Development
Abstract
The developmental abnormalities associated with disruption of signaling by retinoic acid (RA), the biologically active form of vitamin A, have been known for decades from studies in animal models and humans. These include defects in the respiratory system, such as lung hypoplasia and agenesis. However, the molecular events controlled by RA that lead to formation of the lung primordium from the primitive foregut remain unclear. Here, we present evidence that endogenous RA acts as a major regulatory signal integrating Wnt and Tgfβ pathways in the control of Fgf10 expression during induction of the mouse primordial lung. We demonstrated that activation of Wnt signaling required for lung formation was dependent on local repression of its antagonist, Dickkopf homolog 1 (Dkk1), by endogenous RA. Moreover, we showed that simultaneously activating Wnt and repressing Tgfβ allowed induction of both lung buds in RA-deficient foreguts. The data in this study suggest that disruption of Wnt/Tgfβ/Fgf10 interactions represents the molecular basis for the classically reported failure to form lung buds in vitamin A deficiency.
Authors
Felicia Chen, Yuxia Cao, Jun Qian, Fengzhi Shao, Karen Niederreither, Wellington V. Cardoso
Abstract
The molecular mechanisms that govern bone and joint formation are complex, involving an integrated network of signaling pathways and gene regulators. We investigated the role of Hox genes, which are known to specify individual segments of the skeleton, in the formation of autopod limb bones (i.e., the hands and feet) using the mouse mutant synpolydactyly homolog (spdh), which encodes a polyalanine expansion in Hoxd13. We found that no cortical bone was formed in the autopod in spdh/spdh mice; instead, these bones underwent trabecular ossification after birth. Spdh/spdh metacarpals acquired an ovoid shape and developed ectopic joints, indicating a loss of long bone characteristics and thus a transformation of metacarpals into carpal bones. The perichondrium of spdh/spdh mice showed abnormal morphology and decreased expression of Runt-related transcription factor 2 (Runx2), which was identified as a direct Hoxd13 transcriptional target. Hoxd11–/–Hoxd12–/–Hoxd13–/– triple-knockout mice and Hoxd13–/–Hoxa13+/– mice exhibited similar but less severe defects, suggesting that these Hox genes have similar and complementary functions and that the spdh allele acts as a dominant negative. This effect was shown to be due to sequestration of other polyalanine-containing transcription factors by the mutant Hoxd13 in the cytoplasm, leading to their degradation. These data indicate that Hox genes not only regulate patterning but also directly influence bone formation and the ossification pattern of bones, in part via Runx2.
Authors
Pablo Villavicencio-Lorini, Pia Kuss, Julia Friedrich, Julia Haupt, Muhammed Farooq, Seval Türkmen, Denis Duboule, Jochen Hecht, Stefan Mundlos
Abstract
The lymphatic system plays a key role in tissue fluid homeostasis. Lymphatic dysfunction contributes to the pathogenesis of many human diseases, including lymphedema and tumor metastasis. However, the mechanisms regulating lymphangiogenesis remain largely unknown. Here, we show that COUP-TFII (also known as Nr2f2), an orphan member of the nuclear receptor superfamily, mediates both developmental and pathological lymphangiogenesis in mice. Conditional ablation of COUP-TFII at an early embryonic stage resulted in failed formation of pre-lymphatic ECs (pre-LECs) and lymphatic vessels. COUP-TFII deficiency at a late developmental stage resulted in loss of LEC identity, gain of blood EC fate, and impaired lymphatic vessel sprouting. siRNA-mediated downregulation of COUP-TFII in cultured primary human LECs demonstrated that the maintenance of lymphatic identity and VEGF-C–induced lymphangiogenic activity, including cell proliferation and migration, are COUP-TFII–dependent and cell-autonomous processes. COUP-TFII enhanced the pro-lymphangiogenic actions of VEGF-C, at least in part by directly stimulating expression of neuropilin-2, a coreceptor for VEGF-C. In addition, COUP-TFII inactivation in a mammary gland mouse tumor model resulted in inhibition of tumor lymphangiogenesis, suggesting that COUP-TFII also regulates neo-lymphangiogenesis in the adult. Thus, COUP-TFII is a critical factor that controls lymphangiogenesis in embryonic development and tumorigenesis in adults.
Authors
Fu-Jung Lin, Xinpu Chen, Jun Qin, Young-Kwon Hong, Ming-Jer Tsai, Sophia Y. Tsai
Abstract
The receptor tyrosine kinase ret protooncogene (RET) is implicated in the pathogenesis of several diseases and in several developmental defects, particularly those in neural crest–derived structures and the genitourinary system. In order to further elucidate RET-mediated mechanisms that contribute to these diseases and decipher the basis for specificity in the pleiotropic effects of RET, we characterized development of the enteric and autonomic nervous systems in mice expressing RET9 or RET51 isoforms harboring mutations in tyrosine residues that act as docking sites for the adaptors Plcγ, Src, Shc, and Grb2. Using this approach, we found that development of the genitourinary system and the enteric and autonomic nervous systems is dependent on distinct RET-stimulated signaling pathways. Thus, mutation of RET51 at Y1062, a docking site for multiple adaptor proteins including Shc, caused distal colon aganglionosis reminiscent of Hirschsprung disease (HSCR). On the other hand, this mutation in RET9, which encodes an isoform that lacks the Grb2 docking site present in RET51, produced severe abnormalities in multiple organs. Mutations that abrogate RET-Plcγ binding, previously shown to produce features reminiscent of congenital anomalies of kidneys or urinary tract (CAKUT) syndrome, produced only minor abnormalities in the nervous system. Abrogating RET51-Src binding produced no major defects in these systems. These studies provide insight into the basis of organotypic specificity and redundancy in RET signaling within these unique systems and in diseases such as HSCR and CAKUT.
Authors
Sanjay Jain, Amanda Knoten, Masato Hoshi, Hongtao Wang, Bhupinder Vohra, Robert O. Heuckeroth, Jeffrey Milbrandt
Abstract
Various acute and chronic inflammatory stimuli increase the number and activity of pulmonary mucus-producing goblet cells, and goblet cell hyperplasia and excess mucus production are central to the pathogenesis of chronic pulmonary diseases. However, little is known about the transcriptional programs that regulate goblet cell differentiation. Here, we show that SAM-pointed domain–containing Ets-like factor (SPDEF) controls a transcriptional program critical for pulmonary goblet cell differentiation in mice. Initial cell-lineage–tracing analysis identified nonciliated secretory epithelial cells, known as Clara cells, as the progenitors of goblet cells induced by pulmonary allergen exposure in vivo. Furthermore, in vivo expression of SPDEF in Clara cells caused rapid and reversible goblet cell differentiation in the absence of cell proliferation. This was associated with enhanced expression of genes regulating goblet cell differentiation and protein glycosylation, including forkhead box A3 (Foxa3), anterior gradient 2 (Agr2), and glucosaminyl (N-acetyl) transferase 3, mucin type (Gcnt3). Consistent with these findings, levels of SPDEF and FOXA3 were increased in mouse goblet cells after sensitization with pulmonary allergen, and the proteins were colocalized in goblet cells lining the airways of patients with chronic lung diseases. Deletion of the mouse Spdef gene resulted in the absence of goblet cells in tracheal/laryngeal submucosal glands and in the conducting airway epithelium after pulmonary allergen exposure in vivo. These data show that SPDEF plays a critical role in regulating a transcriptional network mediating the goblet cell differentiation and mucus hyperproduction associated with chronic pulmonary disorders.
Authors
Gang Chen, Thomas R. Korfhagen, Yan Xu, Joseph Kitzmiller, Susan E. Wert, Yutaka Maeda, Alexander Gregorieff, Hans Clevers, Jeffrey A. Whitsett
Abstract
Neural crest cells (NCCs) participate in the remodeling of the cardiac outflow tract and pharyngeal arch arteries during cardiovascular development. Focal adhesion kinase (FAK) mediates signal transduction by integrin and growth factor receptors, each of which is important for normal cardiovascular development. To investigate the role of FAK in NCC morphogenesis, we deleted it in murine NCCs using Wnt1cre, yielding craniofacial and cardiovascular malformations resembling those observed in individuals with DiGeorge syndrome. In these mice, we observed normal cardiac NCC migration but reduced differentiation into smooth muscle within the aortic arch arteries and impaired cardiac outflow tract rotation, which resulted in a dextroposed aortic root. Moreover, within the conotruncal cushions, Fak-deficient NCCs formed a less organized mesenchyme, with reduced expression of perlecan and semaphorin 3C, and exhibited disorganized F-actin stress fibers within the aorticopulmonary septum. Additionally, absence of Fak resulted in reduced in vivo phosphorylation of Crkl and Erk1/2, components of a signaling pathway essential for NCC development. Consistent with this, both TGF-β and FGF induced FAK and Crkl phosphorylation in control but not Fak-deficient NCCs in vitro. Our results indicate that FAK plays an essential role in cardiac outflow tract development by promoting the activation of molecules such as Crkl and Erk1/2.
Authors
Ainara Vallejo-Illarramendi, Keling Zang, Louis F. Reichardt
Abstract
Notch signaling is vital for proper cardiovascular development and function in both humans and animal models. Indeed, mutations in either JAGGED or NOTCH cause congenital heart disease in humans and NOTCH mutations are associated with adult valvular disease. Notch typically functions to mediate developmental interactions between adjacent tissues. Here we show that either absence of the Notch ligand Jagged1 or inhibition of Notch signaling in second heart field tissues results in murine aortic arch artery and cardiac anomalies. In mid-gestation, these mutants displayed decreased Fgf8 and Bmp4 expression. Notch inhibition within the second heart field affected the development of neighboring tissues. For example, faulty migration of cardiac neural crest cells and defective endothelial-mesenchymal transition within the outflow tract endocardial cushions were observed. Furthermore, exogenous Fgf8 was sufficient to rescue the defect in endothelial-mesenchymal transition in explant assays of endocardial cushions following Notch inhibition within second heart field derivatives. These data support a model that relates second heart field, neural crest, and endocardial cushion development and suggests that perturbed Notch-Jagged signaling within second heart field progenitors accounts for some forms of congenital and adult cardiac disease.
Authors
Frances A. High, Rajan Jain, Jason Z. Stoller, Nicole B. Antonucci, Min Min Lu, Kathleen M. Loomes, Klaus H. Kaestner, Warren S. Pear, Jonathan A. Epstein
Abstract
Congenital anomalies affecting the ureter-bladder junction are frequent in newborns and are often associated with other developmental defects. However, the molecular and morphological processes underlying these malformations are still poorly defined. In this study, we identified the leukocyte antigen–related (LAR) family protein tyrosine phosphatase, receptor type, S and F (Ptprs and Ptprf [also known as Lar], respectively), as crucially important for distal ureter maturation and craniofacial morphogenesis in the mouse. Embryos lacking both Ptprs and Ptprf displayed severe urogenital malformations, characterized by hydroureter and ureterocele, and craniofacial defects such as cleft palate, micrognathia, and exencephaly. The detailed analysis of distal ureter maturation, the process by which the ureter is displaced toward its final position in the bladder wall, leads us to propose a revised model of ureter maturation in normal embryos. This process was deficient in embryos lacking Ptprs and Ptprf as a result of a marked reduction in intrinsic programmed cell death, thereby causing urogenital system malformations. In cell culture, Ptprs bound and negatively regulated the phosphorylation and signaling of the Ret receptor tyrosine kinase, whereas Ptprs-induced apoptosis was inhibited by Ret expression. Together, these results suggest that ureter positioning is controlled by the opposing actions of Ret and LAR family phosphatases regulating apoptosis-mediated tissue morphogenesis.
Authors
Noriko Uetani, Kristen Bertozzi, Melanie J. Chagnon, Wiljan Hendriks, Michel L. Tremblay, Maxime Bouchard
Abstract
Individuals with the birth defect synpolydactyly (SPD) have 1 or more digit duplicated and 2 or more digits fused together. One form of SPD is caused by polyalanine expansions in homeobox d13 (Hoxd13). Here we have used the naturally occurring mouse mutant that has the same mutation, the SPD homolog (Spdh) allele, and a similar phenotype, to investigate the molecular pathogenesis of SPD. A transgenic approach and crossing experiments showed that the Spdh allele is a combination of loss and gain of function. Here we identify retinaldehyde dehydrogenase 2 (Raldh2), the rate-limiting enzyme for retinoic acid (RA) synthesis in the limb, as a direct Hoxd13 target and show decreased RA production in limbs from Spdh/Spdh mice. Intrauterine treatment with RA restored pentadactyly in Spdh/Spdh mice. We further show that RA and WT Hoxd13 suppress chondrogenesis in mesenchymal progenitor cells, whereas Hoxd13 encoded by Spdh promotes cartilage formation in primary cells isolated from Spdh/Spdh limbs, and that this was associated with increased expression of Sox6/9. Increased Sox9 expression and ectopic cartilage formation in the interdigital mesenchyme of limbs from Spdh/Spdh mice suggest uncontrolled differentiation of these cells into the chondrocytic lineage. Thus, we propose that mutated Hoxd13 causes polydactyly in SPD by inducing extraneous interdigital chondrogenesis, both directly and indirectly, via a reduction in RA levels.
Authors
Pia Kuss, Pablo Villavicencio-Lorini, Florian Witte, Joachim Klose, Andrea N. Albrecht, Petra Seemann, Jochen Hecht, Stefan Mundlos
Abstract
Based on extensive preclinical data, glycogen synthase kinase–3 (GSK-3) has been proposed to be a viable drug target for a wide variety of disease states, ranging from diabetes to bipolar disorder. Since these new drugs, which will be more powerful GSK-3 inhibitors than lithium, may potentially be given to women of childbearing potential, and since it has controversially been suggested that lithium therapy might be linked to congenital cardiac defects, we asked whether GSK-3 family members are required for normal heart development in mice. We report that terminal cardiomyocyte differentiation was substantially blunted in Gsk3b–/– embryoid bodies. While GSK-3α–deficient mice were born without a cardiac phenotype, no live-born Gsk3b–/– pups were recovered. The Gsk3b–/– embryos had a double outlet RV, ventricular septal defects, and hypertrophic myopathy, with near obliteration of the ventricular cavities. The hypertrophic myopathy was caused by cardiomyocyte hyperproliferation without hypertrophy and was associated with increased expression and nuclear localization of three regulators of proliferation — GATA4, cyclin D1, and c-Myc. These studies, which we believe are the first in mammals to examine the role of GSK-3α and GSK-3β in the heart using loss-of-function approaches, implicate GSK-3β as a central regulator of embryonic cardiomyocyte proliferation and differentiation, as well as of outflow tract development. Although controversy over the teratogenic effects of lithium remains, our studies suggest that caution should be exercised in the use of newer, more potent drugs targeting GSK-3 in women of childbearing age.
Authors
Risto Kerkela, Lisa Kockeritz, Katrina MacAulay, Jibin Zhou, Bradley W. Doble, Cara Beahm, Sarah Greytak, Kathleen Woulfe, Chinmay M. Trivedi, James R. Woodgett, Jonathan A. Epstein, Thomas Force, Gordon S. Huggins
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ISSN: 0021-9738 (print), 1558-8238 (online)