic anhydrase 5b, mitochondria, car5b no expression of these two mitochondrial enzymes is detected in the mouse embryo at e. carbonic anhydrase car no expression is detected for the car gene in the e mouse embryo. carbonic anhydrase car this cytoplasmic enzyme shows weak to medium expression throughout the embryo. carbonic anhydrase car expression of this ca-rp is highly regional. transcripts are abundant in cerebellum, thyroid, clavicle primordium, intestine and stomach. car is also detected in heart, dorsal aorta, bronchi, salivary glands and the cortical region of the kidneys. carbonic anhydrase car weak to medium expression can be seen scattered throughout the embryo, although there are also car10-negative tissues such as the axial skeleton. somewhat elevated expression can be seen in scattered neurons of the cns and pns and in the heart. carbonic anhydrase car car is expressed in the cranial and dorsal root ganglia, olfactory bulb, striatum, amygdala and the midbrain. particularly striking is the enrichment of these ca-rp transcripts in the boundary cap of the cranial and dorsal root ganglia. carbonic anhydrase car this is another example of a regionally-expressed ca gene. choroid plexi, salivary glands and facial as well as ventral integument, including the whisker follicles, express this transmembrane protein strongly. a medium level of expression is detected in kidney, lung, cerebral cortex, hindbrain, spinal cord and the pns. carbonic anhydrase car car is strongly expressed in the inner ear and cochlea. however, the most striking expression site of this gene is at a muscle fiber bundle located posterior to the submandibular gland. meninges covering the ventral delineation of the hypothalamus and pons exhibit attenuated expression. carbonic anhydrase car this gene is expressed exclusively in the pigment layer of the retina and choroid plexi. carbonic anhydrase car strong expression is localized in cells scattered throughout the dorsal root ganglia, dorsal spinal cord, pons, tegmentum and inferior colliculus. a few other structures also express the gene, but only weakly. because carbonic anhydrases catalyze a ubiquitous reaction, one might have predicted them to be widely expressed. however, this was not the case, since half the ca genes showed a considerable degree of regionalization. although there have been few reports about expression in the developing mouse embryo, we found our results to be generally consistent with published ca data. at comparable embryonic stages, transcripts of car were reported in skeletal muscle and ureter, and car mrna was seen in cerebellum, gut, kidney, lung and heart. the expression of car in choroid plexus and kidney was also consistent with previous reports. the specific expression sites reported here for car car car and car had not been described previously, although expression had been reported in whole embryos and in adult tissues using northern blots and rt-pcr. ma data for car and showed them to be absent, but we were able to reveal definite and regionalized expression of these genes using ish. ma analysis was apparently not sufficiently sensitive to detect mrnas that are confined to a few cells representing a minute fraction of the total tissue from which the rna was extracted. the developmental expression we report here is also consistent with the expression described in adult tissues. expression of embryonic car,,, and presumably presages that in the adult. for example, adult car protein is abundant in erythrocytes. the expression we describe for car in the embryonic liver presumably marks sites of hematopoiesis because car expression resembles that of hematopoietic maturation-associated c-myb and the hepatocyte differentiation molecular marker hnf4α. furthermore, car was recently suggested as a target of the transcription factor c-myb. embryonic expression of car in muscles and car in glomeruli and blood vessels of the cns presumably presages adult expression in these tissues. in adult rat, car has been proposed as an immunohistochemical marker for the blood-brain barrier and is thought to play an important role in co2/hco3- homeostasis in the brain. expression in blood vessels of the cns is already apparent in the e embryo, suggesting that co2/hco - conversion is established early in development. car has been reported as responsible for supplying hco - in gluconeogenesis and ureagenesis, pathways restricted to liver mitochondria. consistent with this, car protein is only detected in adult liver. despite the substantial activities of all gluconeogenesis enzymes, gluconeogenesis itself is reported to proceed at very low rates in fetal hepatocytes and to be triggered by birth itself. this is consistent with our inability to detect car in the embryo. finally, car is normally secreted into saliva and milk; there is no embryonic expression of car perhaps because these fluids are not yet produced at e, although salivary and mammary gland primordia are present at this stage of development.  the results presented here constitute the first comprehensive description of the expression of all enzymes involved in glycolysis and the tca cycle. global studies such as this are especially useful for revealing higher degree-associations. we provide evidence for a stereotypical energy metabolism pattern of expression. of the total of genes encoding either a glycolysis or a tca cycle enzyme, exhibited a semp. the defining future of a semp is expression in "signature tissue". for the majority of genes classified as semp, expression in all of the signature tissue is observed. examples of exceptions are fh sucla and hk the existence of a semp raises the possibility that many glycolytic and tca cycle genes may be regulated in a coordinated fashion. whether this is also true for other metabolic pathways, such as amino acid or protein syntheses, remains to be seen. our study also makes the point that even so-called housekeeping genes, although quite broadly expressed, may vary significantly among tissues in levels of expression. kinases have received much attention during recent decades owing to their physiological importance and their high potential as drug targets. initial efforts in the study of the kinome have concentrated on three specific aspects: the identification of novel kinases, the identification of ligands to known kinases, and the complete characterization of signal transduction to map the biochemical pathways of the kinome fully. as more data become available, so systematic and interactive databases arise, similar to that presented here for managing gene expression data. thus, our study complements the information gathered by the kinome-research community. at the same time, current trends in kinome research are switching towards structure prediction, structure analysis and gene expression profiling for eventual comparison in mutant or disease conditions. the expression analysis of kinases reported here sets the ground and illustrates the feasibility of a high-throughput expression analysis of the kinome, and provides the expression patterns for over one third of the mouse kinome. with this study we also corroborate the practicality of high-throughput ish. the usefulness of comprehensive data sets is even more apparent when all results are accessible through the genepaint database. contrary to the common expectation that enzymes encoded by "housekeeping" genes will be widely expressed, almost half the carbonic anhydrase gene family shows a regionalized expression pattern and a number of them are so highly regionalized that they are suitable as tissue-specific markers. hence these enzymes show regional diversity in expression, as reported for transcription factors and growth factors, both of which are recognized key components of biological regulation. in summary, this study greatly increases our general knowledge of the expression patterns of many genes that encode enzymes catalyzing various types of metabolic reactions. although the data are from e embryos we believe that information collected at this stage serves as a suitable predictor and starting point for the elucidation of expression patterns in adult mice and presumably also in humans. 
in situ hybridization is a powerful method for visualizing gene expression patterns at the organismal level with cellular resolution. the expression patterns of genes that encode enzymes were determined by ish in the mid-gestation mouse embryo, a stage that models the complexity of the adult organism. the annotated expression patterns were deposited on the genepaint database and are retrievable by user-defined queries including gene name and sites of expression. the expression patterns discussed here comprised gene products with activities associated with catalysis. many mrnas encoding glycolytic and tca cycle enzymes exhibited a characteristic expression pattern. forty-five percent of transcripts encoding metabolic enzymes showed a regional expression pattern. we found no difference in tissue specificity between mrnas encoding housekeeping functions and those encoding components of signal transduction pathways, as exemplified by the kinases.
 in situ hybridization is a powerful method for visualizing gene expression patterns at the organismal level with cellular resolution. when automated, it is capable of determining the expression of a large number of genes.  the expression patterns of genes that encode enzymes were determined by ish in the mid-gestation mouse embryo, a stage that models the complexity of the adult organism. forty-five percent of transcripts encoding metabolic enzymes showed a regional expression pattern. a similar percentage was found for the kinases that were also analyzed. many mrnas encoding glycolytic and tca cycle enzymes exhibited a characteristic expression pattern. the annotated expression patterns were deposited on the genepaint database and are retrievable by user-defined queries including gene name and sites of expression.  the expression patterns discussed here comprised gene products with activities associated with catalysis. preliminary analysis of these data revealed that a significant number of genes encoding housekeeping functions such as biosynthesis and catabolism were expressed regionally, so they could be used as tissue-specific gene markers. we found no difference in tissue specificity between mrnas encoding housekeeping functions and those encoding components of signal transduction pathways, as exemplified by the kinases. 