An in vitro mouse model of cleft palate: defining a critical intershelf distance necessary for palatal clefting.

It is unclear whether cleft palate formation is attributable to intrinsic biomolecular defects in the embryonic elevating palatal shelves or to an inability of the shelves to overcome a mechanical obstruction (such as the tongue in Pierre Robin sequence) to normal fusion. Regardless of the specific mechanism, presumably embryonic palatal shelves are ultimately unable to bridge a critical distance and remain unapproximated, resulting in a clefting defect at birth. We propose to use a palate organ culture system to determine the critical distance beyond which embryonic palatal shelves fail to fuse (i.e., the minimal critical intershelf distance). In doing so, we hope to establish an in vitro cleft palate model that could then be used to investigate the contributions of various signaling pathways to cleft formation and to study novel in utero treatment strategies. Palatal shelves from CD-1 mouse embryos were microdissected on day 13.5 of gestation (E13.5; term = 19.5 days), before fusion. Using a standardized microscope ocular grid, paired palatal shelves were placed on a filter insert at precisely graded distances ranging from 0 (in contact) to 1.9 mm (0, 0.095, 0.19, 0.26, 0.38, 0.48, 0.57, 0.76, 0.95, and 1.9 mm). A total of 68 paired palatal shelves were placed in serum-free organ culture for 96 hours (n = 68). Sample sizes of 10 were used for each intershelf distance up to and including 0.48 mm (n = 60). For intershelf distances of 0.57 mm and greater, two-paired palatal shelves were cultured (n = 8). All specimens were assessed grossly and histologically for palatal fusion. Palatal fusion occurred in our model only when intershelf distances were 0.38 mm or less. At 0.38 mm, eight of 10 palates appeared grossly adherent, whereas six of 10 demonstrated clear fusion histologically with resolution of the medial epithelial seam and continuity of the palatal mesenchyme. None of the 18 palates fused when placed at intershelf distances of 0.48 mm or greater. Using our selected intershelf distances as a guideline, we have established an approximate minimal critical intershelf distance (0.48 mm) at which we can reliably expect no palatal fusion. Culturing palatal shelves at intershelf distances of 0.48 mm or greater results in nonfusion or clefting in vitro. This model will allow us to study biomolecular characteristics of unfused or cleft palatal shelves in comparison with fused shelves. Furthermore, we plan to study the efficacy of grafting with exogenous embryonic mesenchyme or candidate factors to overcome clefting in vitro as a first step toward future in utero treatment strategies.

Immunolocalization of fibroblast growth factor receptors 1 and 2 in mouse palate development.

Recent evidence has implicated mutations of fibroblast growth factor receptors (FGF-R) in the pathogenesis of craniosynostotic syndromes. Cleft palate can be a component of such syndromes. The expression of FGF-R1 and FGF-R2 has been delineated in normally developing cranium, where they seem to regulate cellular differentiation and proliferation, respectively. The specific role of fibroblast growth factor signaling in mammalian palate development is unclear. The authors investigated the patterns of expression of FGF-R1 and FGF-R2 throughout mouse palatal development in the embryo. Time-dated CD-1 mouse heads (n = 135) were harvested at embryonic ages 12.5, 13.5, 14.5, 15.5, and 16.5 days (term gestation = 19.5 days), fixed in paraformaldehyde, embedded in paraffin, and sectioned. In addition, paired palatal shelves (n = 30) were isolated by means of microdissection from embryonic day--13.5 embryos, grown on Millipore filters in serum-free medium in vitro for 24, 48, 72, or 96 hours and processed for histological analysis. Immunohistochemical analysis for FGF-R1 and FGF-R2 was performed on the in vivo and in vitro specimens. FGF-R1 and FGF-R2 were found to be specifically expressed in the epithelium of the developing palatal shelves from the time of their outgrowth from the maxillary processes through completion of fusion in vivo and in vitro. Expression of both receptors was particularly strong during the phases of medial epithelial-medial epithelial contact between the individual shelves, through the formation of the medial epithelial seam, to the ultimate dissolution of the seam. Such a pattern of expression seems to implicate fibroblast growth factor signaling in the regulation of the critical phase of fusion of the bilateral shelves. The expression of both FGF-R1 and FGF-R2 in the lateral palatal mesenchyme, where such secondary structures as tooth primordia and bone begin to appear, also suggests a role for fibroblast growth factor signaling in the induction of ongoing differentiation and maturation of the palate after fusion. These data suggest that fibroblast growth factor signaling may play a role in the epithelial-mesenchymal interactions that dictate fusion and maturation of the developing palate. Furthermore, the data are consistent with the correlation of cleft palate formation with aberrant fibroblast growth factor signaling.

Defective fibroblast growth factor signaling allows for nonbranching growth of the respiratory-derived fistula tract in esophageal atresia with tracheoesophageal fistula.

BACKGROUND/PURPOSE: The fistula tract in esophageal atresia with tracheoesophageal fistula (EA-TEF) appears to arise from a trifurcation of the embryonic lung bud. Subsequently, it does not branch like the other bronchi, which also arise from the lung bud. Previous results have implied that aberrant mesenchymal-epithelial signaling in the developing foregut, possibly involving fibroblast growth factors, may allow for the nonbranching growth of the fistula, and the ultimate development of the fistula tract in TEF. METHODS: Adriamycin injections into pregnant rat dams induced EA-TEF formation in rat embryos. Control and Adriamycin-exposed embryos were harvested on the 13th gestational day, and the developing foregut was isolated with microdissection. mRNA was isolated from the developing fistula tract, embryonic lung, and normal embryonic esophagus. Reverse transcription-polymerase chain reaction (RT-PCR) for the IIIb splice variant of the FGF2R receptor was performed. Foregut specimens also were processed for histologic analysis, and immunofluorescence for FGF1 was performed. RESULTS: FGF2R-IIIb is specifically absent from the developing fistula tract in TEF, whereas it is present in the normal developing lung and esophagus. FGF1 also is uniquely absent from the developing fistula tract, but it is present in the normal lung mesenchyme. CONCLUSIONS: FGF1, FGF7, and FGF10 are critical mesenchymal factors that mediate proliferation and branching morphogenesis by the developing respiratory epithelium. The absence of FGF2R-IIIb, the obligate common receptor for FGF7 and FGF10, from the fistula tract, and the absence of FGF1 in the fistula tract mesenchyme, collectively imply the absence of a specific FGF signaling pathway in the developing fistula tract. This absence of FGF signaling could explain the lack of branching by the developing fistula tract as it grows caudally in the abnormally developing embryo. Downregulation of these components of the FGF signaling pathways may allow for a patterned compensation by the embryo for the proximal foregut atresia in this anomaly. This compensation may then reestablish gastrointestinal continuity as the fistula tract connects to the developing stomach.

Transforming growth factor-beta 1 in the developing mouse pancreas: a potential regulator of exocrine differentiation.

Transforming growth factor-beta 1 (TGF-beta 1) is known to regulate cell growth, differentiation, and function in developing mammalian systems. Altering TGF-beta 1 expression in the developing pancreas has been shown to affect both exocrine and endocrine development, suggesting that it is an important regulator of pancreatic organogenesis. We proposed to examine the ontogeny of TGF-beta 1 mRNA expression in the developing pancreas, as well as characterize the patterns of relative TGF-beta 1 gene expression and activity. We performed in situ hybridization for TGF-beta 1 on pancreas specimens obtained from CD-1 mice on gestational days 12.5 (E12.5), 15.5 (E15.5), and 18.5 (18.5). We also isolated mRNA from the pancreas on each of these days and performed a semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) to assess relative TGF-beta 1 expression as a function of gestational age. Finally, we performed a TGF-beta 1 ELISA with media conditioned by embryonic pancreas from gestational days 15.5 and 18.5. By in situ hybridization, TGF-beta 1 mRNA is expressed exclusively in the E12.5 pancreatic epithelium, sparing the surrounding mesenchyme. As pancreatic organogenesis progresses, TGF-beta 1 mRNA expression localizes predominantly to the developing acini. TGF-beta 1 gene expression appears modest through E15.5 but is upregulated near the end of gestation, at E18.5. TGF-beta 1 activity, by ELISA, is also upregulated at E18.5. TGF-beta 1 may thus be a modulator of pancreatic organogenesis. Modest TGF-beta 1 expression through E15.5 may be permissive for exocrine lineage selection. TGF-beta 1 expression may then become critical for terminal acinar differentiation. Upregulated TGF-beta 1 expression at the end of gestation may be important for islet formation, and it may be necessary to inhibit continued proliferation and differentiation of pluripotent cells within the pancreatic ductal epithelium.

Defective epithelial-mesenchymal interactions dictate the organogenesis of tracheoesophageal fistula.

We have previously suggested that the fistula tract in esophageal atresia with tracheoesophageal fistula (EA/TEF) arises from a trifurcation of the embryonic lung bud. Thus, it appears to be a respiratory-derived structure, and expresses the lung-specific transcription factor TTF-1 in its epithelium. The fistula tract does not give rise to lungs like the other branches from the bud. It grows caudally until it fistulizes with the stomach. We hypothesized that epithelial-mesenchymal interactions (EMI) dictate the differential pattern of growth of the respiratory-derived fistula tract in EA/TEF. EA/TEF was induced in rat embryos via prenatal exposure to adriamycin. Microdissection was performed on E13.5 embryos to isolate developing lung bud, fistula tract, or esophagus from adriamycin-treated or control animals, respectively. The mesenchyme and epithelium from each of these foregut structures were separated. The individual epithelia were recombined with each of the various mesenchymes and grown in culture. They were assayed for relative degrees of branching. Isolated lung-bud epithelia (LBE) or fistula epithelium were also cultured in Matrigel with exogenous fibroblast growth factors (FGF) and subsequently assayed for branching. The fistula-tract mesenchyme relatively inhibited branching of lung epithelium. The epithelium of the fistula tract could be induced to branch by non-fistula (lung or esophageal) mesenchyme. The fistula-tract and adriamycin-treated LBE both branched in response to FGF1. In contrast, neither responded to FGF7 or FGF10. EMI are defective in the developing EA/TEF. The inability to respond to FGF7 and FGF10 suggests an epithelial defect involving the receptor FGF2R-IIIb, to which these mesenchymal factors obligately bind. Thus, the mesenchyme around the developing fistula tract may lack an FGF branching morphogen(s), such as FGF1. Hence, this mesenchyme is unable to induce branching of respiratory epithelia and allows the middle branch of the embryonic tracheal trifurcation to grow caudally as an unbranched tube until it fistulizes into the stomach.

Basement membrane exposure defines a critical window of competence for pancreatic duct differentiation from undifferentiated pancreatic precursor cells.

We previously showed that the undifferentiated pancreatic epithelium can differentiate into islets, ducts, or acini depending on its milieu and that laminin is necessary for pancreatic duct formation. Therefore we wanted to study the plasticity of laminin-induced duct differentiation the better to understand mechanisms of pancreatic duct lineage selection induced by basement membrane. Mouse embryonic pancreases were dissected at gestational day 11 (E11.5), and epithelium was isolated from its surrounding mesenchyme. Some epithelia were cultured in a collagen gel devoid of laminin. These epithelia were "rescued" at days 1-7 of culture by transferring them to a laminin-rich matrix (Matrigel) for 7 additional days. Other epithelia were instead first cultured in Matrigel, and then placed into collagen. Immunohistochemistry was performed for insulin, amylase, and carbonic anhydrase II. Pancreatic epithelia rescued from collagen into laminin during days 1-4 after harvest were still able to form ducts, whereas epithelia deprived of laminin for longer than this 4-day window were not. Pancreatic epithelia exposed to laminin for as little as 1 day, and then placed into collagen, still retained the ability to make ducts. Thus there is a clear cut-off in the development of the pancreatic epithelium at E11.5, after which laminin appears necessary to induce duct formation. We believe that such "windows of competence" in embryonic development imply that developmental programs in the embryo allow some flexibility.

Patterning of the "distal esophagus" in esophageal atresia with tracheo-esophageal fistula: is thyroid transcription factor 1 a player?

BACKGROUND: We have recently proposed that the "distal esophagus" in esophageal atresia with tracheo-esophageal fistula (EA/TEF) is actually embryologically derived from the middle branch of a trifurcation of the embryonic lung bud, which subsequently grows caudally in the foregut to connect with the developing stomach. We hypothesized that differential mRNA expression of the lung-specific patterning transcription factor, thyroid transcription factor 1 (TTF-1), in the developing fistula tract in TEF relative to the bronchi (the other branches of the lung bud trifurcation) might explain the unique nonbranching pattern of growth of the fistula tract. MATERIALS AND METHODS: EA/TEF was induced in Sprague-Dawley rat embryos via intraperitoneal injection of 2.2 mg/kg adriamycin into pregnant dams on Days 6-9 of gestation. The foregut from embryos developing EA/TEF and from control embryos (no adriamycin) were isolated on Gestational Days 13.5, 15.5, and 17.5 (term = 21 days). Some were processed for whole-mount in situ hybridization for TTF-1, while others were embedded and sectioned for histologic analysis via in situ hybridization for TTF-1. RESULTS: The expression of the respiratory-specific transcription factor TTF-1 is conserved in the epithelium of the developing fistula tract in TEF. The pattern of expression of TTF-1 in the fistula tract mirrors the expression in the large airways of the developing lungs, despite the fact that the fistula tract does not form secondary branches to give rise to a lung. CONCLUSIONS: The fistula tract in TEF is a respiratory-derived structure that expresses the lung-specific transcription factor TTF-1 throughout its development in the foregut. Contrary to the patterning role that it normally plays in the developing lung, TTF-1 does not induce branching morphogenesis in the fistula tract. Thus, the nonbranching pattern of growth of the fistula tract may be attributable to local mesenchymal-epithelial interactions that override TTF-1 patterning activity.

Expression and role of laminin-1 in mouse pancreatic organogenesis.

Previous studies have suggested that basement membrane alone may induce ductal differentiation and morphogenesis in the undifferentiated embryonic pancreas. The mechanism by which this induction occurs has not been investigated. Studies of other organ systems such as the lungs and mammary glands, where differentiation has been shown to be induced by basement membrane, have suggested a major role for laminin as a mediator of ductal or tubular morphogenesis and differentiation. We first defined the ontogeny of laminin-1 in the developing mouse pancreas. To determine the specific role of basement membrane laminin in pancreatic ductal morphogenesis and differentiation, we microdissected 11-day mouse embryonic pancreatic epithelium free from its surrounding mesenchyme and then suspended the explants in a 3-dimensional organ culture to allow us to assay cell differentiation and morphogenesis. When the pancreatic epithelium buds off the foregut endoderm, the pancreatic mesenchyme diffusely expresses laminin-1. This laminin subsequently organizes to the interface between the epithelium and the mesenchyme by E12.5. As gestation progresses, epithelial cells in direct contact with laminin-1 seem to differentiate into ducts and acini, whereas those spared intimate contact with laminin-1 appeared to organize into islets. Although basement membrane gel could induce pancreatic ductal morphogenesis of embryonic pancreatic epithelium, this induction was blocked when we added neutralizing antibodies against any of the following: 1) laminin (specifically laminin-1), 2) the "cross-region" of laminin-1, and 3) the alpha6 moiety of the integrin receptor, which is known to bind laminins. Immunohistochemistry, however, showed that pancreatic duct cell-specific differentiation (carbonic anhydrase II) without ductal morphogenesis was still present, despite the blockage of duct morphogenesis by the anti-laminin-1 neutralizing antibodies. Interestingly, there appeared to be a decrease in carbonic anhydrase II expression over time when the epithelia were grown in a collagen gel, rather than in a basement membrane gel. The pattern of laminin-1 expression in the embryonic pancreas supports the conclusion that laminin-1 is important in the induction of exocrine (ducts and acini) differentiation in the pancreas. Furthermore, our data demonstrate that 1) pancreatic ductal morphogenesis appears to require basement membrane laminin-1 and an alpha6-containing integrin receptor; 2) the cross-region of basement membrane laminin is a biologically active locus of the laminin molecule necessary for pancreatic ductal morphogenesis; 3) duct-specific cytodifferentiation, in the form of carbonic anhydrase II expression, is not necessarily coupled to duct morphogenesis; and 4) the basement membrane gel may contain components (e.g., growth factors) other than laminin-1 that can sustain both carbonic anhydrase II expression and, possibly, the capacity to form ducts, despite the absence of duct structures.

In vitro validation of duct differentiation in developing embryonic mouse pancreas.

BACKGROUND: Early embryonic pancreatic epithelia have the capacity for either endocrine or exocrine lineage commitment. Recent studies demonstrated the pluripotential nature of these undifferentiated cells. Isolated pancreatic epithelia grown under the renal capsule formed primarily islets. However, when these same epithelia were grown in a basement-membrane-rich gel (Matrigel) they formed mostly ducts. Currently, there is no model for in vitro pancreatic duct formation and therefore, the mechanism of duct morphogenesis has never been described. The purpose of this study was to provide such a model by characterizing the expression of two duct markers, carbonic anhydrase II (CAII) and the cystic fibrosis transmembrane conductance regulator (CFTR), in isolated undifferentiated pancreatic epithelia grown in vitro. MATERIALS AND METHODS: We microdissected embryonic pancreases at Embryonic Days (E)9.5-11.5 and performed RT-PCR for CAII and CFTR on E9.5 whole pancreases, E10. 5 and E11.5 epithelia, as well as E11.5 epithelia grown for 7 days in Matrigel. Next we performed in situ hybridization for CAII and CFTR and immunohistochemistry for CAII on E11.5 epithelia grown for 7 days in Matrigel. RESULTS: Early, undifferentiated embryonic pancreatic epithelium does not express CAII and CFTR by RT-PCR. When E11.5 epithelia were grown for 7 days in Matrigel, however, gene expression for both markers is upregulated as ducts form. Furthermore, CAII was seen by IHC and both CAII and CFTR were seen by in situ hybridization in the ducts after 7 days in Matrigel. CONCLUSIONS: These data validate our in vitro system as a model for studying the mechanism of normal pancreatic duct differentiation and may potentially help us to understand the faulty mechanism involved in pancreatic ductal carcinogenesis.

Ontogeny of activin B and follistatin in developing embryonic mouse pancreas: implications for lineage selection.

Activin, a member of the transforming growth factor-beta superfamily, has been shown to be a critical regulator in exocrine and endocrine pancreas formation. The purpose of our study was to describe the ontogeny of activin B and its inhibitor, follistatin, in developing pancreas and to elucidate potential mechanisms for exocrine and endocrine lineage selection. Mouse embryonic pancreata were dissected at various ages (day 10 [E10.5] to birth [E18.5]), sectioned, and immunostained for activin B (one of two existing isomers, A and B), follistatin, insulin, and glucagon. In addition, reverse transcriptase-polymerase chain reaction was employed to determine the messenger RNA expression of follistatin in isolated pancreatic epithelia and mesenchyme of various ages. Activin B was first detected at E12.5 in epithelial cells coexpressing glucagon. At E16.5 these coexpressors appeared as clusters in close proximity to early ducts. By E18.5 activin B was localized to forming islets where cells coexpressed glucagon and were arranged in the mantle formation characteristic of mature alpha cells. Follistatin was found to be ubiquitous in pancreatic mesenchyme at early ages by immunohistochemical analysis, disappearing sometime after E12.5. Follistatin reappeared in E18.5 islets and remains expressed in adult islets. Follistatin messenger RNA was first detected in epithelium at E11.5, preceding its protein expression in islets later in gestation. We propose that mesenchyme-derived follistatin inhibits epithelium-derived activin at early embryonic ages allowing for unopposed exocrine differentiation and relative suppression of endocrine differentiation. At later ages the decrease in the amount of mesenchyme relative to epithelium and the subsequent drop in follistatin levels liberates epithelial activin to allow differentiation of endocrine cells to form mature islets by the time of birth.

TTF-1 and HNF-3beta in the developing tracheoesophageal fistula: further evidence for the respiratory origin of the distal esophagus'.

PURPOSE: Using an established rat model of esophageal atresia with tracheoesophageal fistula (EA-TEF), the authors have studied the organogenesis of this congenital anomaly. The authors previously have proposed that the "distal esophagus" actually is of respiratory lineage. In this report this hypothesis is tested by examining the expression of two foregut patterning transcription factors, thyroid transcription factor-1 (TTF-1) and hepatocyte nuclear factor-3beta (HNF-3beta), within the developing TEF. METHODS: Pregnant Sprague-Dawley rats were injected with 2.2 mg/kg of Adriamycin intraperitoneally on days 6 to 9 of gestation. Using microdissection, the trachea, blind-ending esophagus, TEF, and stomach were isolated from embryos of various gestional ages. Immunohistochemistry was performed using polyclonal antibodies to TTF-1 and HNF-3beta. RESULTS: TTF-1 is a homeodomain protein that previously has been shown to be expressed in the lung and trachea but not in the gastrointestinal tract, and which, when deleted in a developing lung, results in a mouse with no peripheral lung parenchyma. TTF-1 was expressed strongly in the lung, fistula, and distal esophagus, but not in the proximal esophagus. HNF-3beta is a forkhead transcription factor important in foregut patterning that binds and activates the TTF-1 promotor sequence. HNF-3beta was expressed globally in the fistula and lung as well as the esophagus. CONCLUSIONS: The expression of the lung-specific transcription factor TTF-1 within the TEF strongly implies that the "distal esophagus" is a respiratory-derived structure and thus supports our theory of TEF organogenesis. The conservation of HNF-3beta expression both in the TEF as well as the normal developing trachea and esophagus suggests that global foregut patterning is intact in the formation of this anomaly, and the defect lies at the level of the respiratory versus gastrointestinal commitment.

Molecular approaches to understanding organogenesis.

The elucidation of the molecular mechanisms of mammalian organogenesis is the foundation on which we can build an improved understanding of organ pathology and pathophysiology. This paper uses the lung and the pancreas as paradigms to demonstrate how advances in basic molecular developmental biology research has translated into new appreciation of, and even novel potential treatment strategies for, congenital anomalies and mature diseases of these organs.

Fetal midgut volvulus presenting at term.

This report describes a rare case of intrauterine midgut volvulus that presented at term. The pregnancy was uncomplicated. There were no signs of fetal distress or polyhydramnios, and the child was delivered vaginally. This patient had the unusual presentation in that at the time of delivery, the patient was distended and acidotic. She immediately required an extensive resection of gangrenous small bowel. Comparing this case to the 10 other cases of fetal intestinal volvulus that have been reported, it seems this child was particularly fortunate that the volvulus occurred at a point in gestation when she was mature enough to tolerate birth and surgery. This case is also the first demonstration that volvulus can present with abdominal distension in the immediate newborn period.

Repair of pediatric pectus deformity and congenital heart disease as a combined procedure.

Coexisting pectus deformity and congenital heart disease is not uncommon. Traditionally, the approach to this problem has been to repair each one with a separate surgical procedure because of fear of increased complications from bleeding, infections, and anesthesia. More recently, many reports of successful combined repair have been published, particularly in adults with coronary artery or aortic pathology. The authors wished to determine the feasibility of this combined procedure in younger patients, particularly those with a severe pectus deformity. Three patients underwent repair, including a 17 year old with Marfan's syndrome and a severe pectus excavatum deformity. The postoperative course was smooth for these patients, and all had good short- and long-term (over 18 months) results.

The ontogeny of TGF-beta1, -beta2, -beta3, and TGF-beta receptor-II expression in the pancreas: implications for regulation of growth and differentiation.

BACKGROUND/PURPOSE: The transforming growth factor-beta (TGF-beta) cytokines are important regulators of growth and differentiation in multiple mammalian organ systems. Recent studies suggest that they may play a significant role in the regulation of pancreatic organogenesis. The authors proposed to examine the ontogeny of expression of the TGF-beta cytokine isoforms (TGF-beta1, beta2, and beta3), as well as that of the type II TGF-beta receptor (TbetaRII), in the pancreas. We hypothesized that their patterns of expression might help to clarify the manner in which they influence the development of this organ. METHODS: Embryos from pregnant CD-1 mice were harvested on gestational days 12.5, 15.5, and 18.5. Microdissection was performed on the embryos to isolate their pancreases. The pancreases were fixed, frozen embedded, and sectioned with a cryostat. Immunohistochemistrywas performed using polyclonal antibodies to TGF-beta1, beta2, and beta3, and TbetaRII. RESULTS: The patterns of expression of TGF-beta1, beta2, and beta3 were similar throughout gestation. They were all present, though weakly, early in the development of the pancreas, in the E12.5 epithelial cells. Their expression persisted and became localized to the acinar cells later in gestation. TbetaRII staining was present in both the E12.5 epithelial cells and the surrounding mesenchyme. As the pancreas developed, TbetaRII became strongly expressed in the ductal epithelial cells with only minimal staining in the acinar and endocrine cells. CONCLUSIONS: TGF-betas may play a role in regulating pancreatic organogenesis. Our data suggest that they may be required for the normal development of acini. As in other cell systems, TGF-beta1 may act as a suppressor of pancreatic cellular growth and differentiation. The localization of TbetaRII to the mature ductal epithelium may indicate a need for ongoing regulation of growth and differentiation in the pancreatic ducts beyond the fetal period.

Epithelio-mesenchymal interactions in the developing mouse pancreas: morphogenesis of the adult architecture.

BACKGROUND/PURPOSE: The mammalian pancreas is thought to develop through a complex interaction between the budding epithelium and the surrounding mesenchyme. The exact nature of this interaction is unclear. Most of what is known to date of these interactions comes from a series of organ culture experiments done in the late 1960s. Nevertheless, these important experiments may have been confounded by less-defined culture media and organ dissection techniques, because the results are not reproducible in our hands. The authors undertook a study to reexplore these basic epithelio-mesenchymal interactions. METHODS: Using previously described organ dissection and culture techniques the authors examined the basic interactions between the embryonic pancreatic epithelium and its mesenchyme with histological and immunohistological techniques. RESULTS: The authors found that, contrary to previous reports, the earliest pancreatic anlage did not possess the intrinsic signaling necessary to support normal growth and differentiation in vitro. Intimate contact between the epithelium and the mesenchyme may be necessary until E11.5 for normal growth and differentiation. The age of the mesenchyme seemed to correlate with the degree of acinar differentiation, and proximity of mesenchyme was important for acinar differentiation. CONCLUSIONS: Previous investigations into the basic epithelio-mesenchymal interactions in the developing mammalian pancreas may have had confounding factors. Extrinsic signals seem necessary for complete pancreatic differentiation, and mesenchymal factors appear important for acinar differentiation.

Vascular development in the mouse embryonic pancreas and lung.

PURPOSE: The purpose of this study was to analyze the formation of blood vessels in the developing mouse pancreas and lung by studying two ligands, angiopoietin-1 (ang1) and angiopoietin-2 (ang2), which are thought to play a role as angiogenesis-activating factors in development. Understanding the role of vasculogenic peptides in normal embryonic development also may have important implications for common clinical problems regarding neonatal pulmonary vasculature. METHODS: Reverse transcriptase-polymerase chain reaction (RT-PCR) as well as Southern blotting was used to determine the ontogeny of angiopoietin-1 and angiopoietin-2 gene expression in the embryonic mouse pancreas and lung. Immunohistochemistry was performed for von Willebrand factor, a known marker of endothelial cells, to chronicle the development of the vasculature in these organs. RESULTS: The authors determined the temporal expression of angiopoietin-1 and angiopoietin-2 as a function of gestational age. RT-PCR data demonstrated expression of ang1 and ang2 in the developing mouse lung between gestational day E9.5 and postnatal day 1, and in the developing pancreas between gestational days E12.5 and E18.5. Southern blot analysis confirmed PCR data for ang2 expression in both the lung and pancreas. The authors also traced the spatial development of the vascular system by von Willebrand factor staining. For both lung and pancreas specimens, no blood vessels were identifiable by immunohistochemistry until embryonic day 12.5. With increased gestational age, the blood vessel networks grew larger. CONCLUSION: The authors have demonstrated that ang1 and ang2 may be involved in the mechanisms of vascular development in the embryonic mouse lung and pancreas.

Esophageal atresia with tracheoesophageal fistula: suggested mechanism in faulty organogenesis.

BACKGROUND/PURPOSE: The organogenesis of esophageal atresia with tracheoesophageal fistula (EA-TEF) is unknown. Using an established model for EA-TEF in rats, the authors proposed to study this aberrancy of development in the hope of gaining insight into its mechanism of formation. METHODS: Pregnant Sprague-Dawley rats were injected with 2.2 mg/kg of Adriamycin intraperitoneally on days 6 through 9 of gestation. Using microdissection, the trachea, blind-ending esophagus, TEF, and stomach were isolated from embryos of various gestional ages. The specimens were analyzed histologically with routine H&E staining. RESULTS: The classic EA-TEF developed in the embryos, with proximal EA and distal TEF. As expected, the atresia formed as a blind-ending pouch, but the distal fistula began as an apparent equal trifurcation of the tracheal anlage into two mainstem bronchi and the fistula tract leading to the stomach. Histological analysis of the fistula tract showed respiratorylike pseudostratified columnar epithelium. CONCLUSIONS: TEF develops as the middle branch of a tracheal trifurcation. EA-TEF occurs by a primary atresia of the esophagus. As a secondary phenomenon, the distal foregut anlage is switched toward the pulmonary phenotype. It trifurcates, and its middle branch grows caudally to fistulize into the stomach.