Unravelling the function of Myf5 in the developing limb musculature

Motivation: Myogenesis is a complex process controlled by different networks, depending on the origin of different muscles, and that’s why there are different types of muscle distrophies. However, the myogenic cascade is always regulated by the same Myogenic Regulatory Factors (MRFs): Myf5, Mrf4, MyoD and MyoG. These transcription factors bind DNA and activate the expression of specific genes in particular progenitor cells that will give rise to the different muscles in the adult body. The MRFs’ cascade is initiated by Myf5, the first MRF to be expressed in the embryo. We and others have extensively studied the complex transcriptional regulation of the Myf5/Mrf4 locus using transgenic mice. Nowadays, we know that there are more than 25 regulatory elements controlling the expression of Mrf4 and Myf5 in a specific time frame and in particular embryonic progenitors.The limb enhancer is located 57 kb upstream of the Myf5 transcriptional start site. This enhancer controls Myf5 expression in limbs during development. While the mechanisms involved in the spatiotemporal regulation of Myf5 have been extensively studied at single-enhancer and global regulatory levels, the function of Myf5 in different subpopulations of muscle progenitor cells is still not clear. This project focuses on the characterization of a new allele in which the limb enhancer has been removed from the genome.Methods: To unravel the function of Myf5 in the developing limb musculature we have generated a new knock-out (KO) allele with CRISPR/Cas9 in which the limb enhancer has been targeted. Then, we have prepared RNA probes for In Situ Hybridisation (ISH) of genes that are expressed in limbs and are potential targets of Myf5 in other muscle progenitors (López-Mayorga et al., unpublished data), to test if the expression patters of these genes are modified in the KO allele. We are going to study three different embryonic stages: 10.5, 11.5 and 12.5 days post-coitum (dpc) by ISH using KO and wild type (WT) embryos. This time window was chosen to maximise the probability of detecting any pattern changes before the phenotype is rescued by the activity of MyoD, as previously shown. We are also preparing total RNA of fore-limbs from KO and WT embryos at 11.5 dpc to perform microarrays, which will give us some information about the genes regulated by Myf5 direct or indirectly. Finally, we will validate the results from microarrays by ISH and qPCR

Evolutionary comparison reveals that diverging CTCF sites are signatures of ancestral topological associating domains borders

Increasing evidence in the last years indicates that the vast amount of regulatory information contained in mammalian genomes is organized in precise 3D chromatin structures. However, the impact of this spatial chromatin organization on gene expression and its degree of evolutionary conservation is still poorly understood. The Six homeobox genes are essential developmental regulators organized in gene clusters conserved during evolution. Here, we reveal that the Six clusters share a deeply evolutionarily conserved 3D chromatin organization that predates the Cambrian explosion. This chromatin architecture generates two largely independent regulatory landscapes (RLs) contained in two adjacent topological associating domains (TADs). By disrupting the conserved TAD border in one of the zebrafish Six clusters, we demonstrate that this border is critical for preventing competition between promoters and enhancers located in separated RLs, thereby generating different expression patterns in genes located in close genomic proximity. Moreover, evolutionary comparison of Six-associated TAD borders reveals the presence of CCCTC-binding factor (CTCF) sites with diverging orientations in all studied deuterostomes. Genome-wide examination of mammalian HiC data reveals that this conserved CTCF configuration is a general signature of TAD borders, underscoring that common organizational principles underlie TAD compartmentalization in deuterostome evolution

Gestión del conocimiento: perspectiva multidisciplinaria. Volumen 11

El libro “Gestión del Conocimiento. Perspectiva Multidisciplinaria”, Volumen 11, de la Colección Unión Global, es resultado de investigaciones. Los capítulos del libro, son resultados de investigaciones desarrolladas por sus autores. El libro cuenta con el apoyo de los grupos de investigación: Universidad Sur del Lago “Jesús María Semprúm” (UNESUR), Zulia – Venezuela; Universidad Politécnica Territorial de Falcón Alonso Gamero (UPTAG), Falcón – Venezuela; Universidad Politécnica Territorial de Mérida Kleber Ramírez (UPTM), Mérida – Venezuela; Universidad Guanajuato (UG) - Campus Celaya - Salvatierra - Cuerpo Académico de Biodesarrollo y Bioeconomía en las Organizaciones y Políticas Públicas (C.A.B.B.O.P.P), Guanajuato – México; Centro de Altos Estudios de Venezuela (CEALEVE), Zulia – Venezuela, Centro Integral de Formación Educativa Especializada del Sur (CIFE - SUR) - Zulia - Venezuela, Centro de Investigaciones Internacionales SAS (CIN), Antioquia - Colombia.y diferentes grupos de investigación del ámbito nacional e internacional que hoy se unen para estrechar vínculos investigativos, para que sus aportes científicos formen parte de los libros que se publiquen en formatos digital e impreso

Skeletal muscle: CRISPR/Cas9 approaches to study embryonic development and human disease

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

Global transcriptional regulation of the Mrf4/Myf5 locus

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.Peer Reviewe

Genetic control of akeletal muscle hypertrophy

Resumen del trabajo presentado en el European Developmental Biology Congress, celebrado en Alicante (España), del 23 al 26 de octubre de 2019The 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

Characterization of new Mrf4 KOs

Motivation: The myogenic regulatory factors (MRFs) are a family of basic-helix-loop-helix (bHLH) transcription factors, essential for skeletal muscle development, homeostasis and regeneration. There are four MRFs: MyoD, Myf5, Mrf4 and MyoG. During this project we will focus on Mrf4. Mrf4 is first expressed in the mouse embryo at 9.5 days post-coitum (dpc) in undifferenciated cells of the ventral somitic region, extending to other somites by 10 dpc. Its expression is down-regulated from 12.5 dpc, up-regulated in a second foetal phase and then maintained in all adult skeletal muscles. Mrf4 is linked to Myf5 in mammals. The organization of the Mrf4/Myf5 locus, in which the regulatory elements are interdigitated, requires a series of equilibria, to maintain the specific expression pattern of each gene and avoid the cross-interaction between their enhancers and promoters. This complex equilibrium has hindered the study of their individual function, as the modification of some of the elements in the locus affect the expression of not only one but both genes. This is specially evident in the case of previous Mrf4 knock-outs, in which three different phenotypes were obtained varying from birth lethality to complete viability. We have previously described the effect of Mrf4 Knock-down mediated by iRNA electroporation of skeletal muscle in adult rats. During this project we have performed a preliminary characterisation of three new Mrf4 KO alleles, recently generated in the lab by CRISPR/Cas9 targeting the Mrf4 minimal promoter (MP4 allele), or its first exon (112-KO alleles). With this approach we expect to unravel the role of Mrf4 during mouse embryonic development and in adult muscles. Methods: To study the effect of inactivating Mrf4 on the expression pattern of the other MRFs we have performed In Situ Hybridisation (ISH) using 10.5 and 11.5 dpc embryos. We will study RNA expression levels by qPCR at four embryonic stages (10.5, 11.5, 12.5 and 16.5 dpc) and we are also looking for possible skeletal phenotypes by bone staining in fetuses at 18.5 dpc. Finally, we are studying how these three different KO affect adult skeletal muscles in normal conditions and an induced atrophy model. We have obtained Soleus, Tibialis Anterior (TA), and Extensor digitorum longus (EDL) leg muscles from adult WT and mutant mice and we are characterising putative fiber type changes usin

Relationship between dorsal muscle progenitors and adult musculature

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.Peer Reviewe

Lack of MRF4 during embryonic development leads to muscle hypertrophy in the adult

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

New roles of MYF5 in dorsal somitic progenitors

Resumen del trabajo presentado en el European Developmental Biology Congress, celebrado en Alicante (España), del 23 al 26 de octubre de 2019Skeletal 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