Conserved regulatory switches for the transition from natal down to juvenile feather in birds.

The transition from natal downs for heat conservation to juvenile feathers for simple flight is a remarkable environmental adaptation process in avian evolution. However, the underlying epigenetic mechanism for this primary feather transition is mostly unknown. Here we conducted time-ordered gene co-expression network construction, epigenetic analysis, and functional perturbations in developing feather follicles to elucidate four downy-juvenile feather transition events. We report that extracellular matrix reorganization leads to peripheral pulp formation, which mediates epithelial-mesenchymal interactions for branching morphogenesis. α-SMA (ACTA2) compartmentalizes dermal papilla stem cells for feather renewal cycling. LEF1 works as a key hub of Wnt signaling to build rachis and converts radial downy to bilateral symmetry. Novel usage of scale keratins strengthens feather sheath with SOX14 as the epigenetic regulator. We show that this primary feather transition is largely conserved in chicken (precocial) and zebra finch (altricial) and discuss the possibility that this evolutionary adaptation process started in feathered dinosaurs.

Transition from natal downs to juvenile feathers: conserved regulatory switches in Neoaves.

The transition from natal downs for heat conservation to juvenile feathers for simple flight is a remarkable environmental adaptation process in avian evolution. However, the underlying epigenetic mechanism for this primary feather transition is mostly unknown. Here we conducted time-ordered gene co-expression network construction, epigenetic analysis, and functional perturbations in developing feather follicles to elucidate four downy-juvenile feather transition events. We discovered that LEF1 works as a key hub of Wnt signaling to build rachis and converts radial downy to bilateral symmetry. Extracellular matrix reorganization leads to peripheral pulp formation, which mediates epithelial -mesenchymal interactions for branching morphogenesis. ACTA2 compartments dermal papilla stem cells for feather cycling. Novel usage of scale keratins strengthens feather sheath with SOX14 as the epigenetic regulator. We found this primary feather transition largely conserved in chicken (precocious) and zebra finch (altricial) and discussed the possibility that this evolutionary adaptation process started in feathered dinosaurs.

The role of HIF-1α-mediated autophagy in ionizing radiation-induced testicular injury.

Testis, as a key organ for maintaining male fertility, are extremely sensitive to ionizing radiation (IR). IR-induced testicular dysfunction and infertility are common adverse effects of radiation therapy in patients with pelvic cancer. To study the phenotype and mechanism of IR-induced testicular injury, the mice were irradiated with different radiation doses (0, 2 and 5 Gy) below the semi-lethal dose for one month. Our present results showed that testicular weight and the serum testosterone levels significantly decreased with the structural injury of the testis in an IR dose-dependent manner, indicating that IR caused not only the structural damage of the testis, but also the functional damage. Further analysis by TUNEL staining and Western blotting found that IR induced the apoptosis in a dose-dependent manner as indicated by increased expressions of cleaved caspase3, p53 and Bax on Day 15 after IR treatment. Combined with significantly increased oxidative stress, these results indicated that IR-induced testicular damage may be a long-term, progressively aggravated process, accompanied by apoptosis. Given the role of autophagy in apoptosis, the present study also detected and analyzed the localization and expressions of autophagy-related proteins LC-3I/II, beclin1, p62 and Atg12 in testicular cells, and found that LC-3II, beclin1 and Atg12 expressions significantly increased in the testicular cells of mice irradiated with 2 Gy and 5 Gy, while p62 expression significantly decreased with 5 Gy, implying autophagy was involved in the apoptosis of testicular cells induced by IR. Furthermore, the expressions of HIF-1α and BNIP3 were significantly enhanced in the testis cells of mice irradiated with 2 Gy and 5 Gy, suggesting the potential role of HIF-1α/BNIP3-mediated autophagy in the apoptosis of testicular cells induced by IR. Taken together, our findings demonstrated that HIF-1α/BNIP3-mediated autophagy not only plays a protective effect on the testicular cells of mice irradiated with 2 Gy, but also induces the apoptosis of the testicular cells of mice irradiated with 5 Gy, indicating the double effects on apoptosis, which will help us further understanding the molecular mechanisms of IR-induced testicular injury, and will facilitate us further studies on testicular radioprotection.

Keratin 17 Impacts Global Gene Expression and Controls G2/M Cell Cycle Transition in Ionizing Radiation-Induced Skin Damage.

Keratin 17 (K17) is a cytoskeletal protein that is part of the intermediate filaments in epidermal keratinocytes. In K17-/- mice, ionizing radiation induced more severe hair follicle damage, whereas the epidermal inflammatory response was attenuated compared with that in wild-type mice. Both p53 and K17 have a major impact on global gene expression because over 70% of the differentially expressed genes in the skin of wild-type mice showed no expression change in p53-/- or K17-/- skin after ionizing radiation. K17 does not interfere with the dynamics of p53 activation; rather, global p53 binding in the genome is altered in K17-/- mice. The absence of K17 leads to aberrant cell cycle progression and mitotic catastrophe in epidermal keratinocytes, which is due to nuclear retention, thus reducing the degradation of B-Myb, a key regulator of the G2/M cell cycle transition. These results expand our understanding of the role of K17 in regulating global gene expression and ionizing radiation-induced skin damage.

Clinical Pathobiology of Radiotherapy-Induced Alopecia: A Guide toward More Effective Prevention and Hair Follicle Repair.

Because hair follicles (HFs) are highly sensitive to ionizing radiation, radiotherapy-induced alopecia (RIA) is a core adverse effect of oncological radiotherapy. Yet, effective RIA-preventive therapy is unavailable because the underlying pathobiology remains underinvestigated. Aiming to revitalize interest in pathomechanism-tailored RIA management, we describe the clinical RIA spectrum (transient, persistent, progressive alopecia) and our current understanding of RIA pathobiology as an excellent model for studying principles of human organ and stem cell repair, regeneration, and loss. We explain that HFs respond to radiotherapy through two distinct pathways (dystrophic anagen or catagen) and why this makes RIA management so challenging. We discuss the responses of different HF cell populations and extrafollicular cells to radiation, their roles in HF repair and regeneration, and how they might contribute to HF miniaturization or even loss in persistent RIA. Finally, we highlight the potential of targeting p53-, Wnt-, mTOR-, prostaglandin E2-, FGF7-, peroxisome proliferator-activated receptor-γ-, and melatonin-associated pathways in future RIA management.

Three amphioxus reference genomes reveal gene and chromosome evolution of chordates.

The slow-evolving invertebrate amphioxus has an irreplaceable role in advancing our understanding of the vertebrate origin and innovations. Here we resolve the nearly complete chromosomal genomes of three amphioxus species, one of which best recapitulates the 17 chordate ancestor linkage groups. We reconstruct the fusions, retention, or rearrangements between descendants of whole-genome duplications, which gave rise to the extant microchromosomes likely existed in the vertebrate ancestor. Similar to vertebrates, the amphioxus genome gradually establishes its three-dimensional chromatin architecture at the onset of zygotic activation and forms two topologically associated domains at the Hox gene cluster. We find that all three amphioxus species have ZW sex chromosomes with little sequence differentiation, and their putative sex-determining regions are nonhomologous to each other. Our results illuminate the unappreciated interspecific diversity and developmental dynamics of amphioxus genomes and provide high-quality references for understanding the mechanisms of chordate functional genome evolution.

Evolutionary analysis of a complete chicken genome.

Microchromosomes are prevalent in nonmammalian vertebrates [P. D. Waters et al., Proc. Natl. Acad. Sci. U.S.A. 118 (2021)], but a few of them are missing in bird genome assemblies. Here, we present a new chicken reference genome containing all autosomes, a Z and a W chromosome, with all gaps closed except for the W. We identified ten small microchromosomes (termed dot chromosomes) with distinct sequence and epigenetic features, among which six were newly assembled. Those dot chromosomes exhibit extremely high GC content and a high level of DNA methylation and are enriched for housekeeping genes. The pericentromeric heterochromatin of dot chromosomes is disproportionately large and continues to expand with the proliferation of satellite DNA and testis-expressed genes. Our analyses revealed that the 41-bp CNM repeat frequently forms higher-order repeats (HORs) at the centromeres of acrocentric chromosomes. The centromere core regions where the kinetochore attaches often encompass telomeric sequence (TTAGGG)n, and in a one of the dot chromosomes, the centromere core recruits an endogenous retrovirus (ERV). We further demonstrate that the W chromosome shares some common features with dot chromosomes, having large arrays of hypermethylated tandem repeats. Finally, using the complete chicken chromosome models, we reconstructed a fine picture of chordate karyotype evolution, revealing frequent chromosomal fusions before and after vertebrate whole-genome duplications. Our sequence and epigenetic characterization of chicken chromosomes shed insights into the understanding of vertebrate genome evolution and chromosome biology.

Chemotherapy suppresses SHH gene expression via a specific enhancer.

Sonic hedgehog (SHH) signaling is a key regulator of embryonic development and tissue homeostasis that is involved in gastrointestinal (GI) cancer progression. Regulation of SHH gene expression is a paradigm of long-range enhancer function. Using the classical chemotherapy drug 5-fluorouracil (5FU) as an example, here we show that SHH gene expression is suppressed by chemotherapy. SHH is downstream of immediate early genes (IEGs), including Early growth response 1 (Egr1). A specific 139 kb upstream enhancer is responsible for its down-regulation. Knocking down EGR1 expression or blocking its binding to this enhancer renders SHH unresponsive to chemotherapy. We further demonstrate that down-regulation of SHH expression does not depend on 5FU's impact on nucleotide metabolism or DNA damage; rather, a sustained oxidative stress response mediates this rapid suppression. This enhancer is present in a wide range of tumors and normal tissues, thus providing a target for cancer chemotherapy and its adverse effects on normal tissues. We propose that SHH is a stress-responsive gene downstream of IEGs, and that traditional chemotherapy targets a specific enhancer to suppress its expression.

Regulation and dysregulation of hair regeneration: aiming for clinical application.

Hair growth and regeneration represents a remarkable example of stem cell function. Recent progress emphasizes the micro- and macro- environment that controls the regeneration process. There is a shift from a stem cell-centered view toward the various layers of regulatory mechanisms that control hair regeneration, which include local growth factors, immune and neuroendocrine signals, and dietary and environmental factors. This is better suited for clinical application in multiple forms of hair disorders: in male pattern hair loss, the stem cells are largely preserved, but androgen signaling diminishes hair growth; in alopecia areata, an immune attack is targeted toward the growing hair follicle without abrogating its regeneration capability. Genome-wide association studies further revealed the genetic bases of these disorders, although the precise pathological mechanisms of the identified loci remain largely unknown. By analyzing the dysregulation of hair regeneration under pathological conditions, we can better address the complex interactions among stem cells, the differentiated progeny, and mesenchymal components, and highlight the critical role of macroenvironment adjustment that is essential for hair growth and regeneration. The poly-genetic origin of these disorders makes the study of hair regeneration an interesting and challenging field.

Recurrent chromosome reshuffling and the evolution of neo-sex chromosomes in parrots.

The karyotype of most birds has remained considerably stable during more than 100 million years' evolution, except for some groups, such as parrots. The evolutionary processes and underlying genetic mechanism of chromosomal rearrangements in parrots, however, are poorly understood. Here, using chromosome-level assemblies of four parrot genomes, we uncover frequent chromosome fusions and fissions, with most of them occurring independently among lineages. The increased activities of chromosomal rearrangements in parrots are likely associated with parrot-specific loss of two genes, ALC1 and PARP3, that have known functions in the repair of double-strand breaks and maintenance of genome stability. We further find that the fusion of the ZW sex chromosomes and chromosome 11 has created a pair of neo-sex chromosomes in the ancestor of parrots, and the chromosome 25 has been further added to the sex chromosomes in monk parakeet. Together, the combination of our genomic and cytogenetic analyses characterizes the complex evolutionary history of chromosomal rearrangements and sex chromosomes in parrots.

Period 2 Suppresses the Malignant Cellular Behaviors of Colorectal Cancer Through the Epithelial-Mesenchymal Transformation Process.

INTRODUCTION: The PER2 (Period circadian regulator 2) gene is related to the circadian clock, and it has been deemed as a suppressor gene in osteosarcoma and lung carcinoma. However, the part of PER2 in CRC (colorectal cancer) needs to be further determined. METHODS: First, we collected clinical samples to detect PER2 expression in CRC. Then, we used cell transfection to knock down PER2 expression in CRC cell lines and performed a series of functional experiments to elucidate the effects of PER2 on CRC cells. We next verified whether PER2 affects the epithelial-mesenchymal transformation (EMT) process in CRC by conducting quantitative real-time PCR and western blotting. RESULTS: In the research, we revealed that the expression of PER2 decreased in CRC clinical samples. In addition, knocking down PER2 expression caused CRC cells to acquire malignant biological features. Finally, we found that PER2 knockdown may activate the Snail/Slug axis through inhibiting p53, therefore promote the activation of the EMT pathway. CONCLUSION: In conclusion, low PER2 expression reinforces migration and activates EMT in CRC, suggesting that PER2 is closely related to CRC development and could be used as a potential treatment site in the clinic.

Programmed gene expression change in mouse skin after ultraviolet radiation damage.

Ultraviolet (UV) radiation is a major cause of skin damage and carcinogenesis. Here, we systematically analyse the acute gene expression change in skin in vivo after UV exposure, aiming to establish the common C57BL/6 mouse strain as a convenient model for future pathological research and drug discovery. The back fur of C57BL/6 mice was depilated, and a mixed UV light source was used to irradiate the skin. Full-thickness skin samples were collected at 0, 0.5, 2, 6, 12 and 24 h. Total RNAs were extracted and subjected to RNA sequencing analysis. We found that the gene expression change in mouse skin is highly similar to previous reports in human skin. These include down-regulation of differentiation-related genes and extracellular matrix genes, and up-regulation of cytokine/chemokine genes. An early wave of activator protein 1 (AP-1) expression is induced, whereas activation of the p53 pathway is not significant. The impact of the AP-1 transcription factors and the antioxidant tea polyphenols is discussed. The analysis of acute gene expression change in skin after UV irradiation provides a starting point to investigate how the skin responds to genotoxic stress.

The global regulatory logic of organ regeneration: circuitry lessons from skin and its appendages.

In organ regeneration, the regulatory logic at a systems level remains largely unclear. For example, what defines the quantitative threshold to initiate regeneration, and when does the regeneration process come to an end? What leads to the qualitatively different responses of regeneration, which restore the original structure, or to repair which only heals a wound? Here we discuss three examples in skin regeneration: epidermal recovery after radiation damage, hair follicle fate choice after chemotherapy damage, and wound-induced feather regeneration. We propose that the molecular regulatory circuitry is of paramount significance in organ regeneration. It is conceivable that defects in these controlling pathways may lead to failed regeneration and/or organ renewal, and understanding the underlying logic could help to identify novel therapeutic strategies.

Inhibition of Shh Signaling through MAPK Activation Controls Chemotherapy-Induced Alopecia.

Chemotherapy-induced hair loss (alopecia) (CIA) remains a major unsolved problem in clinical oncology. CIA is often considered to be a consequence of the antimitotic and apoptosis-promoting properties of chemotherapy drugs acting on rapidly proliferating hair matrix keratinocytes. Here, we show that in a mouse model of CIA, the downregulation of Shh signaling in the hair matrix is a critical early event. Inhibition of Shh signaling recapitulated key morphological and functional features of CIA, whereas recombinant Shh protein partially rescued hair loss. Phosphoproteomics analysis revealed that activation of the MAPK pathway is a key upstream event, which can be further manipulated to rescue CIA. Finally, in organ-cultured human scalp hair follicles as well as in patients undergoing chemotherapy, reduced expression of SHH gene correlates with chemotherapy-induced hair follicle damage or the degree of CIA, respectively. Our work revealed that Shh signaling is an evolutionarily conserved key target in CIA pathobiology. Specifically targeting the intrafollicular MAPK-Shh axis may provide a promising strategy to manage CIA.

How chemotherapy and radiotherapy damage the tissue: Comparative biology lessons from feather and hair models.

Chemotherapy and radiotherapy are common modalities for cancer treatment. While targeting rapidly growing cancer cells, they also damage normal tissues and cause adverse effects. From the initial insult such as DNA double-strand break, production of reactive oxygen species (ROS) and a general stress response, there are complex regulatory mechanisms that control the actual tissue damage process. Besides apoptosis, a range of outcomes for the damaged cells are possible including cell cycle arrest, senescence, mitotic catastrophe, and inflammatory responses and fibrosis at the tissue level. Feather and hair are among the most actively proliferating (mini-)organs and are highly susceptible to both chemotherapy and radiotherapy damage, thus provide excellent, experimentally tractable model systems for dissecting how normal tissues respond to such injuries. Taking a comparative biology approach to investigate this has turned out to be particularly productive. Started in chicken feather and then extended to murine hair follicles, it was revealed that in addition to p53-mediated apoptosis, several other previously overlooked mechanisms are involved. Specifically, Shh, Wnt, mTOR, cytokine signalling and ROS-mediated degradation of adherens junctions have been implicated in the damage and/or reparative regeneration process. Moreover, we show here that inflammatory responses, which can be prominent upon histological examination of chemo- or radiotherapy-damaged hair follicle, may not be essential for the hair loss phenotype. These studies point to fundamental, evolutionarily conserved mechanisms in controlling tissue responses in vivo, and suggest novel strategies for the prevention and management of adverse effects that arise from chemo- or radiotherapy.

Long non-coding RNAs regulate Wnt signaling during feather regeneration.

Long non-coding RNAs (lncRNAs) are non-protein coding transcripts that are involved in a broad range of biological processes. Here, we examine the functional role of lncRNAs in feather regeneration. RNA-seq profiling of the regenerating feather blastema revealed that Wnt signaling is among the most active pathways during feather regeneration, with Wnt ligands and their inhibitors showing distinct expression patterns. Co-expression analysis identified hundreds of lncRNAs with similar expression patterns to either the Wnt ligands (the Lwnt group) or their downstream target genes (the Twnt group). Among these, we randomly picked two lncRNAs in the Lwnt group and three lncRNAs in the Twnt group to validate their expression and function. Members in the Twnt group regulated feather regeneration and axis formation, whereas members in the Lwnt group showed no obvious phenotype. Further analysis confirmed that the three Twnt group members inhibit Wnt signal transduction and, at the same time, are downstream target genes of this pathway. Our results suggest that the feather regeneration model can be utilized to systematically annotate the functions of lncRNAs in the chicken genome.

Coupling of apical-basal polarity and planar cell polarity to interpret the Wnt signaling gradient in feather development.

Sensing a global directional cue to orient cell growth is crucial in tissue morphogenesis. An anterior-posterior gradient of Wnt signaling controls the helical growth of feather branches (barbs), and thus the formation of bilateral feathers. However, it remains unclear how the keratinocytes sense this gradient and orient barb growth. Here, we show that in chicken, owing to feather branching, the global Wnt gradient is subdivided into periodic barbs. Within each barb, the anterior barbule plate cells tilt before the posterior cells. The core planar cell polarity gene Prickle1 is involved, as knockdown of its expression resulted in no cell shape change and no barb tilting. Furthermore, perturbation of the Wnt gradient leads to diffusive Prickle1 expression and loss of barb orientation. Finally, the asymmetric distribution of Wnt6/Fzd10 is coordinated by the apical-basal polarity of the barbule plate keratinocytes, which is in turn regulated by the Par3/aPKC machinery. Our data elucidate a new mechanism through which the global Wnt signaling gradient is interpreted locally to construct complex spatial forms.

Contraction of basal filopodia controls periodic feather branching via Notch and FGF signaling.

Branching morphogenesis is a general mechanism that increases the surface area of an organ. In chicken feathers, the flat epithelial sheath at the base of the follicle is transformed into periodic branches. How exactly the keratinocytes are organized into this pattern remains unclear. Here we show that in the feather follicle, the pre-branch basal keratinocytes have extensive filopodia, which contract and smooth out after branching. Manipulating the filopodia via small GTPases RhoA/Cdc42 also regulates branch formation. These basal filopodia help interpret the proximal-distal FGF gradient in the follicle. Furthermore, the topological arrangement of cell adhesion via E-Cadherin re-distribution controls the branching process. Periodic activation of Notch signaling drives the differential cell adhesion and contraction of basal filopodia, which occurs only below an FGF signaling threshold. Our results suggest a coordinated adjustment of cell shape and adhesion orchestrates feather branching, which is regulated by Notch and FGF signaling.

Ionizing radiation, but not ultraviolet radiation, induces mitotic catastrophe in mouse epidermal keratinocytes with aberrant cell cycle checkpoints.

Ultraviolet radiation (UVR) and ionizing radiation (IR) are common genotoxic stresses that damage human skin, although the specific damages to the genomic DNA are different. Here, we show that in the mouse glabrous skin, both UVR and IR induce DNA damage, cell cycle arrest, and condensed cell nuclei. However, only IR induces mitotic catastrophe (MC) in the epidermis. This is because UVR induces a complete blockage of pRB phosphorylation and cell cycle arrest in the G1 phase, whereas pRB phosphorylation remains positive in a significant portion of the epidermal keratinocytes following IR exposure. Furthermore, Cyclin B1 expression is significantly downregulated only by IR but not UVR. Finally, there are more MC cells in the epidermis of p53-/- mice after IR exposure as compared to wild-type mice. Our results suggest that although both IR and UVR are genotoxic, they show distinct impacts on the cell cycle machinery and thus damage the epidermal keratinocytes via different mechanisms.

Pretreatment Tumor Thickness as a Predictor of Pathologic Complete Response to Neoadjuvant Chemoradiation Therapy for Stage II/III Rectal Adenocarcinoma.

OBJECTIVES: To evaluate pretreatment tumor thickness in predicting pathologic complete response (pCR) of stage II/III rectal adenocarcinoma to neoadjuvant chemoradiation (chemoradiotherapy [CRT]). METHODS: We retrospectively analyzed 185 patients who were diagnosed with stage II or III rectal adenocarcinoma from January 2011 to July 2013 and treated with neoadjuvant intensity-modulated radiation therapy (45 Gy in 1.8-Gy fractions to pelvis and 50 Gy in 2-Gy fractions to rectal tumor as an integrated boost) or 3 dimensionally conformal radiation therapy (45 Gy in 1.8-Gy fractions to pelvis followed by an additional 5.4-Gy to rectal tumor) concurrently with two 3-week cycles of chemotherapy (oxaliplatin 130 mg/m on day 1 and capecitabine 825 mg/m, twice per day from day 1 to 14, cycle 2 starts on week 4). One week after CRT, 36% patients received 1 more cycle of the above chemotherapy and 55% received 1 to 2 cycles of FOLFOX6. Tumor response was categorized as pCR and non-pCR. Tumor thickness measured on magnetic resonance imaging was collected. A multivariate logistic regression model was used to evaluate the association of potential predictors and pCR. RESULTS: Thirty-eight patients (20.5%) reached pCR. Multivariate analysis found the pretreatment tumor thickness to be associated with higher probability of pCR after adjusting for radiation therapy-surgery interval time and pretreatment carcino-embryonic antigen level. The pretreatment carcino-embryonic antigen level was associated with pCR in the univariate analysis but lost the association in the multivatiate model. The pretreatment T or N stage, tumor volume, distance from tumor to anal verge, craniocaudal length of tumor, radiation therapy technique, and patient age and sex were not associated with pCR. CONCLUSIONS: We concluded that pretreatment tumor thickness is an independent predictor for pCR of stage II/III rectal adenocarcinoma to the neoadjuvant CRT.