Macrophages allocate before apoptosis initiation and produce reactive oxygen species during interdigital phagocytosis.

During programmed cell death (PCD), it is commonly accepted that macrophages are recruited by apoptotic cells to complete cell degradation. Interdigital cell death, a classical model of PCD, contributes to digit individualization in limbs of mammals and other vertebrates. Here, we show that macrophages are present in interdigits before significant cell death occurs and remain after apoptosis inhibition. The typical interdigital phagocytic activity was not observed after a partial depletion of macrophages and was markedly reduced by engulfment/phagosome maturation inhibition, as detected by its association with high lysosomal activity. ß-galactosidase activity in this region was also coupled with phagocytosis, against its relationship with cellular senescence. Interdigital phagocytosis correlated with high levels of reactive oxygen species (ROS), common in embryo regions carrying abundant cell death, suggesting that macrophages are the major source of ROS. ROS generation was dependent on NADPH oxidases and blood vessel integrity, but not directly associated with lysosomal activity. Therefore, macrophages prepattern regions where abundant cell death is going to occur, and high lysosomal activity and the generation of ROS by an oxidative burst-like phenomenon are activities of phagocytosis.

Influence of DNA-methylation at multiple stages of limb chondrogenesis.

Precise regulation of gene expression is of utmost importance during cell fate specification. DNA methylation is a key epigenetic mechanism that plays a significant role in the regulation of cell fate by recruiting repression proteins or inhibiting the binding of transcription factors to DNA to regulate gene expression. Limb development is a well-established model for understanding cell fate decisions, and the formation of skeletal elements is coordinated through a sequence of events that control chondrogenesis spatiotemporally. It has been established that epigenetic control participates in cartilage maturation. However, further investigation is required to determine its role in the earliest stages of chondrocyte differentiation. This study investigates how the DNA methylation environment affects cell fate divergence during the early chondrogenic events. Our research has shown for the first time that inhibiting DNA methylation in interdigital tissue with 5-azacytidine results in the formation of an ectopic digit. This discovery suggested that DNA methylation dynamics could regulate the fate of cells between chondrogenesis and cell death during autopod development. Our in vitro findings indicate that DNA methylation at the early stages of chondrogenesis is integral in regulating condensation by controlling cell adhesion and proapoptotic genes. As a result, the dynamics of methylation and demethylation are crucial in governing chondrogenesis and cell death during different stages of limb chondrogenesis.

Current research on mechanisms of limb bud development, and challenges for the next decade.

The developmental mechanisms of limb buds have been studied in developmental biology as an excellent model of pattern formation. Chick embryos have contributed to the discovery of new principles in developmental biology, as it is easy to observe live embryos and manipulate embryonic tissues. Herein, I outline recent findings and future issues over the next decade regarding three themes, based on my research: limb positioning, proximal-distal limb elongation and digit identity determination. First, how hindlimb position is determined at the molecular level is described, with a focus on the transforming growth factor-ß signaling molecule GDF11. Second, I explain how the cell population in the limb bud deforms with developmental progress, shaping the limb bud with elongation along the proximal-distal axis. Finally, I describe the developmental mechanisms that determine digit identity through the interdigits.

Apical ectodermal ridge regulates three principal axes of the developing limb / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Understanding limb development not only gives insights into the outgrowth and differentiation of the limb, but also has clinical relevance. Limb development begins with two paired limb buds (forelimb and hindlimb buds), which are initially undifferentiated mesenchymal cells tipped with a thickening of the ectoderm, termed the apical ectodermal ridge (AER). As a transitional embryonic structure, the AER undergoes four stages and contributes to multiple axes of limb development through the coordination of signalling centres, feedback loops, and other cell activities by secretory signalling and the activation of gene expression. Within the scope of proximodistal patterning, it is understood that while fibroblast growth factors (FGFs) function sequentially over time as primary components of the AER signalling process, there is still no consensus on models that would explain proximodistal patterning itself. In anteroposterior patterning, the AER has a dual-direction regulation by which it promotes the sonic hedgehog (Shh) gene expression in the zone of polarizing activity (ZPA) for proliferation, and inhibits Shh expression in the anterior mesenchyme. In dorsoventral patterning, the AER activates Engrailed-1 (En1) expression, and thus represses Wnt family member 7a (Wnt7a) expression in the ventral ectoderm by the expression of Fgfs, Sp6/8, and bone morphogenetic protein (Bmp) genes. The AER also plays a vital role in shaping the individual digits, since levels of Fgf4/8 and Bmps expressed in the AER affect digit patterning by controlling apoptosis. In summary, the knowledge of crosstalk within AER among the three main axes is essential to understand limb growth and pattern formation, as the development of its areas proceeds simultaneously.

Apical ectodermal ridge regulates three principal axes of the developing limb / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Understanding limb development not only gives insights into the outgrowth and differentiation of the limb, but also has clinical relevance. Limb development begins with two paired limb buds (forelimb and hindlimb buds), which are initially undifferentiated mesenchymal cells tipped with a thickening of the ectoderm, termed the apical ectodermal ridge (AER). As a transitional embryonic structure, the AER undergoes four stages and contributes to multiple axes of limb development through the coordination of signalling centres, feedback loops, and other cell activities by secretory signalling and the activation of gene expression. Within the scope of proximodistal patterning, it is understood that while fibroblast growth factors (FGFs) function sequentially over time as primary components of the AER signalling process, there is still no consensus on models that would explain proximodistal patterning itself. In anteroposterior patterning, the AER has a dual-direction regulation by which it promotes the sonic hedgehog (Shh) gene expression in the zone of polarizing activity (ZPA) for proliferation, and inhibits Shh expression in the anterior mesenchyme. In dorsoventral patterning, the AER activates Engrailed-1 (En1) expression, and thus represses Wnt family member 7a (Wnt7a) expression in the ventral ectoderm by the expression of Fgfs, Sp6/8, and bone morphogenetic protein (Bmp) genes. The AER also plays a vital role in shaping the individual digits, since levels of Fgf4/8 and Bmps expressed in the AER affect digit patterning by controlling apoptosis. In summary, the knowledge of crosstalk within AER among the three main axes is essential to understand limb growth and pattern formation, as the development of its areas proceeds simultaneously.

Changes of Apoptosis After X-irradiation During the Hindlimb Development in the Rat Fetus / 체질인류학회지

Ionizing radiation exerts harmful effect during the limb development, but the exact mechanism is still largely unknown. In this study, 2 Gy of X-ray irradiated to the rat fetuses on gestation day of 13.7 when the hindlimb buds appear, and sacrificed at GD 14.7, GD 15.7 and GD 16.7, respectively. To reveal the changes of apoptotic figures between control and experimental groups, TUNEL immunohistochemistry and confocal laser scanning microscopy were carried. Mean body weight of fetuses of irradiated groups were decreased significantly compared to the control group. Numerical digit anomalies and asymmetries between right and left sides were increased significantly in the irradiated group compared to control group. Some digit anomalies were increased significantly in the right side. Radiation-induced decrement of the density of apoptotic figures on GD 14.7 was presumed to be related with foot and digit anomalies.

EFFECT OF DIFFERENT CONCENTRATION OF TRICHOSTATIN A ON CHICKEN WING BUD DEVELOPMENT / 解剖学报

Objective To investigate the reaction of the limbs treated with different concentration of Trichostatin A(75?mol/L,750 ?mol/L,1.5mmol/L),a histone deacetylase(HDAC)-inhibitor.This may be useful to improve our understanding of the role of chromatin remodelling and epigenetic control of gene expression patterns and ultimately the development of drugs against cancer. Methods Using the chicken embryonic limb as an experimental model.The embryos received grafts of TSA soaked beads or PBS control beads into the right forelimb buds.Then they were submitted to in situ hybridization with probes and apoptosis test.Results TSA could modulate the expression of some myogenesis related genes,MyoD and Myf5 during chicken myogenesis.Using apoptosis staining methods,there was no significant apoptosis in the TSA(75?mol/L) treated embryos.However the induction of morphological changes and apoptosis at specific stage was possible with high concentration of TSA.Conclusion TSA(75?mol/L) regulates certain important transcriptional targets and strongly control muscle differentiation.Increasing the concentration of TSA(≥750?mol/L) can induce apoptosis and embryonic limb malformations.Chicken limb development can serve as a convenient in vivo model for studying the effect of HDAC inhibitors.