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.

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.

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.

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.

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.

The Feather Model for 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.

E-Cadherin-Mediated Cell Contact Controls the Epidermal Damage Response in Radiation Dermatitis.

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 ß-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 ß-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.

p53 Is a Direct Transcriptional Repressor of Keratin 17: Lessons from a Rat Model of Radiation Dermatitis.

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.

Is the irradiated small bowel volume still a predictor for acute lower gastrointestinal toxicity during preoperative concurrent chemo-radiotherapy for rectal cancer when using intensity-modulated radiation therapy?

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/m(2) on day 1 and capecitabine 825 mg/m(2), 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 cm(3) versus without grade ≥2 ALGIT: 58.5 ± 25.2 cm(3), 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.

Circadian gene expression predicts patient response to neoadjuvant chemoradiation therapy for rectal cancer.

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<0.05), whereas Per1, Cry1 and Bmal1 expressions did not differ significantly between groups. Among the downstream genes involved in cell cycle regulation, c-Myc showed significantly higher expression in the pCR group (P<0.05), whereas Wee1 and Chk2 expression did not differ significantly between groups. Circadian genes are potential biomarkers for predicting whether a patient with rectal cancer would benefit from neoadjuvant chemoradiation therapy.

Testing chemotherapeutic agents in the feather follicle identifies a selective blockade of cell proliferation and a key role for sonic hedgehog signaling in chemotherapy-induced tissue damage.

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.

SIRT2 as a new player in epigenetic programming of keratinocyte differentiation and a candidate tumor suppressor.

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.

Dissecting the molecular mechanism of ionizing radiation-induced tissue damage in the feather follicle.

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.

Loss of Msx2 function down-regulates the FoxE3 expression and results in anterior segment dysgenesis resembling Peters anomaly.

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.

Interrogating cell signalling network sensitively monitors cell fate transition during early differentiation of mouse embryonic stem cells.

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.