Epithelial stem/progenitor cells in lung postnatal growth, maintenance, and repair.

The adult lung consists of a trachea leading into a system of branched airways ending in millions of alveolar sacs. It contains many different epithelial cell types arranged in precise patterns along the proximodistal axis. Each region of the lung has the capacity to repair through the proliferation of different epithelial cell types. However, the precise identity of the cells mediating repair is not fully resolved. To address this problem, we are using genetic lineage-labeling techniques in the mouse. The tools we have made will also be useful for understanding how progenitor cell behavior is regulated under normal and pathological conditions.

Summary: present and future challenges for stem cell research.

Stem cell research is being driven forward at an intense pace by creative interactions among scientists working in different fields. These include developmental and reproductive biology, regeneration, genomics, live cell imaging, RNA biology, and cancer biology, to name a few. Numerous model systems and techniques are being exploited, and lab scientists are teaming up with bioengineers and clinicians. The ferment of ideas that makes the field so exciting was in full evidence throughout the Symposium. However, many challenges still need to be overcome to translate basic discoveries into therapeutic outcomes that will save lives and fulfill the promises that have been made. This chapter summarizes some of the highlights of the Symposium and indicates future directions that are being taken by leaders in the field.

Bone morphogenetic protein 4 in the extraembryonic mesoderm is required for allantois development and the localization and survival of primordial germ cells in the mouse.

Evidence suggests that the specification of primordial germ cells (PGCs) in the mammalian embryo does not depend on maternal determinants. Rather, previous genetic analysis in the mouse has shown that bone morphogenetic protein 4 (Bmp4) is required for the formation of both PGCs and allantois. Bmp4 is expressed in both the trophoblast-derived extraembryonic ectoderm (ExE) and in the epiblast-derived extraembryonic mesoderm (ExM), in which the PGCs, allantois primordium, and angioblasts are first detected. We have shown that Bmp4 made in the ExE functions to induce precursors of PGCs and allantois in the adjacent epiblast, resulting in complete lack of both cell types in homozygous null mutants. However, the function of Bmp4 in the ExM is totally unknown. To address this question, we generated tetraploid (4N) chimeras by aggregating Bmp4 null ES cells with wild-type tetraploid embryos. In this combination, wild-type tetraploid cells contribute to the extraembryonic trophoblast and primitive endoderm lineages but are excluded from the epiblast and its derivatives, including the ExM. Our results clearly demonstrate that Bmp4 made in the ExM does not affect the establishment of either PGC or allantois lineages, but is required for PGC localization and survival and for the differentiation of the allantois. These findings suggest that Bmp4 expressed in epiblast-derived tissues plays vital roles in reproduction by regulating both the development of the germ line and the vascular connection between the embryo and the placenta.

Haploinsufficient Bmp4 ocular phenotypes include anterior segment dysgenesis with elevated intraocular pressure.

BACKGROUND: Glaucoma is a blinding disease usually associated with high intraocular pressure (IOP). In some families, abnormal anterior segment development contributes to glaucoma. The genes causing anterior segment dysgenesis and glaucoma in most of these families are not identified and the affected developmental processes are poorly understood. Bone morphogenetic proteins (BMPs) participate in various developmental processes. We tested the importance of Bmp4 gene dosage for ocular development and developmental glaucoma. RESULTS: Bmp4+/- mice have anterior segment abnormalities including malformed, absent or blocked trabecular meshwork and Schlemm's canal drainage structures. Mice with severe drainage structure abnormalities, over 80% or more of their angle's extent, have elevated IOP. The penetrance and severity of abnormalities is strongly influenced by genetic background, being most severe on the C57BL/6J background and absent on some other backgrounds. On the C57BL/6J background there is also persistence of the hyaloid vasculature, diminished numbers of inner retinal cells, and absence of the optic nerve. CONCLUSIONS: We demonstrate that heterozygous deficiency of BMP4 results in anterior segment dysgenesis and elevated IOP. The abnormalities are similar to those in human patients with developmental glaucoma. Thus, BMP4 is a strong candidate to contribute to Axenfeld-Rieger anomaly and other developmental conditions associated with human glaucoma. BMP4 also participates in posterior segment development and wild-type levels are usually critical for optic nerve development on the C57BL/6J background. Bmp4+/- mice are useful for studying various components of ocular development, and may allow identification of strain specific modifiers affecting a variety of ocular phenotypes.

The murine winged helix transcription factors, Foxc1 and Foxc2, are both required for cardiovascular development and somitogenesis.

The murine Foxc1/Mf1 and Foxc2/Mfh1 genes encode closely related forkhead/winged helix transcription factors with overlapping expression in the forming somites and head mesoderm and endothelial and mesenchymal cells of the developing heart and blood vessels. Embryos lacking either Foxc1 or Foxc2, and most compound heterozygotes, die pre- or perinatally with similar abnormal phenotypes, including defects in the axial skeleton and cardiovascular system. However, somites and major blood vessels do form. This suggested that the genes have similar, dose-dependent functions, and compensate for each other in the early development of the heart, blood vessels, and somites. In support of this hypothesis, we show here that compound Foxc1; Foxc2 homozygotes die earlier and with much more severe defects than single homozygotes alone. Significantly, they have profound abnormalities in the first and second branchial arches, and the early remodeling of blood vessels. Moreover, they show a complete absence of segmented paraxial mesoderm, including anterior somites. Analysis of compound homozygotes shows that Foxc1 and Foxc2 are both required for transcription in the anterior presomitic mesoderm of paraxis, Mesp1, Mesp2, Hes5, and Notch1, and for the formation of sharp boundaries of Dll1, Lfng, and ephrinB2 expression. We propose that the two genes interact with the Notch signaling pathway and are required for the prepatterning of anterior and posterior domains in the presumptive somites through a putative Notch/Delta/Mesp regulatory loop.

The winged helix transcription factor Foxc1a is essential for somitogenesis in zebrafish.

Previous studies identified zebrafish foxc1a and foxc1b as homologs of the mouse forkhead gene, Foxc1. Both genes are transcribed in the unsegmented presomitic mesoderm (PSM), newly formed somites, adaxial cells, and head mesoderm. Here, we show that inhibiting synthesis of Foxc1a (but not Foxc1b) protein with two different morpholino antisense oligonucleotides blocks formation of morphological somites, segment boundaries, and segmented expression of genes normally transcribed in anterior and posterior somites and expression of paraxis implicated in somite epithelialization. Patterning of the anterior PSM is also affected, as judged by the absence of mesp-b, ephrinB2, and ephA4 expression, and the down-regulation of notch5 and notch6. In contrast, the expression of other genes, including mesp-a and papc, in the anterior of somite primordia, and the oscillating expression of deltaC and deltaD in the PSM appear normal. Nevertheless, this expression is apparently insufficient for the maturation of the presumptive somites to proceed to the stage when boundary formation occurs or for the maintenance of anterior/posterior patterning. Mouse embryos that are compound null mutants for Foxc1 and the closely related Foxc2 have no morphological somites and show abnormal expression of Notch signaling pathway genes in the anterior PSM. Therefore, zebrafish foxc1a plays an essential and conserved role in somite formation, regulating both the expression of paraxis and the A/P patterning of somite primordia.

Distinct mesodermal signals, including BMPs from the septum transversum mesenchyme, are required in combination for hepatogenesis from the endoderm.

Mesodermal signaling is critical for patterning the embryonic endoderm into different tissue domains. Classical tissue transplant experiments in the chick and recent studies in the mouse indicated that interactions with the cardiogenic mesoderm are necessary and sufficient to induce the liver in the ventral foregut endoderm. Using molecular markers and functional assays, we now show that septum transversum mesenchyme cells, a distinct mesoderm cell type, are closely apposed to the ventral endoderm and contribute to hepatic induction. Specifically, using a mouse Bmp4 null mutation and an inhibitor of BMPs, we find that BMP signaling from the septum transversum mesenchyme is necessary to induce liver genes in the endoderm and to exclude a pancreatic fate. BMPs apparently function, in part, by affecting the levels of the GATA4 transcription factor, and work in parallel to FGF signaling from the cardiac mesoderm. BMP signaling also appears critical for morphogenetic growth of the hepatic endoderm into a liver bud. Thus, the endodermal domain for the liver is specified by simultaneous signaling from distinct mesodermal sources.

Sequence and expression of zebrafish foxc1a and foxc1b, encoding conserved forkhead/winged helix transcription factors.

Mouse Foxc1 (previously Mf1) is a member of the conserved forkhead/winged helix transcription factor gene family. It is expressed in many mesodermal tissues including paraxial mesoderm of the trunk and head, prechondrogenic mesenchyme, branchial arches and developing kidney. Homozygous mutants die perinatally with hydrocephalus and skeletal, cardiovascular, ocular and genitourinary defects. Here, we report the cloning and expression of two zebrafish foxc1 homologues, foxc1a and foxc1b. During gastrulation and somitogenesis both genes have similar expression patterns in the hypoblast, paraxial and presomitic mesoderm, somites and trunk adaxial cells. Expression in the somites is downregulated as they differentiate, but is maintained in the sclerotome. Later, some differences in expression pattern emerge. For example, only foxc1a transcripts are detected in the pronephros primodia and in the head mesoderm around the eyes, while only foxc1b is expressed in the pharyngeal arches and pectoral fins. Early expression of foxc1a in the paraxial mesoderm is modified in chordino, swirl, somitabun, and spadetail mutants.

Cellular and molecular responses of the uterus to embryo implantation can be elicited by locally applied growth factors.

The implantation of a blastocyst into a receptive uterus is associated with a series of events, namely the attachment reaction followed by decidualization of the stroma. Previous studies established that the gene encoding heparin-binding EGF-like growth factor (HB-EGF) is expressed in the luminal epithelium solely at the site of blastocyst apposition preceding the attachment reaction. We report here the expression during implantation of 21 genes encoding other signaling proteins, including those belonging to the Bone morphogenetic protein (BMP), fibroblast growth factor (FGF), WNT, and Hedgehog (HH) pathways. We find that the attachment reaction is associated with a localized stromal induction of genes encoding BMP-2, FGF-2, and WNT-4. Despite efforts by many investigators, a simple in vitro model of implantation is not yet available to study either the hierarchy of the events triggered in the uterus by the embryo or the function of individual signaling proteins. We have therefore approached these questions by introducing beads loaded with purified factors into the receptive uterus. We show that beads soaked in HB-EGF or insulin-like growth factor-1 (IGF-1), but not other proteins, induce many of the same discrete local responses elicited by the blastocyst, including increased localized vascular permeability, decidualization, and expression of Bmp2 at the sites of the beads. By contrast, the expression domains of Indian hedgehog (Ihh), patched, and noggin become restricted as decidualization proceeds. Significantly, beads containing BMP-2 do not themselves elicit an implantation response but affect the spacing of implantation sites induced by blastocysts cotransferred with the beads.

Inhibition of Bmp signaling affects growth and differentiation in the anagen hair follicle.

Growth and differentiation of postnatal hair follicles are controlled by reciprocal interactions between the dermal papilla and the surrounding epidermal hair precursors. The molecular nature of these interactions is largely unknown, but they are likely to involve several families of signaling molecules, including Fgfs, Wnts and Bmps. To analyze the function of Bmp signaling in postnatal hair development, we have generated transgenic mice expressing the Bmp inhibitor, Noggin, under the control of the proximal Msx2 promoter, which drives expression in proliferating hair matrix cells and differentiating hair precursor cells. Differentiation of the hair shaft but not the inner root sheath is severely impaired in Msx2-Noggin transgenic mice. In addition to hair keratins, the expression of several transcription factors implicated in hair development, including Foxn1 and Hoxc13, is severely reduced in the transgenic hair follicles. Proliferating cells, which are normally restricted to the hair matrix surrounding the dermal papilla, are found in the precortex and hair shaft region. These results identify Bmps as key regulators of the genetic program controlling hair shaft differentiation in postnatal hair follicles.

Notch/Delta expression in the developing mouse lung.

Factors controlling the differentiation of the multipotent embryonic lung endoderm and mesoderm are poorly understood. Recent evidence that Delta-like 1 (Dll1) and other genes in the Notch/Delta signaling pathway are expressed in the embryonic mouse lung suggests that this pathway is important for cell fate decisions and/or the differentiation of lung cell types. Here, we report the localization of transcripts of several genes encoding members of the Notch/Delta pathway in the early mouse lung. Most genes are expressed in specific populations and so may contribute to cell diversification.

Development and validation of a liquid chromatographic method for the determination of the related substances of ramipril in Altace capsules.

The development and validation of a reversed-phase liquid chromatographic method for the determination of the related substances of 2-[N-[(S)-1-Ethoxycarbonyl-3-phenylpropyl]-L-alanyl]-(1S, 3S, 5S)-2-azabicyclo[3.3.0]octane-3-carboxylic acid (ramipril) in Altace capsules is described. The method utilizes an ion-pairing agent and a simple two-step gradient for the separation of ramipril and ten related substances from each other in a 40-min run time. Four of the related substances are ramipril diastereomers. To the best of our knowledge, no method described previously in the literature has demonstrated resolution of ramipril from this set of related substances. No method for the determination of the related substances of ramipril is currently described in the United States Pharmacopoeia or the European Pharmacopoeia. The proposed method was validated with respect to accuracy, precision, linearity, and specificity. Also, the method was determined to be robust with regards to the following parameters: mobile phase apparent pH: mobile phase organic content: mobile phase perchlorate concentration; detection wavelength and time dependence of sample and standard stability.

Bmp4 and Fgf10 play opposing roles during lung bud morphogenesis.

Morphogenesis of the mouse lung involves reciprocal interactions between the epithelial endoderm and the surrounding mesenchyme, leading to an invariant early pattern of branching that forms the basis of the respiratory tree. There is evidence that Fibroblast growth factor 10 (Fgf10) and Bone Morphogenetic Protein 4 (Bmp4), expressed in the distal mesenchyme and endoderm, respectively, play important roles in branching morphogenesis. To examine these roles in more detail, we have exploited an in vitro culture system in which isolated endoderm is incubated in Matrigel(TM) substratum with Fgf-loaded beads. In addition, we have used a Bmp4(lacZ) line of mice in which lacZ faithfully reports Bmp4 expression. Analysis of lung endoderm in vivo shows a dynamic pattern of Bmp4(lacZ) expression during bud outgrowth, extension and branching. In vitro, Fgf10 induces both proliferation and chemotaxis of isolated endoderm, whether it is derived from the distal or proximal lung. Moreover, after 48 hours, Bmp4(lacZ) expression is upregulated in the endoderm closest to the bead. Addition of 30-50 ng/ml of exogenous purified Bmp4 to the culture medium inhibits Fgf-induced budding or chemotaxis, and inhibits overall proliferation. By contrast, the Bmp-binding protein Noggin enhances Fgf-induced morphogenesis. Based on these and other results, we propose a model for the combinatorial roles of Fgf10 and Bmp4 in branching morphogenesis of the lung.

Haploinsufficiency of the transcription factors FOXC1 and FOXC2 results in aberrant ocular development.

Anterior segment developmental disorders, including Axenfeld-Rieger anomaly (ARA), variably associate with harmfully elevated intraocular pressure (IOP), which causes glaucoma. Clinically observed dysgenesis does not correlate with IOP, however, and the etiology of glaucoma development is not understood. The forkhead transcription factor genes Foxc1 (formerly Mf1 ) and Foxc2 (formerly Mfh1 ) are expressed in the mesenchyme from which the ocular drainage structures derive. Mutations in the human homolog of Foxc1, FKHL7, cause dominant anterior segment defects and glaucoma in various families. We show that Foxc1 (+/-)mice have anterior segment abnormalities similar to those reported in human patients. These abnormalities include small or absent Schlemm's canal, aberrantly developed trabecular meshwork, iris hypoplasia, severely eccentric pupils and displaced Schwalbe's line. The penetrance of clinically obvious abnormalities varies with genetic background. In some affected eyes, collagen bundles were half normal diameter, or collagen and elastic tissue were very sparse. Thus, abnormalities in extracellular matrix synthesis or organization may contribute to development of the ocular phenotypes. Despite the abnormalities in ocular drainage structures in Foxc1 (+/-)mice, IOP was normal in almost all mice analyzed, on all genetic backgrounds and at all ages. Similar abnormalities were found in Foxc2 (+/-)mice, but no disease-associated mutations were identified in the human homolog FKHL14 in 32 ARA patients. Foxc1 (+/-)and Foxc2 (+/-)mice are useful models for studying anterior segment development and its anomalies, and may allow identification of genes that interact with Foxc1 and Foxc2 (or FKHL7 and FKHL14 ) to produce a phenotype with elevated IOP and glaucoma.

Bone morphogenetic protein 4 regulates the budding site and elongation of the mouse ureter.

In the normal mouse embryo, Bmp4 is expressed in mesenchymal cells surrounding the Wolffian duct (WD) and ureter stalk, whereas bone morphogenetic protein (BMP) type I receptor genes are transcribed either ubiquitously (Alk3) or exclusively in the WD and ureter epithelium (Alk6). Bmp4 heterozygous null mutant mice display, with high penetrance, abnormalities that mimic human congenital anomalies of the kidney and urinary tract (CAKUT), including hypo/dysplastic kidneys, hydroureter, ectopic ureterovesical (UV) junction, and double collecting system. Analysis of mutant embryos suggests that the kidney hypo/dysplasia results from reduced branching of the ureter, whereas the ectopic UV junction and double collecting system are due to ectopic ureteral budding from the WD and accessory budding from the main ureter, respectively. In the cultured metanephros deprived of sulfated glycosaminoglycans (S-GAGs), BMP4-loaded beads partially rescue growth and elongation of the ureter. By contrast, when S-GAGs synthesis is not inhibited, BMP4 beads inhibit ureter branching and expression of Wnt 11, a target of glial cell-derived neurotrophic factor signaling. Thus, Bmp4 has 2 functions in the early morphogenesis of the kidney and urinary tract. One is to inhibit ectopic budding from the WD or the ureter stalk by antagonizing inductive signals from the metanephric mesenchyme to the illegitimate sites on the WD. The other is to promote the elongation of the branching ureter within the metanephros, thereby promoting kidney morphogenesis.

Murine forkhead/winged helix genes Foxc1 (Mf1) and Foxc2 (Mfh1) are required for the early organogenesis of the kidney and urinary tract.

The murine genes, Foxc1 and Foxc2 (previously, Mf1 and Mfh1), encode forkhead/winged helix transcription factors with virtually identical DNA-binding domains and overlapping expression patterns in various embryonic tissues. Foxc1/Mf1 is disrupted in the mutant, congenital hydrocephalus (Foxc1/Mf1(ch)), which has multiple developmental defects. We show here that, depending on the genetic background, most Foxc1 homozygous mutants are born with abnormalities of the metanephric kidney, including duplex kidneys and double ureters, one of which is a hydroureter. Analysis of embryos reveals that Foxc1 homozygotes have ectopic mesonephric tubules and ectopic anterior ureteric buds. Moreover, expression in the intermediate mesoderm of Glial cell-derived neurotrophic factor (Gdnf), a primary inducer of the ureteric bud, is expanded more anteriorly in Foxc1 homozygous mutants compared with wild type. These findings support the hypothesis of Mackie and Stephens concerning the etiology of duplex kidney and hydroureter in human infants with congenital kidney abnormalities (Mackie, G. G. and Stephens, F. G. (1975) J. Urol. 114, 274-280). Previous studies established that most Foxc1(lacZ )Foxc2(tm1) compound heterozygotes have the same spectrum of cardiovascular defects as single homozygous null mutants, demonstrating interaction between the two genes in the cardiovascular system. Here, we show that most compound heterozygotes have hypoplastic kidneys and a single hydroureter, while all heterozygotes are normal. This provides evidence that the two genes interact in kidney as well as heart development.

Out of Eden: stem cells and their niches.

Stem cells are currently in the news for two reasons: the successful cultivation of human embryonic stem cell lines and reports that adult stem cells can differentiate into developmentally unrelated cell types, such as nerve cells into blood cells. Both intrinsic and extrinsic signals regulate stem cell fate and some of these signals have now been identified. Certain aspects of the stem cell microenvironment, or niche, are conserved between tissues, and this can be exploited in the application of stem cells to tissue replacement therapy.

Minimal phenotype of mice homozygous for a null mutation in the forkhead/winged helix gene, Mf2.

Mf2 (mesoderm/mesenchyme forkhead 2) encodes a forkhead/winged helix transcription factor expressed in numerous tissues of the mouse embryo, including paraxial mesoderm, somites, branchial arches, vibrissae, developing central nervous system, and developing kidney. We have generated mice homozygous for a null mutation in the Mf2 gene (Mf2(lacZ)) to examine its role during embryonic development. The lacZ allele also allows monitoring of Mf2 gene expression. Homozygous null mutants are viable and fertile and have no major developmental defects. Some mutants show renal abnormalities, including kidney hypoplasia and hydroureter, but the penetrance of this phenotype is only 40% or lower, depending on the genetic background. These data suggest that Mf2 can play a unique role in kidney development, but there is functional redundancy in this organ and other tissues with other forkhead/winged helix genes.