Gabriela Morosan-Puopolo

Inhibitors of CXCR4 affect the migration and fate of CXCR4+ progenitors in the developing limb of chick embryos

Chemokines and their receptors play major roles in numerous physiological and pathological processes during development and disease. CXCR4 is the most abundantly expressed chemokine receptor during development. In contrast to other chemokine receptors, CXCR4 binds and is activated exclusively by its ligand stromal derived factor‐1 (SDF‐1) or CXCL12. SDF‐1 signaling has a wide range of effects on CXCR4‐expressing cells depending on the cell type ranging from cell growth to adhesion, chemotaxis, and migration. CXCR4 also serves as a co‐receptor for HIV‐1 entry into T‐cells and has been implicated in the pathogenesis of rheumatoid arthritis and cancer growth and invasion. Numerous inhibitors and antagonists of CXCR4 have been produced and are being tested for their efficiency to target its role in pathogenesis. Our initial expression analysis revealed that CXCR4 is expressed by the migrating myogenic and angiogenic precursors in the developing chick limb. In this study, we used the most specific peptidic inhibitors of CXCR4, T140 and its analog TN14003, to analyse the effect of blocking CXCR4/SDF‐1 signaling on the undetermined bioptent migratory progenitors in the developing chick limb. Our results point to defects in migration and an altered differentiation program of these CXCR4‐expressing progenitor pool in the limb. Developmental Dynamics 235:3007–3015, 2006.

Retrograde migration of pectoral girdle muscle precursors depends on CXCR4/SDF-1 signaling

In vertebrates, muscles of the pectoral girdle connect the forelimbs with the thorax. During development, the myogenic precursor cells migrate from the somites into the limb buds. Whereas most of the myogenic precursors remain in the limb bud to form the forelimb muscles, several cells migrate back toward the trunk to give rise to the superficial pectoral girdle muscles, such as the large pectoral muscle, the latissimus dorsi and the deltoid. Recently, this developing mode has been referred to as the “In–Out” mechanism. The present study focuses on the mechanisms of the “In–Out” migration during formation of the pectoral girdle muscles. Combining in ovo electroporation, tissue slice-cultures and confocal laser scanning microscopy, we visualize live in detail the retrograde migration of myogenic precursors from the forelimb bud into the trunk region by live imaging. Furthermore, we present for the first time evidence for the involvement of the chemokine receptor CXCR4 and its ligand SDF-1 during these processes. After microsurgical implantations of CXCR4 inhibitor beads in the proximal forelimb region of chicken embryos, we demonstrate with the aid of in situ hybridization and live-cell imaging that CXCR4/SDF-1 signaling is crucial for the retrograde migration of pectoral girdle muscle precursors. Moreover, we analyzed the MyoD expression in CXCR4-mutant mouse embryos and observed a considerable decrease in pectoral girdle musculature. We thus demonstrate the importance of the CXCR4/SDF-1 axis for the pectoral girdle muscle formation in avians and mammals.

ATOH8, a regulator of skeletal myogenesis in the hypaxial myotome of the trunk

Abstract The embryonic muscles of the axial skeleton and limbs take their origin from the dermomyotomes of the somites. During embryonic myogenesis, muscle precursors delaminate from the dermomyotome giving rise to the hypaxial and epaxial myotome. Mutant studies for myogenic regulatory factors have shown that the development of the hypaxial myotome differs from the formation of the epaxial myotome and that the development of the hypaxial myotome depends on the latter within the trunk region. The transcriptional networks that regulate the transition of proliferative dermomyotomal cells into the predominantly post-mitotic hypaxial myotome, as well as the eventual patterning of the myotome, are not fully understood. Similar transitions occurring during the development of the neural system have been shown to be controlled by the Atonal family of helix-loop-helix transcription factors. Here, we demonstrate that ATOH8, a member of the Atonal family, is expressed in a subset of embryonic muscle cells in the dermomyotome and myotome. Using the RNAi approach, we show that loss of ATOH8 in the lateral somites at the trunk level results in a blockage of differentiation and thus causes cells to be maintained in a predetermined state. Furthermore, we show that ATOH8 is also expressed in cultured C2C12 mouse myoblasts and becomes dramatically downregulated during their differentiation. We propose that ATOH8 plays a role during the transition of myoblasts from the proliferative phase to the differentiation phase and in the regulation of myogenesis in the hypaxial myotome of the trunk.

Diversification and molecular evolution of ATOH8, a gene encoding a bHLH transcription factor.

ATOH8 is a bHLH domain transcription factor implicated in the development of the nervous system, kidney, pancreas, retina and muscle. In the present study, we collected sequence of ATOH8 orthologues from 18 vertebrate species and 24 invertebrate species. The reconstruction of ATOH8 phylogeny and sequence analysis showed that this gene underwent notable divergences during evolution. For those vertebrate species investigated, we analyzed the gene structure and regulatory elements of ATOH8. We found that the bHLH domain of vertebrate ATOH8 was highly conserved. Mammals retained some specific amino acids in contrast to the non-mammalian orthologues. Mammals also developed another potential isoform, verified by a human expressed sequence tag (EST). Comparative genomic analyses of the regulatory elements revealed a replacement of the ancestral TATA box by CpG-islands in the eutherian mammals and an evolutionary tendency for TATA box reduction in vertebrates in general. We furthermore identified the region of the effective promoter of human ATOH8 which could drive the expression of EGFP reporter in the chicken embryo. In the opossum, both the coding region and regulatory elements of ATOH8 have some special features, such as the unique extended C-terminus encoded by the third exon and absence of both CpG islands and TATA elements in the regulatory region. Our gene mapping data showed that in human, ATOH8 was hosted in one chromosome which is a fusion product of two orthologous chromosomes in non-human primates. This unique chromosomal environment of human ATOH8 probably subjects its expression to the regulation at chromosomal level. We deduce that the great interspecific differences found in both ATOH8 gene sequence and its regulatory elements might be significant for the fine regulation of its spatiotemporal expression and roles of ATOH8, thus orchestrating its function in different tissues and organisms.

A novel role of CXCR4 and SDF-1 during migration of cloacal muscle precursors

The cloaca acts as a common chamber into which gastrointestinal and urogenital tracts converge in lower vertebrates. The distal end of the cloaca is guarded by a ring of cloacal muscles or sphincters, the equivalent of perineal muscles in mammals. It has recently been shown that the development of the cloacal musculature depends on hindlimb muscle formation. The signaling molecules responsible for the outward migration of hindlimb myogenic precursors are not known. Based on the expression studies for CXCR4 and SDF‐1, we hypothesized a role of this signaling pair during cloacal muscle precursor migration. The aim of our study was to investigate the role of SDF‐1/CXCR4 during cloacal muscle precursor migration in the chicken embryos. We show that SDF‐1 is expressed in the cloacal region, and by experimentally manipulating the SDF‐1/CXCR4 signaling, we can show that SDF‐1 guides the migration of CXCR4‐expressing cloacal muscle precursors. Developmental Dynamics 239:1622–1631, 2010.

Thymosin β4 induces folding of the developing optic tectum in the chicken (Gallus domesticus)

Thymosin β4 (Tβ4) is a highly conserved G‐actin binding polypeptide with multiple intra‐ and extracellular functions. While stem‐cell activation as well as promotion of cell survival and migration by Tβ4 have been investigated in various in vitro and in vivo studies, there are few data on the implications of Tβ4 in brain development. In the present study we analyzed Tβ4 expression in the developing optic tectum of the chicken (Gallus domesticus) and performed in ovo retroviral transduction and plasmid electroporation for overexpression and knockdown of Tβ4. We found marked Tβ4 expression in the tectal plate and in all neuronal layers of later developmental stages, but not in the ventricular zone where neural stem cells reside and divide. Knockdown of Tβ4 inhibited growth of Tβ4‐depleted hemispheres, whereas overexpression of Tβ4 led to the production of neuroepithelial folds resembling gyri and sulci, which are not normally present in avian brains. The mechanism yielding enhanced growth of Tβ4 overexpressing hemispheres involved enhanced proliferation, thus indicating an impact of Tβ4 on the neural stem cell and/or progenitor cell population. In summary, we found that due to its effects on proliferation, Tβ4 expression has a large impact on neuroepithelial and macroscopic brain development. J. Comp. Neurol. 520:1650–1662, 2012.

Wnt11 is required for oriented migration of dermogenic progenitor cells from the dorsomedial lip of the avian dermomyotome.

The embryonic origin of the dermis in vertebrates can be traced back to the dermomyotome of the somites, the lateral plate mesoderm and the neural crest. The dermal precursors directly overlying the neural tube display a unique dense arrangement and are the first to induce skin appendage formation in vertebrate embryos. These dermal precursor cells have been shown to derive from the dorsomedial lip of the dermomyotome (DML). Based on its expression pattern in the DML, Wnt11 is a candidate regulator of dorsal dermis formation. Using EGFP-based cell labelling and time-lapse imaging, we show that the Wnt11 expressing DML is the source of the dense dorsal dermis. Loss-of-function studies in chicken embryos show that Wnt11 is indeed essential for the formation of dense dermis competent to support cutaneous appendage formation. Our findings show that dermogenic progenitors cannot leave the DML to form dense dorsal dermis following Wnt11 silencing. No alterations were noticeable in the patterning or in the epithelial state of the dermomyotome including the DML. Furthermore, we show that Wnt11 expression is regulated in a manner similar to the previously described early dermal marker cDermo-1. The analysis of Wnt11 mutant mice exhibits an underdeveloped dorsal dermis and strongly supports our gene silencing data in chicken embryos. We conclude that Wnt11 is required for dense dermis and subsequent cutaneous appendage formation, by influencing the cell fate decision of the cells in the DML.

A thymosin beta15‑like peptide promotes intersegmental myotome extension in the chicken embryo

Abstract Beta-thymosins constitute a group of small actin-sequestering peptides. These highly conserved peptides are involved in cytoskeleton dynamics and can influence different cell properties such as motility, substrate adhesion, shape and chemotaxis. As a marker for tumour metastasis, the mammalian thymosin beta15 is believed to have an important diagnostic relevance in cancer prognosis, although little is known about its physiological function. In order to study the role of thymosin beta15avian in embryogenesis, we cloned the chicken and quail orthologues of thymosin beta15 and used the chicken as a model for vertebrate development. Avian thymosin beta15, the first known non-mammalian thymosin beta15-like gene, encodes a peptide that possesses a cysteine at position one after the methionine which is a significant difference compared to its mammalian counterparts. Thymosin beta15avian expression starts at an early stage of development. The expression pattern changes rapidly with development and differs from that of the related thymosin beta4 gene. The most prominent expression domain is seen in developing muscles of limbs and trunk. Gain-of-function experiments revealed that thymosin beta15avian has a function in normal myotome development. Ectopic over-expression of thymosin beta15avian leads to premature elongation of myotome cells trespassing segment borders. We conclude that thymosin beta15avian has a still undescribed function in promoting myocyte elongation.

Combination of in ovo electroporation and time-lapse imaging to study migrational events in chicken embryos.

Background: During embryonic development cell migration plays a principal role in several processes. In past decades, many studies were performed to investigate migrational events, occurring during embryonic organogenesis, neurogenesis, gliogenesis or myogenesis, just to name a few. Although different common techniques are already used for this purpose, one of their major limitations is the static character. However, cell migration is a sophisticated and highly dynamic process, wherefore new appropriate technologies are required to investigate this event in all its complexity. Results and Conclusions: Here we report a novel approach for dynamic analysis of cell migration within embryonic tissue. We combine the modern transfection method of in ovo electroporation with the use of tissue slice culture and state‐of‐the‐art imaging techniques, such as confocal laser scanning microscopy or spinning disc confocal microscopy, and thus, develop a method to study live the migration of myogenic precursors in chicken embryos. The conditions and parameters used in this study allow long‐term imaging for up to 24 hr. Our protocol can be easily adapted for investigations of a variety of other migrational events and provides a novel conception for dynamic analysis of migration during embryonic development. Developmental Dynamics 243:690–698, 2014.

Molecular cloning of chicken Cecr2 and its expression during chicken embryo development

Cecr2 is a transcription factor involved in neurulation and chromatin remodeling. In the present study, the full length of the coding sequence of the chicken orthologue Cecr2 was obtained by RT-PCR. Sequence analysis and alignment showed that it contained an AT hook, as well as a bromodomain which was highly conserved among different species, consistent with its role in chromatin remodeling. The expression pattern of chicken Cecr2 was subsequently investigated during the development of the chicken embryo by in situ hybridization. In addition to its predominant expression in neural tissues during neurulation, Cecr2 was also found to be expressed in the developing somites and in the intermediate zone of the spinal cord, suggesting that it may play a role in somite and neuronal development.