FOXP4 differentially controls cold-induced beige adipocyte differentiation and thermogenesis.

Beige adipocytes have a discrete developmental origin and possess notable plasticity in their thermogenic capacity in response to various environmental cues, but the transcriptional machinery controlling beige adipocyte development and thermogenesis remains largely unknown. By analyzing beige adipocyte-specific knockout mice, we identified a transcription factor, forkhead box P4 (FOXP4), that differentially governs beige adipocyte differentiation and activation. Depletion of Foxp4 in progenitor cells impaired beige cell early differentiation. However, we observed that ablation of Foxp4 in differentiated adipocytes profoundly potentiated their thermogenesis capacity upon cold exposure. Of note, the outcome of Foxp4 deficiency on UCP1-mediated thermogenesis was confined to beige adipocytes, rather than to brown adipocytes. Taken together, we suggest that FOXP4 primes beige adipocyte early differentiation, but attenuates their activation by potent transcriptional repression of the thermogenic program.

Foxp2 regulates anatomical features that may be relevant for vocal behaviors and bipedal locomotion.

Fundamental human traits, such as language and bipedalism, are associated with a range of anatomical adaptations in craniofacial shaping and skeletal remodeling. However, it is unclear how such morphological features arose during hominin evolution. FOXP2 is a brain-expressed transcription factor implicated in a rare disorder involving speech apraxia and language impairments. Analysis of its evolutionary history suggests that this gene may have contributed to the emergence of proficient spoken language. In the present study, through analyses of skeleton-specific knockout mice, we identified roles of Foxp2 in skull shaping and bone remodeling. Selective ablation of Foxp2 in cartilage disrupted pup vocalizations in a similar way to that of global Foxp2 mutants, which may be due to pleiotropic effects on craniofacial morphogenesis. Our findings also indicate that Foxp2 helps to regulate strength and length of hind limbs and maintenance of joint cartilage and intervertebral discs, which are all anatomical features that are susceptible to adaptations for bipedal locomotion. In light of the known roles of Foxp2 in brain circuits that are important for motor skills and spoken language, we suggest that this gene may have been well placed to contribute to coevolution of neural and anatomical adaptations related to speech and bipedal locomotion.

Foxp1/2/4 regulate endochondral ossification as a suppresser complex.

Osteoblast induction and differentiation in developing long bones is dynamically controlled by the opposing action of transcriptional activators and repressors. In contrast to the long list of activators that have been discovered over past decades, the network of repressors is not well-defined. Here we identify the expression of Foxp1/2/4 proteins, comprised of Forkhead-box (Fox) transcription factors of the Foxp subfamily, in both perichondrial skeletal progenitors and proliferating chondrocytes during endochondral ossification. Mice carrying loss-of-function and gain-of-function Foxp mutations had gross defects in appendicular skeleton formation. At the cellular level, over-expression of Foxp1/2/4 in chondroctyes abrogated osteoblast formation and chondrocyte hypertrophy. Conversely, single or compound deficiency of Foxp1/2/4 in skeletal progenitors or chondrocytes resulted in premature osteoblast differentiation in the perichondrium, coupled with impaired proliferation, survival, and hypertrophy of chondrocytes in the growth plate. Foxp1/2/4 and Runx2 proteins interacted in vitro and in vivo, and Foxp1/2/4 repressed Runx2 transactivation function in heterologous cells. This study establishes Foxp1/2/4 proteins as coordinators of osteogenesis and chondrocyte hypertrophy in developing long bones and suggests that a novel transcriptional repressor network involving Foxp1/2/4 may regulate Runx2 during endochondral ossification.

Ablation of Wntless in endosteal niches impairs lymphopoiesis rather than HSCs maintenance.

Osteoblasts and perivascular stromal cells constitute essential niches for HSC self-renewal and maintenance in the bone marrow. Wnt signaling is important to maintain HSC integrity. However, the paracrine role of Wnt proteins in osteoblasts-supported HSC maintenance and differentiation remains unclear. Here, we investigated hematopoiesis in mice with Wntless (Wls) deficiency in osteoblasts or Nestin-positive mesenchymal progenitor cells, which presumptively block Wnt secretion in osteoblasts. We detected defective B-cell lymphopoiesis and abnormal T-cell infiltration in the bone marrow of Wls mutant mice. Notably, no impact on HSC frequency and repopulation in the bone marrow was observed with the loss of osteoblastic Wls. Our findings revealed a supportive role of Wnts in osteoblasts-regulated B-cell lymphopoiesis. They also suggest a preferential niche role of osteoblastic Wnts for lymphoid cells rather than HSCs, providing new clues for the molecular nature of distinct niches occupied by different hematopoietic cells.

Ectodermal Wnt signaling regulates abdominal myogenesis during ventral body wall development.

Defects of the ventral body wall are prevalent birth anomalies marked by deficiencies in body wall closure, hypoplasia of the abdominal musculature and multiple malformations across a gamut of organs. However, the mechanisms underlying ventral body wall defects remain elusive. Here, we investigated the role of Wnt signaling in ventral body wall development by inactivating Wls or ß-catenin in murine abdominal ectoderm. The loss of Wls in the ventral epithelium, which blocks the secretion of Wnt proteins, resulted in dysgenesis of ventral musculature and genito-urinary tract during embryonic development. Molecular analyses revealed that the dermis and myogenic differentiation in the underlying mesenchymal progenitor cells was perturbed by the loss of ectodermal Wls. The activity of the Wnt-Pitx2 axis was impaired in the ventral mesenchyme of the mutant body wall, which partially accounted for the defects in ventral musculature formation. In contrast, epithelial depletion of ß-catenin or Wnt5a did not resemble the body wall defects in the ectodermal Wls mutant. These findings indicate that ectodermal Wnt signaling instructs the underlying mesodermal specification and abdominal musculature formation during ventral body wall development, adding evidence to the theory that ectoderm-mesenchyme signaling is a potential unifying mechanism for the origin of ventral body wall defects.

Ndrg2 regulates vertebral specification in differentiating somites.

It is generally thought that vertebral patterning and identity are globally determined prior to somite formation. Relatively little is known about the regulators of vertebral specification after somite segmentation. Here, we demonstrated that Ndrg2, a tumor suppressor gene, was dynamically expressed in the presomitic mesoderm (PSM) and at early stage of differentiating somites. Loss of Ndrg2 in mice resulted in vertebral homeotic transformations in thoracic/lumbar and lumbar/sacral transitional regions in a dose-dependent manner. Interestingly, the inactivation of Ndrg2 in osteoblasts or chondrocytes caused defects resembling those observed in Ndrg2(-/-) mice, with a lower penetrance. In addition, forced overexpression of Ndrg2 in osteoblasts or chondrocytes also conferred vertebral defects, which were distinct from those in Ndrg2(-/-) mice. These genetic analyses revealed that Ndrg2 modulates vertebral identity in segmented somites rather than in the PSM. At the molecular level, combinatory alterations of the amount of Hoxc8-11 gene transcripts were detected in the differentiating somites of Ndrg2(-/-) embryos, which may partially account for the vertebral defects in Ndrg2 mutants. Nevertheless, Bmp/Smad signaling activity was elevated in the differentiating somites of Ndrg2(-/-) embryos. Collectively, our findings unveiled Ndrg2 as a novel regulator of vertebral specification in differentiating somites.

Deacetylation of FOXP1 by HDAC7 potentiates self-renewal of mesenchymal stem cells.

BACKGROUND: Mesenchymal stem cells (MSCs) are widely used in a variety of tissue regeneration and clinical trials due to their multiple differentiation potency. However, it remains challenging to maintain their replicative capability during in vitro passaging while preventing their premature cellular senescence. Forkhead Box P1 (FOXP1), a FOX family transcription factor, has been revealed to regulate MSC cell fate commitment and self-renewal capacity in our previous study. METHODS: Mass spectra analysis was performed to identify acetylation sites in FOXP1 protein. Single and double knockout mice of FOXP1 and HDAC7 were generated and analyzed with bone marrow MSCs properties. Gene engineering in human embryonic stem cell (hESC)-derived MSCs was obtained to evaluate the impact of FOXP1 key modification on MSC self-renewal potency. RESULTS: FOXP1 is deacetylated and potentiated by histone deacetylase 7 (HDAC7) in MSCs. FOXP1 and HDAC7 cooperatively sustain bone marrow MSC self-renewal potency while attenuating their cellular senescence. A mutation within human FOXP1 at acetylation site (T176G) homologous to murine FOXP1 T172G profoundly augmented MSC expansion capacity during early passages. CONCLUSION: These findings reveal a heretofore unanticipated mechanism by which deacetylation of FOXP1 potentiates self-renewal of MSC and protects them from cellular senescence. Acetylation of FOXP1 residue T172 as a critical modification underlying MSC proliferative capacity. We suggest that in vivo gene editing of FOXP1 may provide a novel avenue for manipulating MSC capability during large-scale expansion in clinical trials.

Forkhead box P1 (Foxp1) in osteoblasts regulates bone mass accrual and adipose tissue energy metabolism.

Adiponectin (AdipoQ), a hormone abundantly secreted by adipose tissues, has multiple beneficial functions, including insulin sensitization as well as lipid and glucose metabolism. It has been reported that bone controls energy metabolism through an endocrine-based mechanism. In this study, we observed that bone also acts as an important endocrine source for AdipoQ, and its capacity in osteoblasts is controlled by the forkhead box P1 (FOXP1) transcriptional factor. Deletion of the Foxp1 gene in osteoblasts led to augmentation of AdipoQ levels accompanied by fueled energy expenditure in adipose tissues. In contrast, overexpression of Foxp1 in bones impaired AdipoQ secretion and restrained energy consumption. Chromatin immunoprecipitation sequencing (ChIP-seq) analysis revealed that AdipoQ expression, which increases as a function of bone age, is directly controlled by FOXP1. Our results indicate that bones, especially aged bones, provide an important source of a set of endocrine factors, including AdipoQ, that control body metabolism. © 2021 American Society for Bone and Mineral Research (ASBMR).

WD40 repeat and FYVE domain containing 3 is essential for cardiac development.

AIMS: WD40 repeat and FYVE domain containing 3 (WDFY3) is an adaptor protein involved in selective degradation of protein aggregates by autophagy. Recent studies have revealed that Wdfy3 is critical in the regulation of brain development and osteoclastogenesis in vivo. However, the function of Wdfy3 in cardiac development remains completely unknown. In this study, we explore the role of Wdfy3 in cardiac morphogenesis using Wdfy3-deficient mice. METHODS AND RESULTS: Wdfy3 was expressed in the developing heart in mice and peaked at embryonic day 12.5 (E12.5). Loss of Wdfy3 in mice led to embryonic and neonatal lethality. Wdfy3-deficient mice displayed various congenital heart defects including membranous ventricular septal defect (VSD), aortic overriding (AO), double outlet right ventricle (DORV), thinning of ventricular wall, ventricular dilation, and disorganized ventricular trabeculation at E14.5. Cell proliferation was reduced in the hearts from Wdfy3-deficient mice at E12.5 and E14.5, which was associated with enhanced p21 expression. Cardiomyocyte differentiation was diminished as demonstrated by reduced Myh6 and MLC2v in Wdfy3-deficient mice at E14.5. In addition, Nkx2-5 and Mef2c, two cardiac transcription factors regulating cardiomyocyte differentiation, were decreased in Wdfy3-deficient mice at E14.5. Apoptotic cell death remained unaltered. These data suggest that reduced cell proliferation and cardiomyocyte differentiation contribute to cardiac defects in Wdfy3-deficient mice. Mechanistically, loss of Wdfy3 led to a reduction in protein levels of Notch 1 intracellular domain and its downstream targets Hes1 and Hey1, which was accompanied with enhanced full-length Notch1 protein levels. In vitro luciferase assay showed that Wdfy3 deficiency induced activity of p21 promoter, while diminished activity of Hes1 promoter through modulation of Notch1 signalling. Moreover, Wdfy3 was co-localized with Notch1 in primary embryonic cardiomyocytes. Endogenous Wdfy3 physically interacted with full-length Notch1 in the developing heart. These results suggest that Notch1 signalling is perturbed in the hearts from Wdfy3-deficient mice. No alteration of autophagy was detected in the hearts from Wdfy3-deficient mice. CONCLUSION: Taken together, our data suggest that Wdfy3 plays an essential role in cardiac development, which may be mediated by modulation of Notch1 signalling.

Foxp1 controls brown/beige adipocyte differentiation and thermogenesis through regulating ß3-AR desensitization.

ß-Adrenergic receptor (ß-AR) signaling is a pathway controlling adaptive thermogenesis in brown or beige adipocytes. Here we investigate the biological roles of the transcription factor Foxp1 in brown/beige adipocyte differentiation and thermogenesis. Adipose-specific deletion of Foxp1 leads to an increase of brown adipose activity and browning program of white adipose tissues. The Foxp1-deficient mice show an augmented energy expenditure and are protected from diet-induced obesity and insulin resistance. Consistently, overexpression of Foxp1 in adipocytes impairs adaptive thermogenesis and promotes diet-induced obesity. A robust change in abundance of the ß3-adrenergic receptor (ß3-AR) is observed in brown/beige adipocytes from both lines of mice. Molecularly, Foxp1 directly represses ß3-AR transcription and regulates its desensitization behavior. Taken together, our findings reveal Foxp1 as a master transcriptional repressor of brown/beige adipocyte differentiation and thermogenesis, and provide an important clue for its targeting and treatment of obesity.

Bone marrow fibrosis with fibrocytic and immunoregulatory responses induced by ß-catenin activation in osteoprogenitors.

Wnt/ß-catenin signaling has been reported to contribute to the development of bone fibrous dysplasia. However, it remains unclear whether fibrocytes and immune cells are involved in this ß-catenin-mediated bone marrow fibrosis. In this study, we showed that constitutive activation of ß-catenin by Col1a1-Cre (3.6-kb) exhibited bone marrow fibrosis, featured with expanded populations of fibrocytes, myofibroblasts and osteoprogenitors. Lineage tracing and IHC examinations showed that Col3.6-Cre display Cre recombinase activity not only in osteoprogenitors, but also in monocyte-derived fibrocytes in the endosteal niches of bones. Additionally, ß-catenin stimulated the secretion of cytokines and pro-fibrotic signals in bone marrow, including GM-CSF, TGFß1 and VEGF. Consequently, the frequency of differentiated immature monocyte-derived dendritic cells and naïve T cells was markedly increased in the mutant bone marrow. These phenotypes were quite different from those following ß-catenin activation in mature osteoblasts driven by Col1a1-Cre (2.3-kb). Our findings suggested that a conserved pro-fibrotic signal cascade might underlie ß-catenin-mediated bone marrow fibrosis, involving TGFß1-enhanced fibrocyte activation and immunoregulatory responses. This study might shed new light on the understanding and development of a therapeutic strategy for bone fibrous dysplasia.

[Establish a transgenic mice model harboring structural genes of hepatitis C virus].

OBJECTIVES: To establish an animal model of HCV transgenic mice to elucidate the pathogenesis of hepatitis C virus infection and function of the viral structural proteins. METHODS: Structural gene of HCV were amplified and recombined into eukaryotic expression vectors, pcDNA4HisMax and pMT/BiP/V5-His A, after their expressive activity was confirmed to detect the structural protein in the transfected COS7 and S2 cells by Western blot. The fertilized expression element, which contained CMV or pMT promoter, structural gene of HCV and polyadenylation signal sequence, was microinjected into 1736 C57BL/6 mouse fertilized ova. The ova were then replanted into the oviducts of 69 pseudopregnant recipient mice. RESULTS: Twenty-five recipient mice were impregnated and later produced 105 newborns; 49 of them died from unknown causes and 57 survived. After the specific HCV structural genes were identified by PCR and Southern blot hybridization, 26 founders were obtained; among them 10 were stable expression mice and 16 were the inducible ones. The rate of founders developed from implanted embryos was only 1.50%. Through hybridization with normal mice, 58 hybrid mice have been obtained at present. CONCLUSION: Two kinds of different transgenic mice of HCV were developed; one is of stable expression, and the other is inducible. This transgenic mice model may create an opportunity for studying the function of the structural gene of HCV and elucidate its pathogenicity.

Foxp1 Regulates the Proliferation of Hair Follicle Stem Cells in Response to Oxidative Stress during Hair Cycling.

Hair follicle stem cells (HFSCs) in the bugle circularly generate outer root sheath (ORS) through linear proliferation within limited cycles during anagen phases. However, the mechanisms controlling the pace of HFSC proliferation remain unclear. Here we revealed that Foxp1, a transcriptional factor, was dynamically relocated from the nucleus to the cytoplasm of HFSCs in phase transitions from anagen to catagen, coupled with the rise of oxidative stress. Mass spectrum analyses revealed that the S468 phosphorylation of Foxp1 protein was responsive to oxidative stress and affected its nucleocytoplasmic translocation. Foxp1 deficiency in hair follicles led to compromised ROS accrual and increased HFSC proliferation. And more, NAC treatment profoundly elongated the anagen duration and HFSC proliferation in Foxp1-deficient background. Molecularly, Foxp1 augmented ROS levels through suppression of Trx1-mediated reductive function, thereafter imposing the cell cycle arrest by modulating the activity of p19/p53 pathway. Our findings identify a novel role for Foxp1 in controlling HFSC proliferation with cellular dynamic location in response to oxidative stress during hair cycling.

Osteoblastic Wnts differentially regulate bone remodeling and the maintenance of bone marrow mesenchymal stem cells.

Wnt signaling has important roles in embryonic bone development and postnatal bone remodeling, but inconsistent impact on bone property is observed in different genetic alterations of Lrp5 and ß-catenin. More importantly, it is still controversial whether Lrp5 regulate bone formation locally or globally through gut-derived serotonin. Here we explored the function of Wnt proteins in osteoblastic niche through inactivation of the Wntless (Wls) gene, which abrogates the secretion of Wnts. The depletion of Wls in osteoblast progenitor cells resulted in severe osteopenia with more profound defects in osteoblastogenesis, osteoclastogenesis and maintenance of bone marrow mesenchymal stem cells (BMSCs) compared to that observed in Lrp5 and ß-catenin mutants. These findings support the point of view that Wnt/Lrp5 signaling locally regulates bone mass accrual through multiple effects of osteoblastic Wnts on osteoblastic bone formation and osteoclastic bone resorption. Moreover, osteoblastic Wnts confer a niche role for maintenance of BMSCs, providing novel cues for the definition of BMSCs niche in bone marrow.

Wnt-mediated reciprocal regulation between cartilage and bone development during endochondral ossification.

The role of Wnt signaling is extensively studied in skeletal development and postnatal bone remodeling, mostly based on the genetic approaches of ß-catenin manipulation. However, given their independent function, a requirement for ß-catenin is not the same as that for Wnt. Here, we investigated the effect of Wnt proteins in both tissues through generating cartilage- or bone-specific Wls null mice, respectively. Depletion of Wls by Col2-Cre, which would block Wnt secretion in the chondrocytes and perichondrium, delayed chondrocyte hypertrophy in the growth plate and impaired perichondrial osteogenesis. Loss of Wls in chondrocytes also disturbed the proliferating chondrocyte morphology and division orientation, which was similar to the defect observed in Wnt5a null mice. On the other hand, inactivation of Wls in osteoblasts by Col1-Cre resulted in a shorter hypertrophic zone and an increase of TRAP positive cell number in the chondro-osseous junction of growth plate, coupled with a decrease in bone mass. Taken together, our studies reveal that Wnt proteins not only modulate differentiation and cellular communication within populations of chondrocytes, but also mediate the cross regulation between the chondrocytes and osteoblasts in growth plate.

Misexpression of Pknox2 in mouse limb bud mesenchyme perturbs zeugopod development and deltoid crest formation.

The TALE (Three Amino acid Loop Extension) family consisting of Meis, Pbx and Pknox proteins is a group of transcriptional co-factors with atypical homeodomains that play pivotal roles in limb development. Compared to the in-depth investigations of Meis and Pbx protein functions, the role of Pknox2 in limb development remains unclear. Here, we showed that Pknox2 was mainly expressed in the zeugopod domain of the murine limb at E10.5 and E11.5. Misexpression of Pknox2 in the limb bud mesenchyme of transgenic mice led to deformities in the zeugopod and forelimb stylopod deltoid crest, but left the autopod and other stylopod skeletons largely intact. These malformations in zeugopod skeletons were recapitulated in mice overexpressing Pknox2 in osteochondroprogenitor cells. Molecular and cellular analyses indicated that the misexpression of Pknox2 in limb bud mesenchyme perturbed the Hox10-11 gene expression profiles, decreased Col2 expression and Bmp/Smad signaling activity in the limb. These results indicated that Pknox2 misexpression affected mesenchymal condensation and early chondrogenic differentiation in the zeugopod skeletons of transgenic embryos, suggesting Pknox2 as a potential regulator of zeugopod and deltoid crest formation.