for most vertebrate organs and tissues , the majority of development occurs during embryogenesis , and postnatal changes are primarily concerned with growth . the central nervous system ( cns ) is unusual in that a considerable amount of morphological development , cell differentiation and acquisition of function takes place during postnatal development . as yet , the molecular mechanisms underlying these complex developmental processes are not well understood . we have recently completed a differential display ( ddrt - pcr )   analysis of genes expressed in the murine postnatal developing brain . this analysis formed the baseline for an ongoing study and produced a series of rna fingerprints representing genes transcribed at specific stages of brain development . for our analysis we compared rna fingerprints at four different timepoints during postnatal development : newborn ( day 1 ) , day 10 , day 20 and adult ( day 42 ) . incidental to our experimental objective , we also identified a large number of genes which were clearly developmentally regulated in the wild - type mouse brain . we submit this data principally to allow others to isolate specific transcripts with developmentally regulated expression in the postnatal brain without the necessity of performing extensive screening procedures . from a total of approximately 25,000 transcripts displayed , around 200 - 300 ( approximately 1% ) exhibited developmentally regulated expression profiles . here we present groups of rna fingerprints displaying a subset of these developmentally regulated genes , along with sufficient technical information to replicate specific fingerprints and recover selected cdnas . the particular fingerprints presented here were selected on the basis that identical ddrt - pcr profiles were obtained with at least two separate batches of rna , and that each fingerprint displayed at least three developmentally regulated transcripts . on each group of fingerprints we have indicated the position of at least three bands representing the more obviously developmentally regulated genes . we have assigned these marked transcripts into three broad categories as follows : genes for which mrna levels increase during brain development ; genes for which mrna levels decrease during development ; and genes exhibiting a peak in mrna levels during this developmental period . to ensure that changes in ddrt - pcr profiles represent genuine changes in expression levels , two cdna fragments were recovered and used in downstream expression analyses . the northern blot expression profiles of both transcripts accurately replicated the original ddrt - pcr expression profiles , confirming the validity of our approach . in this study , we have observed a large number of transcripts which apparently increase and/or decrease in expression level during postnatal murine brain development . the period of development under study spanned from day 1 to day 42 post - partum . figure 2 shows a selection of rna fingerprints containing bands representing such developmentally regulated transcripts . thirty - four groups of rna fingerprints were selected on the basis that identical ddrt - pcr profiles were obtained with at least two separate batches of rna , and that each fingerprint displayed at least three developmentally regulated transcripts . the gel positions of a subset of developmentally regulated transcripts are indicated in figure 2 and individual transcripts are assigned into one of three broad expression categories : a ( + ) , genes for which mrna levels increase during brain development ; b ( - ) , genes for which mrna levels decrease during development ; and c ( ^ ) , genes exhibiting a peak in mrna levels during this developmental period . of a total of 131 transcripts indicated , 61 are assigned to category a , 61 to category b , and 9 to category c. the sequence details of the primers used to produce the rna fingerprints shown in figure 2 are contained in table 1 . a detailed description of the various procedures employed and the animals used in this study are presented in the materials and methods section . to ensure that developmentally dependent changes in intensity of individual ddrt - pcr bands reflected changes in levels of individual mrnas , two apparently up - regulated transcripts were chosen for further study . the cdna fragments represented by these bands were recovered , purified from co - migrating dna species by msscp , cloned and used in northern hybridization studies ( figure 3 ) . for northern analyses , whole brain rna was isolated from a separate batch of animals to those used in the original ddrt - pcr . as can be seen from figure 3 , for both transcripts northern analyses produced expression profiles similar to those observed by ddrt - pcr , thereby validating this approach to identifying developmentally regulated transcripts . sequence analysis revealed that these cdna fragments represented portions of the prion protein ( prp ) and of the thy-1.2 gene transcripts . the cloned dna used as a probe in figure 3a is identical to nucleotides 588 - 1,414 of the prp transcript while that used in figure 3b is identical to nucleotides 1,599 - 1,700 of the thy-1.2 transcript ( figure 4 ) . this shows that the original ddrt - pcr reactions have amplified an internal fragment of the prp transcript and the terminal 3 ' untranslated region ( utr ) of the thy-1.2 transcript . the prp fragment was amplified by a combination of arbitrary primer r4 and anchored primer t12cc while the thy-1.2 fragment was amplified by a combination of arbitrary primer p3 and the anchored primer t12ca . for the prp fragment , the arbitrary primer showed a 100% match while the anchored primer showed 86% homology ( 12/14 ) with the published sequence . in contrast , for the thy 1.2 fragment , while the anchored primer matched with 100% homology , the arbitrary primer showed only 70% homology ( 7/10 ) with the corresponding region of the published sequence . in the course of analyzing gene expression in the postnatal developing murine brain we have observed that approximately 1% of genes transcribed are developmentally regulated . the objective of this report is to permit those interested in such transcripts to selectively isolate specific developmentally regulated transcripts without the necessity of performing an extensive screen . here we present the rna fingerprints containing a subset of these developmentally regulated transcripts and we include all the necessary information to permit individual transcripts to be isolated and identified . we have highlighted a total of 131 developmentally regulated transcripts in three broad categories of expression profiles . of these , approximately 7% ( 9 ) fell into category c while the remainder were equally divided between categories a ( 61 ) and b ( 61 ) . we also present confirmatory evidence that changing ddrt - pcr expression profiles represent genuine alterations in expression levels during brain development . it should be noted that changes in expression profile reflect changes in rna level per microgram total rna in whole brain and , given that the postnatal brain is not a uniform structure , expression profiles should be interpreted accordingly . animals were humanely sacrificed and whole brains from six individual animals at p1 , p10 , p20 and p42 stages of postnatal development were collected , flash frozen in liquid nitrogen and stored at -80c . total rna was extracted using the rnazol b method ( ams biotechnology , oxon , uk ) , based on the guanidinium thiocyanate / phenol / chloroform extraction method of chomczynski and sacchi . following the initial precipitation step , the rna pellet was washed once with 85% ethanol , resuspended in water , and 0.1 volumes of 3 m sodium acetate ( ph 5.2 ) and 2.5 volumes ethanol were added , the tubes mixed briefly and rna stored as ethanol precipitates at -80c . for quantitation of total rna , a small aliquot was removed , pelleted by centrifugation , resuspended in water and the concentration estimated by spectrophotometry . stock rna solutions were adjusted to a concentration of approximately 1 g/l in ethanol suspension . the use of rnazol b and homogenization of tissue with a polytron homogenizer resulted in the isolation of total rna with od260/280 ratios consistently greater than 1.95 . for each sample , a volume of rna stock solution containing approximately 8 g rna was transferred to a fresh tube . samples were centrifuged at 13,000 rpm for 25 minutes and the resulting rna pellet was washed with 85% ethanol , dried at 45c for 2 minutes and resuspended in 7 l rnase - free water . a small portion ( 2 l ) of each sample was re - quantitated by spectrophotometery and 5 g total rna ( in 5 l volume ) was used to synthesize first - strand cdna ( first - strand cdna synthesis kit ; amersham pharmacia biotech , hertfordshire , uk ) . reactions contained 1 l dtt ( 200 mm ) , 5 l of bulk first - strand mix ( containing moloney murine leukemia virus reverse transcriptase ) and 4 l of either d(t)12ma , d(t)12 mg , d(t)12mc , d(t)12mt primer ( 24 m ; m = a , g or c ) . reactions were mixed and incubated at 37c for 1 hour and then heated to 95c for 10 minutes to inactivate reverse - transcriptase . the synthesized cdna was then dispensed into 1 l aliquots and stored at -20c . for differential display pcr reactions , 1 l aliquoted cdna was diluted to 133 l with water and 10 l of this solution was used for each display pcr ( equivalent to the amount of cdna produced from 25 ng rna ) . to each 10 l cdna on ice , 2 l arbitrary 10-mer primer ( 5 m ) was added and the solution overlaid with 30 l mineral oil ( sigma , dorset , uk ) . master mix ( 8 l ) containing 2 l 10x pcr buffer ( boehringer mannheim , east sussex , uk ) , 2 l dntps ( 20 m ; amersham pharmacia biotech , hertfordshire , uk ) , 0.3 l taq polymerase ( 1.5 u , boehringer mannheim , east sussex , uk ) , 2 l ( dt)12mn ( 25 m ) , 1 l [ -s]datp ( 1000 ci / mmol ) and 0.7 l dh2o was added to each tube . tubes were centrifuged briefly and incubated in a biometra unoblock pcr machine at 94c ( 2 minutes ) , followed by 40 cycles of 94c denaturation ( 30 seconds ) , 40c annealing ( 2 minutes ) and 72c extension ( 30 seconds ) , followed by a final extension step at 72c for 5 minutes . type iii   loading dye ( 4 l ) was added to each tube and 8 l of each sample loaded onto a 6% non - denaturing hr-1000 genomyxlr polyacrylamide gel ( beckman instruments ltd . samples were run for 2 hours 15 minutes at 2,700v ( 50c ) on a genomyxlr dna analyzer ( beckman instruments ltd . , bucks , uk ) . the gels were transferred to 3 mm blotting paper , dried and exposed to biomaxmr autoradiography film for 16 hours . gel regions corresponding to bands representing candidate cdnas were excised from all four lanes using sterile scalpels and transferred to sterile 0.5 ml eppendorf tubes . glogos autoradiograph markers ( stratagene , cambridgeshire , uk ) were used to align the gel with the autoradiograph and identical regions were excised from all lanes . the gel fragments were rehydrated at room temperature for 15 minutes in 100 l water , and cdna eluted at 99c for 15 minutes before transfer of the liquid phase to fresh 0.5 ml tubes . cdna was precipitated by the addition of 2.5 volumes ethanol , 0.1 volumes of 3 m sodium acetate ( ph 5.2 ) and 1 l see - dna ( amersham pharmacia biotech , buckinghamshire , uk ) , and stored on dry ice for 1 hour . following centrifugation ( 13,000 rpm , 25 minutes , 4c ) the pellet was washed in 85% ethanol and resuspended in 4 l of water . for the msscp - pcr reaction , 4 l 10x pcr buffer ( boehringer mannheim , east sussex , uk ) , 3.2 l dntps ( 2.5 mm dgtp , dctp , dttp ; 0.025 mm datp ; amersham pharmacia biotech , buckinghamshire , uk ) , 2.5 l anchored primer ( 20 m ) , 2.5 l arbitrary primer ( 20 m ) , 0.3 l taq polymerase ( 1.5 u ; boehringer mannheim , east sussex , uk ) and 0.5 l [ -p ] datp ( 3,000 ci / mmol ) was added to the dna and the reaction volume adjusted to 40 l with water . pcr conditions were similar to those used for display - pcr , with the exception that only five cycles were performed . after removal of mineral oil , pcr products were purified by phenol / chloroform / isoamyl alcohol extraction and precipitated for 1 hour on dry ice . pellets were washed , resuspended in 8 l of msscp loading buffer ( 80% deionized formamide , 0.01% bromophenol blue , 0.01% xylene cyanol , 1 mm edta , 10 mm naoh ) and denatured at 95c for 10 minutes before loading onto a 0.5x mde gel ( flowgen , staffordshire , uk ) . samples were electrophoresed typically for 18 hours at 8w ( 25c ) in 0.6x tbe buffer using the genomyx lr system . following autoradiography , areas of the gel corresponding to candidate cdnas were excised and the cdna eluted and precipitated as described above . a final pcr reamplification of the recovered cdna was performed using modified versions of the arbitrary and anchored primers used in the ddrt - pcr and msscp steps . for both the anchored and arbitrary primers , oligonucleotides were synthesized with an additional 5 ' sequence containing an ecori restriction site to aid cloning ( referred to as extended primers ; see table 1 ) . reactions contained 4 l msscp purified cdna , 4 l 10x buffer , 3.2 l dntps ( 10 mm each datp , dctp , dttp , dgtp ) , 2.5 l extended anchored primer ( 20 m ) , 2.5 l extended arbitrary primer ( 20 m ) and 0.5 l taq polymerase ( boehringer mannheim , east sussex , uk ) in a 40 l volume . samples were denatured at 94c for 2 minutes and a single round of pcr performed at 94c for 30 seconds , 40c for 2 minutes and 72c for 30 seconds . a further five cycles of pcr were then performed at 94c for 30 seconds , 58c for 1 minute and 72c for 30 seconds , followed by a final extension step of 72c for 5 minutes . pcr products were phenol / chloroform extracted , precipitated and then subjected to ecori restriction endonuclease digestion , prior to further purification and cloning into the ecori site of pbluescript skii or ksii ( stratagene , cambridgeshire , uk ) . cloned cdnas were sequenced and the sequences compared to non - redundant genbank / embl protein and nucleotide databases , using either the gcg   or ncbi worldwide web   implementation of the blast algorithm . under standard procedures , sequences would also be compared with the genbank / embl expressed sequence tags ( est ) databases and matching i.m.a.g.e . clones are then fully sequenced in both directions and the resulting sequence re - checked against the databases to confirm their identity . the expression profiles of transcripts represented by isolated clones were confirmed by standard northern procedures . rna fingerprints produced by specific ddrt - pcr primer combinations at four stages of brain development run in parallel . anchored primer / arbitrary primer combinations are shown at the top for each group of rna fingerprints and stages of development are indicated in days ( 1 , 10 , 20 and 42 ) . gel positions of developmentally regulated transcripts in expression category a ( + ) , expression category b ( - ) and expression category c ( ^ ) are highlighted . downstream analysis of two developmentally regulated transcripts - ( a ) and ( b ) are the two transcripts . candidate cdnas were selected on the basis of the original ddrt - pcr profile ( i ) , purified from co - migrating sequences by msscp ( ii ) and used as probes in northern analysis ( iii ) . to correct for variation in rna loading , northern blot membranes were re - probed for expression of the mouse beta actin gene ( iv ) . schematic illustrating the position of ddrt - pcr cdna fragments in relation to full - length transcripts of ( a ) prp and ( b ) thy-1.2 . sequences of regions matching arbitrary and anchored primers are detailed with mismatched pairs in bold . animals were humanely sacrificed and whole brains from six individual animals at p1 , p10 , p20 and p42 stages of postnatal development were collected , flash frozen in liquid nitrogen and stored at -80c . total rna was extracted using the rnazol b method ( ams biotechnology , oxon , uk ) , based on the guanidinium thiocyanate / phenol / chloroform extraction method of chomczynski and sacchi . following the initial precipitation step , the rna pellet was washed once with 85% ethanol , resuspended in water , and 0.1 volumes of 3 m sodium acetate ( ph 5.2 ) and 2.5 volumes ethanol were added , the tubes mixed briefly and rna stored as ethanol precipitates at -80c . for quantitation of total rna , a small aliquot was removed , pelleted by centrifugation , resuspended in water and the concentration estimated by spectrophotometry . stock rna solutions were adjusted to a concentration of approximately 1 g/l in ethanol suspension . the use of rnazol b and homogenization of tissue with a polytron homogenizer resulted in the isolation of total rna with od260/280 ratios consistently greater than 1.95 . for each sample , a volume of rna stock solution containing approximately 8 g rna was transferred to a fresh tube . samples were centrifuged at 13,000 rpm for 25 minutes and the resulting rna pellet was washed with 85% ethanol , dried at 45c for 2 minutes and resuspended in 7 l rnase - free water . a small portion ( 2 l ) of each sample was re - quantitated by spectrophotometery and 5 g total rna ( in 5 l volume ) was used to synthesize first - strand cdna ( first - strand cdna synthesis kit ; amersham pharmacia biotech , hertfordshire , uk ) . reactions contained 1 l dtt ( 200 mm ) , 5 l of bulk first - strand mix ( containing moloney murine leukemia virus reverse transcriptase ) and 4 l of either d(t)12ma , d(t)12 mg , d(t)12mc , d(t)12mt primer ( 24 m ; m = a , g or c ) . reactions were mixed and incubated at 37c for 1 hour and then heated to 95c for 10 minutes to inactivate reverse - transcriptase . for differential display pcr reactions , 1 l aliquoted cdna was diluted to 133 l with water and 10 l of this solution was used for each display pcr ( equivalent to the amount of cdna produced from 25 ng rna ) . to each 10 l cdna on ice , 2 l arbitrary 10-mer primer ( 5 m ) was added and the solution overlaid with 30 l mineral oil ( sigma , dorset , uk ) . master mix ( 8 l ) containing 2 l 10x pcr buffer ( boehringer mannheim , east sussex , uk ) , 2 l dntps ( 20 m ; amersham pharmacia biotech , hertfordshire , uk ) , 0.3 l taq polymerase ( 1.5 u , boehringer mannheim , east sussex , uk ) , 2 l ( dt)12mn ( 25 m ) , 1 l [ -s]datp ( 1000 ci / mmol ) and 0.7 l dh2o was added to each tube . tubes were centrifuged briefly and incubated in a biometra unoblock pcr machine at 94c ( 2 minutes ) , followed by 40 cycles of 94c denaturation ( 30 seconds ) , 40c annealing ( 2 minutes ) and 72c extension ( 30 seconds ) , followed by a final extension step at 72c for 5 minutes . type iii   loading dye ( 4 l ) was added to each tube and 8 l of each sample loaded onto a 6% non - denaturing hr-1000 genomyxlr polyacrylamide gel ( beckman instruments ltd . , samples were run for 2 hours 15 minutes at 2,700v ( 50c ) on a genomyxlr dna analyzer ( beckman instruments ltd . , bucks , uk ) . the gels were transferred to 3 mm blotting paper , dried and exposed to biomaxmr autoradiography film for 16 hours . gel regions corresponding to bands representing candidate cdnas were excised from all four lanes using sterile scalpels and transferred to sterile 0.5 ml eppendorf tubes . glogos autoradiograph markers ( stratagene , cambridgeshire , uk ) were used to align the gel with the autoradiograph and identical regions were excised from all lanes . the gel fragments were rehydrated at room temperature for 15 minutes in 100 l water , and cdna eluted at 99c for 15 minutes before transfer of the liquid phase to fresh 0.5 ml tubes . cdna was precipitated by the addition of 2.5 volumes ethanol , 0.1 volumes of 3 m sodium acetate ( ph 5.2 ) and 1 l see - dna ( amersham pharmacia biotech , buckinghamshire , uk ) , and stored on dry ice for 1 hour . following centrifugation ( 13,000 rpm , 25 minutes , 4c ) the pellet was washed in 85% ethanol and resuspended in 4 l of water . for the msscp - pcr reaction , 4 l 10x pcr buffer ( boehringer mannheim , east sussex , uk ) , 3.2 l dntps ( 2.5 mm dgtp , dctp , dttp ; 0.025 mm datp ; amersham pharmacia biotech , buckinghamshire , uk ) , 2.5 l anchored primer ( 20 m ) , 2.5 l arbitrary primer ( 20 m ) , 0.3 l taq polymerase ( 1.5 u ; boehringer mannheim , east sussex , uk ) and 0.5 l [ -p ] datp ( 3,000 ci / mmol ) was added to the dna and the reaction volume adjusted to 40 l with water . pcr conditions were similar to those used for display - pcr , with the exception that only five cycles were performed . after removal of mineral oil , pcr products were purified by phenol / chloroform / isoamyl alcohol extraction and precipitated for 1 hour on dry ice . pellets were washed , resuspended in 8 l of msscp loading buffer ( 80% deionized formamide , 0.01% bromophenol blue , 0.01% xylene cyanol , 1 mm edta , 10 mm naoh ) and denatured at 95c for 10 minutes before loading onto a 0.5x mde gel ( flowgen , staffordshire , uk ) . samples were electrophoresed typically for 18 hours at 8w ( 25c ) in 0.6x tbe buffer using the genomyx lr system . following autoradiography , areas of the gel corresponding to candidate cdnas were excised and the cdna eluted and precipitated as described above . a final pcr reamplification of the recovered cdna was performed using modified versions of the arbitrary and anchored primers used in the ddrt - pcr and msscp steps . for both the anchored and arbitrary primers , oligonucleotides were synthesized with an additional 5 ' sequence containing an ecori restriction site to aid cloning ( referred to as extended primers ; see table 1 ) . reactions contained 4 l msscp purified cdna , 4 l 10x buffer , 3.2 l dntps ( 10 mm each datp , dctp , dttp , dgtp ) , 2.5 l extended anchored primer ( 20 m ) , 2.5 l extended arbitrary primer ( 20 m ) and 0.5 l taq polymerase ( boehringer mannheim , east sussex , uk ) in a 40 l volume . samples were denatured at 94c for 2 minutes and a single round of pcr performed at 94c for 30 seconds , 40c for 2 minutes and 72c for 30 seconds . a further five cycles of pcr were then performed at 94c for 30 seconds , 58c for 1 minute and 72c for 30 seconds , followed by a final extension step of 72c for 5 minutes . pcr products were phenol / chloroform extracted , precipitated and then subjected to ecori restriction endonuclease digestion , prior to further purification and cloning into the ecori site of pbluescript skii or ksii ( stratagene , cambridgeshire , uk ) . cloned cdnas were sequenced and the sequences compared to non - redundant genbank / embl protein and nucleotide databases , using either the gcg   or ncbi worldwide web   implementation of the blast algorithm . under standard procedures , sequences would also be compared with the genbank / embl expressed sequence tags ( est ) databases and matching i.m.a.g.e . clones are then fully sequenced in both directions and the resulting sequence re - checked against the databases to confirm their identity . the expression profiles of transcripts represented by isolated clones were confirmed by standard northern procedures . rna fingerprints produced by specific ddrt - pcr primer combinations at four stages of brain development run in parallel . anchored primer / arbitrary primer combinations are shown at the top for each group of rna fingerprints and stages of development are indicated in days ( 1 , 10 , 20 and 42 ) . gel positions of developmentally regulated transcripts in expression category a ( + ) , expression category b ( - ) and expression category c ( ^ ) are highlighted . downstream analysis of two developmentally regulated transcripts - ( a ) and ( b ) are the two transcripts . candidate cdnas were selected on the basis of the original ddrt - pcr profile ( i ) , purified from co - migrating sequences by msscp ( ii ) and used as probes in northern analysis ( iii ) . to correct for variation in rna loading , northern blot membranes were re - probed for expression of the mouse beta actin gene ( iv ) . schematic illustrating the position of ddrt - pcr cdna fragments in relation to full - length transcripts of ( a ) prp and ( b ) thy-1.2 . sequences of regions matching arbitrary and anchored primers are detailed with mismatched pairs in bold .
background : for most vertebrate organs and tissues , the majority of development occurs during embryogenesis , and postnatal changes are primarily concerned with growth . 
 the central nervous system is unusual in that a considerable amount of morphological development , cell differentiation and acquisition of function , takes place during postnatal development . 
 as yet , the molecular mechanisms underlying these complex developmental processes are not well understood . in order to identify markers for these developmental processes , 
 we have analyzed the expression profiles , during postnatal murine brain development , of approximately 25,000 transcripts . 
 this analysis , performed at day 1 , day 10 , day 20 and day 42 of postnatal development , identified a large number of developmentally regulated genes which we have assigned into three broad expression categories.results:expression levels at four timepoints during postnatal murine brain development were established for approximately 25,000 gene transcripts . 
 approximately 1% of the genes examined displayed a developmentally regulated pattern of expression and we provide all the necessary information required to easily obtain molecular markers for a subset of these developmentally regulated transcripts . 
 of this subset , 61 showed increasing expression during development , 61 showed decreasing expression during development , and 9 exhibited a peak of expression during this period.conclusions:a small percentage of the genes expressed in the postnatal developing brain show changes in expression level during the newborn to adult phase of development . 
 it is likely that these developmentally regulated transcripts represent molecular markers for the complex developmental process occurring in the postnatal brain .