Xiaoyang Sun

Loss of lysyl oxidase-like 3 causes cleft palate and spinal deformity in mice

In mammals, embryonic development are highly regulated morphogenetic processes that are tightly controlled by genetic elements. Failure of any one of these processes can result in embryonic malformation. The lysyl oxidase (LOX) family genes are closely related to human diseases. In this study, we investigated the essential role of lysyl oxidase-like 3 (LOXL3), a member of the LOX family, in embryonic development. Mice lacking LOXL3 exhibited perinatal lethality, and the deletion of the Loxl3 gene led to impaired development of the palate shelves, abnormalities in the cartilage primordia of the thoracic vertebrae and mild alveolar shrinkage. We found that the obvious decrease of collagen cross-links in palate and spine that was induced by the lack of LOXL3 resulted in cleft palate and spinal deformity. Thus, we provide critical in vivo evidence that LOXL3 is indispensable for mouse palatogenesis and vertebral column development. The Loxl3 gene may be a candidate disease gene resulting in cleft palate and spinal deformity.

Abnormal cerebellar development and Purkinje cell defects in Lgl1-Pax2 conditional knockout mice.

Lgl1 was initially identified as a tumour suppressor in flies and is characterised as a key regulator of epithelial polarity and asymmetric cell division. A previous study indicated that More-Cre-mediated Lgl1 knockout mice exhibited significant brain dysplasia and died within 24h after birth. To overcome early neonatal lethality, we generated Lgl1 conditional knockout mice mediated by Pax2-Cre, which is expressed in almost all cells in the cerebellum, and we examined the functions of Lgl1 in the cerebellum. Impaired motor coordination was detected in the mutant mice. Consistent with this abnormal behaviour, homozygous mice possessed a smaller cerebellum with fewer lobes, reduced granule precursor cell (GPC) proliferation, decreased Purkinje cell (PC) quantity and dendritic dysplasia. Loss of Lgl1 in the cerebellum led to hyperproliferation and impaired differentiation of neural progenitors in ventricular zone. Based on the TUNEL assay, we observed increased apoptosis in the cerebellum of mutant mice. We proposed that impaired differentiation and increased apoptosis may contribute to decreased PC quantity. To clarify the effect of Lgl1 on cerebellar granule cells, we used Math1-Cre to specifically delete Lgl1 in granule cells. Interestingly, the Lgl1-Math1 conditional knockout mice exhibited normal proliferation of GPCs and cerebellar development. Thus, we speculated that the reduction in the proliferation of GPCs in Lgl1-Pax2 conditional knockout mice may be secondary to the decreased number of PCs, which secrete the mitogenic factor Sonic hedgehog to regulate GPC proliferation. Taken together, these findings suggest that Lgl1 plays a key role in cerebellar development and folia formation by regulating the development of PCs.

LKB1 Regulates Cerebellar Development by Controlling Sonic Hedgehog-mediated Granule Cell Precursor Proliferation and Granule Cell Migration.

The Liver Kinase B1 (LKB1) gene plays crucial roles in cell differentiation, proliferation and the establishment of cell polarity. We created LKB1 conditional knockout mice (LKB1Atoh1 CKO) to investigate the function of LKB1 in cerebellar development. The LKB1Atoh1 CKO mice displayed motor dysfunction. In the LKB1Atoh1 CKO cerebellum, the overall structure had a larger volume and morelobules. LKB1 inactivationled to an increased proliferation of granule cell precursors (GCPs), aberrant granule cell migration and overproduction of unipolar brush cells. To investigate the mechanism underlying the abnormal foliation, we examined sonic hedgehog signalling (Shh) by testing its transcriptional mediators, the Gli proteins, which regulate the GCPs proliferation and cerebellar foliation during cerebellar development. The expression levels of Gli genes were significantly increased in the mutant cerebellum. In vitro assays showed that the proliferation of cultured GCPs from mutant cerebellum significantly increased, whereas the proliferation of mutant GCPs significantly decreased in the presence of a Shh inhibitor GDC-0049. Thus, LKB1 deficiency in the LKB1Atoh1 CKO mice enhanced Shh signalling, leading to the excessive GCP proliferation and the formation of extra lobules. We proposed that LKB1 regulates cerebellar development by controlling GCPs proliferation through Shh signalling during cerebellar development.

Loss of Lysyl Oxidase-like 3 Attenuates Embryonic Lung Development in Mice.

Lysyl oxidase-like 3 (LOXL3), a human disease gene candidate, is a member of the lysyl oxidase (LOX) family and is indispensable for mouse palatogenesis and vertebral column development. Our previous study showed that the loss of LOXL3 resulted in a severe cleft palate and spinal deformity. In this study, we investigated a possible role for LOXL3 in mouse embryonic lung development. LOXL3-deficient mice displayed reduced lung volumes and weights, diminished saccular spaces, and deformed and smaller thoracic cavities. Excess elastic fibres were detected in LOXL3-deficient lungs, which might be related to the increased LOXL4 expression. Increased transforming growth factor β1 (TGFβ1) expression might be involved in the up-regulation of LOXL4 in LOXL3-deficient lungs. We concluded that the loss of LOXL3 attenuates mouse embryonic lung development.

Deletion of Brg1 causes abnormal hair cell planer polarity, hair cell anchorage, and scar formation in mouse cochlea.

Hair cells (HCs) are mechanosensors that play crucial roles in perceiving sound, acceleration, and fluid motion. The precise architecture of the auditory epithelium and its repair after HC loss is indispensable to the function of organ of Corti (OC). In this study, we showed that Brg1 was highly expressed in auditory HCs. Specific deletion of Brg1 in postnatal HCs resulted in rapid HC degeneration and profound deafness in mice. Further experiments showed that cell-intrinsic polarity of HCs was abolished, docking of outer hair cells (OHCs) by Deiter’s cells (DCs) failed, and scar formation in the reticular lamina was deficient. We demonstrated that Brg1 ablation disrupted the Gαi/Insc/LGN and aPKC asymmetric distributions, without overt effects on the core planer cell polarity (PCP) pathway. We also demonstrated that Brg1-deficient HCs underwent apoptosis, and that leakage in the reticular lamina caused by deficient scar formation shifted the mode of OHC death from apoptosis to necrosis. Together, these data demonstrated a requirement for Brg1 activity in HC development and suggested a role for Brg1 in the proper cellular structure formation of HCs.

Rcan2 and estradiol independently regulate body weight in female mice

Rcan2 increases food intake and plays an important role in the development of age- and diet- induced obesity in male mice. However, in females, wild-type mice grow almost at a similar rate as Rcan2−/− mice on normal chow diet from 6 weeks of age. Here we showed that the ability of Rcan2 to promote weight gain was attenuated by energy expenditure mediated by 17β-estradiol in female mice. Using ovariectomy-operated models, we found that 17β-estradiol deprivation did not alter food intake, but induced more weight gain in wild-type mice than Rcan2−/− mice. If wild-type mice ingested equally as Rcan2−/− mice, in the same ovarian state they exhibited similar weight changes, but the mice in ovariectomized groups were significantly heavier than the ovarian-intact mice, suggesting that body weight is not only regulated by Rcan2, but also by 17β-estradiol. Furthermore, we demonstrated that Rcan2 and 17β-estradiol independently regulated body weight even on high-fat diets. Therefore, our findings indicate that Rcan2 and 17β-estradiol regulate body weight through different mechanisms. Rcan2 increases food intake, whereas 17β-estradiol promotes energy expenditure. These findings provide novel insights into the sexual dimorphism of body weight regulation.

Tuberous sclerosis complex–mediated mTORC1 overactivation promotes age-related hearing loss

The underlying molecular mechanisms of age-related hearing loss (ARHL) in humans and many strains of mice have not been fully characterized. This common age-related disorder is assumed to be closely associated with oxidative stress. Here, we demonstrate that mTORC1 signaling is highly and specifically activated in the cochlear neurosensory epithelium (NSE) in aging mice, and rapamycin injection prevents ARHL. To further examine the specific role of mTORC1 signaling in ARHL, we generated murine models with NSE-specific deletions of Raptor or Tsc1, regulators of mTORC1 signaling. Raptor-cKO mice developed hearing loss considerably more slowly than WT littermates. Conversely, Tsc1 loss led to the early-onset death of cochlear hair cells and consequently accelerated hearing loss. Tsc1-cKO cochleae showed features of oxidative stress and impaired antioxidant defenses. Treatment with rapamycin and the antioxidant N-acetylcysteine rescued Tsc1-cKO hair cells from injury in vivo. In addition, we identified the peroxisome as the initial signaling organelle involved in the regulation of mTORC1 signaling in cochlear hair cells. In summary, our findings identify overactive mTORC1 signaling as one of the critical causes of ARHL and suggest that reduction of mTORC1 activity in cochlear hair cells may be a potential strategy to prevent ARHL.

Tprn is essential for the integrity of stereociliary rootlet in cochlear hair cells in mice

Tprn encodes the taperin protein, which is concentrated in the tapered region of hair cell stereocilia in the inner ear. In humans, TPRN mutations cause autosomal recessive nonsyndromic deafness (DFNB79) by an unknown mechanism. To determine the role of Tprn in hearing, we generated Tprn-null mice by clustered regularly interspaced short palindromic repeat/Cas9 genome-editing technology from a CBA/CaJ background. We observed significant hearing loss and progressive degeneration of stereocilia in the outer hair cells of Tprn-null mice starting from postnatal day 30. Transmission electron microscopy images of stereociliary bundles in the mutant mice showed some stereociliary rootlets with curved shafts. The central cores of the stereociliary rootlets possessed hollow structures with surrounding loose peripheral dense rings. Radixin, a protein expressed at stereocilia tapering, was abnormally dispersed along the stereocilia shafts in Tprn-null mice. The expression levels of radixin and β-actin significantly decreased.We propose that Tprn is critical to the retention of the integrity of the stereociliary rootlet. Loss of Tprn in Tprn-null mice caused the disruption of the stereociliary rootlet, which resulted in damage to stereociliary bundles and hearing impairments. The generated Tprn-null mice are ideal models of human hereditary deafness DFNB79.

Oocyte-specific deletion of Gsα induces oxidative stress and deteriorates oocyte quality in mice

&NA; The stimulatory heterotrimeric Gs protein alpha subunit (Gs&agr;) is a ubiquitous guanine nucleotide‐binding protein that regulates the intracellular cAMP signaling pathway and consequently participates in a wide range of biological events. In the reproductive system, despite Gs&agr; being associated with oocyte meiotic arrest in vitro, the exact role of Gs&agr; in female fertility in vivo remains largely unknown. Here, we generated oocyte‐specific Gs&agr; knockout mice by using the Cre/LoxP system. We observed that the deletion of Gs&agr; caused complete female infertility. Exclusion of post‐implantation abnormalities, oogenesis, fertilization, and early embryo development was subsequently monitored; meiosis in Gs&agr;‐deficient oocytes precociously resumed in only 43% of antral follicles from mutant mice, indicating that alteration of meiotic pause was not the key factor in infertility. Ovulation process and number were normal, but the rate of morphological abnormal oocytes was apparently increased; spindle organization, fertilization, and early embryo development were impaired. Furthermore, the level of ROS (reactive oxygen species) and the mitochondrial aggregation increased, and antioxidant glutathione (GSH) content, ATP level, mtDNA copy number, and mitochondrial membrane potential decreased in Gs&agr;‐deficient oocytes. GV oocytes from mutant mice showed early‐stage apoptosis. Meanwhile, the Gs&agr; knockout‐induced decline in oocyte quality and low developmental potential was partially rescued by antioxidant supplementation. To sum up, our results are the first to reveal that the profile of Gs&agr; oocyte‐specific deletion caused female infertility in vivo, and oxidative stress plays an important role in this event. HighlightsOocyte‐specific disruption of Gs&agr; leads to female infertility in mice.Deletion of Gs&agr; in oocytes causes poor oocyte quality and impaired early embryo development.Gs&agr; deficiency in oocytes leads to oxidative stress.

A disputed evidence on obesity: comparison of the effects of Rcan2−/− and Rps6kb1−/− mutations on growth and body weight in C57BL/6J mice

It is widely accepted that body weight and adipose mass are tightly regulated by homeostatic mechanisms, in which leptin plays a critical role through hypothalamic pathways, and obesity is a result of homeostatic disorder. However, in C57BL/6J mice, we found that Rcan2 increases food intake and plays an important role in the development of age- and diet-induced obesity through a leptin-independent mechanism. RCAN2 was initially identified as a thyroid hormone (T3)-responsive gene in human fibroblasts. Expression of RCAN2 is regulated by T3 through the PI3K-Akt/PKB-mTOR-Rps6kb1 signaling pathway. Intriguingly, both Rcan2−/− and Rps6kb1−/− mutations were reported to result in lean phenotypes in mice. In this study we compared the effects of these two mutations on growth and body weight in C57BL/6J mice. We observed reduced body weight and lower fat mass in both Rcan2−/− and Rps6kb1−/− mice compared to the wild-type mice, and we reported other differences unique to either the Rcan2−/− or Rps6kb1−/− mice. Firstly, loss of Rcan2 does not directly alter body length; however, Rcan2−/− mice exhibit reduced food intake. In contrast, Rps6kb1−/− mice exhibit abnormal embryonic development, which leads to smaller body size and reduced food intake in adulthood. Secondly, when fed a normal chow diet, Rcan2−/− mice weigh significantly more than Rps6kb1−/− mice, but both Rcan2−/− and Rps6kb1−/− mice develop similar amounts of epididymal fat. On a high-fat diet, Rcan2−/− mice gain body weight and fat mass at slower rates than Rps6kb1−/− mice. Finally, using the double-knockout mice (Rcan2−/−Rps6kb1−/−), we demonstrate that concurrent loss of Rcan2 and Rps6kb1 has an additive effect on body weight reduction in C57BL/6J mice. Our data suggest that Rcan2 and Rps6kb1 mutations both affect growth and body weight of mice, though likely through different mechanisms.中文概要目的通过比较Rcan2−/−和Rps6kb1−/−两种突变对肥胖 动物模型C57BL/6J 小鼠生长和体重的影响,确 定它们是否参与肥胖的发生。创新点明确了Rcan2−/−和Rps6kb1−/−两种基因突变影响 体重的机制,进一步证实了Rcan2 基因在小鼠肥 胖发生中的重要作用,同时发现高脂肪食物以一 种与基因无关的方式促进体重增长。方法在严格控制的饲养条件下,从4 周龄开始给野生 小鼠、Rcan2−/−小鼠和Rps6kb1−/−小鼠分别喂食普 通饲料和高脂肪饲料,连续16 周监测小鼠的体 重增长曲线。体重监测结束后解剖小鼠并测定其 胫骨长、脂肪及肝脏重量,对各组的脂肪和肝脏 进行组织学分析和比较。通过野生小鼠和 Rcan2−/−小鼠的高脂肪饲料配对喂养实验,确定 二者正常摄食条件下的体重差别是否由摄食量 不同所致。通过研究双突变(Rcan2−/−Rps6kb1−/−) 小鼠与Rps6kb1−/−小鼠的相关指标,进一步确定 Rcan2 和Rps6kb1 是否经过不同机制影响体重。结论Rcan2−/−和Rps6kb1−/−两种突变对小鼠的生长和 体重均产生了影响,但二者通过不同的机制参与 该过程。Rps6kb1 基因的缺失首先影响了小鼠的 胚胎发育进而造成小鼠体型的变小,其摄食量的 减少及体重减轻可能源于体型的改变,这表明 Rps6kb1 基因是调节生长发育的一个重要基因; Rcan2 基因的缺失不影响小鼠的胚胎发育,而是 直接减少了小鼠的摄食量,进而对小鼠的生长和 体重产生了影响,这些结果进一步证明Rcan2 可 能是一种增加摄食量的基因。此外,我们发现高 脂肪食物以一种与 Rcan2 基因无关的方式促进 体重增长, Rcan2 基因和高脂肪食物共同存在时 能够导致C57BL/6J 小鼠体重的快速增加,这与 流行病学的研究结论相吻合。该发现与体重的稳 态控制理论相悖,但可为肥胖的发生和流行成因 提供一种新的解释。