BACKGROUND
even before neurulation begins, the neural plate is broadly patterned into domains with anterior and posterior molecular character.
as development proceeds, the founding pattern is further refined upon until the morphologically characteristic structures of the forebrain, midbrain, hindbrain and spinal cord arise  <cit> .
the molecular mechanism by which the acquisition of the anterior-posterior  identity is achieved has been the subject of intense study.
in recent years many fundamental patterning processes have been elucidated but relatively little is understood about the complete set of signalling influences or genetic networks used to achieve this neuronal diversity.
however, it is known that the specification and subsequent patterning of the neural tube uses hierarchies of transcription factors working in combination with the activity of signalling centres located at discrete ap positions.
during gastrulation, otx <dig> and gbx <dig> are expressed in opposing domains in the neural plate, anteriorly and posteriorly respectively.
within the otx <dig> positive domain the forebrain vesicle develops and there is good evidence for transcriptional regulation playing a deterministic patterning role of that region .
the interface between the zones of expression of otx <dig> and gbx <dig> uniquely determines the future position of the midbrain-hindbrain organizer {mhb, also referred to as isthmus), a structure intimately associated with correct patterning of the mid-hindbrain region .
subsequently, a series of transcription factors including pax <dig>  pax <dig>  pax <dig>  en <dig>  en <dig> are expressed at the otx2-gbx <dig> boundary that are required for maintenance of the mhb organizer and specification of the midbrain territory.
these transcription factors then participate together with the secreted signals fgf <dig> and wnt <dig> in a cross-regulatory network to maintain the mhb organizer and direct patterning of the midbrain and cerebellar structures .
multiple lines of evidence show that fgf <dig>  which is expressed in the temporally appropriate manner at the mhb in all vertebrate classes  <cit> , constitutes an important component of the isthmic-patterning signal.
introduction of ectopic fgf <dig> protein into the avian brain can respecify posterior forebrain to become midbrain and anterior midbrain to develop posterior midbrain characteristics  <cit> .
taken together, these data demonstrate the existence of a complex interplay of genetic regulation and local signalling that are required to specify and pattern the midbrain territory .
the hindbrain becomes organized into a repeated series of cell lineage-restricted metametric units defined as rhombomeres  .
these compartments facilitate a coordination of ap and dorsoventral  signals to regulate the pattern of the neuronal subtypes born within them such that each rhombomere can be considered to have a unique identity.
rhombomere  <dig> is notable in respect of its lack of branchial motor neurons and being the precursor region for the cerebellum.
the anterior rhombic lip of r <dig>  generate a large migratory cell population that forms the external germinal layer and later the internal granule cell layer of the cerebellum  <cit> .
it is now well established that the hindbrain is patterned by the combinatorial action of the homeodomain-containing hox transcription factor code.
within the region that is initially defined as gbx <dig> positive in the neural plate, the initial hox code in the hindbrain is first set up under the influence of ap signals such as retinoic acid  .
given the distribution of the hox transcripts and their ability to act in a co-operative fashion, it has been suggested that individual rhombomere identity is conferred by a combinatorial code of the hox proteins  <cit> .
functional evidence for the role of hox genes in the patterning of the hindbrain can be observed from interference studies on the hoxb <dig> gene that is normally highly expressed in r <dig>  disruption of the hoxb <dig> gene in mice leads to transformation of the r <dig> territory into an r2-like state  <cit> , whereas retroviral-mediated over expression of hoxb <dig> in r <dig> causes homeotic transformation of r <dig> to an r4-like condition  <cit> .
evidence is now also emerging about some of the other factors that act upstream of hox genes themselves to activate them at appropriate ap levels.
as well as cross-regulation between the various hox genes, upstream regulators of the hox genes include mafb and krox <dig> which, in addition to controlling segmentation of the neuroepithelium act in a parallel but related process to regulate the hox genes.
thus, mafb directly modulates expression of paralogue group  <dig> hox genes in r <dig>  <cit> , and krox <dig> is a direct activator of both hoxa <dig> and hoxb <dig>  <cit>  and a repressor of hoxb <dig>  <cit> .
given the prevalence of transcriptional regulation in the specification of ap pattern of the neuraxis, the aim of this study was to identify novel candidate or known genes with potential roles regulating the ap pattern of the neural tube.
many methodologies have been developed that are designed to identify and/or sample the array of genes expressed by a cell or tissue type, commonly defined as the transcriptome.
the technologies vary both in the number of genes they are designed to profile and if a priori knowledge of gene identity is required.
for example, technologies employing subtractive hybridization of cdna libraries have been successfully used to find regulated changes in gene transcription during development of the neural tube  <cit> .
a current method of choice is microarrays.
however, due to the lack of availability of chick-based systems at the time of onset of this study, we chose to implement a large-scale messenger mrna differential display screen  <cit> .
despite a prevalent false positive rate, recent advances in primer technology, gel electrophoresis and cdna cloning of the appropriate differentially expressed gene has dramatically decreased such error  <cit> .
an advantage of dd is that it can be used simply and relatively cheaply to assay the gene expression profiles of multiple cell or tissue types simultaneously, to detect both up and down transcriptional regulation.
when taken together with the capacity to precisely stage and dissect the developing chick embryo the methodology presents a unique opportunity to accurately assay transcriptional regulation of the early cns.
in addition, despite the prevailing idea that all genes can be identified computationally once a genome has been sequenced, there is now clear evidence that a substantial amount of genome annotation is still required and novel genes are still being identified by other approaches  <cit> .
thus, there is still good reason to find novel genes using empirical screening technologies and place them into an appropriate biological context.
we present the findings of an exhaustive differential display screen for regionally restricted transcripts during early brain development.
clones were sequenced and compared to the latest est, genomic, known gene and predicted gene databases to characterize differentially expressed candidates.
results were confirmed by in situ hybridization and the embryonic expression of several of the isolated genes is described for the first time.
taken together this study provides a foundation from which genetic networks and hierarchies can be inferred and further explored.
RESULTS
efficacy of the messenger rna differential display screen
preliminary data from this screen was reported in .
we now report the results from the completed screen as well as presenting analysis, identity and expression for many of the candidate cdnas isolated.
we have used all  <dig> primer combinations available  <dig> t <dig> anchor primers in combination with  <dig> arbitrary primers; hieroglyph kit, beckman coulter), as well as including some custom arbitrary primers .
according to manufacturer's data, this approach should survey >95% of the transcriptome.
we recorded  <dig> reproducible changes in differential display gel profile from the  <dig> primer combinations tried in total .
the changes observed were robust in that they appeared in the series  <dig> and  <dig> rna pools and were reproducible in subsequent identical differential display experiments .
overall, this represents a 14% incidence of difference in gene transcription across neural tube regions tested.
the clone designation, identity, function and recorded gel profile of each of the differentially expressed clones are listed in table  <dig>  fig.
2b shows a schematic representation of the molecular functions of the differentially expressed candidates whilst fig.
2c shows how the changes were distributed across the neural tube.
a large proportion of the changes were recorded as being present in the midbrain or rhombomere  <dig> regions  which is consistent with this being previously known to be a site of active transcriptional events.
patterns of gene expression unique to certain areas were also seen which is compatible with prior knowledge of patterning of that region .
other profiles were also present involving combinations of transcriptional activation .
where an unequivocal identity is found for the differential display clone in bbsrc, genbank or swissprot databases, it is shown in the appropriate column.
sp = swiss-prot database, tf = transcription factor, rtp = receptor tyrosine phosphatase, fgfr = fibroblast growth factor receptor, glp = glugacon-like peptide, ef = elongation factor, lrp = laminin receptor protein, l1cam = l1-like cell adhesion molecule, mafb ~ kreisler, 5' race = rapid amplification of 5' cdna end.
where no identity for a cdna is found in the sequence repositories, the sequence is given in supplementary information.
f = forebrain, m = midbrain, r <dig> = rhombomere  <dig>  h = hindbrain .
* denotes clones whose identity has been derived from significant matches with the chick genome sequence .
+ denotes identity derived from similarity to an ortholog.
where a 100% match with an est from the umist collection was found the fragment sequence was not submitted to genbank.
coverage of messenger rna screen
a differentiated eukaryotic cell is thought to contain  <dig> – <dig>  distinct mrna species .
in the study described here  <dig> primer combinations were used, each displaying around  <dig> pcr products  to generate over  <dig>  amplified fragments.
this figure broadly correlates with the current estimate of gene number for a higher eukaryotic organisms  <cit> .
thus, in principle we surveyed the majority of genes expressed during the transcriptional specification of the fore-, mid- and hindbrain regions of the developing avian cns.
however, the total number of bands displayed does not compensate for the observed redundancy of the displays.
that is, many mrnas would have been displayed more than once, whereas others, particularly low abundance messages, would not have been detected at all.
this is an intrinsic property of the differential display methodology and contributes to the restricted coverage generated.
an example of redundancy was observed with clone c, where the same differential display gel profile  and subsequent cdna identity was observed from candidate bands of a different size obtained using a different arbitrary primer .
by contrast, a slight under-estimation of the number of distinct pcr products is possible due to the existence of more than one species of pcr product of the same size in an individual band .
in these circumstances, a single stranded conformational polymorphism  strategy was adopted to accurately determine the differentially expressed cdna .
an additional consideration to the restricted coverage of the technique is that where a fixed number of pcr cycles are used , it is likely that small differences in the levels of some regulated transcripts will be missed  <cit> .
compiling identities of the chick differential display cdna clones
of the  <dig> differentially expressed genes identified,  <dig> could be assigned an unequivocal identity either directly from the sequence of the displayed fragment or following the isolation of a longer cdna clone.
for the remaining clones, often after the addition of more upstream sequence from cdna libraries, an identity, putative function or membership to a gene or protein family could not be assigned.
it is likely that, despite our efforts to isolate large cdna fragments, the 3' bias of the methodology resulted in preferential isolation of 3' untranslated  sequences.
for many clones, the recent release of the chick est database was similarly insufficient to assimilate an identity.
the chick ests were generated from  <dig> different embryonic and adult tissues, ranging from a complete early developmental stage hh st <dig> embryos to a single adult tissue e.g.
the limb.
despite the fact that the individual cdna libraries constructed from each point were poly t-primed and therefore generated from the 3' end of an mrna transcript, a similarity of the  <dig>  usable est sequences with the clones described here was not always found.
this may be a function of the representation of the est libraries or that only the 5' ends of the directionally cloned libraries have so far been sequenced  thus, for those longer clones in the est repository, the sequence immediately upstream of the poly a tail may be under represented.
these types of issues also imply that studies like those described here have a useful role in annotating genomes and contributing to an accurate description of transcriptomes.
in the absence of a similarity match from the cdna databases, we searched the recently released chick draft sequence  for potential information .
subsequent to a significant match  against the chick genome, we identified the appropriate sense strand and investigated up to  <dig> kb upstream for exons that might be informative.
this approach, although helpful, has the caveat that associated exons must first be shown to physically spliced to the cdna query sequence, for example by rt-pcr, before an identity can be unequivocal.
current data suggests that the average 3'utr is within the range of  <dig>  to  <dig>  kb .
where possible, a northern blot was performed to indicate the appropriate size of cdna to look for in the genome.
however, for some clones no matches could be obtained from the genome sequence confirming that sequence remains to be finalised.
validation of the experimental technique: isolation of en <dig>  mafb, spry <dig>  fgfr <dig>  robo1
the strategy described here used a microsurgical approach and a subsequent pooling strategy prior to the screen.
proof of principle was confirmed by the isolation and identification of five genes known to be expressed in embryonic patterns faithful to the differential display gel profile and also to play important roles during neural tube development.
analysis of the display profile from the subset of mrnas amplified with anchor and arbitrary primer  <dig> revealed a pcr product, clone a, that was reproducibly seen in the m and r <dig> samples but not in the f and h samples ).
subsequent isolation, reamplification and cloning of this band gave a  <dig> bp cdna sequence in all  <dig> clones tested.
comparison of this sequence using blastn showed it to be 100% identical to the 3' utr region of chick engrailed  <dig> cdna .
further confirmation was obtained by in situ hybridisation to chick hh st <dig> embryos where the expression pattern observed was identical that previously described.
en <dig> has previously been reported as being a primary genetic determinant of the developing midbrain and rhombomere <dig> regions and mice homozygous for an en <dig> loss of function allele lack these entire structures .
clone b derived from an anchor primer  <dig> – arbitrary primer  <dig> combination-generated  <dig> bp hindbrain-enriched pcr band , was found to be 94% identical to the 3'utr of the mouse mafb  cdna.
in situ hybridisation with this cdna demonstrated that expression was fully concordant with the previously described expression in rhombomeres  <dig> and  <dig> in the hh st <dig> neural tube .
available data demonstrates the developmental significance of this gene since animals lacking functional mafb protein show, amongst other developmental defects, a deletion of the rhombomere  <dig> region  <cit> .
additional evidence for the efficacy of the screen described here was obtained from the reamplification, cloning, sequencing and expression analysis of clone  <dig> that displayed a midbrain-enriched gel profile .
this  <dig> bp clone showed 100% identity to the 3'utr of the fgfr <dig>  a transmembrane receptor tyrosine kinase that was previously described as being expressed in the midbrain vesicle of a hh st <dig> chick embryo  <cit> .
the cloning, expression and interplay with fgf <dig> signalling of sprouty <dig>  was also reported from this screen .
a detailed functional analysis of the role of sprouty proteins in development is being performed in our laboratory .
taken together, these data demonstrate that the screen was reflective of the transcriptomes surveyed and was capable identifying a range of genes with varied and important development roles.
as such, the data presented in table  <dig> is likely to describe a true picture of gene expression changes in the developing neural tube.
differential display has an efficacy lower than that statistically predicted
despite the established efficacy of the screen, the detection of fewer than  <dig> differentially expressed bands is indicative of a relatively small proportion of mrnas with altered expression across the ap neuraxis of a hh st <dig> developing chick embryo.
that we did not identify any of the large number of previously characterised differentially expressed genes associated with hindbrain patterning , strongly suggests that the coverage was less than anticipated.
this may be due in part to the low level of mrna abundance of the hox transcription factors.
however, this is largely countered by the observation that they can be readily detected by wholemount in situ hybridisation.
a survey of the accumulated literature shows that greater than  <dig> genes have been reported as being differentially expressed in the tissues under study here .
it is clear from this study that messenger rna differential display operates at efficiency much lower than that statistically predicted by binding of the arbitrary primers to their cognate sequence alone.
detailed expression analysis
following validation of the screen, we selected several identified genes for detailed expression analyses; sparc-related modular calcium binding protein  <dig>  laminin receptor protein  <dig>  clone β, hb gam, clone g, translation elongation factor 1α, receptor protein tyrosine phosphatase zeta and l1cam.
these clones were selected based upon their identity and possibility that they may interact with other known pathways that pattern the mid-hindbrain territories at this developmental time; for example, to ask if rtpζ and hb gam are expressed in the appropriate place to modulate/interact with fgf signalling around the isthmus organiser?
although others showed no obvious identity , they were selected based upon their differential display profile  that suggested that they were expressed in domains known to be important in patterning the areas under study .
similarly, we chose clones who have a know role in cell adhesion to see if they could contribute to either boundary formation or cell lineage restriction in the mid-hindbrain region.
in addition, for some of the clones isolated it was not possible to produce a definitive in situ hybridisation pattern.
this may be due to the 3' utr nature of the sequence.
the identities of the cdnas not used for further in situ hybridisation studies, where known, is presented in table  <dig>
clone c: sparc-related modular calcium binding protein  1
using arbitrary and anchor primers  <dig> and  <dig> respectively, as well as  <dig> and  <dig> respectively, a  <dig> bp pcr band was reproducibly enriched in the series  <dig> and  <dig> r <dig> and h mrna pools.
reamplification, cloning and sequencing of the cdna clone on both strands failed to produce a significant match within the databases.
the 3'utr sequences showed a typical poly a addition consensus site  proximal to the site of anchor primer binding .
this clone was not represented in either of the cdna libraries screened.
using a nested 5' race approach, a further  <dig>  kb of sequence immediately upstream of this 3'utr sequence was acquired.
this sequence lacked any obvious orfs, partial or otherwise , and no identity was derived suggesting that it was the 3'utr region.
scanning of the chick genome with clone c sequence revealed a region of 98%  identity on chromosome  <dig> .
immediately adjacent to this match, exist a previously annotated protein coding exon coding for 'similar to sparc-related modular calcium binding protein 1'  .
the immediacy of the clone c-derived sequence to this region implies that clone c codes for the protein described as for 'similar to sparc-related modular calcium binding protein 1'.
sparc  has an extracellular ca2+ binding domain  and is a multifunctional glycoprotein that functions to regulate cell-matrix interactions.
it binds to such proteins as collagen and vitronectin and can binds to endothelial cells and inhibit cellular proliferation.
the extracellular  domain interacts with a follistatin-like  domain that appears to stabilize ca2+ binding.
the two ef-hands interact canonically but their conserved disulfide bonds confer a tight association between the ef-hand pair and an acid/amphiphilic n-terminal helix.
proposed active form involves a ca2+ dependent symmetric homodimerization of ec-fs modules  .
srmcbp <dig> has dynamic expression in the developing embryo ; expression is first seen in the neural plate followed by staining in the primitive streak.
at the onset of somitogenesis, srmcbp <dig> becomes restricted to presumptive tail bud and newly formed somites.
later at 3–6s, the transcripts are more abundant in lateral tail bud regions but still present in the somites.
at 12s, srmcbp <dig> transcripts are seen in rhombomere  <dig>  ectodermal regions surrounding the otic vesicle and medial somites.
at hh st <dig> the hindbrain expression has expanded into all rhombomeres but is excluded from the boundary zones.
clone q: laminin receptor protein  37
analysis of the differential display gel profile obtained using anchor primer  <dig> combined with arbitrary primer  <dig> consistently and reproducibly identified a cdna of  <dig> base pairs in series  <dig> and  <dig> mrna pools  whose expression was switched on in the posterior hindbrain cdna pool but not in the rhombomere <dig> cdna pool.
excision of this candidate cdna, cloning, library screening, sequencing and blast analysis revealed it to be 100% identical at the nucleotide level to the previously identified gallus gallus laminin receptor protein  <dig> gene .
the laminins are a family of glycoproteins that form a critical component of the basement membranes of most organisms and are known to be an important factor in cell migration, axon pathfinding and modulation of cell survival as well as other cellular processes.
laminins self assemble from α, β and γ subunit chains and are secreted into the extracellular space where they can interact with a range of other extracelluar matrix molecules.
to date, twelve different laminin heterotrimers have been identified in mammals as well as other variants being found in hydra, drosophila and c. elegans.
it has been reported that the laminin family members have tissue specific, but overlapping, distributions during embryonic development .
critically, laminins have been shown to have a diverse and important and role during embryonic development to later post natal development.
for examples and a recent review see .
the primary mechanism by which cells recognize and interact with laminins is through the integrins, a family of heterodimeric transmembrane receptors consisting of α and β subunits.
integrins are capable of exerting regulatory effects on both the cytoskeleton and cellular signaling apparatus.
lrp has been described as a multifunctional molecule involved in both cell adhesion via laminin and formation of the polysome in the cytosol.
in situ hybridisation reveals that the lrp transcripts are expressed very early in embryonic development in regions fated to form mainly trunk structures.
as development proceeds, the expression of lrp is observed in the neural tube, somites, ectodermal and endodermal tissues, but only immediately posterior to the rhombomere  6/ <dig> border.
interestingly, this gene has not been previously reported to be expressed embryonically and its function in development remains to be elucidated
clone β: pax2
given the differential display gel profile of clone a and its identification to be the developmentally significant en <dig> cdna, it was of particular interest that clone β had an identical gel profile .
reamplification, cloning and sequencing produced  <dig> identical subclones that gave  <dig> bp of sequence.
similar to other clones described here, this clone gave no identity or no expression pattern in hh st <dig> chick embryos.
a high stringency library screen of the hh st <dig> chick λzapii library produced  <dig> independent clones ranging in size from  <dig> to  <dig>  kb .
the longest of these clones was selected and sequenced on both strands.
despite the large amount of information no clear identity or orf greater than  <dig> amino acids could b obtained.
to determine the size of the endogenous transcript we performed a northern blot on chick whole embryo hh st <dig> total rna and observed two bands of ~ <dig> and  <dig> kb .
given the size of the full length mrna, it was likely that our sequence information may still have been in the 3'utr.
using the elongated  <dig>  kb clone β, we obtained the expression during the formation of the neural tube .
notably, the expression observed in the neural tube was similar that seen for clone a  and consistent with the gel profile.
in fact, the expression of clone β was recorded very early in the ectoderm destined to become the mhb.
to ascertain the identity of clone β and discern the potential early function in mhb formation, we scanned the genomic sequence upstream of the existing  <dig>  kb.
using this approach we located an exon that codes for a region of the paired homeodomain transcription factor pax <dig>  this observation is consistent with the known pax <dig> that is the earliest transcription factor expressed in the hierarchy of mhb formation .
however, although clone β is now known to represent the previously well-characterized pax <dig>  it should be noted that the  <dig>  kb in situ fragment provides extremely definitive patterns that may be useful in future studies.
the methodology described here also serves as model to elucidate the identity of other cdnas.
clone f: similar to heparin-binding growth-associated molecule
clone f is a reproducibly expressed hindbrain-enriched  <dig> bp display fragment.
the available sequence was insufficient to discern identity or expression for in situ studies.
following screening of the chick hh st <dig> λzapii cdna library, four independent clones were obtained.
verification of clone f identity by southern blotting  and sequencing revealed an additional  <dig>  kb of sequence had been attained.
the largest construct showed no extended open reading frames or blastn/x identity with the existing sequence warehouses.
however, submission of the extended clone f sequence to the chick genome blastn algorithm revealed a 97% identical match to the nw_ <dig>  contig derived from chromosome w sequencing .
further analysis of this locus  showed the presence of the previously annotated gene defined as 'similar to heparin-binding growth-associated molecule '         ), immediately adjacent to the clone f sequence match.
given the appropriate orientation and 3'utr nature of the clone f sequence, it is reasonable to conclude that clone f is the gene described as 'similar to hbgam'.
heparin-binding growth-associated molecule itself is a basement membrane-associated protein that was initially isolated as a neurite outgrowth-promoting factor from perinatal rat brain  <cit> .
hb-gam is composed of two thrombospondin type  <dig>  domains flanked by lysine-rich n- and c-termini of undefined structure.
the tsr domains may mediate binding to heparan sulfates and to the cell surface, while the role of the lysine tails is unknown  <cit> .
hb-gam also has another close relative; midkine  that was isolated and cloned as a retinoic acid induced differentiation factor  <cit> .
however, although the in vivo functions of hb-gam are still not generally understood, recent work by  <cit>  has demonstrated a clear role for hb-gam in the selective inhibition of fgf2-mediated activation of fgfr <dig> by restricting the interaction between ligand and the proteoglycan co-receptors required for normal activation .
in this context, hb-gam inhibited the proliferation and promoted the differentiation of neural stem cells by restricting the signalling activities of fgfs.
the capacity of hb-gam  to negatively regulate fgf-stimulation of their cognate receptors is of significance given the expression of clone f in the developing embryo.
at hh st11- clone f transcripts are seen in rhombomere  <dig>  medial somites and the presomitic mesoderm .
the pattern observed in the presomitic mesoderm is of particular interest as it is known that fgf8-mediated signalling is required here to maintain proliferation and inhibit inappropriate maturation of this zone .
thus, the fgf8-mediated presomitic signalling pathway may be mechanistically regulated by hb-gam  in an analogous fashion to that described by .
we are currently conducting a series of functional interference studies to determine the precise role of this protein during early patterning of the neural tube.
clone g: unknown
amplification of the mrna pools with anchor primer  <dig> and arbitrary primer  <dig> revealed a  <dig> bp pcr product whose expression was recorded principally in the forebrain, midbrain and rhombomere  <dig> samples but absent from the hindbrain group.
sequence analysis followed by comparison to databases showed no significant homologies.
using this cdna fragment as a probe,  <dig> independent positive plaques were isolated from the chick hh st <dig> λzapii cdna library.
excision of the insert and subsequent southern blot analysis determined the longest of these clones to be ~ <dig> bp.
the sequence of this clone is presented in supplementary information and was shown to be 100% identical to the clone g probe sequence at the 3' end.
the extended clone g sequence provided no further insight into the identity of the resultant protein.
the umist chick est database does not contain the equivalent cdna sequence.
furthermore, this sequence is as yet unrepresented in the available draft of the chick genome.
however, northern analysis shows that for total rna derived from a hh st <dig> embryo that there are two transcripts of equal abundance at  <dig>  and  <dig>  kb .
thus with  <dig>  kb of sequence and the expected size of the coding rna, the open reading frame and potential function can be predicted upon completion of the genome sequence.
using the longest library clone to generate an antisense probe, the expression of clone g in a chick embryo was shown to mirror the differential display gel profile .
at hh st 10+, clone g transcripts were seen in the forebrain, midbrain and rhombomere  <dig> regions but completely absent from the hindbrain  region of the neural tube.
notably, there was an obvious gap in the expression at both the anterior neuropore and mhb.
these are both sites of fgf8-signalling and raise the possibility that clone g is repressed by fgf <dig>  this supported by the observation that there are no gaps between the zones of expression of fgf <dig> and clone g as assessed by a single colour 'fill in' in situ hybridization .
the capacity of fgf <dig> protein to repress the transcription of clone g is currently under investigation using fgf8-coated beads implanted into the anterior midbrain.
transverse section of hh st10+ embryos in the rostral midbrain show that expression in the dv axis is excluded from the roofplate and floorplate areas  suggesting that clone g may also be repressed by factors expressed in these regions such as bmps and shh respectively.
clone 4: translation elongation factor 1α
using a similar cloning and sequencing approach to that described elsewhere, we demonstrated that a  <dig> bp pcr product enriched in the forebrain pools corresponded to a  <dig> bp library cdna with 100% identity to gallus gallus translation elongation factor 1α .
even at early stages, ef1α are more abundant in the anterior neural plate.
as development proceeds up to hh st <dig>  ef1α expression becomes most evident towards the rostral end of the embryo.
the function of this type of pattern remains to be determined.
however, there is now increasing evidence that mrna-specific translational control, whereby the translation of a defined group of mrnas is modulated without affecting general protein biosynthesis or the translational status of the cellular transcriptome as a whole is an important factor in the regulation of cellular identity.
in addition, global control of protein synthesis can also be achieved by changes in the phosphorylation state of initiation factors or the regulators that interact with them .
in addition, previous studies have also implicated the differential distribution of protein coding machinery in the developing neural tube.
the translational-regulatory component eif2a kinase-related is expressed exclusively in r <dig> of the developing hindbrain  <cit> .
here, a potential role in the r5-specific regulation of apoptosis has been speculated.
it is of note that the two translation factors reported here, ef <dig> and eif3zeta, showed a robust hindbrain-specific gel profile .
taken together, these data are suggestive of a neural tube specific combinatorial translation factor code operating alongside the well-established transcription factor patterning events.
clone 2: receptor protein tyrosine phosphatase zeta
previous work reported from this study  <cit>  has helped to elucidate some of the molecular regulation of fgf-signaling at the mhb.
it was therefore of interest when the identity and expression of clone  <dig> was elucidated.
using the acaatttcacacaggattttggctcc arbitrary primer in conjunction with ggaaaaaaaaaaagccctatagtgagtcgt anchor primer, a  <dig> bp pcr consistently upregulated in midbrain and forebrain pools of series  <dig> and  <dig> mrnas was obtained.
reamplification, cloning and sequencing of the differential display band followed by database queries produced no clear identity.
to establish an identity we submitted the sequence to the chick est database and commenced a 5' walking strategy  <cit> .
this approach gave an exact match to the 603788872f1-designated assembled sequence contig .
using the longer sequence an unequivocal match  to gallus domesticus phosphotyrosyl phosphatase mrna complete cds  was found, which itself is othologous to the rattus norvegicus receptor type protein tyrosine phosphatase zeta/beta .
thus it has been established that clone  <dig> is chick ortholog of the receptor type protein tyrosine phosphatase zeta/beta.
receptor protein tyrosine phosphatases  constitute a large family of structurally related proteins that are responsible for the regulated removal of phosphates from tryrosine residues.
tyrosine phosphatases are classified into  <dig> subfamilies spread across three groups: cytoplasmic, receptor-type, and dual specificity phosphatases that dephosphorylate serine threonine residues, and tyrosine residues that are in close proximity  receptor protein tyrosine phosphatase zeta is a member of the receptor type group with an extracellular domain similar to that of the cell adhesion molecules  .
evidence from genetic studies in c. elegans has demonstrated that one function of rptps is to antagonize receptor tyrosine kinase signalling.
the rptp, clr- <dig>  functions to inhibit signalling by the fgf receptor-related rtk, egl- <dig>  to regulate the migration of sex myoblasts  <cit> .
in addition, a mitogen-activated protein kinase phosphatase  gene, encoding a dual specificity phosphatase, has been shown to directly antagonise fgf8-mediated receptor activation during limb development  <cit> .
given the potential function of clone  <dig> and a midbrain-enriched gel profile we selected this clone for further expression analysis by in situ hybridisation.
other than a report on the expression of rptpγ  <cit> , there is no description of the expression of rtps in early embryogenesis
at hh st10+  the expression of rtpζ was observed in the midbrain and forebrain vesicles.
the mrna distribution in the midbrain is potentially in a caudal to rostral gradient.
no expression was observed in the neural tube caudal to the mhb.
additional weak expression was also seen in the ectoderm immediately adjacent to r <dig> .
both the midbrain territory and otic ectoderm are known sites of fgf-signaling .
thus we hypothesize that rtpζ may be involved in modulating the fgf-signaling events that are occurring in those regions.
alternatively, other than being involved in the control of fgf8-mediated signal transduction at the mhb, rtpζ may be involved in another independent process such axonal guidance in retinotectal mapping.
the functional role of rtps in these types of events has previously been documented .
clone l: l1cam
data presented in table  <dig> shows that following the library screening, sequencing and in situ analysis, clone l was the gallus gallus close homolog of l <dig> cell adhesion molecule  with a distinctive transcript distribution.
at hh10+, l1cam was seen in the spinal cord, a caudal to rostral graded midbrain expression and some mrna localized to the posterior forebrain.
the gene expression pattern is fully correlative with the observed gel profile of on in all regions other the r <dig> pool .
l1cam is a  <dig> kda transmembrane cell adhesion molecule with six extracellular ig-like and five fibronectin iii domains that enable l <dig> to homophilically bind to opposing cells as well as to heterophilically bind to tag- <dig>  β1-integrins, f11/contactin, neural cell adhesion molecule , and proteoglycans .
l1cam is capable of activating a map kinase signalling cascade through the intermediates src, phospohinositide- <dig> kinase, rac <dig>  and p21-activated kinase, leading to neurite growth .
studies also suggest that retinal axons require the function of l <dig> in addition to repellent epha guidance receptors to achieve proper topographic mapping  <cit>  thus this early, previously unreported, graded expression of l1cam in the midbrain may play a role in establishing the appropriate projections of the retino-tectal system.
a lack cdna representation in current chick databases
the observation that neither the identity nor a clear wholemount expression pattern could be obtained from the differential display cdnas cloned into the pcr-topo vector lead us to the search for longer more informative clones.
however, for clones h, j, n, p, u, x, y, z &  <dig> no alternatives cdnas were obtained from either screening of the chick λzapii library, rzpd neural tube-specific gridded library , our in house 5' race library or the chick est database  .
together, this implies that these transcripts are expressed at a very low abundance or are refractory to traditional cdna cloning methodologies.
furthermore, these sequences were not represented in the present draft of the chick genome sequence.
thus, following completion of the chick genome the identity and potential role of these genes in development can be re-assessed
discussion
taken together, the confirmed expression of the known and novel genes describe here fully validates the efficacy and content of the screen .
hence we provide a description of gene expression during early development of the neural tube that provides novel insights into some of the molecular pathways employed .
the data provided here can thus be used as a foundation for future functional studies and valedictory support for forthcoming microarray studies.
in particular, several of the potential patterning mechanisms alluded to here are being functionally pursued.
for example, the midbrain-enriched expression of rtpz is being investigated for its capacity to negatively regulate isthmic-fgf <dig> signalling and its subsequent impact on midbrain patterning.
using the latest advances in messenger rna differential display technology described herein, as many as  <dig> different cdnas were amplified per primer pair.
an innate consequence of the technique is that any cdna isolated, novel or otherwise, is anchored at the 3' poly a tail end of an rna transcript.
given that the 3' untranslated  region of transcripts are generally greater than  <dig> bp and maybe up to  <dig> kb long , it has been traditionally difficult to obtain definitive information of a cdna's identity.
this has usually required a laborious cdna-walking strategy to obtain further sequence information ).
however, recent advances in whole genome sequencing and large scale est projects have provided a large substrate of 3' sequence information that can be accessed and searched using standard blast methodologies.
the availability of this resource has dramatically increased the ease with which an identity or probable function can be assigned to a cdna obtained by dd.
conversely, the work described in this study highlights a short-coming of differential display.
namely, this technique is restricted in the percentage coverage of the mrna pool it samples.
using over  <dig> primer combinations  only  <dig> differentially expressed cdnas were identified and characterized.
this is at odds with previously known number of over  <dig> genes that are differentially expressed throughout the developing neural tube .
therefore, dd in its latest guise has been shown to be capable of accurately and reproducibly monitoring gene expression, albeit in a restricted fashion.
using short cdna sequences obtained from this screen, it has been possible to walk up to a further  <dig>  kb 5' by reiterative blast comparison  <cit>  with sequences deposited from the chick est project .
overall, the stringent technological advances and increased sequence data from genome wide studies have given dd a further lifespan as a useful cdna screening technology for obtaining small numbers  of differentially expressed genes in species where high density microarrays are not yet available
the data provided in this study will also be beneficial for the implementation and validation of the similar types of screens using the newly released chick high density oligonucleotide arrays .
not only does it serve as a methodological platform for large scale screening purposes but also the gene expression data described provides an instant read out of the efficacy of the genechip approaches.
despite the relatively restricted coverage of messenger rna differential display, it still remains an extremely useful tool for the interrogation of transcriptomes.
in particular for those species where high density microarrays are unavailable at present, it can be applied to the study of differential gene expression and used to isolate important regulatory transcriptional events.
for example, in the quail retinoic acid-deficient system,  successfully used differential display to identify a large set of candidate genes whose expression was altered in the dv axis of the spinal cord compared to that of a normal spinal cord.
CONCLUSIONS
this study provides an insight into some of the varied and novel molecular networks that operate during the regionalization of embryonic neural tissue and expands our knowledge of molecular repertoire used during development.

 even before neurulation begins, the neural plate is broadly patterned into domains with anterior and posterior molecular character. as development proceeds, the founding pattern is further refined upon until the morphologically characteristic structures of the forebrain, midbrain, hindbrain and spinal cord arise. the molecular mechanism by which the acquisition of the anterior-posterior identity is achieved has been the subject of intense study. in recent years many fundamental patterning processes have been elucidated but relatively little is understood about the complete set of signalling influences or genetic networks used to achieve this neuronal diversity. however, it is known that the specification and subsequent patterning of the neural tube uses hierarchies of transcription factors working in combination with the activity of signalling centres located at discrete ap positions. during gastrulation, otx and gbx are expressed in opposing domains in the neural plate, anteriorly and posteriorly respectively. within the otx positive domain the forebrain vesicle develops and there is good evidence for transcriptional regulation playing a deterministic patterning role of that region. the interface between the zones of expression of otx and gbx uniquely determines the future position of the midbrain-hindbrain organizer {mhb, also referred to as isthmus), a structure intimately associated with correct patterning of the mid-hindbrain region. subsequently, a series of transcription factors including pax pax pax en en are expressed at the otx2-gbx boundary that are required for maintenance of the mhb organizer and specification of the midbrain territory. these transcription factors then participate together with the secreted signals fgf and wnt in a cross-regulatory network to maintain the mhb organizer and direct patterning of the midbrain and cerebellar structures. multiple lines of evidence show that fgf which is expressed in the temporally appropriate manner at the mhb in all vertebrate classes, constitutes an important component of the isthmic-patterning signal. introduction of ectopic fgf protein into the avian brain can respecify posterior forebrain to become midbrain and anterior midbrain to develop posterior midbrain characteristics. taken together, these data demonstrate the existence of a complex interplay of genetic regulation and local signalling that are required to specify and pattern the midbrain territory. the hindbrain becomes organized into a repeated series of cell lineage-restricted metametric units defined as rhombomeres. these compartments facilitate a coordination of ap and dorsoventral signals to regulate the pattern of the neuronal subtypes born within them such that each rhombomere can be considered to have a unique identity. rhombomere is notable in respect of its lack of branchial motor neurons and being the precursor region for the cerebellum. the anterior rhombic lip of r generate a large migratory cell population that forms the external germinal layer and later the internal granule cell layer of the cerebellum. it is now well established that the hindbrain is patterned by the combinatorial action of the homeodomain-containing hox transcription factor code. within the region that is initially defined as gbx positive in the neural plate, the initial hox code in the hindbrain is first set up under the influence of ap signals such as retinoic acid. given the distribution of the hox transcripts and their ability to act in a co-operative fashion, it has been suggested that individual rhombomere identity is conferred by a combinatorial code of the hox proteins. functional evidence for the role of hox genes in the patterning of the hindbrain can be observed from interference studies on the hoxb gene that is normally highly expressed in r disruption of the hoxb gene in mice leads to transformation of the r territory into an r2-like state, whereas retroviral-mediated over expression of hoxb in r causes homeotic transformation of r to an r4-like condition. evidence is now also emerging about some of the other factors that act upstream of hox genes themselves to activate them at appropriate ap levels. as well as cross-regulation between the various hox genes, upstream regulators of the hox genes include mafb and krox which, in addition to controlling segmentation of the neuroepithelium act in a parallel but related process to regulate the hox genes. thus, mafb directly modulates expression of paralogue group hox genes in r, and krox is a direct activator of both hoxa and hoxb and a repressor of hoxb. given the prevalence of transcriptional regulation in the specification of ap pattern of the neuraxis, the aim of this study was to identify novel candidate or known genes with potential roles regulating the ap pattern of the neural tube. many methodologies have been developed that are designed to identify and/or sample the array of genes expressed by a cell or tissue type, commonly defined as the transcriptome. the technologies vary both in the number of genes they are designed to profile and if a priori knowledge of gene identity is required. for example, technologies employing subtractive hybridization of cdna libraries have been successfully used to find regulated changes in gene transcription during development of the neural tube. a current method of choice is microarrays. however, due to the lack of availability of chick-based systems at the time of onset of this study, we chose to implement a large-scale messenger mrna differential display screen. despite a prevalent false positive rate, recent advances in primer technology, gel electrophoresis and cdna cloning of the appropriate differentially expressed gene has dramatically decreased such error. an advantage of dd is that it can be used simply and relatively cheaply to assay the gene expression profiles of multiple cell or tissue types simultaneously, to detect both up and down transcriptional regulation. when taken together with the capacity to precisely stage and dissect the developing chick embryo the methodology presents a unique opportunity to accurately assay transcriptional regulation of the early cns. in addition, despite the prevailing idea that all genes can be identified computationally once a genome has been sequenced, there is now clear evidence that a substantial amount of genome annotation is still required and novel genes are still being identified by other approaches. thus, there is still good reason to find novel genes using empirical screening technologies and place them into an appropriate biological context. we present the findings of an exhaustive differential display screen for regionally restricted transcripts during early brain development. clones were sequenced and compared to the latest est, genomic, known gene and predicted gene databases to characterize differentially expressed candidates. results were confirmed by in situ hybridization and the embryonic expression of several of the isolated genes is described for the first time. taken together this study provides a foundation from which genetic networks and hierarchies can be inferred and further explored.  efficacy of the messenger rna differential display screen preliminary data from this screen was reported in. we now report the results from the completed screen as well as presenting analysis, identity and expression for many of the candidate cdnas isolated. we have used all primer combinations available t anchor primers in combination with arbitrary primers; hieroglyph kit, beckman coulter), as well as including some custom arbitrary primers. according to manufacturer's data, this approach should survey >95% of the transcriptome. we recorded reproducible changes in differential display gel profile from the primer combinations tried in total. the changes observed were robust in that they appeared in the series and rna pools and were reproducible in subsequent identical differential display experiments. overall, this represents a 14% incidence of difference in gene transcription across neural tube regions tested. the clone designation, identity, function and recorded gel profile of each of the differentially expressed clones are listed in table fig. 2b shows a schematic representation of the molecular functions of the differentially expressed candidates whilst fig. 2c shows how the changes were distributed across the neural tube. a large proportion of the changes were recorded as being present in the midbrain or rhombomere regions which is consistent with this being previously known to be a site of active transcriptional events. patterns of gene expression unique to certain areas were also seen which is compatible with prior knowledge of patterning of that region. other profiles were also present involving combinations of transcriptional activation. where an unequivocal identity is found for the differential display clone in bbsrc, genbank or swissprot databases, it is shown in the appropriate column. sp = swiss-prot database, tf = transcription factor, rtp = receptor tyrosine phosphatase, fgfr = fibroblast growth factor receptor, glp = glugacon-like peptide, ef = elongation factor, lrp = laminin receptor protein, l1cam = l1-like cell adhesion molecule, mafb ~ kreisler, 5' race = rapid amplification of 5' cdna end. where no identity for a cdna is found in the sequence repositories, the sequence is given in supplementary information. f = forebrain, m = midbrain, r = rhombomere h = hindbrain. * denotes clones whose identity has been derived from significant matches with the chick genome sequence. + denotes identity derived from similarity to an ortholog. where a 100% match with an est from the umist collection was found the fragment sequence was not submitted to genbank. coverage of messenger rna screen a differentiated eukaryotic cell is thought to contain – distinct mrna species. in the study described here primer combinations were used, each displaying around pcr products to generate over amplified fragments. this figure broadly correlates with the current estimate of gene number for a higher eukaryotic organisms. thus, in principle we surveyed the majority of genes expressed during the transcriptional specification of the fore-, mid- and hindbrain regions of the developing avian cns. however, the total number of bands displayed does not compensate for the observed redundancy of the displays. that is, many mrnas would have been displayed more than once, whereas others, particularly low abundance messages, would not have been detected at all. this is an intrinsic property of the differential display methodology and contributes to the restricted coverage generated. an example of redundancy was observed with clone c, where the same differential display gel profile and subsequent cdna identity was observed from candidate bands of a different size obtained using a different arbitrary primer. by contrast, a slight under-estimation of the number of distinct pcr products is possible due to the existence of more than one species of pcr product of the same size in an individual band. in these circumstances, a single stranded conformational polymorphism strategy was adopted to accurately determine the differentially expressed cdna. an additional consideration to the restricted coverage of the technique is that where a fixed number of pcr cycles are used, it is likely that small differences in the levels of some regulated transcripts will be missed. compiling identities of the chick differential display cdna clones of the differentially expressed genes identified, could be assigned an unequivocal identity either directly from the sequence of the displayed fragment or following the isolation of a longer cdna clone. for the remaining clones, often after the addition of more upstream sequence from cdna libraries, an identity, putative function or membership to a gene or protein family could not be assigned. it is likely that, despite our efforts to isolate large cdna fragments, the 3' bias of the methodology resulted in preferential isolation of 3' untranslated sequences. for many clones, the recent release of the chick est database was similarly insufficient to assimilate an identity. the chick ests were generated from different embryonic and adult tissues, ranging from a complete early developmental stage hh st embryos to a single adult tissue e.g. the limb. despite the fact that the individual cdna libraries constructed from each point were poly t-primed and therefore generated from the 3' end of an mrna transcript, a similarity of the usable est sequences with the clones described here was not always found. this may be a function of the representation of the est libraries or that only the 5' ends of the directionally cloned libraries have so far been sequenced thus, for those longer clones in the est repository, the sequence immediately upstream of the poly a tail may be under represented. these types of issues also imply that studies like those described here have a useful role in annotating genomes and contributing to an accurate description of transcriptomes. in the absence of a similarity match from the cdna databases, we searched the recently released chick draft sequence for potential information. subsequent to a significant match against the chick genome, we identified the appropriate sense strand and investigated up to kb upstream for exons that might be informative. this approach, although helpful, has the caveat that associated exons must first be shown to physically spliced to the cdna query sequence, for example by rt-pcr, before an identity can be unequivocal. current data suggests that the average 3'utr is within the range of to kb. where possible, a northern blot was performed to indicate the appropriate size of cdna to look for in the genome. however, for some clones no matches could be obtained from the genome sequence confirming that sequence remains to be finalised. validation of the experimental technique: isolation of en mafb, spry fgfr robo1 the strategy described here used a microsurgical approach and a subsequent pooling strategy prior to the screen. proof of principle was confirmed by the isolation and identification of five genes known to be expressed in embryonic patterns faithful to the differential display gel profile and also to play important roles during neural tube development. analysis of the display profile from the subset of mrnas amplified with anchor and arbitrary primer revealed a pcr product, clone a, that was reproducibly seen in the m and r samples but not in the f and h samples ). subsequent isolation, reamplification and cloning of this band gave a bp cdna sequence in all clones tested. comparison of this sequence using blastn showed it to be 100% identical to the 3' utr region of chick engrailed cdna. further confirmation was obtained by in situ hybridisation to chick hh st embryos where the expression pattern observed was identical that previously described. en has previously been reported as being a primary genetic determinant of the developing midbrain and rhombomere regions and mice homozygous for an en loss of function allele lack these entire structures. clone b derived from an anchor primer – arbitrary primer combination-generated bp hindbrain-enriched pcr band, was found to be 94% identical to the 3'utr of the mouse mafb cdna. in situ hybridisation with this cdna demonstrated that expression was fully concordant with the previously described expression in rhombomeres and in the hh st neural tube. available data demonstrates the developmental significance of this gene since animals lacking functional mafb protein show, amongst other developmental defects, a deletion of the rhombomere region. additional evidence for the efficacy of the screen described here was obtained from the reamplification, cloning, sequencing and expression analysis of clone that displayed a midbrain-enriched gel profile. this bp clone showed 100% identity to the 3'utr of the fgfr a transmembrane receptor tyrosine kinase that was previously described as being expressed in the midbrain vesicle of a hh st chick embryo. the cloning, expression and interplay with fgf signalling of sprouty was also reported from this screen. a detailed functional analysis of the role of sprouty proteins in development is being performed in our laboratory. taken together, these data demonstrate that the screen was reflective of the transcriptomes surveyed and was capable identifying a range of genes with varied and important development roles. as such, the data presented in table is likely to describe a true picture of gene expression changes in the developing neural tube. differential display has an efficacy lower than that statistically predicted despite the established efficacy of the screen, the detection of fewer than differentially expressed bands is indicative of a relatively small proportion of mrnas with altered expression across the ap neuraxis of a hh st developing chick embryo. that we did not identify any of the large number of previously characterised differentially expressed genes associated with hindbrain patterning, strongly suggests that the coverage was less than anticipated. this may be due in part to the low level of mrna abundance of the hox transcription factors. however, this is largely countered by the observation that they can be readily detected by wholemount in situ hybridisation. a survey of the accumulated literature shows that greater than genes have been reported as being differentially expressed in the tissues under study here. it is clear from this study that messenger rna differential display operates at efficiency much lower than that statistically predicted by binding of the arbitrary primers to their cognate sequence alone. detailed expression analysis following validation of the screen, we selected several identified genes for detailed expression analyses; sparc-related modular calcium binding protein laminin receptor protein clone β, hb gam, clone g, translation elongation factor 1α, receptor protein tyrosine phosphatase zeta and l1cam. these clones were selected based upon their identity and possibility that they may interact with other known pathways that pattern the mid-hindbrain territories at this developmental time; for example, to ask if rtpζ and hb gam are expressed in the appropriate place to modulate/interact with fgf signalling around the isthmus organiser? although others showed no obvious identity, they were selected based upon their differential display profile that suggested that they were expressed in domains known to be important in patterning the areas under study. similarly, we chose clones who have a know role in cell adhesion to see if they could contribute to either boundary formation or cell lineage restriction in the mid-hindbrain region. in addition, for some of the clones isolated it was not possible to produce a definitive in situ hybridisation pattern. this may be due to the 3' utr nature of the sequence. the identities of the cdnas not used for further in situ hybridisation studies, where known, is presented in table clone c: sparc-related modular calcium binding protein 1 using arbitrary and anchor primers and respectively, as well as and respectively, a bp pcr band was reproducibly enriched in the series and r and h mrna pools. reamplification, cloning and sequencing of the cdna clone on both strands failed to produce a significant match within the databases. the 3'utr sequences showed a typical poly a addition consensus site proximal to the site of anchor primer binding. this clone was not represented in either of the cdna libraries screened. using a nested 5' race approach, a further kb of sequence immediately upstream of this 3'utr sequence was acquired. this sequence lacked any obvious orfs, partial or otherwise, and no identity was derived suggesting that it was the 3'utr region. scanning of the chick genome with clone c sequence revealed a region of 98% identity on chromosome. immediately adjacent to this match, exist a previously annotated protein coding exon coding for'similar to sparc-related modular calcium binding protein 1'. the immediacy of the clone c-derived sequence to this region implies that clone c codes for the protein described as for'similar to sparc-related modular calcium binding protein 1'. sparc has an extracellular ca2+ binding domain and is a multifunctional glycoprotein that functions to regulate cell-matrix interactions. it binds to such proteins as collagen and vitronectin and can binds to endothelial cells and inhibit cellular proliferation. the extracellular domain interacts with a follistatin-like domain that appears to stabilize ca2+ binding. the two ef-hands interact canonically but their conserved disulfide bonds confer a tight association between the ef-hand pair and an acid/amphiphilic n-terminal helix. proposed active form involves a ca2+ dependent symmetric homodimerization of ec-fs modules. srmcbp has dynamic expression in the developing embryo; expression is first seen in the neural plate followed by staining in the primitive streak. at the onset of somitogenesis, srmcbp becomes restricted to presumptive tail bud and newly formed somites. later at 3–6s, the transcripts are more abundant in lateral tail bud regions but still present in the somites. at 12s, srmcbp transcripts are seen in rhombomere ectodermal regions surrounding the otic vesicle and medial somites. at hh st the hindbrain expression has expanded into all rhombomeres but is excluded from the boundary zones. clone q: laminin receptor protein 37 analysis of the differential display gel profile obtained using anchor primer combined with arbitrary primer consistently and reproducibly identified a cdna of base pairs in series and mrna pools whose expression was switched on in the posterior hindbrain cdna pool but not in the rhombomere cdna pool. excision of this candidate cdna, cloning, library screening, sequencing and blast analysis revealed it to be 100% identical at the nucleotide level to the previously identified gallus gallus laminin receptor protein gene. the laminins are a family of glycoproteins that form a critical component of the basement membranes of most organisms and are known to be an important factor in cell migration, axon pathfinding and modulation of cell survival as well as other cellular processes. laminins self assemble from α, β and γ subunit chains and are secreted into the extracellular space where they can interact with a range of other extracelluar matrix molecules. to date, twelve different laminin heterotrimers have been identified in mammals as well as other variants being found in hydra, drosophila and c. elegans. it has been reported that the laminin family members have tissue specific, but overlapping, distributions during embryonic development. critically, laminins have been shown to have a diverse and important and role during embryonic development to later post natal development. for examples and a recent review see. the primary mechanism by which cells recognize and interact with laminins is through the integrins, a family of heterodimeric transmembrane receptors consisting of α and β subunits. integrins are capable of exerting regulatory effects on both the cytoskeleton and cellular signaling apparatus. lrp has been described as a multifunctional molecule involved in both cell adhesion via laminin and formation of the polysome in the cytosol. in situ hybridisation reveals that the lrp transcripts are expressed very early in embryonic development in regions fated to form mainly trunk structures. as development proceeds, the expression of lrp is observed in the neural tube, somites, ectodermal and endodermal tissues, but only immediately posterior to the rhombomere 6/ border. interestingly, this gene has not been previously reported to be expressed embryonically and its function in development remains to be elucidated clone β: pax2 given the differential display gel profile of clone a and its identification to be the developmentally significant en cdna, it was of particular interest that clone β had an identical gel profile. reamplification, cloning and sequencing produced identical subclones that gave bp of sequence. similar to other clones described here, this clone gave no identity or no expression pattern in hh st chick embryos. a high stringency library screen of the hh st chick λzapii library produced independent clones ranging in size from to kb. the longest of these clones was selected and sequenced on both strands. despite the large amount of information no clear identity or orf greater than amino acids could b obtained. to determine the size of the endogenous transcript we performed a northern blot on chick whole embryo hh st total rna and observed two bands of ~ and kb. given the size of the full length mrna, it was likely that our sequence information may still have been in the 3'utr. using the elongated kb clone β, we obtained the expression during the formation of the neural tube. notably, the expression observed in the neural tube was similar that seen for clone a and consistent with the gel profile. in fact, the expression of clone β was recorded very early in the ectoderm destined to become the mhb. to ascertain the identity of clone β and discern the potential early function in mhb formation, we scanned the genomic sequence upstream of the existing kb. using this approach we located an exon that codes for a region of the paired homeodomain transcription factor pax this observation is consistent with the known pax that is the earliest transcription factor expressed in the hierarchy of mhb formation. however, although clone β is now known to represent the previously well-characterized pax it should be noted that the kb in situ fragment provides extremely definitive patterns that may be useful in future studies. the methodology described here also serves as model to elucidate the identity of other cdnas. clone f: similar to heparin-binding growth-associated molecule clone f is a reproducibly expressed hindbrain-enriched bp display fragment. the available sequence was insufficient to discern identity or expression for in situ studies. following screening of the chick hh st λzapii cdna library, four independent clones were obtained. verification of clone f identity by southern blotting and sequencing revealed an additional kb of sequence had been attained. the largest construct showed no extended open reading frames or blastn/x identity with the existing sequence warehouses. however, submission of the extended clone f sequence to the chick genome blastn algorithm revealed a 97% identical match to the nw_ contig derived from chromosome w sequencing. further analysis of this locus showed the presence of the previously annotated gene defined as'similar to heparin-binding growth-associated molecule' ), immediately adjacent to the clone f sequence match. given the appropriate orientation and 3'utr nature of the clone f sequence, it is reasonable to conclude that clone f is the gene described as'similar to hbgam'. heparin-binding growth-associated molecule itself is a basement membrane-associated protein that was initially isolated as a neurite outgrowth-promoting factor from perinatal rat brain. hb-gam is composed of two thrombospondin type domains flanked by lysine-rich n- and c-termini of undefined structure. the tsr domains may mediate binding to heparan sulfates and to the cell surface, while the role of the lysine tails is unknown. hb-gam also has another close relative; midkine that was isolated and cloned as a retinoic acid induced differentiation factor. however, although the in vivo functions of hb-gam are still not generally understood, recent work by has demonstrated a clear role for hb-gam in the selective inhibition of fgf2-mediated activation of fgfr by restricting the interaction between ligand and the proteoglycan co-receptors required for normal activation. in this context, hb-gam inhibited the proliferation and promoted the differentiation of neural stem cells by restricting the signalling activities of fgfs. the capacity of hb-gam to negatively regulate fgf-stimulation of their cognate receptors is of significance given the expression of clone f in the developing embryo. at hh st11- clone f transcripts are seen in rhombomere medial somites and the presomitic mesoderm. the pattern observed in the presomitic mesoderm is of particular interest as it is known that fgf8-mediated signalling is required here to maintain proliferation and inhibit inappropriate maturation of this zone. thus, the fgf8-mediated presomitic signalling pathway may be mechanistically regulated by hb-gam in an analogous fashion to that described by. we are currently conducting a series of functional interference studies to determine the precise role of this protein during early patterning of the neural tube. clone g: unknown amplification of the mrna pools with anchor primer and arbitrary primer revealed a bp pcr product whose expression was recorded principally in the forebrain, midbrain and rhombomere samples but absent from the hindbrain group. sequence analysis followed by comparison to databases showed no significant homologies. using this cdna fragment as a probe, independent positive plaques were isolated from the chick hh st λzapii cdna library. excision of the insert and subsequent southern blot analysis determined the longest of these clones to be ~ bp. the sequence of this clone is presented in supplementary information and was shown to be 100% identical to the clone g probe sequence at the 3' end. the extended clone g sequence provided no further insight into the identity of the resultant protein. the umist chick est database does not contain the equivalent cdna sequence. furthermore, this sequence is as yet unrepresented in the available draft of the chick genome. however, northern analysis shows that for total rna derived from a hh st embryo that there are two transcripts of equal abundance at and kb. thus with kb of sequence and the expected size of the coding rna, the open reading frame and potential function can be predicted upon completion of the genome sequence. using the longest library clone to generate an antisense probe, the expression of clone g in a chick embryo was shown to mirror the differential display gel profile. at hh st 10+, clone g transcripts were seen in the forebrain, midbrain and rhombomere regions but completely absent from the hindbrain region of the neural tube. notably, there was an obvious gap in the expression at both the anterior neuropore and mhb. these are both sites of fgf8-signalling and raise the possibility that clone g is repressed by fgf this supported by the observation that there are no gaps between the zones of expression of fgf and clone g as assessed by a single colour'fill in' in situ hybridization. the capacity of fgf protein to repress the transcription of clone g is currently under investigation using fgf8-coated beads implanted into the anterior midbrain. transverse section of hh st10+ embryos in the rostral midbrain show that expression in the dv axis is excluded from the roofplate and floorplate areas suggesting that clone g may also be repressed by factors expressed in these regions such as bmps and shh respectively. clone 4: translation elongation factor 1α using a similar cloning and sequencing approach to that described elsewhere, we demonstrated that a bp pcr product enriched in the forebrain pools corresponded to a bp library cdna with 100% identity to gallus gallus translation elongation factor 1α. even at early stages, ef1α are more abundant in the anterior neural plate. as development proceeds up to hh st ef1α expression becomes most evident towards the rostral end of the embryo. the function of this type of pattern remains to be determined. however, there is now increasing evidence that mrna-specific translational control, whereby the translation of a defined group of mrnas is modulated without affecting general protein biosynthesis or the translational status of the cellular transcriptome as a whole is an important factor in the regulation of cellular identity. in addition, global control of protein synthesis can also be achieved by changes in the phosphorylation state of initiation factors or the regulators that interact with them. in addition, previous studies have also implicated the differential distribution of protein coding machinery in the developing neural tube. the translational-regulatory component eif2a kinase-related is expressed exclusively in r of the developing hindbrain. here, a potential role in the r5-specific regulation of apoptosis has been speculated. it is of note that the two translation factors reported here, ef and eif3zeta, showed a robust hindbrain-specific gel profile. taken together, these data are suggestive of a neural tube specific combinatorial translation factor code operating alongside the well-established transcription factor patterning events. clone 2: receptor protein tyrosine phosphatase zeta previous work reported from this study has helped to elucidate some of the molecular regulation of fgf-signaling at the mhb. it was therefore of interest when the identity and expression of clone was elucidated. using the acaatttcacacaggattttggctcc arbitrary primer in conjunction with ggaaaaaaaaaaagccctatagtgagtcgt anchor primer, a bp pcr consistently upregulated in midbrain and forebrain pools of series and mrnas was obtained. reamplification, cloning and sequencing of the differential display band followed by database queries produced no clear identity. to establish an identity we submitted the sequence to the chick est database and commenced a 5' walking strategy. this approach gave an exact match to the 603788872f1-designated assembled sequence contig. using the longer sequence an unequivocal match to gallus domesticus phosphotyrosyl phosphatase mrna complete cds was found, which itself is othologous to the rattus norvegicus receptor type protein tyrosine phosphatase zeta/beta. thus it has been established that clone is chick ortholog of the receptor type protein tyrosine phosphatase zeta/beta. receptor protein tyrosine phosphatases constitute a large family of structurally related proteins that are responsible for the regulated removal of phosphates from tryrosine residues. tyrosine phosphatases are classified into subfamilies spread across three groups: cytoplasmic, receptor-type, and dual specificity phosphatases that dephosphorylate serine threonine residues, and tyrosine residues that are in close proximity receptor protein tyrosine phosphatase zeta is a member of the receptor type group with an extracellular domain similar to that of the cell adhesion molecules. evidence from genetic studies in c. elegans has demonstrated that one function of rptps is to antagonize receptor tyrosine kinase signalling. the rptp, clr- functions to inhibit signalling by the fgf receptor-related rtk, egl- to regulate the migration of sex myoblasts. in addition, a mitogen-activated protein kinase phosphatase gene, encoding a dual specificity phosphatase, has been shown to directly antagonise fgf8-mediated receptor activation during limb development. given the potential function of clone and a midbrain-enriched gel profile we selected this clone for further expression analysis by in situ hybridisation. other than a report on the expression of rptpγ, there is no description of the expression of rtps in early embryogenesis at hh st10+ the expression of rtpζ was observed in the midbrain and forebrain vesicles. the mrna distribution in the midbrain is potentially in a caudal to rostral gradient. no expression was observed in the neural tube caudal to the mhb. additional weak expression was also seen in the ectoderm immediately adjacent to r. both the midbrain territory and otic ectoderm are known sites of fgf-signaling. thus we hypothesize that rtpζ may be involved in modulating the fgf-signaling events that are occurring in those regions. alternatively, other than being involved in the control of fgf8-mediated signal transduction at the mhb, rtpζ may be involved in another independent process such axonal guidance in retinotectal mapping. the functional role of rtps in these types of events has previously been documented. clone l: l1cam data presented in table shows that following the library screening, sequencing and in situ analysis, clone l was the gallus gallus close homolog of l cell adhesion molecule with a distinctive transcript distribution. at hh10+, l1cam was seen in the spinal cord, a caudal to rostral graded midbrain expression and some mrna localized to the posterior forebrain. the gene expression pattern is fully correlative with the observed gel profile of on in all regions other the r pool. l1cam is a kda transmembrane cell adhesion molecule with six extracellular ig-like and five fibronectin iii domains that enable l to homophilically bind to opposing cells as well as to heterophilically bind to tag- β1-integrins, f11/contactin, neural cell adhesion molecule, and proteoglycans. l1cam is capable of activating a map kinase signalling cascade through the intermediates src, phospohinositide- kinase, rac and p21-activated kinase, leading to neurite growth. studies also suggest that retinal axons require the function of l in addition to repellent epha guidance receptors to achieve proper topographic mapping thus this early, previously unreported, graded expression of l1cam in the midbrain may play a role in establishing the appropriate projections of the retino-tectal system. a lack cdna representation in current chick databases the observation that neither the identity nor a clear wholemount expression pattern could be obtained from the differential display cdnas cloned into the pcr-topo vector lead us to the search for longer more informative clones. however, for clones h, j, n, p, u, x, y, z & no alternatives cdnas were obtained from either screening of the chick λzapii library, rzpd neural tube-specific gridded library, our in house 5' race library or the chick est database. together, this implies that these transcripts are expressed at a very low abundance or are refractory to traditional cdna cloning methodologies. furthermore, these sequences were not represented in the present draft of the chick genome sequence. thus, following completion of the chick genome the identity and potential role of these genes in development can be re-assessed discussion taken together, the confirmed expression of the known and novel genes describe here fully validates the efficacy and content of the screen. hence we provide a description of gene expression during early development of the neural tube that provides novel insights into some of the molecular pathways employed. the data provided here can thus be used as a foundation for future functional studies and valedictory support for forthcoming microarray studies. in particular, several of the potential patterning mechanisms alluded to here are being functionally pursued. for example, the midbrain-enriched expression of rtpz is being investigated for its capacity to negatively regulate isthmic-fgf signalling and its subsequent impact on midbrain patterning. using the latest advances in messenger rna differential display technology described herein, as many as different cdnas were amplified per primer pair. an innate consequence of the technique is that any cdna isolated, novel or otherwise, is anchored at the 3' poly a tail end of an rna transcript. given that the 3' untranslated region of transcripts are generally greater than bp and maybe up to kb long, it has been traditionally difficult to obtain definitive information of a cdna's identity. this has usually required a laborious cdna-walking strategy to obtain further sequence information ). however, recent advances in whole genome sequencing and large scale est projects have provided a large substrate of 3' sequence information that can be accessed and searched using standard blast methodologies. the availability of this resource has dramatically increased the ease with which an identity or probable function can be assigned to a cdna obtained by dd. conversely, the work described in this study highlights a short-coming of differential display. namely, this technique is restricted in the percentage coverage of the mrna pool it samples. using over primer combinations only differentially expressed cdnas were identified and characterized. this is at odds with previously known number of over genes that are differentially expressed throughout the developing neural tube. therefore, dd in its latest guise has been shown to be capable of accurately and reproducibly monitoring gene expression, albeit in a restricted fashion. using short cdna sequences obtained from this screen, it has been possible to walk up to a further kb 5' by reiterative blast comparison with sequences deposited from the chick est project. overall, the stringent technological advances and increased sequence data from genome wide studies have given dd a further lifespan as a useful cdna screening technology for obtaining small numbers of differentially expressed genes in species where high density microarrays are not yet available the data provided in this study will also be beneficial for the implementation and validation of the similar types of screens using the newly released chick high density oligonucleotide arrays. not only does it serve as a methodological platform for large scale screening purposes but also the gene expression data described provides an instant read out of the efficacy of the genechip approaches. despite the relatively restricted coverage of messenger rna differential display, it still remains an extremely useful tool for the interrogation of transcriptomes. in particular for those species where high density microarrays are unavailable at present, it can be applied to the study of differential gene expression and used to isolate important regulatory transcriptional events. for example, in the quail retinoic acid-deficient system, successfully used differential display to identify a large set of candidate genes whose expression was altered in the dv axis of the spinal cord compared to that of a normal spinal cord.  this study provides an insight into some of the varied and novel molecular networks that operate during the regionalization of embryonic neural tissue and expands our knowledge of molecular repertoire used during development. 
this study provides an insight into some of the varied and novel molecular networks that operate during the regionalization of embryonic neural tissue and expands our knowledge of molecular repertoire used during development. however, it is clear that much remains to be learned about the complete set of molecular cues that are employed to establish the identity and intrinsic neuronal diversity of these territories. others represent genes whose embryonic expression has not been previously reported. during early development the vertebrate neural tube is broadly organized into the forebrain, midbrain, hindbrain and spinal cord regions.
 during early development the vertebrate neural tube is broadly organized into the forebrain, midbrain, hindbrain and spinal cord regions. each of these embryonic zones is patterned by a combination of genetic pathways and the influences of local signaling centres. however, it is clear that much remains to be learned about the complete set of molecular cues that are employed to establish the identity and intrinsic neuronal diversity of these territories. in order to address this, we performed a high-resolution messenger rna differential display screen to identify molecules whose expression is regionally restricted along the anteroposterior neuraxis during early chick development, with particular focus on the midbrain and hindbrain vesicles.  this approach identified different genes, with both known and unknown functions, whose transcription is differentially regulated along the ap axis. the identity and ontological classification of these genes is presented. the wide variety of functional classes of transcripts isolated in this screen reflects the diverse spectrum of known influences operating across these embryonic regions. of these genes, several have been selected for detailed in situ hybridization analysis to validate the screen and accurately define the expression domains. many of the identified cdnas showed no identity to the current databases of known or predicted genes or ests. others represent genes whose embryonic expression has not been previously reported. expression studies confirmed the predictions of the primary differential display data. moreover, the nature of identified genes, not previously associated with regionalisation of the brain, identifies novel potential mechanisms in that process.  this study provides an insight into some of the varied and novel molecular networks that operate during the regionalization of embryonic neural tissue and expands our knowledge of molecular repertoire used during development. 