Immobilization secondary to cell death of muscle precursors with a dual transcriptional signature contributes to the emu wing skeletal pattern.

Limb reduction has occurred multiple times in tetrapod history. Among ratites, wing reductions range from mild vestigialization to complete loss, with emus (Dromaius novaehollandiae) serving as a model for studying the genetic mechanisms behind limb reduction. Here, we explore the developmental mechanisms underlying wing reduction in emu. Our analyses reveal that immobilization resulting from the absence of distal muscles contributes to skeletal shortening, fusion and left-right intraindividual variation. Expression analysis and single cell-RNA sequencing identify muscle progenitors displaying a dual lateral plate mesodermal and myogenic signature. These cells aggregate at the proximal region of wing buds and undergo cell death. We propose that this cell death, linked to the lack of distal muscle masses, underlines the morphological features and variability in skeletal elements due to reduced mechanical loading. Our results demonstrate that differential mobility during embryonic development may drive morphological diversification in vestigial structures.

Air-breathing behavior underlies the cell death in limbs of Rana pirica tadpoles.

Amphibians shape their limbs by differential outgrowth of digits and interdigital regions. In contrast, amniotes employ cell death, an additional developmental system, to determine the final shape of limbs. Previous work has shown that high oxygen availability is correlated with the induction of cell death in developing limbs. Given the diversity of life histories of amphibians, it is conceivable that some amphibians are exposed to a high-oxygen environment during the tadpole phase and exhibit cell death in their limbs. Here, we examined whether air-breathing behavior underlies the cell death in limbs of aquatic tadpoles of the frog species Rana pirica. Our experimental approach revealed that R. pirica tadpoles exhibit cell death in their limbs that is likely to be induced by oxidative stress associated with their frequent air-breathing behavior.

Intrinsic Angiogenic Potential and Migration Capacity of Human Mesenchymal Stromal Cells Derived from Menstrual Blood and Bone Marrow.

Several therapies are being developed to increase blood circulation in ischemic tissues. Despite bone marrow-derived mesenchymal stromal cells (bmMSC) are still the most studied, an interesting and less invasive MSC source is the menstrual blood, which has shown great angiogenic capabilities. Therefore, the aim of this study was to evaluate the angiogenic properties of menstrual blood-derived mesenchymal stromal cells (mbMSC) in vitro and in vivo and compared to bmMSC. MSC's intrinsic angiogenic capacity was assessed by sprouting and migration assays. mbMSC presented higher invasion and longer sprouts in 3D culture. Additionally, both MSC-spheroids showed cells expressing CD31. mbMSC and bmMSC were able to migrate after scratch wound in vitro, nonetheless, only mbMSC demonstrated ability to engraft in the chick embryo, migrating to perivascular, perineural, and chondrogenic regions. In order to study the paracrine effects, mbMSC and bmMSC conditioned mediums were capable of stimulating HUVEC's tube-like formation and migration. Both cells expressed VEGF-A and FGF2. Meanwhile, PDGF-B was expressed exclusively in mbMSC. Our results indicated that mbMSC and bmMSC presented a promising angiogenic potential. However, mbMSC seems to have additional advantages since it can be obtained by non-invasive procedure and expresses PDGF-B, an important molecule for vascular formation and remodeling.

Environmental Oxygen is a Key Modulator of Development and Evolution: From Molecules to Ecology: Oxygen-sensitive pathways pattern the developing organism, linking genetic and environmental components during the evolution of new traits.

Oxygen is a key regulator of both development and homeostasis and a promising candidate to bridge the influence of the environment and the evolution of new traits. To clarify the various ways in which oxygen may modulate embryogenesis, its effects are reviewed at distinct organizational levels. First, the role of pathways that sense dioxygen levels and reactive oxygen species are reviewed. Then, the effects of microenvironmental oxygen on metabolism, stemness, and differentiation throughout embryogenesis are discussed. Last, the interplay between ecology and development are reexamined with a focus on the evolution of tetrapods, including during the emergence of a novel mechanism that shapes amniote limbs-interdigital cell death. Both genetic and environmental components work together during the formation of organisms, highlighting the importance of a multidisciplinary approach for understanding the evolution of new traits.

Evolution of the avian digital pattern.

Variation in digit number has occurred multiple times in the history of archosaur evolution. The five digits of dinosaur limbs were reduced to three in bird forelimbs, and were further reduced in the vestigial forelimbs of the emu. Regulation of digit number has been investigated previously by examining genes involved in anterior-posterior patterning in forelimb buds among emu (Dromaius novaehollandiae), chicken (Gallus gallus) and zebra finch (Taeniopygia guttata). It was described that the expression of posterior genes are conserved among these three birds, whereas expression of anterior genes Gli3 and Alx4 varied significantly. Here we re-examined the expression pattern of Gli3 and Alx4 in the forelimb of emu, chicken and zebra finch. We found that Gli3 is expressed in the anterior region, although its range varied among species, and that the expression pattern of Alx4 in forelimb buds is broadly conserved in a stage-specific manner. We also found that the dynamic expression pattern of the BMP antagonist Gremlin1 (Grem1) in limb buds, which is critical for autopodial expansion, was consistent with the digital pattern of emu, chicken and zebra finch. Furthermore, in emu, variation among individuals was observed in the width of Grem1 expression in forelimb buds, as well as in the adult skeletal pattern. Our results support the view that the signalling system that regulates the dynamic expression of Grem1 in the limb bud contributes substantially to variations in avian digital patterns.

Environmental Oxygen Exposure Allows for the Evolution of Interdigital Cell Death in Limb Patterning.

Amphibians form fingers without webbing by differential growth between digital and interdigital regions. Amniotes, however, employ interdigital cell death (ICD), an additional mechanism that contributes to a greater variation of limb shapes. Here, we investigate the role of environmental oxygen in the evolution of ICD in tetrapods. While cell death is restricted to the limb margin in amphibians with aquatic tadpoles, Eleutherodactylus coqui, a frog with terrestrial-direct-developing eggs, has cell death in the interdigital region. Chicken requires sufficient oxygen and reactive oxygen species to induce cell death, with the oxygen tension profile itself being distinct between the limbs of chicken and Xenopus laevis frogs. Notably, increasing blood vessel density in X. laevis limbs, as well as incubating tadpoles under high oxygen levels, induces ICD. We propose that the oxygen available to terrestrial eggs was an ecological feature crucial for the evolution of ICD, made possible by conserved autopod-patterning mechanisms.

Cux2 refines the forelimb field by controlling expression of Raldh2 and Hox genes.

In vertebrates, two pairs of buds that give rise to the fore- and hindlimbs form at discrete positions along the rostral-caudal axis of the body. The mechanism responsible for the positioning of the limb buds is still largely unknown. Here we show a novel function for Cut homeobox transcription factor 2 (Cux2), the ortholog of Drosophila cut, in refining the forelimb field during chick development. Cux2 is expressed in the forelimb field before the emergence of the limb buds. Knocking down the expression of Cux2 using small interfering RNA (siRNA) resulted in a caudal shift of the forelimb bud, whereas misexpression of Cux2 or the constitutively active Cux2-VP16 caused a rostral shift of the forelimb bud or reduction of the forelimb field along the anterior-posterior axis. Further functional analyses revealed that expression of Hoxb genes and retinaldehyde dehydrogenase 2 (Raldh2), which are involved in limb positioning, are directly activated by Cux2 in the lateral plate mesoderm. Our data suggest that Cux2 in the lateral plate mesoderm refines the forelimb field via regulation of Raldh2 and Hoxb genes in chicken embryos.