the regulated expression of different families of retrotransposons and dna transposons has been described in different tissues and species . however , a detailed analysis of the transcripts or their temporal and spatial expression patterns has been reported in only a few cases . during drosophila embryogenesis ,
spatial differential expression of the retrotransposon 412 has been described , specifically in the gonadal mesoderm . in mouse
, retrotransposons make a high contribution to the pool of maternal mrnas in early embryos , and the expression of these elements is developmentally regulated . in xenopus ,
the 1a11 retrotransposon - like element is specifically expressed in the mesoderm and its expression is regulated by fgf .
likewise , the expression of the retrotransposon family xretpos is restricted to ventro - posterior regions during development .
we extended these observations by showing that a dna transposable element of the tc1/mariner family is differentially expressed during x. tropicalis development .
transcripts for some tc1-like elements have been detected in other est databases , but no detailed analyses has been performed .
our studies showed that the tc1-like element tc12_xt mrna ( named according to the repeatmasker nomenclature ) is specifically expressed at the gastrula stage in the spemann s organizer .
this region is required for the proper dorso - ventral and anterior - posterior patterning of the embryo and the neuroectoderm , the tissue that will give rise to the nervous system ( fig . 
then as development progresses , tc12_xt mrna is found restricted to neural tissue regions . both tc12_xt sense and antisense strands present similar expression patterns .
the length of the mrnas suggests that these transcripts are not included in other genes .
pirnas are specifically derived from tc12_xt , and analysis of the expression of selected tc12_xt - derived pirnas suggests that these elements control its temporal expression .
( a ) the sequence analysis of the 116 copies of the tc12_xt transposable element in the x. tropicalis genome showed the typical structure of tc1-like elements .
it is flanked by two inverted repeats ( ir ) containing two direct repeats ( white triangles ) for the binding of the transposase .
the transposase orf ( green ) contains a putative dna binding domain ( blue ) and the catalytic triad ( red , aspartic - aspartic - glutamic residues ( dde ) ) .
( b ) schematic representation of the expression of tc12_xt during x. tropicalis early development .
the expression of tc12_xt is shown in blue for early stages of x. tropicalis development .
no expression is detected before the beggining of the zygotic transcription ( st.6 is shown as an example , lateral view ) .
expression is clearly detected from gastrula stage ( st.10 ) in the spemann s organizer and then in anterior and neural tissues .
figures were downloaded from http://www.xenbase.org/anatomy/static/nf/nf-all.jsp and modified according to the expression pattern obtained by in situ hybridization . in the x. tropicalis genome ,
72% of transposable elements correspond to dna transposons with seven families of tc1-like elements characterized .
our analyses for these elements , albeit not detailed , have shown that several of these families are transcribed during x. tropicalis development .
interestingly , we could not find tc12_xt in x. laevis by using rt - pcr or in situ hybridization . whether this is explained because this element invaded the x. tropicalis genome after both species diverged , or to a high divergence in the sequence in the x. laevis genome is not known .
the availability of the x. laevis genome could be very useful to study these alternatives .
however , in x. laevis , two tc1-like elements have been described in the genome , txr and txz .
our studies have demonstrated that txr and txz are also expressed during x. laevis development .
importantly , the expression pattern of these elements is very similar to tc12_xt in x. tropicalis and both strands are also expressed . at present , we do not know how the expression of these elements is controlled and whether one or several loci are being transcribed .
the presence of regulatory elements controlling gene expression in the sequences of these tc1-like elements has not been studied .
however , in contrast to the expression of tc1 elements in c. elegans , which occurs by fortuitous read - through transcription , most tc12_xt rna is not included in other protein - coding transcripts , for which read - through transcription is unlikely to explain the bulk of tc12_xt rnas .
another alternative is the presence of clusters of several copies of transposable elements under the control of a single promoter .
the transcription of clusters of transposable elements to generate pirnas has been described in drosophila .
it is possible that these clusters can also be regulated during development to produce specific expression patterns .
another explanation is that transcription is ubiquitous , and that tc12_xt rnas are degraded in ventral and posterior regions by a pirna dependent - mechanism . in any case , the specific temporal and spatial expression of all these elements strongly suggests that they could play a role during nervous system development .
in the x. tropicalis genome , 111 out of 116 tc12_xt copies contain frame - shifts and mutations rendering putative transposase - inactive copies of the element .
consistently , the analysis of 20 tc12_xt cdnas from gastrula stage embryos indicates that none of them codes for an active transposase .
these results suggest that tc12_xt could play a role as a non - coding rna . in mouse , the transcription of the retrotransposon sine b2 is necessary for gene activation in the growth hormone locus . in this case
, the expression of this retrotransposon regulates the formation of a euchromatin / heterochromatin boundary , resulting in the expression of genes in proximal regions .
it is still unknown whether this is the case for tc12_xt expression , but we can propose that some of the tc12_xt loci may be regulating chromatin architecture , allowing the expression of genes involved in the formation of the nervous system . considering that pirnas derived from tc12_xt were detected , another possible function is to generate pirnas .
the expression of both strands is consistent with the ping - pong model of amplification of pirnas .
if this is the case , it is worth noting that the tc12_xt rna is stable enough to be detected by rt - pct , rna gel blot and in situ hybridization , suggesting that the pirnas produced are most likely not enough to degrade all of the tc12_xt rna during early development .
in addition , the expression of sense and antisense strands is specifically detected in neural tissues , and therefore , the amplification of pirnas could occur in these tissues .
alternatively , pirnas derived from tc12_xt may not be involved in the degradation of tc12_xt itself . rather than that
, they may regulate endogenous genes that contain sites complementary to pirnas . although our analysis for two tc12_xt - derived pirnas showed that all the pirna sequences in the genome are included only in tc12_xt sequences , the possibility that other tc12_xt - derived pirnas map to genes can not be ruled out .
for example , pirnas derived from transposable elements regulate the expression of endogenous genes and allow the clearance of maternal mrnas during early drosophila development .
finally , recently it has been published that pirnas derived from non - repetitive regions have a role in spine morphogenesis in the central nervous system in mice .
although all the tc12_xt cdna copies we analyzed do not contain a functional transposase open reading frame , 5 out of 116 tc12_xt complete sequences in the genome could putatively code for an open reading frame containing the catalytic triad and perhaps an active transposase ( fig . 
we do nt know if these five copies contain all the other residues required for transposition , such us the binding to dna domains .
however , the presence of active copies in the genome and its possible expression can not be ruled out .
interestingly , work from the gage laboratory showed that endogenous line-1 retrotransposition can occur during mouse development .
in addition , line-1 retrotransposition in the vicinity of neural genes can alter the expression of these genes in neural precursor cells .
furthermore , the same group showed that line-1 retrotranspositions can also occur in neural progenitor cells isolated from human fetal brain , suggesting that de novo line-1 retrotransposition events may occur in the human brain .
these events produce mosaicism in the neurons due to different genomic modifications on different neurons in the same individual .
this has been suggested as a novel mechanism involved in the generation of the astonishing neuronal diversity required for nervous system formation .
therefore , the specific expression of tc12_xt ( and txr and txz ) in dorsal and neural tissues allows speculation about a similar role in xenopus . as active elements for transposition
, these transposons could be involved in the generation of heterogeneity during xenopus nervous system development .
in this section we will briefly discuss possible experimental approaches to evaluate transposition of tc1 elements during nervous system development and determine its possible contribution to produce neuronal diversity .
one of the first questions is to determine whether expression of an active dna transposase occurs during early development . after cloning the possible candidates , transposase activity
must first be determined by in vitro assays . for this , a plasmid reporter for excision events needs to be prepared . based upon the white - peach allele studied in drosophila
the egfp open reading frame contains an insertion of a random sequence ( same length as the transposase ) in between of the inverted repeats the putative transposase recognizes .
therefore , when co - expressed with the transposase mrna in an exogenous system ( e.g. , cell culture ) , successful translation into an active transposase would render egfp+ cells .
the reporter construct contains an egfp open reading frame with an insertion of a random sequence ( same length as the original transposase ) .
when transposase mrna is present ( either exogenous , when working in vitro , or endogenous , when in vivo ) and an active transposase is being translated , transposition will occur , rendering egfp+ cells .
the random sequence insertion sites can then be characterized using deep sequencing of reverse pcr amplicons .
experiments to demonstrate in vivo transposition could be performed . for this purpose , transgenic xenopus embryos containing
a tissue - specific promoter would allow us to follow transposition in the central nervous system .
the presence of an endogenously active transposase would render egfp+ cells that we could observe in later developmental stages , such as stage 50 tadpoles .
furthermore , it would be possible to characterize transposon insertion sites in neural tissues of individual tadpoles using deep sequencing of reverse pcr amplicons ( see fig .  2 )
. the comparison of insertion sites in neural tissues with non - neural tissues could be an indicator of in vivo transposition events .
all of these approaches can be useful to determine whether transposition events occur during xenopus development .
although host cells contain mechanisms to avoid the expression of transposable elements , they could also be using these elements for cellular functions .
a proper balance must exist to control these positive and negative effects of transposable elements .
our work has shown that the specific expression of tc1 elements in neural tissues and suggests that transposable elements may play a role during the formation of the nervous system in vertebrates .
 transposable elements ( retrotransposons and dna transposons ) comprise a large proportion of animal genomes , for example 20% in d. melanogaster , 36% in x. tropicalis and 45% in humans . after invading a new genome , the transposable element increases its copy number and subsequently accumulates mutations . 
 these may eventually result in inactive copies . 
 until recent days transposons have been considered  junk  dna and no clear function have been assigned for this important amount of information on genomes . 
Expression of Transposable Elements from the 
Possible Roles of Transposable Elements as Non-Coding RNAs
Possible Roles of Transposable Elements as Active Copies
Experimental Approaches to Study the Role of Transposable Elements during Development