Inactivation of Tsc2 in Mesoderm-Derived Cells Causes Polycystic Kidney Lesions and Impairs Lung Alveolarization

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The American Journal of Pathology, Vol. -, No. -, - 2016

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Inactivation of Tsc2 in Mesoderm-Derived Cells Causes Polycystic Kidney Lesions and Impairs Lung Alveolarization Q34

Siying Ren,*y Yongfeng Luo,y Hui Chen,y David Warburton,yz Hilaire C. Lam,x Larry Wang,y Ping Chen,* Elizabeth P. Henske,x and Wei Shiyz From the Department of Respiratory Medicine,* The Second Xiangya Hospital, Central-South University, Changsha, People’s Republic of China; the Developmental Biology and Regenerative Medicine Program,y The Saban Research Institute, Children’s Hospital Los Angeles, Los Angeles, California; the Department of Surgery,z Keck School of Medicine, University of Southern California, Los Angeles, California; and the Division of Pulmonary and Critical Care Medicine,x Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts Accepted for publication August 29, 2016.

Address correspondence to Wei Shi, Children’s Hospital Los Angeles, 4650 Sunset Blvd, MS 35, Los Angeles, CA 90027; or Elizabeth P. Henske, Division of Pulmonary and Critical Care Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA Q4 Q5 02115. E-mail: [email protected]. Q6 edu or [email protected]. edu.

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The tuberous sclerosis complex (TSC) proteins are critical negative regulators of the mTORC1 pathway. Germline mutations of TSC1 or TSC2 cause TSC, affecting multiple organs, including the kidney and lung, and causing substantial morbidity and mortality. The mechanisms of organ-specific disease in TSC remain incompletely understood, and the impact of TSC inactivation on mesenchymal lineage cells has not been specifically studied. We deleted Tsc2 specifically in mesoderm-derived mesenchymal cells of multiple organs in mice using the Dermo1-Cre driver. The Dermo1-Creedriven Tsc2 conditional knockout mice had body growth retardation and died approximately 3 weeks after birth. Significant phenotypes were observed in the postnatal kidney and lung. Inactivation of Tsc2 in kidney mesenchyme caused polycystic lesions starting from the second week of age, with increased cell proliferation, tubular epithelial hyperplasia, and epithelial-mesenchymal transition. In contrast, Tsc2 deletion in lung mesenchyme led to decreased cell proliferation, reduced postnatal alveolarization, and decreased differentiation with reduced numbers of alveolar myofibroblast and type II alveolar epithelial cells. Two major findings thus result from this model: inactivation of Tsc2 in mesoderm-derived cells causes increased cell proliferation in the kidneys but reduced proliferation in the lungs, and inactivation of Tsc2 in mesoderm-derived cells causes epithelial-lined renal cysts. Therefore, Tsc2-mTOR signaling in mesenchyme is essential for the maintenance of renal structure and for lung alveolarization. (Am J Pathol 2016, -: 1e12; http://dx.doi.org/10.1016/j.ajpath.2016.08.013)

Tuberous sclerosis complex (TSC) is an autosomal dominant genetic disorder resulting from germline loss-offunction mutations in the TSC1 or TSC2 gene.1,2 The TSC1/TSC2 protein complex has GTPase-activating protein activity toward Rheb, an upstream regulator of mTOR, and thus negatively regulates mTORC1 signaling activity by converting active Rheb-GTP to inactive Rheb-GDP. Therefore, abrogation of TSC1 or TSC2 causes dysregulation of mTORC1-dependent pathways, including protein translation, pyrimidine and purine biosynthesis, and autophagy. In addition to neurological disease, which includes seizures and autism, TSC patients have clinical manifestations in multiple organs, including the kidney, heart, lung,

and skin. In the kidney, a variety of lesions, such as angiomyolipoma, isolated or multiple renal cysts, and renal cell carcinoma, can occur in TSC patients. In the lung, TSC patients can develop multifocal micronodular pneumocyte hyperplasia and lymphangioleiomyomatosis (LAM).3,4 LAM is a progressive disease affecting almost exclusively

Supported by NIH grants HL068597 (W.S.), HL118760 (E.P.H.), and DK096556 (E.P.H.); a Tuberous Sclerosis Alliance Research grant (W.S.); the Hunan Province Health Department Research Fund B2014-020 (S.R.); the Hunan Province Natural Science Foundation of China 14JJ7012 (S.R.); and the Engles Program in TSC and LAM Research (E.P.H.). Disclosures: None declared.

Copyright ª 2016 Published by Elsevier Inc. on behalf of the American Society for Investigative Pathology. http://dx.doi.org/10.1016/j.ajpath.2016.08.013

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Ren et al women and resulting in cystic lung degeneration. Up to 80% of women with TSC have multiple lung cysts by the age of 40 years, although only a minority will develop symptomatic LAM. Men with TSC can also develop cystic lung disease, although it is almost always asymptomatic. LAM also occurs in a sporadic form in women who do not have TSC. Sporadic LAM is associated with somatic inactivation of the TSC1 or TSC2 gene.5,6 The pathogenic mechanisms underlying the development of the clinical manifestations of TSC, in particular the pathogenesis of LAM, are not completely understood. One major challenge is the limitation of existing TSC disease models because conventional homozygous Tsc1 or Tsc2 knockout mice are embryonic lethal, and heterozygous Tsc1 or Tsc2 knockout mice develop epithelial renal lesions and tumors as their primary phenotypes.7e9 This is a striking contrast to human TSC, in which mesenchymal lesions of the kidney (angiomyolipomas) and lung (LAM) are major causes of morbidity and mortality. It is still unclear how the TSC protein complex functions in different cell compartments, such as epithelial cells versus mesenchymal cells, during organ development and during post-developmental tissue homeostasis. Although genetic deletions of Tsc1 or Tsc2 in distinct cell lineages, such as neurons, neural crest-derived cells, endothelia, cardiomyocytes, and differentiated smooth muscle cells, have been reported in mice,10e14 the consequences of Tsc1 or Tsc2 inactivation in pan-mesenchymal cells has remained unknown. We have investigated the specific function of Tsc2 in mesoderm-derived cells from the beginning of development in vivo. We achieved this by crossing a mesoderm-specific Cre-expressing mouse line Dermo1-Cre with floxed-Tsc2 mice to generate mesenchyme-specific Tsc2 conditional knockout mice.15,16 Although these Tsc2 conditional knockout mice were alive postnatally, postnatal growth retardation, polycystic kidney pathology, and defective lung alveolarization were detected during postnatal development. These mice died approximately 3 weeks after birth, consistent with an indispensible role of Tsc2 in mesenchymal cells in regulating both organ development and tissue homeostasis.

Materials and Methods Mouse Strains and Breeding Q9 Q10

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Mesenchyme-specific Dermo1-Cre mice were provided by Dr. David Ornitz.15 Floxed-Tsc2 (Tsc2fx/fx) mice were originally generated in Dr. Michael Gambello’s laboratory.16 mT-mG double fluorescence reporter mice and Gt(ROSA)26Sortm1Sor LacZ reporter mice were obtained from Jackson Laboratory.17,18 All mice were bred in C57BL/6 stain background, and housed in pathogen-free conditions at the animal facility of Children’s Hospital Los Angeles. The procedures for mouse breeding and tissue harvest were approved by the Institutional Animal Care and

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Use Committee at the Saban Research Institute of Children’s Hospital Los Angeles. Mouse genotypes were determined by genomic DNA PCR using tail biopsy samples. The genotyping PCR primers were described in previous publications.16,19

Histology and Morphometric Analysis Fresh tissues at different ages were harvested from euthanized mice, and examined under dissecting microscope. Samples were then either fixed in 4% buffered paraformaldehyde or frozen in liquid nitrogen. The fixed tissues were dehydrated and embedded in paraffin, and divided into sections (5 mm thick). The tissue sections were stained with hematoxylin and eosin or periodic acid-Schiff (Sigma Aldrich, St. Louis, MO). Lung elastin was stained using Q12 Hart’s resorcin-fuchsin method, as described in our previous publication.19 For lung morphometric analysis, five sections of each tissue were randomly chosen at approximately 200-mm intervals and stained with hematoxylin and eosin. The mean linear intercept was used to measure average size of alveoli, as described previously.19 Results were analyzed with unpaired t-tests to compare the differences between mean values, and considered significant if P < 0.05. To avoid the sex differences in organ maturation, this quantitative comparison was also performed among fetuses with different sex at each time point.

Immunostaining and Detection Tissue sections were deparaffinized and hydrated, and antigen recovery was performed by boiling tissue slides in Tris-EDTA buffer (pH 9.0) for 30 minutes. The tissue sections were blocked in 5% donkey normal serum and 0.5% Triton X-100 for 1 hour, and then incubated with the following primary antibodies overnight at 4 C: mouse antiegreen fluorescent protein (GTX628528; GeneTex); Q13 goat antieplatelet endothelial cell adhesion molecule 1 (sc-1506), goat anti-CC10 (sc-9772), and goat antieSp-C (sc-7706; Santa Cruz Biotechnology, Dallas, TX); rabbit Q14 antiered fluorescent protein for Tomato protein (600-401379; Rockland); rat anti-Cdh1 (13-1900; ThermoFisher), Q15 Q16 mouse antieb-tubulin IV (MU178-UC; BioGenex); fluo- Q17 rescein-Lotus tetragonobolus lectin and rhodamineDolichos biflorus agglutinin (FL-1321 and RL-1032; Vector Laboratories); mouse anti-cytokeratin and mouse antiea- Q18 smooth muscle actin (C2562 and A2547; Sigma Aldrich); rabbit antiephospho-S6 (Ser235/236) and rabbit antivimentin (4858 and 5741; Cell Signaling Technology); Q19 and hamster anti-T1a (DSHB at the University of Iowa, Iowa City). Alexa Fluro 488e, Alexa Fluro 594e, or Alexa Fluro 647econjugated donkey secondary antibodies (Molecular Probes) were used, and cell nuclei were coun- Q20 terstained by DAPI in the mounting medium (Vector Laboratories). Fluorescence images were taken using the Zeiss LSM710 confocal microscope at the Imaging Core Q21

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Tsc2 in Kidney and Lung Lesions Q1 Facility of Children’s Hospital Los Angeles. Active caspase 3 staining was performed by immunohistochemistry using rabbit anti-cleaved caspase 3 antibody (9661; Cell Signaling Technology) and second anti-rabbit antibodyehorseradish peroxidase conjugate, followed by color development with 3,30 -diaminobenzidine and hematoxylin counterstaining.

Western Blot

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Detection of proteins in tissue lysate by Western blot has been previously described with the following minor modification.20 Equal amounts (50 mg) of total tissue lysate proteins were separated in mini tris-glycine extended precast gels (4% to 15% gradient; Bio-Rad) and transferred into polyvinylidene difluoride membrane using Bio-Rad’s Trans-Blot Turbo Transfer System. Proteins of interest were detected using the following specific antibodies: rabbit antiTsc2 (GTX61245; GeneTex); rabbit antiephospho-S6 (Ser235/236) and mouse anti-S6 (4858 and 2317; Cell Signaling Technology); and rabbit antieb-actin (G046; Applied Biological Materials).

Cell Proliferation

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To detect proliferating cells in vivo, cell nuclear 50 -ethynyl20 -deoxyuridine (EdU) labeling was used.21 Briefly, mice were i.p. injected with EdU (5 mg/kg body weight; Life Technologies) 3 hours before harvesting tissue samples. EdU is a thymidine analogue that is incorporated into newly synthesized chromosome DNA. Incorporated EdU was detected using Alexa Fluor azide (Life Technologies), and cell nuclei were counterstained with DAPI. The image analysis was performed in four random fields per slide from a total of four slides per mouse by an experienced observer blind to the mouse genotype. The ratios of EdUlabeled nuclei/total DAPI-stained nuclei were used to evaluate cell proliferation, and the experiments were repeated in more than three mice within each genotype group.

Serum Cystatin C Measurement

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Blood was collected from five Tsc2 conditional knockout (CKO) and wild-type (WT) littermate control pairs around postnatal day 20 (P20). The sera were diluted 200-fold, and used for cystatin C measurement using a commerical enzyme-linked immunosorbent assay kit (Thermo). Paired sample t-test was used for the analysis.

Statistical Analysis All experiments were repeated at least three times. The quantitative data were presented as means  SD. Statistical analyses were performed using t-tests, with P  0.05 considered significant.

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311 312 313 Conditional Deletion of Tsc2 in Mesoderm-Derived Cells 314 Results in Postnatal Growth Retardation and Lethality 315 316 It has been reported that Dermo1 promoter-driven Cre 317 expression is specific for certain lineages of mesoderm318 derived cells in a variety of organs in the Dermo1-Cre 319 knockin fetal mice,15 and we verified this Cre expression 320 pattern in E14.5 Dermo1-Cre fetuses using Gt(ROSA) Q28 321 26Sortm1Sor LacZ reporter mice (Figure 1A). We crossed ½F1 322 Dermo1-Cre with floxed-Tsc2 mice (Tsc2fx/fx) to specifically 323 delete Tsc2 gene in Dermo1-expressing lineages of mesen324 325 chymal cells within multiple organs/systems during devel326 opment. The mice were born with the expected mendelian 327 ratio of genotypes and sexes. All control pups had normal 328 development and weight gain with normal survival through 329 fx/fx 12 months of age. Dermo1-Cre/Tsc2 conditional 330 knockout (abbreviated as Tsc2 CKO) mice were born with 331 notably smaller body size and lower body weight than their 332 littermate controls. These differences became more obvious 333 during postnatal growth (Figure 1, B and C), suggesting 334 significant postnatal growth retardation in Tsc2 CKO mice. 335 Heterozygous Tsc2 CKO (Dermo1-Cre/Tsc2fx/þ) mice had 336 337 body weight and size comparable to their WT littermates, 338 and did not develop noticeable phenotypic abnormalities up 339 to 5 months of age (data not shown). 340 The homozygous Tsc2 CKO mice died approximately 3 341 weeks after birth, with median survival of P20 (Figure 1D). 342 No sex difference was observed for Tsc2 CKO mouse sur343 vival. Unlike the phenotypic changes seen in liver, cardiac, 344 and central neuronal systems of germline Tsc2 mutant ani345 mals,8,22,23 there were no histologically obvious abnormal346 ities in brain, liver, or heart in the Tsc2 CKO mice at P14 347 (Figure 2). In contrast, the kidney and lung were clearly ½F2 348 349 abnormal at this age (see Results below for detail). The 350 potential causes for the lethality in Tsc2 CKO mice are 351 further analyzed and discussed below. 352 353 Mesodermal Tsc2 Abrogation Results in Severe 354 355 Polycystic Kidney Lesions 356 357 Dermo1-Creedriven gene deletion, shown by green fluores358 cent protein expression in a mT-mG reporter line,17 occurred in 359 renal mesenchymal cells of both the cortical and medullary re360 gions, but not in tubular epithelial cells or endothelial cells, 361 shown by Cdh1þ (E-cadherinþ) and platelet endothelial cell 362 þ adhesion molecule 1 staining, respectively (Figure 3, A and ½F3 363 B). Reduction of Tsc2 protein and activation of the mTORC1 364 pathway (increased phosphorylation of ribosomal protein S6) 365 were verified by Western blotting of the entire Tsc2 CKO kid366 ney tissue lysate (Figure 3C). Examination of the moribund 367 Tsc2 CKO mice sacrificed at approximately 3 weeks after birth 368 369 revealed obvious changes in kidney size and appearance, which 370 looked pale, polycystic, and enlarged by gross view 371 (Figure 3D). We then analyzed the dynamic changes of kidney 372

Results

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morphology in the Tsc2 CKO mice. Interestingly, no significant alterations of kidney structure and size were observed in neonatal Tsc2 CKO mice at P1 (Figure 4, A and C). Microscopic Q29½F4 cystic pathology was observed in the renal cortex from the second week after birth (P14) (Figure 4). The cystic lesions rapidly progressed and expanded into the medulla, destroying the normal renal cortex-medulla structural relationships at 3 weeks of age in all Tsc2 CKO mice (Figure 4). Glycogen accumulation was detected in the cysts by periodic acid-Schiff staining. In addition, multiple foci of tubular epithelial hyperplasia and adenomatous changes adjacent to the cysts were seen in Tsc2 CKO kidney after 2 weeks of age (Figure 4). Consistently, overall cell proliferation in Tsc2 CKO kidneys was doubled compared to that in WT littermates, shown by nucleotide analogue EdU labeling (9.0  2.4 in Tsc2 CKO versus 4.1  1.1 in WT) (Figure 5, A and B) at P7 before obvious ½F5 morphological changes occurred. The cystic changes were further analyzed by staining the tissue with L. tetragonobolus lectin (a proximal tubular marker) and D. biflorus agglutinin (a collecting duct marker). Interestingly, some of the lining epithelial cells within the cystic structures were positive for D. biflorus agglutinin (Figure 5C), but not for L. tetragonobolus lectin, which suggests that the cystic pathology originates in the distal collecting ducts. The renal endothelial networks were comparable between Tsc2 CKO and WT controls (Figure 5C). Some of the tubular epithelial cells were positively stained with vimentin in the Tsc2 CKO kidney at P21, with significant reduction of Cdh1 expression (Figure 5D). Increased vimentin protein level in the Tsc2 CKO kidney was verified by Western blot (Figure 5E), suggesting an epithelial-mesenchymal transition as a consequence of Tsc2 inactivation. In addition, increased apoptotic cells, detected by active caspase 3 immunostaining, were seen in P21 Tsc2 CKO kidney sections (Figure 5, F and G). These apoptotic cells in the cysts appeared to be shed into the lumens. Interestingly, only minor increase of serum cystatin C was detected in the live Tsc2 CKO mice around the age of P20 (10  3% greater than the littermate WT control; *P < 0.05, n Z 5 pairs), suggesting that kidney function is not severely impaired.

Mesodermal Tsc2 Deletion Causes Retardation of Pulmonary Alveolar Development

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Using mT-mG fluorescence reporter, we confirmed that most lung mesenchymal cells, except pulmonary endothelial cells, were targeted by the Dermo1-Cre driver during

Figure 1

Deletion of Tsc2 in mesoderm-derived cells resulted in reduced body growth and lethality at early life. A: LacZ staining (blue) of E14.5 Dermo1-Cre/Gt(ROSA)26Sortm1Sor reporter mice to visualize the pattern of Cre expression. B: Comparison of body sizes between Tsc2fx/fx/Dermo1-Cre [Tsc2 conditional knockout (CKO)] mice and their wild-type (WT) littermate controls. C: Comparison of whole body weights between Tsc2 CKO and their littermate controls at different ages. D: Altered survival was detected in Tsc2 CKO mice compared to the WT controls. n Z 4 per genotype (C); n Z 13 (4 females and 9 males; D). *P < 0.05. P, postnatal day.

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559 labeling (6.7  1.2% in Tsc2 CKOs versus 11.0  1.8% in WT controls) (Figure 7, A and B). Differentiation analysis ½F7 560 561 using immunostaining revealed that lung alveolar myofi562 broblasts in the Tsc2 CKO mice were markedly reduced, as 563 shown by a-smooth muscle actinepositive cells in alveolar 564 septa (Figure 7C), whereas smooth muscle cells of both 565 airways and vasculatures appeared similar between Tsc2 566 CKO and the controls. Interestingly, type II alveolar 567 epithelial cells, shown by Sp-C staining, were also signifi568 569 cantly decreased, whereas type I alveolar epithelial cells, 570 shown by T1a staining, were not changed (Figure 7C). The 571 proximal airway epithelial cells, including ciliated cells and 572 Club cells, shown by b-tubulin IV and CC10 immuno573 staining, respectively, were comparable between Tsc2 CKO 574 and WT mice. The number and distribution of alveolar 575 capillary endothelial cells, detected by platelet endothelial 576 cell adhesion molecule 1 staining, appeared to be normal 577 (Figure 7C). Finally, elastin fiber deposition in the tips of 578 alveolar septa and alveolar walls was attenuated in the Tsc2 579 CKO lung during alveolar stage (P14) (Figure 7, D and E). 580 581 Therefore, reduced lung alveolarization in lung 582 mesenchyme-specific Tsc2 knockout mice is likely because 583 of both altered cell proliferation and myofibroblast differ584 entiation in the peripheral lung.

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Tsc2 in Kidney and Lung Lesions

Figure 2

Histological comparison of brain (A), liver (B), and heart (C) between Tsc2fx/fx/Dermo1-Cre female mice and wild-type (WT) female control mice at postnatal day 14 is shown by hematoxylin and eosinestained tissue sections. Scale bars: 100 mm (A and B); 1 mm (C). CKO, conditional knockout.

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development, whereas no epithelial cells were Cre positive (Figure 6, A and B). The deletion of Tsc2 in the lung tissue of Dermo1-Cre/Tsc2fx/fx mice was then validated by reduced Tsc2 protein expression and increased mTOR pathway activity (phosphorylation of S6) using Western blot and immunostaining (Figure 6, C and D). Lung development was examined by comparing morphological structures between Tsc2 CKO mice and their WT littermate controls. Although prenatal development of the lung in Tsc2 CKO mice was not significantly affected, shown by their lung morphology at P1, significant deficiency of lung alveolarization, which normally occurs from P5 to P30 in mice,24 was detected (Figure 6E). In the Tsc2 CKO mouse lung, growth of alveolar septa was markedly decreased, which resulted in larger alveolar spaces, within the lungs from P7 to P21. This altered histology was then verified by morphometric measurement of average alveolar size (mean linear intercept) (Figure 6F). Therefore, lung mesenchymal Tsc2 has an important functional role in promoting lung alveolarization during postnatal development. To understand the potential mechanisms by which mesenchymal Tsc2 deletion inhibited lung alveolarization, we analyzed cell proliferation and differentiation. Interestingly, a significant reduction of cell proliferation was detected in P7 Tsc2 CKO mouse lungs using nuclear EdU

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Discussion TSC is a disease affecting multiple organs and systems. Germline homozygous deletion of Tsc2 in mice results in embryonic lethality, and Tsc2 heterozygous deletion results in liver, kidney, and lung tumors as the primary phenotypes in adult mice.7e9 The manifestations of human TSC include mesenchymal-lineage tumors of the kidney (angiomyolipomas) and lung (lymphangioleiomyomatosis or LAM). To elucidate the consequences of TSC2 inactivation in multiple mesenchymal lineages, we used the Dermo1-promoter-Cre that drives Cre expression in mesoderm-derived cells of multiple organs.15 Herein, we report the novel findings that pan-mesenchymal Dermo1-Creedriven homozygous Tsc2 CKO mice are viable but have growth retardation, postnatal progressive polycystic kidney disease, and defective alveolarization of the lung. Mesenchymal cells are heterogeneous, including a variety of smooth muscle cells, endothelial cells, fibroblasts, osteoblasts, and chondrocytes. The specific mesenchymal cell types and mechanisms underlying the reduced body size and weight in our Tsc2 conditional knockout mice are not known. Increased craniofacial bone mass in neural crestespecific Tsc1 knockout mice and increased postnatal skeletal bone acquisition in osteoblast-specific Tsc2 knockout mice have been reported previously.25,26 However, retardation of body growth (size or weight) was not found in those prior models. The reduced body size in our mesenchymal knockout model may be the result of aberrant bone development because of loss of Tsc2 function in both

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Figure 3 Dermo1-Cre driven Tsc2 deletion in mouse kidney. A: Dermo1-Cre targeted cells in the kidney were characterized in mT-mG fluorescence protein reporter mice at postnatal day 14 (P14), in which membrane green fluorescent protein (GFP) expression (green) replaced membrane Tomato expression (red) only in cells in which Cremediated loxP DNA recombination occurred. B: Dermo1-Creemediated loxP DNA recombination in mT-mG reporter mice (green) was not detected in P14 kidney epithelial cells (Cdh1þ) or in endothelial cells [platelet endothelial cell adhesion molecule (PECAM)þ]. C: Alterations of Tsc2 protein and phosphorylation of S6, a downstream target of mTOR pathway, were detected in P7 Tsc2 conditional knockout (CKO) kidneys by Western blot. D: Gross view of kidneys isolated from Tsc2 CKO mice and wild-type (WT) control mice at P25. Scale bars: 50 mm (A and B); 1 mm (D).

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chrondrocytes and osteoblasts, and/or the consequence of impaired renal or respiratory function. In our Dermo1-Cre mice, Cre is expressed in condensed mesenchyme from which both chondrocytes and osteoblasts are derived during embryonic endochondral ossification (E11.5).15 At later stages, except in bone marrow cells and osteoclasts, Cre expression in Dermo1-Cre mice is also detected in chondrocytes of growth plate cartilage and osteoblasts of the perichondrium, periosteum, and endosteum.15 A future detailed analysis of Tsc2 in these targeted cells and skeletal development will be needed to understand the relevant mechanisms underlying the phenotype of retarded body growth. Previous studies found that deletion of Tsc1 in smooth muscle cells and cardiomyocytes using an SM22-driven Cre resulted in severe biventricular hypertrophy starting from embryonic day 15, with median survival of these Tsc1 conditional knockout mice of approximately 24 days after birth.12,13 Interestingly, modest cystadenoma pathology was also detected in the kidney and thought to be caused by minor leakage of Cre expression into renal epithelial cells. In the lung, no significant changes in alveolarization were reported; instead, significant muscularization of small peripheral pulmonary vessels was noted. In our Dermo1-Creedriven Tsc2

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knockout mice, no morphological change in heart development was detected (Figure 2), which may be because of lack of cardiomyocyte targeting in our Dermo1-Cre driver line (Figure 1). However, in the kidney, our Tsc2 CKO mice developed severe polycystic pathology within 3 weeks after birth, resulting in destruction of normal kidney structure. In addition, tubular epithelial hyperplasia and epithelialmesenchymal transition, but no angiomyolipoma-like lesions, appeared by 3 weeks of age. Using the fluorescence reporter to track the Dermo1-Cre targeted cells, we found that Cre was expressed specifically in kidney mesenchymal cells, and not in glomerular endothelial cells nor tubular epithelial cells (Figure 3). In addition, overall cell proliferation was increased in the Tsc2 CKO kidney, consistent with epithelial hyperplasia. The potential mechanisms through which mesenchymal Tsc2 deletion results in tubular epithelial hyperplasia and cystic change may include a physical blockage of flow by these hyperplastic tubular structures. These findings suggest that Tsc2 functions within renal mesenchymal cells to maintain normal renal tubular structural homeostasis after development rather than regulating kidney morphogenesis per se. Kidney lesions occur in most TSC patients and are a common cause of morbidity. These include both

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Figure 4 Renal pathology in the Tsc2 conditional knockout (CKO) mice. A: Comparison of overall kidney anatomical structures between the Tsc2 CKO mice and their littermate controls at different postnatal ages. B: Hematoxylin and eosinestained kidney tissue sections at high magnification. Black dotted line highlights a hyperplastic tubule. C: Kidney cortex histology shown by periodic acid-Schiff staining. Arrow indicates multiple layers of hyperplastic tubular epithelia lining a cystic structure; arrowhead, a single layer of epithelium lining a cystic structure. Scale bars: 1 mm (A); 50 mm (B); 100 mm (C). P, postnatal day.

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Tsc2 in Kidney and Lung Lesions

mesenchymal and epithelial lesions: mesenchymal angiomyolipomas and epithelial polycystic kidney disease, oncocytoma, and renal cell carcinoma.27,28 The mechanisms of renal cystic disease in TSC, including whether they arise because of epithelial versus mesenchymal inactivation of TSC2, are not yet completely understood. Several findings have focused on an epithelial pathogenesis model, including that TSC2 is required for the membrane

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localization of polycystin 1 protein and that TSC2 physically interacts with polycystin 1 to regulate the primary cilium in epithelia.29,30 Our data suggest, for the first time, that some of the epithelial cystic lesions in TSC may arise through mesenchymal loss of TSC2 function, supporting a new model for the cyst pathogenesis in TSC. A contiguous gene syndrome, with severe early-onset cystic disease, occurs in human with germline deletions of both TSC2 and

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Figure 5 Cell proliferation and cystic epithelial cells in Tsc2 conditional knockout (CKO) kidney. A and B: Proliferative cells were detected by 3-hour 50 -ethynyl20 -deoxyuridine (EdU) labeling, and the percentage of EdU-positive nuclei (green) was analyzed based on DAPI-nuclear counterstaining (blue). C: Endothelial cells in the renal cortex were detected by platelet endothelial cell adhesion molecule (PECAM) 1 immunostaining. Different tubular epithelial cells were also labeled with Lotus tetragonobolus lectin (LTL; green) or Dolichos biflorus agglutinin (DBA; red)-specific binding. DBA-positive cells in the cystic epithelia are indicated with an arrow. D: Coimmunostaining of Cdh1 (green), vimentin (red), and DBA (gray) for postnatal day 21 (P21) kidneys with indicated Tsc2 genotypes. Vimentin-positive cells in tubules are indicated with arrowheads. E: Increased vimentin in total kidney tissue lysate at P21 was detected by Western blot. b-Actin is a loading control. F: Activated caspase 3 immunostaining for apoptotic cells (brown, shown with arrows) in P21 kidneys. G: The numbers of apoptotic cells per 100 mm2 area of kidney tissue sections are compared. n Z 3 in each genotype (-). *P < 0.05. Scale bar Z 50 mm (-). WT, wild type.

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Figure 6 Lung mesenchyme-specific knockout of Tsc2 inhibited postnatal alveolarization. A and B: Lung cells targeted by the Dermo1-Cre driver at postnatal day 7 (P7) were analyzed in mT-mG fluorescence reporter mice. C and D: Altered Tsc2 protein expression and mTOR activation in P7 Tsc2 conditional knockout (CKO) lungs were analyzed by Western blot and immunostaining. E: Lung histological changes were evaluated by comparing hematoxylin and eosinestained lung tissue structures between Tsc2 CKO and wild-type (WT) control at different postnatal ages. F: Alveolar sizes were quantitatively analyzed by measuring mean linear intercept. n Z 4 per genotype (F). *P < 0.05. Scale bars: 50 mm (AeD); 200 mm (E). a, airway; GFP, green fluorescent protein; PECAM, platelet endothelial cell adhesion molecule; v, vasculature.

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the adjacent polycystic kidney disease 1 (PKD1) gene.31 More important, specific deletion of Tsc2 exon 2 to 4 in our model confirms that polycystic renal pathology can be caused by mesenchymal loss of Tsc2,16 without simultaneous deletion of the adjacent Pkd1 gene,32 which experimentally supports clinical observations that renal cysts can

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occur in patients with TSC1 or TSC2 mutation but no PKD1 deletion.33 In our Tsc2 CKO mouse lung, a striking reduction of pulmonary alveolarization was observed, without obvious changes in pulmonary vascular and airway smooth muscle cells. Yet, by examining the alveolar septa structure,

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Figure 7

Altered lung proliferation and differentiation in Tsc2 conditional knockout (CKO) mice. A and B: Proliferative cells were detected by 3-hour 50 -ethynyl-20 -deoxyuridine (EdU) labeling (green) in postnatal day 7 (P7) mice, and quantified based on cell nuclear counterstaining with DAPI (blue). C: Lung myofibroblasts/smooth muscle cells, endothelial cells, type II and type I alveolar epithelial cells, airway club cells, and ciliated cells were immunostained with the related protein markers. D: Comparison of elastin fibers (black) in lung tissues between Tsc2 CKO and wild-type (WT) control mice at P7 and P14. E: Semiquantification of elastin deposition in P14 lung tissues, measured by the percentage of stained elastin area over total lung tissue area of four tissue sections. *P < 0.05. Scale bars Z 50 mm (AeD). a, airway; PECAM, platelet endothelial cell adhesion molecule; SMA, a-smooth muscle actin; v, vasculature.

we found that a-smooth muscle actinepositive myofibroblasts in our Tsc2 CKO lung were significantly reduced, particularly in the tips of the alveolar septa. In addition, overall lung cell proliferation was significantly decreased in the Tsc2 CKO lung, which is opposite to the

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increased cell proliferation detected in the Tsc2 CKO kidney. Therefore, it appears that loss-of-function mutation in Tsc2 and subsequent mTORC1 hyperactivation result in strikingly diverse cellular responses depending on cell lineages, organs, and stages of development and

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Tsc2 in Kidney and Lung Lesions homeostasis. Lung alveolarization occurs postnatally in mice, with the growth of secondary alveolar septa into primary alveolar sacs. Alveolar myofibroblasts play a critical role in driving alveolar septa formation by proliferating and migrating into the tips of the septa and by producing elastin extracellular matrices.34 Defects in alveolar myofibroblast migration to the tip because of abrogation of platelet-derived growth factor signaling results in arrest of pulmonary alveolarization.35 In our Tsc2 CKO lung, the overall number of alveolar myofibroblasts was significantly reduced, accompanied by decreased elastin deposition within the septa. Therefore, reduced proliferation, differentiation, and function of alveolar myofibroblasts because of loss of Tsc2 function and the resulting hyperactivation of mTORC1 pathway is one of the pivotal mechanisms underlying the alveolar hypoplastic lung phenotype. In contrast, alveolar capillaries were not significantly altered based on immunostaining, suggesting that alveolar angiogenesis, another driving force for pulmonary alveolarization, may not be a critical Tsc2-dependent mechanism for alveolarization. Whether defective alveolarization in these Tsc2 CKO mice contributes to pulmonary cystic lesions in adult remains unknown, because of their shortened life span. It has been reported that decreased alveolar growth in early life has a negative impact on alveolar homeostasis in an aging population, associated with alveolar destruction and emphysema.36 Therefore, it is possible that reduced alveolarization during lung development may contribute to the pulmonary cystic lesions observed in TSC patients. More important, no LAM-like nodules were detected in the female or male Tsc2 CKO mice, which died before adulthood. We therefore speculate that the high incidence pulmonary cysts in women with TSC, estimated at 80% by the age of 40 years,37 may reflect both a developmental alveolar defect involving TSC1/2 inactivation in the lung mesenchyme that is not sex specific, and sexspecific LAM celleproduced proteases and cytokines that mediate tissue destruction. This model could explain the fact that most TSC patients (both male and female) with lung cysts will not progress to symptomatic pulmonary disease. We further speculate that the mesenchymal impact of TSC2 loss cooperates with LAM celleproduced proteases to enhance the lung destruction. To address these hypotheses, further investigation of the relationship between lung mesenchymal Tsc2 inactivation and LAM-like pathology is needed.

Acknowledgments We thank Dr. David M. Ornitz for providing Dermo1-Cre mice; Dr. Michael Gambello for providing floxed-Tsc2 mice; Dr. Esteban Fernandez (Cell Imaging Core of Children’s Hospital Los Angeles) for helping with confocal imaging; Dr. Kevin Lemley for initial discussion about

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mouse kidney phenotype; and Dr. Roderick Bronson (Harvard Medical School) for review of the renal pathology.

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