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Additional file 1: Fig. S1 The Tceal7 gene is derived from the domestication of L1 retrotransposon fragments. Fig. S2 The Bex/Tceal gene cluster was established before the diversification of extant eutherians. Fig. S3 Highly diverged BEX/TCEAL proteins share a coiled coil domain. Fig. S4 BEX/TCEAL proteins might have inherited some of their structural properties from the ancestral transposon. Fig. S5 Selection pressure analyses reveal signatures of positive selection in the Bex/Tceal genes. Fig. S6 A BGW-like sequence was already present in the GLA promoter of the last therian common ancestor. Fig. S7 Bex/Tceal genes show tissue-enriched expression patterns during development. Fig. S8 Bex3 and Tceal7 genes, but not the ancestral HALEX element, induce cell proliferation in chicken neural tube. Fig. S9 The deletions introduced using CRISPR-Cas9 technology can be observed in the mRNA expressed from the Bex3 mutant alleles. Fig. S10 CRISPR-Cas9-generated Bex3 mutant alleles show subtle skull abnormalities. Fig. S11 Bex3 mutant mice show normal acoustic startle reflex. Fig. S12 Bex3 deficiency leads to aberrant mTOR signaling in the brain. Table S1 Coding genes putatively derived from transposable elements in the human and mouse genomes. Table S2 Altered expression of BEX and TCEAL genes in subjects with autism spectrum disorder or schizophrenia. Table S3 Enrichment of differential gene expression in BEX and TCEAL gene families in subjects with autism spectrum disorder or schizophrenia. Table S4 Primers and reconstructed gene sequences used in this work. Supplementary references.

Additional file 2. Review history.

Resumen del trabajo presentado en el European Developmental Biology Congress, celebrado en Alicante (España), del 23 al 26 de octubre de 2019, Skeletal muscles originate from different mesoderms: presomitic (limb and trunk muscles), head (extraocular muscles) and pharyngeal mesoderm (facial muscles). Independently from their origin, all muscles develop under the control of the four Myogenic Regulatory Factors: Myf5, Mrf4, MyoD and MyoG. Myf5 is the first to be expressed, and controls myogenic specification. MyoD function overlaps with that of Myf5, and rescues the Myf5-KO phenotype. Myf5/MyoD double mutants have a general lack of skeletal muscles as myogenic precursors are not specified. MyoG drives terminal differentiation and in its absence adult muscles are not formed. Mrf4 plays roles in specification and differentiation, although its function remains poorly understood. The Early Epaxial Enhancer (EEE) directs the earliest Myf5 expression, starting in the somitic dorsomedial lip. We have generated a new mouse allele in which the EEE has been removed. This new mutant only loses Myf5 expression in the dorsal part of the first 5-6 somites. RNA-seq reveals differences in myogenic, innervation and limb, neurogenesis and chondrogenesis gene-networks. We have now validated several of the identified genes by qPCR and/or ISH. Because EEE-KO animals lack an overt phenotype, we generated EEE/MyoD double mutants, thus abolishing MyoD rescue in dorsal somitic progenitors. In these animals, we observe severe defects in diaphragm, ribcage and posture (kyphosis), which lead us to study the epaxial musculature more in detail to see if there is a particular group of muscles affected. Also, we are trying to elucidate how absence of Myf5 impacts upon Pax1 expression, presumably causing the ribcage defects.

Resumen del trabajo presentado en el European Developmental Biology Congress, celebrado en Alicante (España), del 23 al 26 de octubre de 2019, The myogenic regulatory factors (MRFs) Myf5, Mrf4, MyoD and MyoG are transcription factors that control the determination, specification and differentiation of skeletal muscle during development. Mrf4 specific functions remain elusive to date in spite of the three knock-out (KO) models available for over two decades. This can be explained by the disparity in their phenotypes, which ranges from complete lethality at birth to full viability into the adult. By studying the long-range interactions established in the locus, we show that the selectable marker cassette introduced upon generation of the mutants completely sequesters the neighbouring Myf5 promoter, only 8.7 Kb apart. Thus, these models behave phenotypically as partial or full compound Mrf4/Myf5 mutants, obscuring Mrf4 function. Using CRISPR/Cas9 technology, we created two novel KO alleles that do not affect Myf5 expression in cis. Preliminary analyses reveal that the expression patterns of the other MRFs are not affected during development and that, unlike previous KO models, no skeletal defects are observed unless Myf5 is additionally inactivated. Mrf4 KO animals show fibre hypertrophy and, surprisingly, cardiac hypertrophy even if none of the MRFs is ever expressed in the heart. Deep morphologic and transcriptomic characterization of the novel KOs under the paradigms of muscle regeneration, natural hypertrophy and denervation-induced atrophy will shed some light into how Mrf4 controls muscle growth and homeostasis.

Trabajo presentado en la 9th Conference of the International Coenzyme Q10 Association, celebrada en Nueva York (Estados Unidos) del 21 al 24 de junio de 2018., Sarcopenia, or muscle atrophy, is characterised by the progressive loss of muscle strength and activity. In an effort to identify modifiers of muscle atrophy, we have recently found that abolishing the function of the transcription factor Mrf4 in adult animals blocks denervation-induced muscle atrophy. Overexpression of Mrf4 has the opposite effect, resulting in the induction of muscle hypertrophy. By analysing transcriptomic profiles, we have identified the transcription factor Mef2C as the most repressed transcript following Mrf4 knockdown, with most of the genes downregulated corresponding to know Mef2C targets. In addition, we find that some of the genes differentially regulated in our knockdown experiments are involved in mitochondrial function, opening a window onto mitochondrial pathways directly implicated in muscle atrophy and hypertrophy. We have now generated several alleles KO for the Mrf4 gene in order to study the effect of Mrf4 loss from embryonic development. Preliminary data reinforce our findings in adult musculature but curiously shows that the effects upon the skeletal muscle transcriptome are very different, pointing towards a development-specific network regulated by Mrf4. These data show that Mrf4 is essential in muscle homeostasis and function, revealing new pathways in muscle atrophy/ hypertrophy and opening new opportunities for therapeutic intervention in muscle disease.

Trabajo presentado en Muscle Development, Regeneration and Disease Meeting, celebrado en Berlín (Alemania) del 22 al 27 de abril de 2018.

Resumen del póster presentado al 11th Meeting of the Spanish Society for Developmental Biology, celebrado en Girona (España) del 19 al 21 de octubre de 2016., Myogenic regulatory factors (MRFs) control the determination, specification and differentiation of skeletal muscle during development. Two of these factors, Myf5 and Mrf4, are closely linked in the genome of all vertebrates analyzed. Their transcriptional regulation relies on multiple enhancers that are intermingled and scattered throughout the locus. In this work, we try to unveil the mechanisms by which precise long-range interactions between enhancers and promoters are established; interactions that change during development and differentiation in order to allow the genes to be accurately expressed according to their highly specific spatiotemporal expression pattern. We hypothesize the existence of a novel type of regulatory element named TRABS (transcriptional balancing sequences) which, together with the promoters and enhancers of the locus, create a series of equilibria states that ensure the establishment of the correct enhancer-promoter regulatory interactions. To reveal the three-dimensional organization of the locus, 4C experiments were carried out in the muscle-derived cell line C2C12 both in growing and differentiating conditions, as well in embryos at different developmental stages and genotypes (including KOs for Mrf4 or Myf5 promoters and TRABS). The integration of all the generated datasets would improve our understanding of how this locus is organized and regulated during the formation of skeletal muscle throughout development.

Resumen del póster presentado al 2nd Meeting of the Portuguese Society for Developmental Biology, celebrado en Lisboa (Portugal) del 24 al 26 de octubre de 2013., The determination and specification of skeletal muscle in vertebrates is orchestrated by the myogenic regulatory factors (MRFs): Myf5, Mrf4, MyoD and Myogenin. Myf5 is the first to be expressed in the embryo, initiating and co-ordinating the myogenic cascade. In absence of Myf5 progenitors fail to be specified at the correct developmental stage. Activation of MyoD rescues the phenotype and myogenesis progresses. In Myf5/MyoD KO animals rescue does not take place and animals lack all skeletal muscle. We and others have shown that Myf5 transcription is controlled by over 25 regulatory elements, most of them drive expression in a specific spatiotemporal context. The Early Epaxial Enhancer (EEE) operates in the dermomyotomal dorsomedial lip (DML) and is the first enhancer to activate Myf5 expression at E8.5 Although the contribution of the different regulatory elements to the expression pattern is well defined, we still lack an understanding on the contribution of the different subpopulations of muscle progenitor cells to adult musculature. Furthermore, there is still no connection between the spatiotemporal activation of Myf5 and its function within the particular set of myogenic precursors in which it is active. In order to address these questions we are following two strategies: 1/ Generation of transgenic strains to analyse the function of the EEE by means of lineage tracing and cell ablation experiments and 2/ Generation of a new mouse knockout strain in which the EEE has been targeted. Comparison of Evolutionary Conserved Regions between different species shows two highly conserved peaks within the experimentally defined EEE element (EEE1 and EEE2). As these could represent different functions within the enhancer, we have cloned each peak in our standard vector and used to generate transgenic animals. Preliminary data show that EEE2 drives reporter gene expression at E8.5 and is turned off at E9.5, while EEE1 activates gene expression at E9.5. We are now transferring these refined enhancers into cell ablation and lineage tracing constructs. We are in the process of analyzing the data obtained by RNAseq from wild type and Myf5EEE-/EEE- embryos in order to determine the downstream targets of Myf5 in the DML. Crucially, we have crossed this new allele into the MyoD KO and show that in the absence of MyoD rescue, some muscles are lost (or severely reduced), linking for the first time a particular set of progenitors to specific adult muscles.

Resumen del póster presentado al 2nd Meeting of the Portuguese Society for Developmental Biology, celebrado en Lisboa (Portugal) del 24 al 26 de octubre de 2013.-- et al., Myogenic Regulatory Factors are a family of transcription factors essential for the determination, specification and differentiation of skeletal muscle during embryonic development. Two members of this family, Mrf4 and Myf5, are closely linked in all vertebrates analysed. We have studied the transcriptional regulation of Mrf4 and Myf5 extensively in transgenic mice and shown that a multitude of interdigitated enhancers regulate gene activation at different times and anatomical locations. Despite this complex architecture the enhancers are able to recognize their respective core promoters, and we have shown that a series of equilibria-states between transcription balancing sequences (trabs), promoters and the enhancers present in the locus is active in order to drive correct expression and transcriptional initiation. The proposed model is well supported by the cis-effects seen in the different knock out Mrf4 and Myf5 alleles created independently. We are investigating the nature of this new class of transcription element by standard cell culture and transgenic approaches. We are testing the hypothesis of the equilibria states by using chromosomal conformation capture (4C-seq) techniques on mouse embryos at different developmental stages. In addition, we are using the muscle cell line C2C12 (differentiated and undifferentiated) to identify nuclear matrix attachment regions within the locus and the possible changes in structure that it may experiment as differentiation takes place. The integration of the data sets obtained by these complementary approaches should provide a clear view of the contribution of trabs towards the global regulation of the locus as well as unveil other elements that may be involved in the global regulation.