Characterization of diethylstilbestrol-induced hypospadias in female mice.

The urethral duct and vagina are formed from the urogenital sinus (UGS) during the early neonatal period in mice. Neonatal estrogen exposure results in hypospadias, or the malpositioning of vaginal and urethral openings, with wide cleft clitoris. We sought to characterize diethylstilbestrol (DES) influence on UGS morphogenesis and hypospadias formation. Newborn (day 0) and 1-4-day-old female mice (ICR/Jcl) were given (s.c.) oil or 3.0 microg DES. Animals were killed 24 hr later; then hypospadias formation and epithelial apoptosis and proliferation within the developing UGS were assessed. DES did not alter normal UGS morphogenesis by day 1, in comparison with controls. However, hypospadias formation was observed in DES-treated mice by day 3. In these mice, the distal dorsal urethral duct appeared to fuse with and open into the lower vaginal solid cord region. Further, DES treatment produced a gradual significant increase in dorsal urethral epithelial apoptosis (P < 0.05) just prior to and during fusion and hypospadias formation. DES-induced urethral epithelial and sinus cord proliferation appeared significantly increased (P < 0.05) and unchanged, respectively, just prior to fusion. By day 5, DES-treated mice exhibited wide cleft clitoris. In addition, if DES was given on day 3 or 5, a gradual, distinct caudal shift in the vaginal-urethral junction was observed compared to mice treated on days 0-2. Although hypospadias was not induced when neonates were given DES on day 7, these mice continued to display early vaginal opening. Dose-response analysis indicated that 0.03 microg DES for 5 days is the lowest known critical dose for hypospadias induction. We have shown for the first time that DES-induced hypospadias onset may primarily be the result of changes in developing dorsal urethral epithelial cell apoptotic and proliferative activity, and that the location of DES-induced hypospadias formation is dependent on age at time of exposure.     

Analysis of the effect of estrogen/androgen perturbation on penile development in transgenic and diethylstilbestrol‐Treated mice

Because both androgens and estrogens have been implicated in penile morphogenesis, we evaluated penile morphology in transgenic mice with known imbalance of androgen and estrogen signaling using scanning electron microscopy (SEM), histology, and immunohistochemistry of androgen and estrogen receptors α/β. Penises of adult wild‐type, estrogen receptor‐α knockout (αERKO), estrogen receptor‐β knockout (βERKO), aromatase knockout (Arom‐KO), and aromatase overexpression (Arom+) mice were evaluated, as well as adult mice treated with diethylstilbestrol (DES) from birth to day 10. Adult penises were examined because the adult pattern is the endpoint of development. The urethral orifice is formed by fusion of the MUMP (male urogenital mating protuberance) with the MUMP ridge, which consists of several processes fused to each other and to the MUMP. Similarly, the internal prepuce is completed ventrally by fusion of a ventral cleft. In adult murine penises the stromal processes that form the MUMP ridge are separated from their neighbors by clefts. αERKO, βERKO, and Arom‐KO mice have penises with a MUMP ridge clefting pattern similar to that of wild‐type mice. In contrast, Arom+ mice and neonatally DES‐treated mice exhibit profound malformations of the MUMP, MUMP ridge clefting pattern, and internal prepuce. Abnormalities observed in Arom+ and neonatally DES‐treated mice correlate with the expression of estrogen receptor‐beta (ERβ) in the affected structures. This study demonstrates that formation of the urethal orifice and internal prepuce is due to fusion of separate epithelial‐surfaced mesenchymal elements, a process dependent upon both androgen and estrogen signaling, in which ERβ signaling is strongly implicated. Anat Rec, 296:1127–1141, 2013. © 2013 Wiley Periodicals, Inc.     

Histone acetylation regulates prostate ductal morphogenesis through a bone morphogenetic protein–dependent mechanism

Background: Epigenetic factors influence stem cell function and other developmental events but their role in prostate morphogenesis is not completely known. We tested the hypothesis that histone deacetylase (HDAC) activity is required for prostate morphogenesis. Results: We identified the presence of class I nuclear HDACs in the mouse urogenital sinus (UGS) during prostate development and found that Hdac 2 mRNA abundance diminishes as development proceeds which is especially evident in prostatic epithelium. Blockade of HDACs with the inhibitor trichostatin A (TSA) decreased the number of prostatic buds formed in UGS explant cultures but not the number of buds undergoing branching morphogenesis. In the latter, TSA promoted an extensive branching phenotype that was reversed by exogenous NOGGIN protein, which functions as a bone morphogenetic protein (BMP) inhibitor. TSA also increased Bmp2 promoter H3K27ac abundance, Bmp2 and Bmp4 mRNA abundance, and the percentage of epithelial cells marked by BMP‐responsive phosphorylated SMAD1/5/8 protein. TSA exposed UGS explants grafted under the kidney capsule of untreated host mice for continued development achieved a smaller size without an obvious difference in glandular histology compared with control treated grafts. Conclusions: These results are consistent with an active role for HDACs in shaping prostate morphogenesis by regulating Bmp abundance. Developmental Dynamics 244:1404–1414, 2015. © 2015 Wiley Periodicals, Inc.     

Androgenic regulation of ventral epithelial bud number and pattern in mouse urogenital sinus

The ventral urogenital sinus (UGS) of control male mice has two rows of 3–4 prostatic buds at birth, but how androgens regulate ventral bud (VB) number and patterning is unclear. VBs in both sexes appeared to be a mixture of prostatic and urethral buds. UGSs from Tfm male and antiandrogen (flutamide)‐exposed mice had small VBs, suggesting that initiation of some VBs is androgen independent. Tfm male mice are widely considered completely androgen insensitive yet their UGSs were 5α‐dihydrotestosterone (DHT)‐ responsive. VBs (6–8) were generally distributed bimodally on the left‐right axis at both minimal and normal male androgen signaling. Yet control females and DHT‐exposed Tfm males had 13–14 VBs, whose left‐right distribution was fairly uniform. These results suggest that VB number and distribution respond biphasically as androgen signaling increases from minimal, and that androgens regulate bud specification. Complete VB agenesis by the selective budding inhibitor 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD) required high androgen signaling. Developmental Dynamics 239:373–385, 2010. © 2009 Wiley‐Liss, Inc.     

Cellular dynamics of epithelial clefting during branching morphogenesis of the mouse submandibular gland

We cultured the rudimental submandibular gland (SMG) of mice with a non–cell‐permeable fluorescent tracer, and observed cell behavior during epithelial branching morphogenesis using confocal time‐lapse microscopy. We traced movements of individual cells as shadowgraph movies. Individual epithelial cells migrated dynamically but erratically. The epithelial cleft extended by wiggling and separated a cluster of cells into two buds during branching. We examined the ultrastructure of the clefts in SMG rudiments treated with the laminin peptide A5G77f, which induces epithelial clefting. A short cytoplasmic shelf with a core of microfilaments was found at the deep end of the cleft. We propose that epithelial clefting involves a dynamic movement of cells at the base of the cleft, and the formation of a shelf within a cleft cell. The shelf might form a matrix attachment point at the base of the cleft with a core of microfilaments driving cleft elongation. Developmental Dynamics 239:1739–1747, 2010. © 2010 Wiley‐Liss, Inc.     

Anatomy of mole external genitalia: Setting the record straight

Anatomy of male and female external genitalia of adult mice (Mus musculus) and broad‐footed moles (Scapanus latimanus) was re‐examined to provide more meaningful anatomical terminology. In the past the perineal appendage of male broad‐footed moles has been called the penis, while the female perineal appendage has been given several terms (e.g. clitoris, penile clitoris, peniform clitoris and others). Histological examination demonstrates that perineal appendages of male and female broad‐footed moles are the prepuce, which in both sexes are covered externally with a hair‐bearing epidermis and lacks erectile bodies. The inner preputial epithelium is non‐hair‐bearing and defines the preputial space in both sexes. The penis of broad‐footed moles lies deep within the preputial space, is an “internal organ” in the resting state and contains the penile urethra, os penis, and erectile bodies. The clitoris of broad‐footed moles is defined by a U‐shaped clitoral epithelial lamina. Residing within clitoral stroma encompassed by the clitoral epithelial lamina is the corpus cavernosum, blood‐filled spaces and the urethra. External genitalia of male and female mice are anatomically similar to that of broad‐footed moles with the exception that in female mice the clitoris contains a small os clitoridis and lacks defined erectile bodies, while male mice have an os penis and a prominent distal cartilaginous structure within the male urogenital mating protuberance (MUMP). Clitori of female broad‐footed moles lack an os clitoridis but contain defined erectile bodies, while male moles have an os penis similar to the mouse but lack the distal cartilaginous structure. Anat Rec, 299:385–399, 2016. © 2015 Wiley Periodicals, Inc.     

Progressive proliferative changes in the oviduct of mice following developmental exposure to diethylstilbestrol

Structural malformation of the oviduct has been reported in experimental animal models and women following prenatal exposure to diethylstilbestrol (DES). To study histological changes in the oviduct in the absence of gross structural malformation, neonatal CD-1 mice were treated with DES (2 micrograms/pup/day) on days 1-5 of age. Focal epithelial hyperplasia was present at 1 month of age in 16 out of 18 (89%) of the DES-treated mice. At 4 months of age, general epithelial hyperplasia with multiple gland-like structures into and through the muscle wall of the oviduct was observed in 90% of the treated mice; by 12 months of age, epithelial hyperplasia and pseudogland formation were seen in 100% of the DES-exposed animals. Epithelial hyperplasia and gland formation were not observed in control mice. The alteration induced by DES in the differentiation and proliferation of mouse oviductal epithelium suggests that the oviduct is a target for DES toxic effects. In addition, there was a progression of the epithelial changes with age. The histological changes described in this study may be partially responsible for the decreased fertility previously reported in this mouse model. Similar changes in the oviduct of DES-exposed women remain to be determined.     

Podoplanin deficient mice show a rhoa‐related hypoplasia of the sinus venosus myocardium including the sinoatrial node

We investigated the role of podoplanin in development of the sinus venosus myocardium comprising the sinoatrial node, dorsal atrial wall, and primary atrial septum as well as the myocardium of the cardinal and pulmonary veins. We analyzed podoplanin wild‐type and knockout mouse embryos between embryonic day 9.5–15.5 using immunohistochemical marker podoplanin; sinoatrial‐node marker HCN4; myocardial markers MLC‐2a, Nkx2.5, as well as Cx43; coelomic marker WT‐1; and epithelial‐to‐mesenchymal transformation markers E‐cadherin and RhoA. Three‐dimensional reconstructions were made and myocardial morphometry was performed. Podoplanin mutants showed hypoplasia of the sinoatrial node, primary atrial septum, and dorsal atrial wall. Myocardium lining the wall of the cardinal and pulmonary veins was thin and perforated. Impaired myocardial formation is correlated with abnormal epithelial‐to‐mesenchymal transformation of the coelomic epithelium due to up‐regulated E‐cadherin and down‐regulated RhoA, which are controlled by podoplanin. Our results demonstrate an important role for podoplanin in development of sinus venosus myocardium. Developmental Dynamics 238:183–193, 2009. © 2008 Wiley‐Liss, Inc.     

Fibroblast growth factor 2 decreases bleomycin‐induced pulmonary fibrosis and inhibits fibroblast collagen production and myofibroblast differentiation

Fibroblast growth factor (FGF) signaling has been implicated in the pathogenesis of pulmonary fibrosis. Mice lacking FGF2 have increased mortality and impaired epithelial recovery after bleomycin exposure, supporting a protective or reparative function following lung injury. To determine whether FGF2 overexpression reduces bleomycin‐induced injury, we developed an inducible genetic system to express FGF2 in type II pneumocytes. Double‐transgenic (DTG) mice with doxycycline‐inducible overexpression of human FGF2 (SPC‐rtTA;TRE‐hFGF2) or single‐transgenic controls were administered intratracheal bleomycin and fed doxycycline chow, starting at either day 0 or day 7. In addition, wild‐type mice received intratracheal or intravenous recombinant FGF2, starting at the time of bleomycin treatment. Compared to controls, doxycycline‐induced DTG mice had decreased pulmonary fibrosis 21 days after bleomycin, as assessed by gene expression and histology. This beneficial effect was seen when FGF2 overexpression was induced at day 0 or day 7 after bleomycin. FGF2 overexpression did not alter epithelial gene expression, bronchoalveolar lavage cellularity or total protein. In vitro studies using primary mouse and human lung fibroblasts showed that FGF2 strongly inhibited baseline and TGFβ1‐induced expression of alpha smooth muscle actin (αSMA), collagen, and connective tissue growth factor. While FGF2 did not suppress phosphorylation of Smad2 or Smad‐dependent gene expression, FGF2 inhibited TGFβ1‐induced stress fiber formation and serum response factor‐dependent gene expression. FGF2 inhibition of stress fiber formation and αSMA requires FGF receptor 1 (FGFR1) and downstream MEK/ERK, but not AKT signaling. In summary, overexpression of FGF2 protects against bleomycin‐induced pulmonary fibrosis in vivo and reverses TGFβ1‐induced collagen and αSMA expression and stress fiber formation in lung fibroblasts in vitro, without affecting either inflammation or epithelial gene expression. Our results suggest that in the lung, FGF2 is antifibrotic in part through decreased collagen expression and fibroblast to myofibroblast differentiation. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Regulation of external genitalia development by concerted actions of FGF ligands and FGF receptors

Members of the fibroblast growth factor (FGF) family play diverse roles during the development and patterning of various organs. In human and mice, 22 FGFs and four receptors derived from several splice variants are present. Redundant expression and function of FGF genes in organogenesis have been reported, but their roles in embryonic external genitalia, genital tubercle (GT), development have not been studied in detail. To address the role of FGF during external genitalia development, we have analyzed the expression of FGF genes (Fgf8, 9, 10) and receptor genes (Fgfr1, r2IIIb, r2IIIc) in GT of mice. Furthermore, Fgf10 and Fgfr2IIIb mutant mice were analyzed to elucidate their roles in embryonic external genitalia development. Fgfr2IIIb was expressed in urethral plate epithelium during GT development. Fgfr2IIIb mutant mice display urethral dysmorphogenesis. Marker gene analysis for urethral plate and bilateral mesenchymal formation suggests the existence of epithelial-mesenchymal interaction during urethral morphogenesis. Therefore, FGF10/FGFR2IIIb signals seem to constitute a developmental cascade for such morphogenesis.Y. Satoh and R. Haraguchi contributed equally to this work     

Atypical Fetal Prostate Development is Associated with Ipsilateral Hypoplasia of the Wolffian Ducts in the ACI Rat

For over a half century, the ACI (August × Copenhagen) rat has been a primary model for studying renal agenesis and ipsilateral hypoplasia (IHP) of the Wolffian‐derived structures (WDS). Because the ACI rat is also used as a model for prostate research, it is important to examine the relationship of IHP and urogenital sinus (UGS) development. The prostate is dependent on androgens for proper growth and differentiation. Alteration in androgen production and/or delivery to the UGS has the potential to perturbate normal development. In this study, we investigate whether the ipsilateral loss of the WDS is associated with altered prostate development. Digital images of serial‐sectioned fetal ACI rat UGS were used to create three‐dimensional (3‐D) surface‐rendered models of the developing prostate, seminal vesicle, vas deferens, and utricle on gestational day 21. The number and volume of prostate ducts developing from the UGS were calculated from the 3‐D model data. Animals exhibiting IHP had a significant decrease in total fetal prostate volume (40%; P < 0.005) with significant regional specific differences when compared with normal male ACI rats. Anatomical and histological differences in the utricle, abnormal histology of the ipsilateral testes, and a truncation of the ipsilateral Wolffian ductal mesenchyme were also seen in the animals with IHP. Additional research is needed to further understand the mechanisms and consequences of IHP on prostate growth and development. Alterations to normal prenatal development of the male accessory sex organs can have important consequences for the growth and morphology of the adult gland. Anat Rec, 2010. © 2010 Wiley‐Liss, Inc.     

Development of thymus,parathyroids, and ultimo-branchial bodies in NMRI and nude mice

In the NMRI mouse embryo, the thymus develops from the third endodermal pouch and the third ectodermal cleft. The cervical vesicle, formed not by the closure of the sinus cervicalis but by an invagination of the dorsal segment of the ectoderm between the third and fourth branchial clefts, contributes to the formation of the thymus. The intense proliferation of the ectoderm of the third cleft on the eleventh day covers the endodermal part. The thymus is thus composed of a central endodermal region and a peripheral ectodermal region. The normal adult thymus, then, has a mixed origin, the cortical cells being ectodermal, and the medullary cells endodermal in origin. The fourth endodermal pouch gives rise to the ultimo-branchial body, which becomes entirely incorporated within the thyroid on the fourteenth day. There is no formation of a thymus IV nor of a second pair of parathyroids from the fourth endodermal pouch. In the Nude mouse embryo, the third branchial pouch and cleft, as well as the cervical vesicle, develop normally for the first 11½ days. From this point on, the ectoderm of the third cleft ceases to develop further. The endoderm is, now, no longer covered by ectoderm and, deprived of its normal inducing agent, ceases to develop further. Thymic dysgenesis is thus ectodermal in origin. The fourth endodermal pouch develops normally. No development of an accessory “thymus-like” structure from the fourth pouch was observed. The dysgenetic thymus originates entirely from the third branchial pouches and clefts. In both Nude and NMRI embryos, the parathyroids develop at about 11½ days from a very limited area in the dorsal region of the cranial wall of the third endodermal pouch between the pharyngo-branchial and ecto-branchial ducts. Morphometric analysis shows that the volume of the parathyroids is the same in both strains of mice at each stage of development; nor does their microscopic appearance differ. Thus, mutation in the Nude mouse does not affect the development of the parathyroids from the third pouch, even though the first anomalies in the development of the thymus are observed at the precise moment at which the parathyroid primordium appears.     

Essential role of Wnt5a‐Ror1/Ror2 signaling in metanephric mesenchyme and ureteric bud formation

Spatiotemporally regulated interaction between the metanephric mesenchyme (MM) and Wolffian duct (WD) is essential for the induction of a single ureteric bud (UB). The MM then interacts with the tip of the UB to induce outgrowth and branching of the UB, which in turn promotes growth of the adjacent MM. The Ror family receptor tyrosine kinases, Ror1 and Ror2, have been shown to act as receptors for Wnt5a to mediate noncanonical Wnt signaling. Previous studies have shown that Ror2‐mutant mice exhibit ectopic formation of the UB, due to abnormal juxtaposition of the MM to the WD. We show here that both Ror1 and Ror2 are expressed in the mesenchyme between the MM and WD during UB formation. Although Ror1‐mutant mice show no apparent defects in UB formation, Ror1;Ror2‐double‐mutant mice exhibit either defects in UB outgrowth and branching morphogenesis, associated with the loss of the MM from the UB domain, or ectopic formation of the UB. We also show genetic interactions between Ror1 and Wnt5a during UB formation. These findings suggest that Wnt5a‐Ror1/Ror2 signaling regulates cooperatively the formation of the MM at the proper position to ensure normal development of the UB.     

Primary vaginal adenocarcinoma arising from the metanephric duct remnant

Primary vaginal adenocarcinoma unrelated to in utero exposure to diethylstilbestrol (DES) is very uncommon. We report a case of 65-year-old Japanese woman who presented with primary adenocarcinoma in the anterior wall of the vagina, where the left ureter-like metanephric duct remnant abnormally terminated. Histological examination in serial sections revealed the direct connection between the carcinoma and the metanephric duct remnant. Moreover, the remnant epithelium showed varying degrees of dysplastic changes, including carcinoma in situ in close proximity to the carcinoma. This patient also had a bicornate uterus and left renal aplasia. To our knowledge, this is the first reported case of a primary vaginal adenocarcinoma arising from the metanephric duct remnant. Although the precise mechanism involved in carcinogenesis in this clinicopathological setting remains unknown, adenocarcinoma should be included in the differential diagnosis of vaginal tumors in patients with renal aplasia and/or an ectopic termination of the ureter or metanephric duct remnant, especially when the tumor is in the anterior wall.     

Induction of estrogen-independent persistent vaginal cornification in cyproterone acetate (CA)-induced feminized male mice

Summary Pregnant ICR/JCL mice were treated with 6 mg of cyproterone acetate (CA) from days 14 to 20 of pregnancy to feminize male offspring. Feminized males delivered on day 20 of pregnancy by cesarean section were castrated the same day, injected with estradiol-17(E₂) or sesame oil from the day of delivery (=day 1) to day 10 and sacrificed on day 60. In oil-injected feminized males, the vaginal epithelium was atrophic and did not show cornification. In feminized males given 20 g E₂ neonatally, the vaginal epithelium exhibited well-differentiated stratified squamous organization, but was not cornified in seven out of the nine mice of this group. In the mice treated with 50 g E₂, persistent cornification was recognized most frequently in the posterior two-thirds of the vaginal epithelium which is considered to originate from the which may contain the epithelial cells of müllerian duct was low. These results provide supporting evidence for the possible participation of epithelial cells which come from the urogenital sinus in the development of estrogen-independent persistent vaginal cornification in neonatally estrogenized mice.     

人胚心静脉窦和传导系统的早期发育

目的 探讨早期人胚心静脉窦及传导系的发生发育机制. 方法 用抗α-平滑肌肌动蛋白(α-SMA)、抗α-横纹肌肌动蛋白(α-SCA)和抗结蛋白(DES)抗体对29例C10～C16期人胚心连续切片行免疫组织化学染色. 结果 人胚发育C12～C13期,系统静脉汇集形成的静脉窦出现于心包腔尾端原始横膈间充质中,静脉窦壁间充质细胞逐渐分化为α-SCA阳性的静脉窦心肌细胞.C14期,心包腔的扩张使静脉窦进入心包腔内,参与了右心房的形成.DES阳性传导系心肌的分化始于C10期心房室管右侧壁,随发育逐渐向室间沟心肌扩展,发育为房室传导系的希氏束、左右束支及心室腔面的小梁心肌.在心房,DES表达首先出现于C11期心房背侧壁,在C13期,可见静脉窦左背侧壁α-SCA、α-SMA、DES阳性心肌带与左心房底部、房室管背侧壁相延续,这条心肌带可能参与了人胚心静脉窦至房室管传导系的发育.C14～C16期,DES强阳性染色从窦房结经左、右静脉瓣及心房的背、腹侧壁延伸至房室管右侧壁,可能是原始的心房传导通路. 结论 心包腔尾端原始横膈间充质是人胚静脉窦心肌发生区,原始横膈间充质细胞逐渐分化为心肌细胞,添加到人胚心管静脉端,形成心静脉窦心肌.人胚心传导系心肌的分化始于房室管,随心管发育逐渐向动、静脉端扩展,在C16期,已分化为形态清晰可辨的DES阳性胚胎心传导系.     

Matrigel‐Induced Tubular Morphogenesis of Human Eccrine Sweat Gland Epithelial Cells

Human eccrine sweat glands are tubule‐structured glands of the skin that are vital in thermoregulation, secretion, and excretion of water and electrolytes. A study of tubular morphogenesis in vitro would facilitate the development of a tissue engineering model for eccrine sweat glands and other tubule‐structured glands. Matrigel, a basement membrane matrix, has been shown to promote differentiation and morphogenesis of many different cell types, including tubular cells. This study investigated the growth, differentiation, and tubular morphogenesis of human eccrine sweat gland epithelial cells cultured in Matrigel. Human eccrine gland epithelial cells were isolated and cultured in vitro. The cell growth in Matrigel was evidenced by the formation of cell clusters, which were observed under an inverted microscope. The internal structure of the cell clusters was further investigated by hematoxylin–eosin (HE) staining and confocal laser scanning microscopy (CLSM) of propidium iodide‐stained nuclei. The results demonstrated that although on a plastic surface or in a collagen gel the cells could not form tubular structures, they formed tubular structures when cultured in Matrigel. Consequently, we conclude that Matrigel can promote tubular morphogenesis of human eccrine sweat gland epithelial cells. Anat Rec, 2011. © 2011 Wiley‐Liss, Inc.     

Timeless in lung morphogenesis.

The Clock gene, timeless, regulates circadian rhythm in Drosophila, but its vertebrate homolog is critical to embryonic development. Timeless was shown to be involved in murine urethral bud branching morphogenesis. We generated a polyclonal antibody to mouse TIMELESS (mTIM) and studied its distribution and its potential role during lung development, which also requires branching morphogenesis. In the early mouse embryo, TIM was localized to all organs, especially the neural epithelium. In embryonic day (E) 9.5 embryos, TIM was present in both epithelial and mesenchymal cells at the onset of lung morphogenesis. In E15 embryos, TIM decreased in the mesenchyme but remained pronounced in the epithelium of both large and small airways. Later, TIM was localized to a specific subset of epithelial cells with alveolar type 2 phenotype. This finding was verified by immunostaining of isolated alveolar type 2 cells. In the proximal airways, TIM was colocalized with CCSP to nonciliated columnar epithelial cells. Antisense oligonucleotides to mTim specifically inhibited branching morphogenesis of embryonic lungs in explant culture without affecting SpC expression an alveolar type 2 cell marker. In cultured lung cells, expression of TIM is independent of cell cycle and proliferation. These studies indicate that the function of Timeless is highly conserved in organs whose formation requires branching morphogenesis.