Notable sequential alterations in notochord volume during development in the Adriamycin rat model

Background/Purpose

The Adriamycin rat model (ARM) is a well-established model of the vertebral, anorectal, cardiac, tracheoesophageal, renal, and limb association. An important finding in the ARM is that Adriamycin induces abnormal notochord morphology in the region of the foregut. Having recently demonstrated notochord hypertrophy in ARM embryos, the authors designed this study to assess notochord volume sequentially from gestational days 10 to 14 (E10-E14) to test the hypothesis that notochord hypertrophy occurs maximally soon after Adriamycin administration.

Methods

Adriamycin (1.75 mg/kg) was administered intraperitoneally to pregnant rats on E7, E8, and E9. Control animals were given saline. Embryos were recovered at E10, E11, E12, E13, and E14 and embedded in paraffin. Quantitative morphology using the Cavalieri technique was performed on hematoxylin and eosin-stained transverse serial sections to determine total embryo and total notochord volume.

Results

The percentage volume of notochord per embryo was significantly increased (P < .05) in Adriamycin-treated embryos at all gestational time frames from E10 to E14 when compared with equivalent controls. This increased volume of notochord was found to be maximal at E11.

Conclusions

These data support the authors' previous finding that Adriamycin induces notochord hypertrophy and suggest that notochord volume is increased relative to embryo volume soon after Adriamycin administration and is maximal on E11. The abnormal increase in notochord volume during the critical phase of development may interfere with organogenesis, resulting in the vertebral, anorectal, cardiac, tracheoesophageal, renal, and limb association.     

Adriamycin disruption of the Shh-Gli pathway is associated with abnormalities of foregut development

Background

The hedgehog signalling pathway appears to have a crucial role in the embryogenesis of the foregut in vertebrates. Sonic hedgehog (Shh) protein and gene are involved in the differentiation of many organ systems such as notochord, floor plate, and limbs; development of the left-right axis in vertebrates; and differentiation of trachea and esophagus from the primitive foregut.

Methods

The prenatal exposure of Sprague-Dawley fetal rats to Adriamycin between days 6 and 9 of gestation was used to induce esophageal atresia and tracheoesophageal fistula. Embryos were harvested by cesarean section on gestational days 11 to 15. Embryos were examined by microscopy, dissection, and serial histology. In situ hybridization was used to study Shh gene expression in the rat embryos.

Results

In situ hybridization showed that the pattern and level of Shh gene expression are affected by Adriamycin. Adriamycin-treated rats have deformed notochord and undivided foregut, and some of the embryos had a lack of the dorso-ventral patterning of Shh expression seen in control embryos.

Conclusions

These results are consistent with the hypothesis that Adriamycin influences the Shh signalling pathway, resulting in disruption of normal development of the foregut.     

Notochord anomalies in the adriamycin rat model: A morphologic and molecular basis for the VACTERL association

Background/Purpose: The Adriamycin rat model (ARM) is a reliable model of the VACTERL association. The notochord is structurally abnormal in the region of the foregut, midgut, and hindgut in the ARM. The authors hypothesised that notochord anomalies allow ectopic expression of molecular signals in the developing embryo and thus lead to VACTERL malformations. This study was designed to investigate this hypothesis. Methods: Adriamycin (1.75 mg/kg) was administered intraperitoneally to pregnant rats on days 7, 8, and 9 of gestation. Control animals were given saline. Embryos were recovered on gestational days 10.5 to 14 at [frac12]-day intervals and at full term. The first group of embryos were embedded in resin, and sagittal sections stained with Toluidine blue were studied for morphologic abnormalities. The second group of embryos were examined using in situ hybridization for the expression of Sonic Hedgehog (Shh), a patterning gene implicated in the etiology of the VACTERL association. Results: Twenty-seven of the 28 (96.4%) full-term embryos showed VACTERL anomalies. Forty-five of the 50 (90%) experimental embryos (gestational days 10.5 to 14) showed notochord abnormalities. Abnormal ventral branches from the notochord toward the gut were a commonly observed abnormality. These were seen to impinge on the developing foregut, midgut, dorsal aorta, and kidney. In situ hybridization for Shh showed that these branches from the notochord expressed Shh in 66.6% of experimental embryos. This abnormal Shh expression was not seen in the control embryos. Conclusions: Adriamycin diffusely induces altered notochord morphology in the rat embryo. The abnormal notochord morphology may allow ectopic expression of Sonic Hedgehog, and, thus, contribute to the malformations found in the VACTERL association. J Pediatr Surg 38:469-473.     

Formation of intestinal atresias in the Fgfr2IIIb-/- mice is not associated with defects in notochord development or alterations in Shh expression

PurposeThe etiology of intestinal atresia remains elusive but has been ascribed to a number of possible events including in utero vascular accidents, failure of recanalization of the intestinal lumen, and mechanical compression. Another such event that has been postulated to be a cause in atresia formation is disruption in notochord development. This hypothesis arose from clinical observations of notochord abnormalities in patients with intestinal atresias as well as abnormal notochord development observed in a pharmacologic animal model of intestinal atresia. Atresias in this model result from in utero exposure to Adriamycin, wherein notochord defects were noted in up to 80% of embryos that manifested intestinal atresias. Embryos with notochord abnormalities were observed to have ectopic expression of Sonic Hedgehog (Shh), which in turn was postulated to be causative in atresia formation. We were interested in determining whether disruptions in notochord development or Shh expression occurred in an established genetic model of intestinal atresia and used the fibroblast growth factor receptor 2IIIb homozygous mutant (Fgfr2IIIb?/?) mouse model. These embryos develop colonic atresias (100% penetrance) and duodenal atresias (42% penetrance).MethodsWild-type and Fgfr2IIIb?/? mouse embryos were harvested at embryonic day (E) 10.5, E11.5, E12.5, and E13.5. Whole-mount in situ hybridization was performed on E10.5 embryos for Shh. Embryos at each time point were harvested and sectioned for hematoxylin-eosin staining. Sections were photographed specifically for the notochord and resulting images reconstructed in 3-D using Amira software. Colons were isolated from wild-type and Fgfr2IIIb?/? embryos at E10.5, then cultured for 48 hours in Matrigel with FGF10 in the presence or absence of exogenous Shh protein. Explants were harvested, fixed in formalin, and photographed.ResultsFgfr2IIIb?/? mouse embryos exhibit no disruptions in Shh expression at E10.5, when the first events in atresia formation are known to occur. Three-dimensional reconstructions failed to demonstrate any anatomical disruptions in the notochord by discontinuity or excessive branching. Culture of wild-type intestines in the presence of Shh failed to induce atresia formation in either the duodenum or colon. Cultured Fgfr2IIIb?/? intestines developed atresias of the colon in either the presence or absence of Shh protein.ConclusionsAlthough disruptions in notochord development can be associated with intestinal atresia formation, in the Fgfr2IIIb?/? genetic animal model neither disruptions in notochord development nor the presence of exogenous Shh protein are causative in the formation of these defects.     

Midgut atresias result from abnormal development of the notochord in an Adriamycin rat model

Background/Purpose: Prenatal exposure to Adriamycin in a rat model (ARM) has been reported to lead to a spectrum of tracheoesophageal and associated malformations of the gastrointestinal tract, including multiple intestinal atresias. An abnormal relationship of the notochord with the foregut has been implicated in the formation of esophageal atresias. The authors hypothesised that midgut atresias arise from abnormal notochord development in the region of the midgut. This study was designed to examine the gut-notochord relationship during early embryonic development. Methods: Timed pregnant Wistar rats were given 1.75 mg/kg of Adriamycin intraperitoneally on days 7, 8, and 9 of gestation. Embryos were recovered at 12-hour intervals from days 9.5 to 14, and at term. A control group was given saline instead of Adriamycin. Embryos were embedded in resin or wax, sectioned, and studied using light microscopy, paying particular attention to the notochord and surrounding structures. Results: The notochord appeared identical in controls and experimental embryos on day 9.5. However, on day 10.5 the notochord was diffusely abnormal in ARM, distorted, and tethered to foregut as well as midgut compared with controls. This abnormality was not seen in control embryos. On day 12 the notochord abnormalities were more exaggerated in the region of the midgut in ARM embryos. Full-term ARM animals had esophageal and multiple intestinal atresias. Conclusions: The notochord is abnormal in the region of the developing midgut, and this may account for the occurrence of atresias found in this region.     

Adriamycin produces a reproducible teratogenic model of vertebral, anal, cardiovascular, tracheal, esophageal, renal, and limb anomalies in the mouse

Background/Purpose

The Adriamycin rat model is an established model for vertebral, anal, cardiac, tracheal, esophageal, renal, and limb (VACTERL) anomalies and gastrointestinal atresias. Mice are the foremost mammal studied by developmental biologists, providing greater availability of molecular probes, antibodies, and transferable knowledge with transgenic studies. Only tracheoesophageal malformations have been previously described in the Adriamycin mouse model. The aim of this study was to carry out a dose-response analysis of the teratogenicity of Adriamycin in the mouse to determine the effect of the dose and timing of exposure in producing tracheoesophageal malformations and show if it causes other VACTERL anomalies.

Methods

CBA/Ca mice were accurately time mated (n = 30). Four different doses (0 [saline], 4, 5, and 6 mg/kg) of Adriamycin (EBEWE Pharma Ges.m.b.H. Nfg.KG, A-4866 Unterach, Austria) at 3 different timings of injections were compared. Dams received 2 intraperitoneal injections, 24 hours apart, commencing on day 7, 7.5, or 8. Fetuses were harvested on day 18. Anomalies were examined using a dissecting microscope and serial transverse sections.

Results

Administering Adriamycin at 6 mg/kg on days 7 and 8 had the most teratogenic effect, with 80% of fetuses having 3 or more VACTERL anomalies: anorectal malformation, 100%; tracheoesophageal malformation, 50%; right-sided aortic arch, 58.3%; bladder agenesis/bilateral hydronephrosis, 100%.

Conclusion

This study establishes a mouse model that should provide insights into the cellular and molecular mechanisms underlying VACTERL anomalies.     

The competency of foregut mesenchyme in islet mesenchyme-to-epithelial transition during embryonic development

Background/Purpose

Potential for curative stem-cell treatments of juvenile-onset diabetes has focussed research into pancreatic islet development. Islets were previously thought to originate solely from embryonic pancreatic epithelium, but we have demonstrated that islets can originate from mesenchyme, that is, islet mesenchyme-to-epithelial transition. The aim of this study was to establish the competence of foregut mesenchyme during mesenchymal islet development.

Methods

Embryonic chick pancreatic epithelium of gestational stage Hamburger-Hamilton (HH) 22 (J Morphol. 1951;88:49-92) was combined with quail stomach mesenchyme of increasing gestation (stage HH22 [n = 6], HH26 [n = 6], HH28 [n = 4], or HH31 [n = 6]). Recombinants were cultured and analysed by immunocytochemistry for coexpression of insulin and quail-specific antigen to determine the embryonic origin of islets.

Results

Recombinants constructed using stage HH22 mesenchyme yielded 34 islets, of which 35% were mesenchymal. However, when recombinants were constructed using stage HH26 mesenchyme, 24% of 25 islets were mesenchymal. When using mesenchyme, 13% of 15 islets were mesenchymal. All islets (n = 35) in recombinants constructed using stage HH31 mesenchyme were epithelial derived. Islet mesenchyme-to-epithelial transition diminished significantly with increasing mesenchymal gestational stage (P = .002).

Conclusions

These data show foregut mesenchyme is competent to form islets between stages HH22 and HH28. Developmental competence of foregut mesenchyme in islet mesenchyme-to-epithelial transition diminishes as gestation increases. This may have important implications for identifying stem cells to treat juvenile-onset diabetes.     

Utility and limits of Hprt-Cre technology in generating mutant mouse embryos

Background

Hprt-Cre doubles the prevalence of homozygous null embryos per litter versus heterozygous breedings without decreasing litter size. Resulting mutant embryos are genotypically and phenotypically equivalent between strategies. We set out to confirm the effectiveness of this approach with other alleles and hypothesized that it would increase efficiency in generating compound mutants.

Materials and methods

Null mutants for Cyp26b1, Pitx2, and Shh were generated with Hprt-Cre from conditional alleles as were double and triple allelic combinations of Fgfr2IIIb, Raldh2, and Cyp26b1. Embryos were genotyped and phenotyped by whole mount photography, histology, and immunohistochemistry.

Results

Fifty percent of Hprt-Cre litters were homozygous null for Cyp26b1 (15/29) and Pitx2 (75/143), with phenotypic and genotypic equivalence to mutants from standard heterozygous breedings. In multi-allele breedings, mutant embryos constituted half of litters without significant embryo loss. In contrast, Shh breedings yielded a smaller ratio of embryos carrying two recombined alleles (6 of 16), with a significant litter size reduction because of early embryonic lethality (16 live embryos from 38 deciduae).

Conclusions

Hprt-Cre can be used to efficiently generate large numbers of mutant embryos with a number of alleles. Compound mutant generation was equally efficient. However, efficiency is reduced for genes whose protein product potentially interacts with the Hprt pathway (e.g., Shh).     

Role of Sonic hedgehog in the development of the trachea and oesophagus

Backround/Purpose: The secreted glycoprotein, Sonic hedgehog (Shh) plays an important patterning role in the development of many organ systems. The authors aimed to study the temporal and spatial pattern of expression of Shh and its receptor Ptc1 during the development of the anterior foregut and to test the hypothesis that the Shh expression pattern is disturbed during the development of oesophageal atresia (OA) and tracheo-oesophageal fistula (TOF) in Adriamycin-treated mouse embryos. Methods: Saline and Adriamycin-treated (4 mg/kg) CBA/Ca embryos were harvested between embryonic days (E) 10.5 and 12.5, and Shh and Ptc1 expression was studied by whole-mount and section in situ hybridisation using digoxygenin-labelled riboprobes. Results: At E10.5, saline-treated embryos had an undivided foregut in which the ventrally placed prospective tracheal epithelium was positive for Shh, whereas the dorsal part was negative. At E11.5, this pattern had reversed with the separated trachea becoming negative and the oesophagus gaining expression of Shh. Ptc1 was expressed in the mesoderm adjacent to Shh expressing endoderm at both stages. Affected Adriamycin-treated embryos had an undivided foregut at E11.5, the epithelium of which showed diffuse Shh staining that lacked the dorso-ventral patterning seen in controls. Conclusions: The reversal in the dorso-ventral pattern of Shh expression during the narrow embryologic window in which tracheo-oesophageal separation is initiated suggests that Shh may play an important role in this process. Transient disturbance of this pattern may underlie the abnormal organogenesis in the Adriamycin model. J Pediatr Surg 38:29-36.     

Faulty bone morphogenetic protein signaling in esophageal atresia with tracheoesophageal fistula

Background

The organogenesis of esophageal atresia with tracheoesophageal fistula remains unclear. We have previously demonstrated that the fistula tract develops from a trifurcation of the embryonic lung bud and displays pulmonary lineage traits. Unlike the lung, the fistula grows without branching. Bone morphogenetic proteins (BMPs) are known to be important in lung branching. We studied possible BMP signaling defects as a potential cause for the absence of branching in the fistula tract.

Methods

Adriamycin was administered to pregnant rats on days 6-9 of gestation to induce tracheoesophageal fistula. Microdissection was performed at E13 and E17 isolating the foregut. Tissues were analyzed using immunohistochemistry for BMP ligand (BMP2, BMP4, BMP7) and receptor (BMPRIA, BMPRIB, BMPRII) expression.

Results

Immunohistochemistry revealed the presence of all 3 BMP ligands at E13, localized specifically to the esophageal mucosa but absent in the fistula and lung. At E17, the ligands were again present in the esophageal mucosa, and additionally in the fistula tract mucosa, but remained absent in the lung. At E17, all of the BMP receptors were also localized to the luminal surface of esophagus and fistula. However, in the lung epithelium, only BMPRII was found, whereas BMPRIA and BMPRIB remained absent.

Conclusions

The normal expression pattern of BMP4 was increased at the branch tips and low between branches. Among other results, we show here a constant expression level of BMP ligands throughout the entire epithelium of the fistula tract. This diffuse expression suggests defective BMP signaling in the fistula tract and explains its nonbranching phenotype.     

Retrieval of immature oocytes from unstimulated ovaries followed by in vitro maturation and vitrification: A novel strategy of fertility preservation for breast cancer patients

Background

We report a novel fertility preservation strategy that may be useful for young breast cancer patients who present with time constraints or concerns about the effect of ovarian stimulation.

Methods

The protocol involves retrieval of immature oocyte from unstimulated ovaries followed by in vitro maturation (IVM), and vitrification of oocytes or embryos.

Results

Thirty-eight patients (age 24-45 years) underwent vitrification of oocytes (n = 18) or embryos (n = 20). The mean ages were 33.1 ± 5.0 years and 34.7 ± 4.8 years, respectively. The mean days required to complete the egg collection was 13 days. The median numbers of vitrified oocytes and embryos per retrieval were 7 (range 1-22) and 4 (range 1-13), respectively.

Conclusions

The strategy of immature oocyte retrieval without ovarian stimulation followed by IVM and oocyte or embryo vitrification, which does not increase the serum estradiol level and delay cancer treatment, represents an attractive option of fertility preservation for many breast cancer patients.     

Developmental study of tethered spinal cord in murine embryos with anorectal malformations

Background/Purpose

Tethered spinal cord is frequently associated with anorectal malformations (ARMs). However, it remains unknown how the tethered spinal cord develops and relates to the severity of ARM. We studied the development of the spinal cord in ARM mouse embryos induced by all-trans retinoic acid (ATRA).

Methods

Pregnant ICR-Slc mice were administered 100 mg/kg of ATRA on the ninth embryonic day (E9.0). Embryonic specimens were obtained from the uteri between E11.0 and E18.5. Midsagittal histologic sections focusing on the spinal cord and pelvis were prepared for immuonhistochemistry specific for neurofilament and Protein Gene Product 9.5 molecules.

Results

More than 98% of ATRA-treated embryos demonstrated ARM with rectourethral or rectocloacal fistula. Normal embryos exhibited progressive ascent of the spinal cord from E14.5. However, in ARM embryos, the distal spinal cord ended with meningomyelocelelike or atypical hamartomatous lesions at E11.5 to E13.5, which later caused stretch force that damaged the spinal cord, resulting in tethered cord between E16.0 and E16.5.

Conclusions

In ATRA-induced ARM mouse embryos, tethered spinal cord was mostly established, accompanied by caudal neural maldevelopment, during early fetal development. This experimental model may be useful for researching detailed neuropathologic conditions in ARM children accompanied with tethered spinal cord.     

Pancreatic alpha-cell differentiation by mesenchyme-to-epithelial transition: implications for cell-based therapies in children

Purpose

Stem cell-derived tissue may provide a curative treatment for children with type 1 diabetes. Using an avian model, we have previously shown that foregut mesenchyme is able to differentiate into insulin-positive β-cell islets (B islets). Successful clinical islet transplantation, however, is reliant on graft tissue containing both insulin- and glucagon-secreting cells. Therefore, in this study, we assessed the ability of foregut mesenchyme to differentiate into glucagon-positive α-cell islets (A islets).

Methods

Chimeric recombinants (n = 14) were constructed using chick pancreatic epithelium combined with quail stomach mesenchyme from day 4 avian embryos and then cultured in 3 dimensions for 7 days. Cryosectioned recombinants were analyzed using immunocytochemistry against glucagon, insulin, and the quail-specific nucleolar antigen. The A islets and B islets were determined to be of solely epithelial, solely mesenchymal, or mixed origin according to the coexpression of the quail-specific nucleolar antigen.

Results

Forty-eight A islets and 34 B islets were analyzed. Eighty-five percent of the A islets were solely derived from the epithelium, but, notably, 5% were solely derived from the mesenchyme and 10% were of mixed origin. A-islet differentiation from foregut mesenchyme was reduced as compared with B islets (P = .03).

Conclusion

We demonstrate that foregut mesenchyme is able to differentiate into both α and β cells, albeit with quantitative differences. These findings may have important implications for the derivation of islet tissue from mesenchymal stem cells to cure juvenile-onset diabetes.     

Medical students' knowledge about organ transplantation: a South African perspective

Background

Educating physicians about transplantation during undergraduate training can improve organ procurement rates. The aim of this study was to evaluate and analyze the knowledge of medical students regarding transplantation.

Methods

A previously validated self-administered anonymous questionnaire was distributed to all medical students.

Results

Of the 346 participants, 217 (63%) were preclinical students. Their mean age was 21 years (range, 18-33) and 62% were women. Twenty-nine (8%) students were registered as organ donors. One third of all study participants received formal transplantation teaching; a greater proportion of clinical students received teaching compared with the preclinical group (52% vs 22%, P < .05). Knowledge was frequently reported for kidney (88%), liver (81%), bone marrow (78%), and heart (76%) transplantation. Small Intestine (13%), pancreas (9%), and pancreatic islets (4%) were the least recognized organs/tissues. Ninety-six percent and 62% of respondents were aware of kidney and liver living-donor transplants, respectively; the 27% of students with an interest in a surgical career had better knowledge of living-donor transplantation (P < .05). Only 22 (6%) students knew which solid organ transplants were performed in South Africa.

Conclusion

Medical students have limited knowledge about organ transplantation; there is a need for educational intervention early in the medical curriculum.     

Abnormal separation of the respiratory primordium in the adriamycin mouse model of tracheoesophageal malformations

Background/Purpose

Organogenesis relies on temperospatially coordinated signaling systems. The adriamycin rat model provided insights into the dysmorphogenesis of tracheoesophageal malformations. An adriamycin mouse model (AMM) would facilitate the investigation of their molecular pathogenesis. To transfer the knowledge gained from the rat, we describe a histological account of the critical period of organogenesis of these malformations in the AMM.

Method

CBA/Ca mice were accurately time-mated (n = 18). Dams received intraperitoneal injections of adriamycin (6 mg/kg) (n = 12) or saline control (n = 6) on days 7 and 8. Fetuses were harvested on days 9, 9.5, 10, 11, 12, and 13, resin embedded, and 1-μm sections of the developing foregut were examined.

Results

Day 11 control fetuses showed normal separation of the respiratory primordium, with apoptotic bodies at the point of separation. A more caudal point of separation of the distal foregut without apoptotic bodies was found in 4 of 10 AMM fetuses. Day 13 AMM fetuses had dorsal or ventral outpouchings of the foregut, indicating which malformation they would develop. Abnormal branching of the notochord was seen from day 9.5 in AMM fetuses. This was not always associated with abnormal tracheoesophageal development.

Conclusion

This study confirms that the abnormal observations made in the rat model apply to the mouse.     

Conditional mutation of fibroblast growth factor receptors 1 and 2 results in an omphalocele in mice associated with disruptions in ventral body wall muscle formation

Background/Purpose

We observed that fibroblast growth factor receptors 1 and 2 (Fgfr1, Fgfr2) are expressed during abdominal wall development in mice and hypothesized that conditional mutation of these genes would result in abdomial wall defects.

Methods

Section in situ hybridizations were performed for Fgfr1 and Fgfr2 on wild-type embryos at embryonic day (E) 11.5 and E13.5. Conditional mutation of Fgfr1and Fgfr2 was achieved with a tamoxifen inducible Cre at E8.5. Litters were harvested at E17.5, whole mount photographs were taken, and paraffin sections were generated and stained with hematoxylin and eosin.

Results

Fgfr1 was expressed in ectoderm, lateral plate mesoderm, and myoblasts, whereas Fgfr2 was expressed almost exclusively in the early dermis and ectoderm of the abdominal wall. Conditional mutation of both Fgfr2 alleles and one Fgfr1 allele resulted in omphalocele in 38.7% of mutants. Histologic examination in mutants demonstrated disruptions in dermal and muscle development.

Conclusions

Mutant embryos with omphalocele arising from mutation in Fgfr1 and Fgfr2 exhibit disruptions in the development of the secondary abdominal wall structures. These findings are consistent with a model of ventral abdominal wall development in which organization of the muscles and connective tissue (secondary abdominal wall structures) is influenced by positional information emanating from the primary abdominal wall.     

Omphalocele induction in the chick embryo by administration of cadmium

Background

Ventral body wall (VBW) defects occur in 1:2000 live births. We examined the association of VBW defect with somite abnormality and lordosis in the chick using in vitro and in ovo methods.

Methods

Explanted chick embryos were treated at 60 hours with 50 μL sodium acetate or 0.001% cadmium acetate solution to produce VBW defects. Mortality and abnormality rates were assessed. A further cohort of chicks was treated in ovo by dropping 50 μL 0.001% to 0.01% cadmium acetate onto the embryo and allowing development to 16.5 days for further assessment of the defect and skeletal staining with alcian blue and alizarin red.

Results

Cadmium treatment at 24 hours induced VBW defects in chicks treated in both shell-less culture and in ovo. Material herniating through the VBW defects was covered by a membrane in all fresh specimens. Membrane removal revealed large defects containing liver and bowel. These criteria clearly indicate that the defect observed is an omphalocele. Affected embryos had reduced somite numbers within 24 hours. Chicks exhibiting exomphalos at 16.5 days invariably had lumbosacral lordosis.

Conclusions

The cadmium-treated chick embryo is a reliable model for exomphalos. A positive association was found between exomphalos and lumbar lordosis in the chick.     

Dorsoventral patterning in oesophageal atresia with tracheo-oesophageal fistula: Evidence from a new mouse model

Background/Purpose: The well-established Adriamycin rat model of oesophageal atresia (OA) and tracheo-oesophageal fistula (TOF) complements recently described mouse genetic models in which loss of function mutations in foregut patterning genes, such as Nkx2.1 (Ttf 1), lead to OA/TOF. The authors aimed to integrate the 2 systems by adapting the Adriamycin model to the mouse to study molecular aspects of tracheo-oesophageal development. Methods: Pregnant CBA/Ca mice were injected intraperitoneally with 4 mg/kg of Adriamycin on embryonic days 7.5 and 8.5. Embryos and fetuses of various gestational ages were subjected to morphologic or histologic examination. Sections were stained with H [amp ] E or processed for immunohistochemistry using an antibody specific for Nkx2.1. Results: Tracheo-oesophageal malformations were observed in 47% of Adriamycin-treated embryos. Early foregut development was similar in Adriamycin-exposed and control embryos but, by E11.5, many treated embryos had an undivided oesophago-trachea, which gave rise to the lung buds and a fistula to the stomach. The fistula originated from the dorsal aspect of the undivided tube and was negative for Nkx2.1, or showed only transient Nkx2.1 expression, compared to the strongly positive bronchi ventrally. Conclusions: The Adriamycin model of OA is adaptable to the mouse. In the absence of tracheo-oesophageal separation, the dorsal fistula retains its nonrespiratory commitment suggesting that dorsoventral patterning of foregut development is undisturbed by Adriamycin exposure.