Lúcia Elvira Alvares

Intrinsic, Hox-dependent cues determine the fate of skeletal muscle precursors.

It is generally held that vertebrate muscle precursors depend totally on environmental cues for their development. We show that instead, somites are predisposed toward a particular myogenic program. This predisposition depends on the somites axial identity: when flank somites are transformed into limb-level somites, either by shifting somitic boundaries with FGF8 or by overexpressing posterior Hox genes, they readily activate the program typical for migratory limb muscle precursors. The intrinsic control over myogenic programs can only be overridden by FGF4 signals provided by the apical ectodermal ridge of a developing limb.

Neural tube derived signals and Fgf8 act antagonistically to specify eye versus mandibular arch muscles

Recent knockout experiments in the mouse generated amazing craniofacial skeletal muscle phenotypes. Yet none of the genes could be placed into a molecular network, because the programme to control the development of muscles in the head is not known. Here we show that antagonistic signals from the neural tube and the branchial arches specify extraocular versus branchiomeric muscles. Moreover, we identified Fgf8 as the branchial arch derived signal. However, this molecule has an additional function in supporting the proliferative state of myoblasts, suppressing their differentiation, while a further branchial arch derived signal, namely Bmp7, is an overall negative regulator of head myogenesis.

Evolutionarily conserved morphogenetic movements at the vertebrate head–trunk interface coordinate the transport and assembly of hypopharyngeal structures

The vertebrate head–trunk interface (occipital region) has been heavily remodelled during evolution, and its development is still poorly understood. In extant jawed vertebrates, this region provides muscle precursors for the throat and tongue (hypopharyngeal/hypobranchial/hypoglossal muscle precursors, HMP) that take a stereotype path rostrally along the pharynx and are thought to reach their target sites via active migration. Yet, this projection pattern emerged in jawless vertebrates before the evolution of migratory muscle precursors. This suggests that a so far elusive, more basic transport mechanism must have existed and may still be traceable today. Here we show for the first time that all occipital tissues participate in well-conserved cell movements. These cell movements are spearheaded by the occipital lateral mesoderm and ectoderm that split into two streams. The rostrally directed stream projects along the floor of the pharynx and reaches as far rostrally as the floor of the mandibular arch and outflow tract of the heart. Notably, this stream leads and engulfs the later emerging HMP, neural crest cells and hypoglossal nerve. When we (i) attempted to redirect hypobranchial/hypoglossal muscle precursors towards various attractants, (ii) placed non-migratory muscle precursors into the occipital environment or (iii) molecularly or (iv) genetically rendered muscle precursors non-migratory, they still followed the trajectory set by the occipital lateral mesoderm and ectoderm. Thus, we have discovered evolutionarily conserved morphogenetic movements, driven by the occipital lateral mesoderm and ectoderm, that ensure cell transport and organ assembly at the head–trunk interface.

An evolutionarily conserved Myostatin proximal promoter/enhancer confers basal levels of transcription and spatial specificity in vivo.

Myostatin (Mstn) is a negative regulator of skeletal muscle mass, and Mstn mutations are responsible for the double muscling phenotype observed in many animal species. Moreover, Mstn is a positive regulator of adult muscle stem cell (satellite cell) quiescence, and hence, Mstn is being targeted in therapeutic approaches to muscle diseases. In order to better understand the mechanisms underlying Mstn regulation, we searched for the gene’s proximal enhancer and promoter elements, using an evolutionary approach. We identified a 260-bp-long, evolutionary conserved region upstream of tetrapod Mstn and teleost mstn b genes. This region contains binding sites for TATA binding protein, Meis1, NF-Y, and for CREB family members, suggesting the involvement of cAMP in Myostatin regulation. The conserved fragment was able to drive reporter gene expression in C2C12 cells in vitro and in chicken somites in vivo; both normally express Mstn. In contrast, the reporter construct remained silent in the avian neural tube that normally does not express Mstn. This suggests that the identified element serves as a minimal promoter, harboring some spatial specificity. Finally, using bioinformatic approaches, we identified additional genes in the human genome associated with sequences similar to the Mstn proximal promoter/enhancer. Among them are genes important for myogenesis. This suggests that Mstn and these genes may form a synexpression group, regulated by a common signaling pathway.

Elastic Fiber Assembly in the Adult Mouse Pubic Symphysis During Pregnancy and Postpartum

ABSTRACT Impairment of pelvic organ support has been described in mice with genetic modifications of the proteins involved in elastogenesis, such as lysyl oxidase-like 1 (LOXL1) and fibulin 5. During pregnancy, elastic fiber-enriched pelvic tissues are modified to allow safe delivery. In addition, the mouse pubic symphysis is remodeled in a hormone-controlled process that entails the modification of the fibrocartilage into an interpubic ligament (IpL) and the relaxation of this ligament. After first parturition, recovery occurs to ensure pelvic tissue homeostasis. Because ligaments are the main supports of the pelvic organs, this study aimed to evaluate elastogenesis in the IpL during mouse pregnancy and postpartum. Accordingly, virgin, pregnant, and postpartum C57BL/6 mice were studied using light, confocal, and transmission electron microscopy as well as Western blots and real-time PCR. Female mice exhibited the separation of the pubic bones and the formation, relaxation, and postpartum recovery of the IpL. By the time the IpL was formed, the elastic fibers had increased in profile length and diameter, and they consisted of small conglomerates of amorphous material distributed among the bundles of microfibrils. Our analyses also indicated that elastin/tropoelastin, fibrillin 1, LOXL1/Loxl1, and fibulin 5 were spatially and temporally regulated, suggesting that these molecules may contribute to the synthesis of new elastic fibers during IpL development. Overall, this work revealed that adult elastogenesis may be important to assure the elasticity of the pelvic girdle during preparation for parturition and postpartum recovery. This finding may contribute to our understanding of pathological processes involving elastogenesis in the reproductive tract.

Conservation of gene linkage in dispersed vertebrate NK homeobox clusters

Nk homeobox genes are important regulators of many different developmental processes including muscle, heart, central nervous system and sensory organ development. They are thought to have arisen as part of the ANTP megacluster, which also gave rise to Hox and ParaHox genes, and at least some NK genes remain tightly linked in all animals examined so far. The protostome–deuterostome ancestor probably contained a cluster of nine Nk genes: (Msx)–(Nk4/tinman)–(Nk3/bagpipe)–(Lbx/ladybird)–(Tlx/c15)–(Nk7)–(Nk6/hgtx)–(Nk1/slouch)–(Nk5/Hmx). Of these genes, only NKX2.6–NKX3.1, LBX1–TLX1 and LBX2–TLX2 remain tightly linked in humans. However, it is currently unclear whether this is unique to the human genome as we do not know which of these Nk genes are clustered in other vertebrates. This makes it difficult to assess whether the remaining linkages are due to selective pressures or because chance rearrangements have “missed” certain genes. In this paper, we identify all of the paralogs of these ancestrally clustered NK genes in several distinct vertebrates. We demonstrate that tight linkages of Lbx1–Tlx1, Lbx2–Tlx2 and Nkx3.1–Nkx2.6 have been widely maintained in both the ray-finned and lobe-finned fish lineages. Moreover, the recently duplicated Hmx2–Hmx3 genes are also tightly linked. Finally, we show that Lbx1–Tlx1 and Hmx2–Hmx3 are flanked by highly conserved noncoding elements, suggesting that shared regulatory regions may have resulted in evolutionary pressure to maintain these linkages. Consistent with this, these pairs of genes have overlapping expression domains. In contrast, Lbx2–Tlx2 and Nkx3.1–Nkx2.6, which do not seem to be coexpressed, are also not associated with conserved noncoding sequences, suggesting that an alternative mechanism may be responsible for the continued clustering of these genes.

Identification of enhancer-like elements in the ribosomal intergenic spacer of Odontophrynus americanus 2n and 4n (Amphibia, Anura).

Repetitive elements are found in the ribosomal intergenic spacer (IGS) of most organisms. A particularly complex pattern of internal repetition occurs in the IGSs of O. americanus 2n and 4n, which are composed of several types of BamHI subrepeats (B-SRs). The most repetitive one is approximately 87 bp long, and is highly represented in the IGS variants of these amphibians. Sequence analyses of six diploid and two tetraploid B-SRs show 87% and 86% homology, respectively, and related secondary structure predictions. The comparison of the 2n and 4n B-SR sequences aligned with the 81 bp enhancer of Xenopus laevis reveals 36% homology. Furthermore, other B-SR features like size, number, and secondary structures resemble those of Xenopus enhancers, suggesting that B-SRs may function as regulators of O. americanus rDNA transcription. The present data also corroborate the close evolutionary relationship between 2n and 4n O. americanus species.

Chicken Dapper genes are versatile markers for mesodermal tissues, embryonic muscle stem cells, neural crest cells, and neurogenic placodes

Dapper (Dpr) proteins are context‐dependent regulators of Wnt and Tgfβ signaling. However, although inroads into their molecular properties have been made, their expression and biological function are not understood. Searching for avian Dpr genes, we found that the chicken harbors a Dpr1 and a Dpr2 paralogue only. The genes are expressed in distinct patterns at gastrulation, neurulation, and organogenesis stages of development with key expression domains being the posterior primitive streak, anterior node and notochord, presomitic mesoderm (segmental plate), lateral and cardiac mesoderm, limb mesenchyme, and neurogenic placodes for Dpr1, and anterior primitive streak, node, epithelial somites, embryonic muscle stem cells, oral ectoderm and endoderm, neural crest cells, limb ectoderm, and lung buds for Dpr2. Expression overlaps in a few tissues; however, in several tissues, expression is complementary. Developmental Dynamics 238:1166–1178, 2009.

Dact genes are chordate specific regulators at the intersection of Wnt and Tgf-β signaling pathways

BackgroundDacts are multi-domain adaptor proteins. They have been implicated in Wnt and Tgfβ signaling and serve as a nodal point in regulating many cellular activities. Dact genes have so far only been identified in bony vertebrates. Also, the number of Dact genes in a given species, the number and roles of protein motifs and functional domains, and the overlap of gene expression domains are all not clear. To address these problems, we have taken an evolutionary approach, screening for Dact genes in the animal kingdom and establishing their phylogeny and the synteny of Dact loci. Furthermore, we performed a deep analysis of the various Dact protein motifs and compared the expression patterns of different Dacts.ResultsOur study identified previously not recognized dact genes and showed that they evolved late in the deuterostome lineage. In gnathostomes, four Dact genes were generated by the two rounds of whole genome duplication in the vertebrate ancestor, with Dact1/3 and Dact2/4, respectively, arising from the two genes generated during the first genome duplication. In actinopterygians, a further dact4r gene arose from retrotranscription. The third genome duplication in the teleost ancestor, and subsequent gene loss in most gnathostome lineages left extant species with a subset of Dact genes. The distribution of functional domains suggests that the ancestral Dact function lied with Wnt signaling, and a role in Tgfβ signaling may have emerged with the Dact2/4 ancestor. Motif reduction, in particular in Dact4, suggests that this protein may counteract the function of the other Dacts. Dact genes were expressed in both distinct and overlapping domains, suggesting possible combinatorial function.ConclusionsThe gnathostome Dact gene family comprises four members, derived from a chordate-specific ancestor. The ability to control Wnt signaling seems to be part of the ancestral repertoire of Dact functions, while the ability to inhibit Tgfβ signaling and to carry out specialized, ortholog-specific roles may have evolved later. The complement of Dact genes coexpressed in a tissue provides a complex way to fine-tune Wnt and Tgfβ signaling. Our work provides the basis for future structural and functional studies aimed at unraveling intracellular regulatory networks.

Dact gene expression profiles suggest a role for this gene family in integrating Wnt and TGF‐β signaling pathways during chicken limb development

Background: Dact gene family encodes multifunctional proteins that are important modulators of Wnt and TGF‐β signaling pathways. Given that these pathways coordinate multiple steps of limb development, we investigated the expression pattern of the two chicken Dact genes (Dact1 and Dact2) from early limb bud up to stages when several tissues are differentiating. Results: During early limb development (HH24‐HH30) Dact1 and Dact2 were mainly expressed in the cartilaginous rudiments of the appendicular skeleton and perichondrium, presenting expression profiles related, but distinct. At later stages of development (HH31–HH35), the main sites of Dact1 and Dact2 expression were the developing synovial joints. In this context, Dact1 expression was shown to co‐localize with regions enriched in the nuclear β‐catenin protein, such as developing joint capsule and interzone. In contrast, Dact2 expression was restricted to the interzone surrounding the domains of bmpR‐1b expression, a TGF‐β receptor with crucial roles during digit morphogenesis. Additional sites of Dact expression were the developing tendons and digit blastemas. Conclusions: Our data indicate that Dact genes are good candidates to modulate and, possibly, integrate Wnt and TGF‐β signaling during limb development, bringing new and interesting perspectives about the roles of Dact molecules in limb birth defects and human diseases. Developmental Dynamics 243:428–439, 2014.