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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 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 mechanism 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.

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.

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.

The slow-evolving invertebrate amphioxus has an irreplaceable role in advancing our understanding into 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 (WGDs), which gave rise to the extant microchromosomes likely existed in the vertebrate ancestor. Similar to vertebrates, the amphioxus genome gradually establishes its 3D 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.

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.

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.

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.

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.

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., Keratinocytes are organised into a periodic pattern in feather branching, but how this is regulated is unclear. Here, the authors show that there is a coordinated change in cell shape and adherence, mediated by Notch, FGF signalling and Rho GTPases, which in turn regulates feather branching.

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.

Chemotherapy-induced hair loss (alopecia, CIA) remains a major unsolved problem in clinical oncology. CIA is often considered to be a consequence of the anti-mitotic and apoptosis-promoting properties of chemotherapy drugs acting on rapidly proliferating hair matrix keratinocytes. Here we show that in a mouse model of CIA, down-regulation 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. Phospho-proteomics 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.

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.

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.

Long noncoding RNAs (lncRNAs) are non-protein coding transcripts that are involved in a broad range of biological processes. Here, we examined the functional roles of lncRNAs in feather regeneration. RNA-seq profiling of the regenerating feather blastema revealed that the Wnt signaling is among the most active pathways during feather regeneration, with the 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 down-stream target genes of this pathway. Our results suggested that the feather regeneration model can be utilized to systematically annotate the functions of lncRNAs in the chicken genome.

Chemotherapeutic agents induce complex tissue responses in vivo and damage normal organ functions. Here we use the feather follicle to investigate details of this damage response. We show that cyclophosphamide treatment, which causes chemotherapy-induced alopecia in mice and man, induces distinct defects in feather formation: feather branching is transiently and reversibly disrupted, thus leaving a morphological record of the impact of chemotherapeutic agents, whereas the rachis (feather axis) remains unperturbed. Similar defects are observed in feathers treated with 5-fluorouracil or taxol but not with doxorubicin or arabinofuranosyl cytidine (Ara-C). Selective blockade of cell proliferation was seen in the feather branching area, along with a downregulation of sonic hedgehog (Shh) transcription, but not in the equally proliferative rachis. Local delivery of the Shh inhibitor, cyclopamine, or Shh silencing both recapitulated this effect. In mouse hair follicles, those chemotherapeutic agents that disrupted feather formation also downregulated Shh gene expression and induced hair loss, whereas doxorubicin or Ara-C did not. Our results reveal a mechanism through which chemotherapeutic agents damage rapidly proliferating epithelial tissue, namely via the cell population–specific, Shh-dependent inhibition of proliferation. This mechanism may be targeted by future strategies to manage chemotherapy-induced tissue damage.

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

Chemo- and radiation therapy are the main modalities for cancer treatment. A major limiting factor is their toxicity to normal tissue, thus reducing the dose and duration of the therapy. The hair follicle, gastrointestinal tract, and hematopoietic system are among the target organs that often show side effects in cancer therapy . Although these organs are highly mitotic in common, the molecular mechanism of the damage remains unclear. The feather follicle is a fast-growing mini-organ, which allows observation and manipulation on each follicle individually. As a model system, the feather follicle is advantageous because of the following reasons: (1) its complex structure is regulated by a set of evolutionarily conserved molecular pathways, thus facilitating the effort to dissect the specific signaling events involved; (2) its morphology allows the continuity of normal-perturbed-normal structure in a single feather, thus "recording" the damaging effect of chemo- and radiation therapy; (3) further histological and molecular analysis of the damage response can be performed on each plucked feather; thus, it is not necessary to sacrifice the experimental animal. Here, we describe methods of applying the feather model to study the molecular mechanism of chemo- and radiation therapy-induced tissue damage.

Chemo- and radiation therapy are the main modalities for cancer treatment. A major limiting factor is their toxicity to normal tissue, thus reducing the dose and duration of the therapy. The hair follicle, gastrointestinal tract, and hematopoietic system are among the target organs that often show side effects in cancer therapy . Although these organs are highly mitotic in common, the molecular mechanism of the damage remains unclear. The feather follicle is a fast-growing mini-organ, which allows observation and manipulation on each follicle individually. As a model system, the feather follicle is advantageous because of the following reasons: (1) its complex structure is regulated by a set of evolutionarily conserved molecular pathways, thus facilitating the effort to dissect the specific signaling events involved; (2) its morphology allows the continuity of normal-perturbed-normal structure in a single feather, thus "recording" the damaging effect of chemo- and radiation therapy; (3) further histological and molecular analysis of the damage response can be performed on each plucked feather; thus, it is not necessary to sacrifice the experimental animal. Here, we describe methods of applying the feather model to study the molecular mechanism of chemo- and radiation therapy-induced tissue damage.

Avian feathers have robust growth and regeneration capability. To evaluate the contribution of signaling molecules and pathways in these processes, we profiled gene expression in the feather follicle using an absolute quantification approach. We identified hundreds of genes that mark specific components of the feather follicle: the dermal papillae (DP) which controls feather regeneration and axis formation, the pulp mesenchyme (Pp) which is derived from DP cells and nourishes the feather follicle, and the ramogenic zone epithelium (Erz) where a feather starts to branch. The feather DP is enriched in BMP/TGF-β signaling molecules and inhibitors for Wnt signaling including Dkk2/Frzb. Wnt ligands are mainly expressed in the feather epithelium and pulp. We find that while Wnt signaling is required for the maintenance of DP marker gene expression and feather regeneration, excessive Wnt signaling delays regeneration and reduces pulp formation. Manipulating Dkk2/Frzb expression by lentiviral-mediated overexpression, shRNA-knockdown, or by antibody neutralization resulted in dual feather axes formation. Our results suggest that the Wnt signaling in the proximal feather follicle is fine-tuned to accommodate feather regeneration and axis formation.

Radiotherapy is a primary oncological treatment modality that also damages normal tissue, including the skin, and causes radiation dermatitis (RD). Here, we explore the mechanism of acute epidermal damage in radiation dermatitis. Two distinctive phases in the damage response were identified: an early destructive phase, where a burst of reactive oxygen species induces loss of E-cadherin–mediated cell contact, followed by a regenerative phase, during which Wnt and Hippo signaling are activated. A blocking peptide, as well as a neutralizing antibody to E-cadherin, works synergistically with ionizing radiation to promote the epidermal damage. In addition, ROS disassembles adherens junctions in epithelial cells via posttranslational mechanisms, that is, activation of Src/Abl kinases and degradation of β-catenin/E-cadherin. The key role of tyrosine kinases in this process is further substantiated by the rescue effect of the tyrosine kinase inhibitor genistein, and the more specific Src/Abl kinase inhibitor dasatinib: both reduced ROS-induced degradation of β-catenin/E-cadherin in vitro and ameliorated skin damage in rodent models. Finally, we confirm that the same key molecular events are also seen in human radiation dermatitis. Therefore, we propose that loss of cell contact in epidermal keratinocytes through reactive oxygen species-mediated disassembly of adherens junctions is pivotal for the acute epidermal damage in radiation dermatitis.

In a feather, there are distinct morphologies along the proximal-distal axis. The proximal part is a cylindrical stalk (calamus), whereas the distal part has barb and barbule branches. Here we focus on what molecular signaling activity can modulate feather stem cells to generate these distinct morphologies. We demonstrate the drastic tissue remodeling during feather cycling which includes initiation, growth and resting phases. In the growth phase, epithelial components undergo progressive changes from the collar growth zone to the ramogenic zone, to maturing barb branches along the proximal- distal axis. Mesenchymal components also undergo progressive changes from the dermal papilla, to the collar mesenchyme, to the pulp along the proximal- distal axis. Over-expression of Spry4, a negative regulator of receptor tyrosine kinases, promotes barb branch formation at the expense of the epidermal collar. It even induces barb branches from the follicle sheath (equivalent to the outer root sheath in hair follicles). The results are feathers with expanded feather vane regions and small or missing proximal feather shafts (the calamus). Spry4 also expands the pulp region while reducing the size of dermal papillae, leading to a failure to regenerate. In contrast, over-expressing Fgf10 increases the size of the dermal papillae, expands collar epithelia and mesenchyme, but also prevents feather branch formation and feather keratin differentiation. These results suggest that coordinated Sprouty/FGF pathway activity at different stages is important to modulate feather epidermal stem cells to form distinct feather morphologies along the proximal-distal feather axis.

Epidermal keratinocytes undergo a continuous process of terminal differentiation, which is accompanied by a dramatic change in the expression and composition of keratins. This complex and carefully orchestrated process is regulated by a large number of signal transduction events and transcriptional factors as well as by epigenetic regulatory mechanisms, namely by histone methylation/acetylation and DNA methylation. In a recent issue of Exp Dermatol, Ming et al. provide evidence that sirtuin-2 (SIRT2), a NAD+-dependent deacetylase, inhibits the expression of keratin 15 and keratin 19, epidermal stem cell markers, while it stimulates the expression of loricrin, a marker of terminal keratinocyte differentiation. Human skin cancer cells show downregulation of SIRT2, and its deletion increases tumor growth in mice. Overall, these findings suggest that this deacetylase is involved in the epigenetic regulation of keratinocyte differentiation and exerts intracutaneous tumor suppressor functions.

The different cell types in an animal are often considered to be specified by combinations of transcription factors, and defined by marker gene expression. This paradigm is challenged, however, in stem cell research and application. Using a mouse embryonic stem cell (mESC) culture system, here we show that the expression level of many key stem cell marker genes/transcription factors such as Oct4, Sox2 and Nanog failed to monitor cell status transition during mESC differentiation. On the other hand, the response patterns of cell signalling network to external stimuli, as monitored by the dynamics of protein phosphorylation, changed dramatically. Our results also suggest that an irreversible alternation in cell signalling network precedes the adjustment of transcription factor levels. This is consistent with the notion that signal transduction events regulate cell fate specification. We propose that interrogating cell signalling network can assess the cell property more precisely, and provide a sensitive measurement for the early events in cell fate transition. We wish to bring up attention to the potential problem of cell identification using a few marker genes, and suggest a novel methodology to address this issue.

Background The small bowel (SB) represents the most important dose-limiting structure in pelvic radiotherapy (RT). However, we observed that the majority of rectal cancer patients who received preoperative pelvic intensity modulated RT (IMRT) developed acute tenesmus without watery diarrhea. The objective of this study is to determine if the RT dose to SB affects the acute lower gastrointestinal toxicity (ALGIT) in rectal cancer patients who received neoadjuvant concurrent chemotherapy-IMRT. We will also evaluate if patient and tumor factors affect the ALGIT. Methods We retrospectively analyzed 63 rectal cancer patients that consecutively received preoperative IMRT (45 Gy for pelvis and 50 Gy for gross tumor in 25 fractions) with concurrent chemotherapy (oxaliplatin 130 mg/m2 on day 1 and capecitabine 825 mg/m2, twice per day from day 1 to day 14, week 1 and 4) between May 2012 and May 2013. The ALGIT was assessed with Common Terminology Criteria for Adverse Events version 3. The patients were stratified into two groups (with and without grade ≥2 ALGIT). The effect of SB volume receiving 5 to 40 Gy (V5 to V40) at a 5 Gy interval dose level on grade ≥2 ALGIT was evaluated. The volume of small bowel is defined as the volume of the small bowel loop. Other factors evaluated include patient’s age and gender, tumor size and location and preexisting number of daily bowel movements. Results Overall, grade ≥2 ALGIT occurred in 57 % (36/63) patients. There was no significant difference between the two groups of patients (with and without grade ≥2 ALGIT) in SB V5 to V40, patient’s age and gender, tumor location and preexisting number of daily bowel movements. There was a significant difference between the two groups of patients in tumor volume (with grade ≥2 ALGIT: 115.5 ± 85.5 cm3 versus without grade ≥2 ALGIT: 58.5 ± 25.2 cm3, p = 0.0001). Multivariate analysis revealed no association between the dose SB received (V5 to V40) and the grade ≥2 ALGIT after adjusting for the tumor volume. Conclusions With IMRT technique used in rectal cancer patients undergoing preoperative chemo-radiotherapy, the acute lower GI toxicity is not associated with small bowel V5 to V40; instead it is associated with rectal tumor size.

Preoperative neoadjuvant chemoradiation therapy may be useful in patients with operable rectal cancer, but treatment responses are variable. We examined whether expression levels of circadian clock genes could be used as biomarkers to predict treatment response. We retrospectively analyzed clinical data from 250 patients with rectal cancer, treated with neoadjuvant chemoradiation therapy in a single institute between 2011 and 2013. Gene expression analysis (RT-PCR) was performed in tissue samples from 20 patients showing pathological complete regression (pCR) and 20 showing non-pCR. The genes analyzed included six core clock genes (Clock, Per1, Per2, Cry1, Cry2 and Bmal1) and three downstream target genes (Wee1, Chk2 and c-Myc). Patient responses were analyzed through contrast-enhanced pelvic MRI and endorectal ultrasound, and verified by histological assessment. pCR was defined histologically as an absence of tumor cells. Among the 250 included patients, 70.8% showed regression of tumor size, and 18% showed pCR. Clock, Cry2 and Per2 expressions were significantly higher in the pCR group than in the non-pCR group (P

The feather is a complex epidermal organ with hierarchical branches and represents a multi-layered topological transformation of keratinocyte sheets. Feathers are made in feather follicles. The basics of feather morphogenesis were previously described (Lucas and Stettenheim, 1972). Here we review new molecular and cellular data. After feather buds form (Jiang et al., this issue), they invaginate into the dermis to form feather follicles. Above the dermal papilla is the proliferating epidermal collar. Distal to it is the ramogenic zone where the epidermal cylinder starts to differentiate into barb ridges or rachidial ridge. These neoptile feathers tend to be downy and radially symmetrical. They are replaced by teleoptile feathers which tend to be bilateral symmetrical and more diverse in shapes. We have recently developed a "transgenic feather" protocol that allows molecular analyses: BMPs enhance the size of the rachis, Noggin increases branching, while anti- SHH causes webbed branches. Different feather types formed during evolution (Wu et al., this issue). Pigment patterns along the body axis or intra-feather add more colorful distinctions. These patterns help facilitate the analysis of melanocyte behavior. Feather follicles have to be connected with muscles and nerve fibers, so they can be integrated into the physiology of the whole organism. Feathers, similarly to hairs, have the extraordinary ability to go through molting cycles and regenerate. Some work has been done and feather follicles might serve as a model for stem cell research. Feather phenotypes can be modulated by sex hormones and can help elucidate mechanisms of sex hormone-dependent growth control. Thus, the developmental biology of feather follicles provides a multi-dimension research paradigm that links molecular activities and cellular behaviors to functional morphology at the organismal level.

The intermediate filament protein keratin 17 (Krt17) shows highly dynamic and inducible expression in skin physiology and pathology. Because Krt17 exerts physiologically important functions beyond providing structural stability to keratinocytes whereas abnormal Krt17 expression is a key feature of dermatoses such as psoriasis and pachyonychia congenita, the currently unclear regulation of Krt17 expression needs to be better understood. Using a rat model of radiation dermatitis, we report here that Krt17 expression initially is down-regulated but later is strongly up-regulated by ionizing radiation. The early down-regulation correlates with the activation of p53 signaling. Deletion of p53 abolishes the initial down-regulation but not its subsequent up-regulation, suggesting that p53 represses Krt17 transcription. Because previous work reported up-regulation of Krt17 by ultraviolet irradiation, which also activates p53 signaling, the effect of ultraviolet radiation was reexamined. This revealed that the initial down-regulation of Krt17 is conserved, but the up-regulation comes much faster. Chromatin immunoprecipitation analysis in vivo and electromobility shift assay in vitro identified two p53-binding sites in the promoter region of Krt17. Thus, p53 operates as a direct Krt17 repressor, which invites therapeutic targeting in dermatoses characterized by excessive Krt17 expression.

In the process of organogenesis, different cell types form organized tissues and tissues are integrated into an organ. Most organs form in the developmental stage, but new organs can also form in physiological states or following injuries during adulthood. Feathers are a good model to study post-natal organogenesis because they regenerate episodically under physiological conditions and in response to injuries such as plucking. Epidermal stem cells in the collar can respond to activation signals. Dermal papilla located at the follicle base controls the regenerative process. Adhesion molecules (e.g., neural cell adhesion molecule (NCAM), tenascin), morphogens (e.g., Wnt3a, sprouty, fibroblast growth factor [FGF]10), and differentiation markers (e.g., keratins) are expressed dynamically in initiation, growth and resting phases of the feather cycle. Epidermal cells are shaped into different feather morphologies based on the molecular micro-environment at the moment of morphogenesis. Chicken feather variants provide a rich resource for us to identify genetic determinants involved in feather regeneration and morphogenesis. An example of using genome-wide single nucleotide polymorphism (SNP) analysis to identify alpha keratin 75 as the mutation in frizzled chickens is demonstrated. Due to its accessibility to experimental manipulation and observation, results of regeneration can be analyzed in a comprehensive way. The layout of time dimension along the distal (formed earlier) to proximal (formed later) feather axis makes the morphological analyses easier. Therefore feather regeneration can be a unique model for understanding organogenesis: from activation of stem cells under various physiological conditions to serving as the Rosetta stone for deciphering the language of morphogenesis.

Complex molecular interactions dictate the developmental steps that lead to a mature and functional cornea and lens. Peters anomaly is one subtype of anterior segment dysgenesis especially due to abnormal development of the cornea and lens. MSX2 was recently implicated as a potential gene that is critical for anterior segment development. However, the role of MSX2 within the complex mechanisms of eye development remains elusive. Our present study observed the morphologic changes in conventional Msx2 knockout (KO) mice and found phenotypes consistent with Peters anomaly and microphthalmia seen in humans. The role of Msx2 in cornea and lens development was further investigated using IHC, in situ hybridization, and quantification of proliferative and apoptotic lens cells. Loss of Msx2 down-regulated FoxE3 expression and up-regulated Prox1 and crystallin expression in the lens. The FoxE3 and Prox1 malfunction and precocious Prox1 and crystallin expression contribute to a disturbed lens cell cycle in lens vesicles and eventually to cornea-lentoid adhesions and microphthalmia in Msx2 KO mice. The observed changes in the expression of FoxE3 suggest that Msx2 is an important contributor in controlling transcription of target genes critical for early eye development. These results provide the first direct genetic evidence of the involvement of MSX2 in Peters anomaly and the distinct function of MSX2 in regulating the growth and development of lens vesicles.

Kinases control many important aspects of cell behavior, such as signal transduction, growth/differentiation, and tumorogenesis. Current methods for assessing kinase activity often require specific antibodies, and/or radioactive labeling. Here we demonstrated a novel detection method to assess kinase activity based on surface enhanced Raman spectroscopy (SERS). Raman signal was obtained after amplification by silver nanoparticles. The sensitivity of this method was comparable to fluorescence measurement of peptide concentration. When purified kinase enzyme was used, the detection limit was comparable to conventional radio-labeling method. We further demonstrated the feasibility to measure kinase activity in crude cell lysate. We suggested this SERS-based kinase activity assay could be a new tool for biomedical research and application.

The evolution of bilaterally symmetric feathers is a fundamental process leading toward flight. One major unsolved mystery is how the feathers of a single bird can form radially symmetric downy feathers and bilaterally symmetric flight feathers. In developing downy feather follicles, barb ridges are organized parallel to the long axis of the feather follicle. In developing flight-feather follicles, the barb ridges are organized helically toward the anterior region, leading to the fusion and creation of a rachis. Here we discover an anterior–posterior molecular gradient of wingless int (Wnt3)a in flight but not downy feathers. Global inhibition of the Wnt gradient transforms bilaterally symmetric feathers into radially symmetric feathers. Production of an ectopic local Wnt3a gradient reoriented barb ridges toward the source and created an ectopic rachis. We further show that the orientation of the Wnt3a gradient is dictated by the dermal papilla (DP). Swapping DPs between wing covert and breast downy feathers demonstrates that both feather symmetry and molecular gradients are in accord with the origin of the DP. Thus the fates of feather epidermal cells are not predetermined through some molecular codes but can be modulated. Together, our data suggest feathers are shaped by a DP→ Wnt gradient→helical barb ridge organization→creation of rachis→bilateral symmetry sequence. We speculate diverse feather forms can be achieved by adjusting the orientation and slope of molecular gradients, which then shape the topological arrangements of feather epithelia, thus linking molecular activities to organ forms and novel functions.

It is important to know how different organs ‘manage’ their stem cells. Both hair and feather follicles show robust regenerative powers that episodically renew the epithelial organ. However, the evolution of feathers (from reptiles to birds) and hairs (from reptiles to mammals) are independent events and their follicular structures result from convergent evolution. Because feathers do not have the anatomical equivalent of a hair follicle bulge, we are interested in determining where their stem cells are localized. By applying long-term label retention1, transplantation2 and DiI tracing to map stem cell activities, here we show that feather follicles contain slow-cycling long-term label-retaining cells (LRCs), transient amplifying cells and differentiating keratinocytes. Each population, located in anatomically distinct regions, undergoes dynamic homeostasis during the feather cycle. In the growing follicle, LRCs are enriched in a ‘collar bulge’ niche. In the moulting follicle, LRCs shift to populate a papillar ectoderm niche near the dermal papilla. On transplantation, LRCs show multi-potentiality. In a three-dimensional view, LRCs are configured as a ring that is horizontally placed in radially symmetric feathers but tilted in bilaterally symmetric feathers. The changing topology of stem cell activities may contribute to the construction of complex feather forms.

Skin appendage formation represents a process of regulated new growth. Bromodeoxyuridine labeling of developing chicken skin demonstrated the presence of localized growth zones, which first promote appendage formation and then move within each appendage to produce specific shapes. Initially, cells proliferate all over the presumptive skin. During the placode stage they are organized to form periodic rings. At the short feather bud stage, the localized growth zones shifted to the posterior and then the distal bud. During the long bud stage, the localized growth zones descended through the flank region toward the feather collar (equivalent to the hair matrix). During feather branch formation, the localized growth zones were positioned periodically in the basilar layer to enhance branching of barb ridges. Wnts were expressed in a dynamic fashion during feather morphogenesis that coincided with the shifting localized growth zones positions. The expression pattern of Wnt 6 was examined and compared with other members of the Wnt pathway. Early in feather development Wnt 6 expression overlapped with the location of the localized growth zones. Its function was tested through misexpression studies. Ectopic Wnt 6 expression produced abnormal localized outgrowths from the skin appendages at either the base, the shaft, or the tip of the developing feathers. Later in feather filament morphogenesis, several Wnt markers were expressed in regions undergoing rearrangements and differentiation of barb ridge keratinocytes. These data suggest that skin appendages are built to specific shapes by adding new cells from well-positioned and controlled localized growth zones and that Wnt activity is involved in regulating such localized growth zone activity.

Ionizing radiation (IR) is a common therapeutic agent in cancer therapy. It damages normal tissue and causes side effects including dermatitis and mucositis. Here we use the feather follicle as a model to investigate the mechanism of IR-induced tissue damage, because any perturbation of feather growth will be clearly recorded in its regular yet complex morphology. We find that IR induces defects in feather formation in a dose-dependent manner. No abnormality was observed at 5 Gy. A transient, reversible perturbation of feather growth was induced at 10 Gy, leading to defects in the feather structure. This perturbation became irreversible at 20 Gy. Molecular and cellular analysis revealed P53 activation, DNA damage and repair, cell cycle arrest and apoptosis in the pathobiology. IR also induces patterning defects in feather formation, with disrupted branching morphogenesis. This perturbation is mediated by cytokine production and Stat1 activation, as manipulation of cytokine levels or ectopic Stat1 over-expression also led to irregular feather branching. Furthermore, AG-490, a chemical inhibitor of Stat1 signaling, can partially rescue IR-induced tissue damage. Our results suggest that the feather follicle could serve as a useful model to address the in vivo impact of the many mechanisms of IR-induced tissue damage.

Stem cells have the remarkable potential to develop into many different cell types in a well organized spatial pattern. How adult stem cells regenerate and pattern themselves are mostly unknown. Chicken feather is a good model for studying regeneration because feathers undergo repetitive molting and regeneration under physiological conditions. The epithelial cells are laid out in an exquisite hierarchical branching pattern, down to single cell. It is a highly sensitive assay because any perturbation of the patterning process can be revealed in this high resolution