Spatial transcriptomics reveals gene interactions and signaling pathway dynamics in rat embryos with anorectal malformation.

Anorectal malformation (ARM) is a prevalent early pregnancy digestive tract anomaly. The intricate anatomy of the embryonic cloaca region makes it challenging for traditional high-throughput sequencing methods to capture location-specific information. Spatial transcriptomics was used to sequence libraries of frozen sections from embryonic rats at gestational days (GD) 14 to 16, covering both normal and ARM cases. Bioinformatics analyses and predictions were performed using methods such as WGCNA, GSEA, and PROGENy. Immunofluorescence staining was used to verify gene expression levels. Gene expression data was obtained with anatomical annotations of clusters, focusing on the cloaca region's location-specific traits. WGCNA revealed gene modules linked to normal and ARM cloacal anatomy development, with cooperation between modules on GD14 and GD15. Differential gene expression profiles and functional enrichment were presented. Notably, protein levels of Pcsk9, Hmgb2, and Sod1 were found to be downregulated in the GD15 ARM hindgut. The PROGENy algorithm predicted the activity and interplay of common signaling pathways in embryonic sections, highlighting their synergistic and complementary effects. A competing endogenous RNA (ceRNA) regulatory network was constructed from whole transcriptome data. Spatial transcriptomics provided location-specific cloaca region gene expression. Diverse bioinformatics analyses deepened our understanding of ARM's molecular interactions, guiding future research and providing insights into gene regulation in ARM development.

Coactivation of NF-κB and Notch signaling is sufficient to induce B-cell transformation and enables B-myeloid conversion.

NF-κB and Notch signaling can be simultaneously activated in a variety of B-cell lymphomas. Patients with B-cell lymphoma occasionally develop clonally related myeloid tumors with poor prognosis. Whether concurrent activation of both pathways is sufficient to induce B-cell transformation and whether the signaling initiates B-myeloid conversion in a pathological context are largely unknown. Here, we provide genetic evidence that concurrent activation of NF-κB and Notch signaling in committed B cells is sufficient to induce B-cell lymphomatous transformation and primes common progenitor cells to convert to myeloid lineage through dedifferentiation, not transdifferentiation. Intriguingly, the converted myeloid cells can further transform, albeit at low frequency, into myeloid leukemia. Mechanistically, coactivation of NF-κB and Notch signaling endows committed B cells with the ability to self renew. Downregulation of BACH2, a lymphoma and myeloid gene suppressor, but not upregulation of CEBPα and/or downregulation of B-cell transcription factors, is an early event in both B-cell transformation and myeloid conversion. Interestingly, a DNA hypomethylating drug not only effectively eliminated the converted myeloid leukemia cells, but also restored the expression of green fluorescent protein, which had been lost in converted myeloid leukemia cells. Collectively, our results suggest that targeting NF-κB and Notch signaling will not only improve lymphoma treatment, but also prevent the lymphoma-to-myeloid tumor conversion. Importantly, DNA hypomethylating drugs might efficiently treat these converted myeloid neoplasms.

MAPbBr3nanocrystals from aqueous solution for poly(methyl methacrylate)-MAPbBr3nanocrystal films with compression-resistant photoluminescence.

In this work, we develop an environmental-friendly approach to produce organic-inorganic hybrid MAPbBr3(MA = CH3NH3) perovskite nanocrystals (PeNCs) and PMMA-MAPbBr3NC films with excellent compression-resistant PL characteristics. Deionized water is used as the solvent to synthesize MAPbBr3powder instead of conventionally-used hazardous organic solvents. The MAPbBr3PeNCs derived from the MAPbBr3powder exhibit a high photoluminescence quantum yield (PLQY) of 93.86%. Poly(methyl methacrylate) (PMMA)-MAPbBr3NC films made from the MAPbBr3PeNCs retain ∼97% and ∼91% of initial PL intensity after 720 h aging in ambient environment at 50 °C and 70 °C, respectively. The PMMA-MAPbBr3NC films also exhibit compression-resistant photoluminescent characteristics in contrast to the PMMA-CsPbBr3NC films under a compressive stress of 1.6 MPa. The PMMA-MAPbBr3NC film integrated with a red emissive film and a blue light emitting source achieves an LCD backlight of ∼114% color gamut of National Television System Committee (NTSC) 1953 standard.

Abnormal expression of TBX4 during anorectal development in rat embryos with ethylenethiourea-induced anorectal malformations.

BACKGROUND: To assess the expression of T-box transcription factor 4 (TBX4) during the anorectal development in normal and ethylenethiourea (ETU)-induced anorectal malformations (ARM) rat embryos. METHODS: Anorectal malformations was induced by ETU on the 10th gestational day (E10) in rat embryos. Spatio-temporal expression of TBX4 was evaluated in normal (n = 490) and ETU-induced ARM rat embryos (n = 455) from E13 to E16 by immunohistochemical staining, Western blot analysis and real-time RT-PCR. RESULTS: In the normal embryos, immunohistochemical staining revealed that TBX4 expression was detected in the epithelium of hindgut and urorectal septum (URS) on E13. TBX4-immunopositive cells were increased significantly in the epithelium of hindgut and URS, the future anal orifice part of cloacal membrane on E14. On E15, abundant stained cells were observed in the rectum, URS and dorsal cloacal membrane and the expression of positive cells reached its peak. On E16, only sporadic positive cells were distributed in the epithelium of the distal rectum. In the ARM embryos, the hindgut/rectum, URS and dorsal cloacal membrane were faint for TBX4 immunohistochemical staining. In the normal group, TBX4 protein and mRNA expression showed time-dependent changes in the hindgut/rectum from E13 to E16 on Western blot and real-time RT-PCR. On E13 and E15, the expression level of TBX4 mRNA in the ARM group was significantly lower than that in the normal group (P < 0.05). On E15, the expression level of TBX4 protein in the ARM group was significantly lower than that in the normal group (P < 0.05). CONCLUSIONS: The expression of TBX4 was downregulated in ETU-induced ARM embryos, which may play important roles in the pathogenesis of anorectal development.

Real-time monitoring system with accelerator controlling: An improvement of radiotherapy monitoring based on binocular location and classification.

Real-time monitoring and amendment of patient position is important for the radiotherapy. However, using electronic portal imaging device (EPID) and cone beam computer tomography (CBCT) in the clinical practice generate different degrees of delay, so that they cannot achieve the purpose of real-time application. Meanwhile, a few products come with the function of the real-time monitoring and amendment, such as CyberKnife, which is too expensive for the common people. The objective of this study is to develop and test a novel independent system to monitor treatment center and amend the position of patient, which is applicable to most accelerators, based on binocular location. The system monitors the treatment center by tracking the markers attached to the patient. Once the treatment center shifts, the system uses the magic finger, which is developed to control the treatment bet automatically to adjust the treatment bed position. To improve the monitoring accuracy, we trained the data collected from the clinic based on SVM (Support Vector Machine). Thus, the training results assist users to adjust the feasible degree of the monitoring. The experiment results showed that using this new monitoring system, the monitoring resolution reached 0.5 mm, and the error ratio of the judgment was less than 1.5%.

Distinct roles of Ape1 protein, an enzyme involved in DNA repair, in high or low linear energy transfer ionizing radiation-induced cell killing.

High linear energy transfer (LET) radiation from space heavy charged particles or a heavier ion radiotherapy machine kills more cells than low LET radiation, mainly because high LET radiation-induced DNA damage is more difficult to repair. Relative biological effectiveness (RBE) is the ratio of the effects generated by high LET radiation to low LET radiation. Previously, our group and others demonstrated that the cell-killing RBE is involved in the interference of high LET radiation with non-homologous end joining but not homologous recombination repair. This effect is attributable, in part, to the small DNA fragments (≤40 bp) directly produced by high LET radiation, the size of which prevents Ku protein from efficiently binding to the two ends of one fragment at the same time, thereby reducing non-homologous end joining efficiency. Here we demonstrate that Ape1, an enzyme required for processing apurinic/apyrimidinic (known as abasic) sites, is also involved in the generation of small DNA fragments during the repair of high LET radiation-induced base damage, which contributes to the higher RBE of high LET radiation-induced cell killing. This discovery opens a new direction to develop approaches for either protecting astronauts from exposure to space radiation or benefiting cancer patients by sensitizing tumor cells to high LET radiotherapy.

Ultraviolet electroluminescence of light-emitting diodes based on single n-ZnO/p-AlGaN heterojunction nanowires.

We present successful fabrication of single n-ZnO/p-AlGaN heterojunction nanowires with excellent optoelectronic properties. Because of the formation of high-quality interfacial structure, heterojunction nanowire showed a diodelike rectification behavior and an electroluminescence (EL) ultraviolet (UV) emission centered at 394 nm from a single nanowire was observed when the injection current is 4 µA due to high exciton efficiency in the interfacial layer between ZnO and AlGaN. With the increase of the applied current, the EL peak at 5 µA becomes weaker revealing an optimal injection current of less than 5 µA. These results are expected to open up new application possibilities in nanoscale UV light-emitting devices based on single ZnO heterostructure.

Green-route manufacturing towards future industrialization of metal halide perovskite nanocrystals.

Perovskite nanocrystals (PeNCs) with excellent optical properties have attracted tremendous research interests and have been considered as promising candidates for new-generation optoelectronic devices. Over the past few years, numerous efforts have been made to overcome the challenges in terms of sustainable manufacturing of PeNCs and related devices and systems, including the solvents used in precursor preparation, antisolvents and perovskite materials for the fabrication of devices and systems, and remarkable progress has been made. However, the usage of toxic, organic solvents in the synthesis of PeNCs poses a threat to the ecosystem and human health, which has hindered the progress in the commercialization and industrialization of PeNCs. This has promoted the development of green solvents for the sustainable manufacturing of PeNCs. In this Feature Article, a state-of-the-art green method for the synthesis of PeNCs is presented, in which the solvents of low toxicities are underlined in contrast to the reported Reviews which focus on toxic solvents for the preparation of precursor solutions. We then focus on green, aqueous methods for the preparation of PeNCs, including conventional perovskite and double PeNCs, by summarizing our previous research efforts and studies. In particular, pure water as the greenest solvent is introduced for the preparation of PeNCs, and the parameters affecting the size and optical characteristics of PeNCs, such as sonication time and ligands for post-treatment, are discussed. The strategies of using a passivation layer to improve the aqueous stability of PeNCs are reviewed, which are grouped into organic polymers and inorganic semiconductors. We highlight the challenges and possible solutions in the green manufacturing and applications of PeNCs. The green routes discussed in this article for the synthesis of PeNCs are expected to be a major step forward for the commercialization and industrialization of the fabrication of PeNCs. It is anticipated that green manufacturing will continue to be the mainstream in the synthesis and fabrication of PeNCs.

Rack1-mediated ferroptosis affects hindgut development in rats with anorectal malformations: Spatial transcriptome insights.

Anorectal malformation (ARM), a common congenital anomaly of the digestive tract, is a result of insufficient elongation of the urorectal septum. The cytoplasmic protein Receptor of Activated C-Kinase 1 (Rack1) is involved in embryonic neural development; however, its role in embryonic digestive tract development and ARM formation is unexplored. Our study explored the hindgut development and cell death mechanisms in ARM-affected rats using spatial transcriptome analysis. We induced ARM in rats by administering ethylenethiourea via gavage on gestational day (GD) 10. On GDs 14-16, embryos from both normal and ARM groups underwent spatial transcriptome sequencing, which identified key genes and signalling pathways. Rack1 exhibited significant interactions among differentially expressed genes on GDs 15 and 16. Reduced Rack1 expression in the ARM-affected hindgut, verified by Rack1 silencing in intestinal epithelial cells, led to increased P38 phosphorylation and activation of the MAPK signalling pathway. The suppression of this pathway downregulated Nqo1 and Gpx4 expression, resulting in elevated intracellular levels of ferrous ions, reactive oxygen species (ROS) and lipid peroxides. Downregulation of Gpx4 expression in the ARM hindgut, coupled with Rack1 co-localisation and consistent mitochondrial morphology, indicated ferroptosis. In summary, Rack1, acting as a hub gene, modulates ferrous ions, lipid peroxides, and ROS via the P38-MAPK/Nqo1/Gpx4 axis. This modulation induces ferroptosis in intestinal epithelial cells, potentially influencing hindgut development during ARM onset.

Microfluidics-enabled intelligent manufacturing of metal halide perovskite nanocrystals.

Large-scale and controllable fabrication is an indispensable step for the industrialization and commercialization of halide perovskite nanocrystals, which are new-generation semiconductor materials for optoelectronic applications. Microfluidics, which provides continuous and precise synthesis, has been considered as a promising technique to fulfill this aspect. The research studies over the past decades have witnessed the advancement of microfluidics as a powerful tool in the fabrication of halide perovskite nanocrystals. In this Perspective, the state-of-the-art research based on microfluidics is introduced initially, including the synthesis of functional structures and materials, devices, as well as the interdisciplinary interactions between microfluidics and artificial intelligence and machine learning, etc. We then detail the issues and challenges in hindering progress in the above areas. Finally, we provide future directions and trends for the technology to achieve its full potential. This Perspective is expected to benefit the collective efforts between the field of nanomaterials and microfluidics in advanced manufacturing.

Kinetic analysis of the growth behavior of perovskite CsPbBr3 nanocrystals in a microfluidic system.

Understanding the growth behavior of nanoparticles and semiconductor nanocrystals under dynamic environments is of profound importance in controlling the sizes and uniformity of the prepared nanoparticles and semiconductor nanocrystals. In this work, we develop a relation between the bandgap (the photoluminescence peak wavelength) of semiconductor nanocrystals and the total flow rate for the synthesis of semiconductor nanocrystals in microfluidic systems under the framework of the quantum confinement effect without the contribution of Coulomb interaction. Using this relation, we analyze the growth behavior of CsPbBr3 nanocrystals synthesized in a microfluidic system by an antisolvent method in the temperature range of 303 to 363 K. The results demonstrate that the square of the average size of the CsPbBr3 nanocrystals is inversely proportional to the total flow rate and support the developed relation. The activation energy for the rate process controlling the growth of the CsPbBr3 nanocrystals in the microfluidic system is 2.05 kJ mol-1. Increasing the synthesis temperature widens the size distribution of the CsPbBr3 NCs prepared in the microfluidic system. The method developed in this work provides a simple approach to use photoluminescent characteristics to in situ monitor and analyze the growth of semiconductor nanocrystals under dynamic environments.

Ultra-stable blue-emitting lead-free double perovskite Cs2SnCl6 nanocrystals enabled by an aqueous synthesis on a microfluidic platform.

Blue emitting Sn-based lead-free halide perovskite nanocrystals (NCs) are considered to be a promising material in lighting and displays. However, industrialised fabrication of blue-emitting NCs still remains a significant challenge due to the use of toxic solvents and optical instability, not mentioning in large-scale synthesis. In this work, a green-route synthesis of blue-emitting lead-free halide perovskite Cs2SnCl6 powders is developed, in which deionized water with a small amount of inorganic acid is used as the solvent and the synthesis of the Cs2SnCl6 powders is achieved on a microfluidic platform. Using the Cs2SnCl6 powders, we prepare Cs2SnCl6 NCs via an ultrasonication process. Changing the volume ratio of the ligands (oleic acid to oleylamine) can alter the photoluminescence (PL) characteristics of the prepared NCs, including the PL-peak wavelength, PL-peak intensity and quantum yield. The highest photoluminescence quantum yield (PLQY) of 13.4% is achieved by the Cs2SnCl6 NCs prepared with the volume ratio of oleic acid to oleylamine of 40 µL to 10 µL. A long-term PL stability test demonstrates that the as-synthesized Cs2SnCl6 NCs can retain a stable PLQY over a period of 60 days. This work opens up a new path for a large-scale green-route synthesis of blue-emitting Sn-based lead-free NCs, such as Cs2SnX6 (Cl, Br and I), towards their applications in optoelectronics.

In Situ Growth Mechanism for High-Quality Hybrid Perovskite Single-Crystal Thin Films with High Area to Thickness Ratio: Looking for the Sweet Spot.

The development of in situ growth methods for the fabrication of high-quality perovskite single-crystal thin films (SCTFs) directly on hole-transport layers (HTLs) to boost the performance of optoelectronic devices is critically important. However, the fabrication of large-area high-quality SCTFs with thin thickness still remains a significant challenge due to the elusive growth mechanism of this process. In this work, the influence of three key factors on in situ growth of high-quality large-size MAPbBr3 SCTFs on HTLs is investigated. An optimal "sweet spot" is determined: low interface energy between the precursor solution and substrate, a slow heating rate, and a moderate precursor solution concentration. As a result, the as-obtained perovskite SCTFs with a thickness of 540 nm achieve a record area to thickness ratio of 1.94 × 104  mm, a record X-ray diffraction peak full width at half maximum of 0.017°, and an ultralong carrier lifetime of 1552 ns. These characteristics enable the as-obtained perovskite SCTFs to exhibit a record carrier mobility of 141 cm2 V-1 s-1 and good long-term structural stability over 360 days.

p27(KIP1) regulates neurogenesis in the rostral migratory stream and olfactory bulb of the postnatal mouse.

Neuronal progenitor cells of the anterior subventricular zone (SVZa) migrate along the rostral migratory stream (RMS) to the olfactory bulb, where they exit the cell cycle and differentiate. The molecular mechanisms that regulate SVZa progenitor proliferation and cell-cycle exit are largely undefined. We investigated the role of p27(KIP1) in regulating cell proliferation and survival in the RMS and olfactory bulb between postnatal day 1 (P1) and P14, the peak period of olfactory bulb neuron generation. A large proportion of cells in the RMS and the olfactory bulb express cytoplasmic p27(KIP1), but a small percentage display high nuclear p27(KIP1) immunostaining, which exhibit a caudal(low)-rostral(high) gradient: lowest in the SVZa and highest in the glomerular layer of the olfactory bulb. p27(KIP1) is also present in the nucleus and/or the cytoplasm of neuron-specific type III beta-tubulin(+) cells. Cells with strong nuclear p27(KIP1) expression are BrdU(-) and Ki67(-). The percentage of BrdU(+) cells in the SVZa, RMS, and olfactory bulb is higher in p27(KIP1) null than wild-type (WT) mice at all ages analyzed. Consistent with these findings, p27(KIP1) overexpression in cultured p27(KIP1) null and WT SVZ cells reduced cell proliferation and self-renewal. Finally, in p27(KIP1) null mice, the diameter of the horizontal limb of the RMS is larger than in WT mice, and development of the olfactory bulb granule cell layer is delayed, together with increased apoptotic cell density. Our results indicate that in the postnatal brain, p27(KIP1) regulates the proliferation and survival of neuronal cells in the RMS and olfactory bulb.

Involvement of proliferative and apoptotic factors in the development of hindgut in rat fetuses with ethylenethiourea-induced anorectal malformations.

BACKGROUND: Anorectal malformations (ARMs) are common congenital malformations of the terminal digestive tract, but little is known regarding their pathogenesis. Aberrant cell proliferation/apoptosis are believed to be involved in ARMs. However, there are no studies on proliferation/apoptosis-related genes. PURPOSE: We aimed to investigate the spatiotemporal expression patterns of two proliferation/apoptosis-related genes (MYC proto-oncogene and tumor protein p53) and explore their potential functions in the hindguts of ethylene thiourea-induced ARMs rat fetuses. METHODS: MYC and p53 expression was evaluated using immunohistochemical staining, western blotting, and quantitative real-time polymerase chain reaction (RT-qPCR). Terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) and p53 costaining were performed to assay the colocalization of apoptotic and p53-expressing cells. RESULTS: Rat fetuses with ARMs displayed fusion failure of the urogenital septum and cloacal membrane. In the control group, MYC was persistently expressed from gestational day (GD)14 to GD16 and distributed throughout the hindgut, while p53 was weakly detected in the terminal segment of the urethra and hindgut; in the ARMs group, MYC expression was obviously reduced, while p53 was widely and highly expressed in the urethra and hindgut. Western blotting and RT-qPCR confirmed the decrease in MYC and increase in p53 expression in ARMs. TUNEL and p53 co-staining revealed considerable overlap between apoptotic and p53-expressing cells. CONCLUSION: The expression patterns of c-myc and p53 were disrupted in ARMs rat embryos, and the downregulation of c-myc and upregulation of p53 might be related to the development of ARMs at the key time points of ARMs morphogenesis.

Printable CsPbBr3 perovskite quantum dot ink for coffee ring-free fluorescent microarrays using inkjet printing.

Printable perovskite quantum dot (QD) ink is very important for achieving high quality coffee ring-free fluorescent microarrays for different kinds of emerging perovskite optoelectronic applications using inkjet printing. In this work, we prepared a printable CsPbBr3 perovskite QD ink by mixing high-boiling point dodecane with low-boiling point toluene as a solvent. The evaporation rate, viscosity and surface tension of the ink were carefully optimized by tuning the volume ratio of these two solvents for forming appropriate Marangoni flow, so as to balance the capillary flow and eliminate the coffee ring effect further. Successfully, CsPbBr3 perovskite microarrays with uniform surface, low roughness and no coffee rings were achieved by inkjet printing the optimized perovskite QD ink on a PVK (poly-(9-vinylcarbazole)) layer. Furthermore, we patterned the CsPbBr3 perovskite QD ink, and the printed patterns were only visible under ultraviolet (UV) light, which can be applied in invisible anti-counterfeiting labels and encryption in the future.

In Situ Growth of All-Inorganic Perovskite Single Crystal Arrays on Electron Transport Layer.

Directly growing perovskite single crystals on charge carrier transport layers will unravel a promising route for the development of emerging optoelectronic devices. Herein, in situ growth of high-quality all-inorganic perovskite (CsPbBr3) single crystal arrays (PeSCAs) on cubic zinc oxide (c-ZnO) is reported, which is used as an inorganic electron transport layer in optoelectronic devices, via a facile spin-coating method. The PeSCAs consist of rectangular thin microplatelets of 6-10 µm in length and 2-3 µm in width. The deposited c-ZnO enables the formation of phase-pure and highly crystallized cubic perovskites via an epitaxial lattice coherence of (100)CsPbBr3∥(100)c-ZnO, which is further confirmed by grazing incidence wide-angle X-ray scattering. The PeSCAs demonstrate a significant structural stability of 26 days with a 9 days excellent photoluminescence stability in ambient environment, which is much superior to the perovskite nanocrystals (PeNCs). The high crystallinity of the PeSCAs allows for a lower density of trap states, longer carrier lifetimes, and narrower energetic disorder for excitons, which leads to a faster diffusion rate than PeNCs. These results unravel the possibility of creating the interface toward c-ZnO heterogeneous layer, which is a major step for the realization of a better integration of perovskites and charge carrier transport layers.

Identification of optimal endogenous reference RNAs for RT-qPCR normalization in hindgut of rat models with anorectal malformations.

BACKGROUND: Quantitative real-time polymerase chain reaction (RT-qPCR) is a sensitive method for quantifying mRNA abundance. With relative expression analysis, however, reliable data output is dependent on stably expressed reference genes across the samples being studied. In anorectal malformations (ARMs), there is limited data on the selection of appropriate reference genes. PURPOSE: This study was aimed to investigate the optimal reference genes for PCR in ARM rat models. METHODS: We selected 15 commonly used reference genes (Rps18, Actb, B2m, Gapdh, Ppia, Hprt1, Pgk1, Ywhaz, Tbp, Ubc, Rps16, Rpl13a, Rplp1, Sdha, and Hmbs) as candidate reference genes and detected their mRNA expression in ARM samples by RT-qPCR. The expression stability and variability of these transcripts were subsequently evaluated using four methods (geNorm, NormFinder, comparative ΔCt, and BestKeeper). RESULTS: The abundance of the candidate reference genes was qualified by RT-qPCR and the cycle threshold (Ct) values ranged between 14.07 (Rplp1) and 21.89 (Sdha). In the overall candidate genes, different variations existed across the different algorithms. A comprehensive analysis revealed that Rpl13a ranked first among the relatively stable genes, followed by Ywhaz, Rps18, Sdha, and Hmbs. CONCLUSIONS: The most stable reference genes for RT-qPCR were Rpl13a, Ywhaz, and Rps18 in ETU-induced ARMs in rat fetus. This study provided a foundation for reference gene selection for future gene expression analyses.

Abnormal Expression of Fgf9 during the Development of the Anorectum in Rat Embryos with Anorectal Malformations.

The study objective was to investigate the role of fibroblast growth factor 9 (Fgf9) in normal and anorectal malformation (ARM) embryos during the development of the anorectum. Fgf9 expression was assayed in both normal rat embryos and embryos with ARM induced by exposure to ethylenethiourea (ETU) on embryonic day 10 (E10). Fgf9 expression was assayed by immunohistochemical staining, Western blotting, and real-time quantitative polymerase chain reaction (qRT-PCR). Immunohistochemical staining revealed spatiotemporal changes in Fgf9 expression between E13 and E16. Fgf9-positive cells predominated in the mesenchyme of the cloaca on E13 and E14 and at the fusion site of the urorectal septum and cloacal membrane, rectal epithelium, and anal membrane on E15. Fgf9-positive cells were obviously decreased after the anal membrane ruptured on E16. Fgf9-positive staining was significantly decreased in embryos with ARM compared with normal embryos from E13 to E15. The results of Western blots and qRT-PCR were consistent, with significantly increased Fgf9 expression in the hindgut and rectum of normal embryos than in embryos with ARM from E13 to E15. However, there was no difference between the two groups on E16. These results suggested that the anorectal embryogenesis might depend on the induction of Fgf9 signal. The expression of Fgf9 was downregulated in ETU-induced ARM embryos, which might be related to the development of ARM.