Testicular descent revisited: a micro-computed tomography study in fetal rats.

PURPOSE: There is a long history of research dealing with the embryology of the testicular descent. However, important aspects like the role of the gubernaculum and the development of the processus vaginalis peritonei are not understood. Micro-computed tomography (µCT) is an established tool for anatomical studies in rodents. Our study applied µCT imaging to visualize the testicular descent in rats and focused on the role of the gubernacular bulb and the development of the processus vaginalis peritonei. METHODS: Rats from embryonic day 15 (ED15) to ED21 and newborns (N0) were fixed and dried using the "critical point" technique. We ran a SkyScan® µCT system and scans were analyzed for gender-specific differentiation of the genital ridge and used for 3D visualization of relevant anatomic structures. RESULTS: µCT imaging confirmed the intraperitoneal location of the testicles from ED15 to N0. The components of the inner genital moved closer together while the intestinal volume expanded. The gubernacular bulb seemed to be involved in the formation of the processus vaginalis peritonei. CONCLUSION: Here, we utilized µCT imaging to visualize the testicular descent in the rat. Imaging provides new morphologic aspects on the development of the processus vaginalis peritonei.

Development of the foregut and the formation of the trachea and esophagus in rat embryos. A symphony of confusion.

Introduction: During embryonic development, the trachea emerges from an area of the foregut, which is often referred to as "anterior" or "common" foregut tube or simply foregut. To explain this process of differentiation, four competing models exist to date. The outgrowth and watershed models propose a foregut that remains constant in length. In the outgrowth model, the trachea buds off and elongates from the foregut, while in the watershed model, a mesenchymal wedge splits the growing foregut into the trachea and esophagus. In contrast, the septation model proposes a cranial splitting and thus a shortening of the "common" foregut tube into the trachea and esophagus by an emerging septum. Finally, the splitting and extension model describes an interaction of cranial splitting of the foregut and simultaneous caudal tracheal and esophageal growth. Methods: Here we examine the development of the undifferentiated foregut by micro computed tomography, which allows precise measurements. Results: Our results show that this area of the foregut transforms into the larynx, a process, which is independent from tracheal and esophageal development. Discussion: These observations are only consistent with the outgrowth model.

Development of the Urinary Tract in Fetal Rats: A Micro-CT Study.

INTRODUCTION: Micro-computed tomography (micro-CT) is an established tool to study fetal development in rodents. This study aimed to use micro-CT imaging to visualize the development of the urinary tract in fetal rats. MATERIALS AND METHODS: Fetal rats from embryonic day (ED) 15, ED17, ED19, ED21, and N0 (newborn) (n = 6 per group; 3 males) were fixed and desiccated using the "critical point" technique. We utilized the micro-CT system (SkyScan) and analyzed the resulting scans with CTAn, DataViewer, and ImageJ to visualize the morphology and quantify the volumes of kidney, bladder, adrenal gland, as well as length of the ureter. RESULTS: High-resolution micro-CT showed continuous growth of both kidneys from ED15 to N0, with the highest increase between ED19 and ED21. The length of the ureter increased from ED15 to ED21 and remained stable until birth. The volume of the bladder steadily increased from ED15 to N0.In females, a statistically higher volume of the adrenal gland on ED21 was observed, whereas no sex-specific differences were seen for kidney, ureter, and bladder development. CONCLUSION: Micro-CT depicts an excellent tool to study urinary tract development in the fetal and neonatal rat. It enables the metric quantification of longitudinal anatomic changes in high definition without previous destructive tissue preparation. The present study revealed sex-specific differences of the adrenal gland development and provides comprehensive data for the understanding of fetal urinary tract development, inspiring future research on congenital urological malformations.

Pediculated Accessory Liver Lobe with Gallbladder in a Preterm with Umbilical Cord Hernia.

(1) Background: Accessory liver lobes are a rare finding and only a few case reports of accessory liver lobes in abdominal wall defects have been reported so far. In the case of a congenital wall defect including liver parenchyma, there is still an ongoing debate on the definition of the abdominal wall defect and best care practice. Even though congenital abdominal wall defects are frequently diagnosed in prenatal screenings, controversy on the underlying etiology, embryology and underlying anatomy remains. Prenatal distinction between omphalocele and hernia into the cord cannot always be obtained; however, due to its clinical relevance for postnatal management and counseling of parents, accurate diagnosis is essential. (2) Case Presentation: We describe the uncommon postnatal finding of a pediculated accessory liver lobe with gallbladder in a preterm with umbilical cord hernia, which was prenatally diagnosed as omphalocele. Postnatal examination revealed an amniotic sac with a diameter of six and a small abdominal wall defect of three centimeters in diameter. Postnatal management included resection of the accessory liver lobe and gallbladder and closure of the defect. (3) Results and (4) Conclusions: Throughout the literature, the distinction between umbilical cord hernia and omphalocele has been variable. This has led to confusion and difficulties regarding postnatal treatment options. In order to achieve an accurate prenatal and/or postnatal diagnosis, the morphological differences and clinical manifestation of umbilical cord hernia and omphalocele need to be assessed. Further embryological studies are warranted to understand the underlying embryological pathology of omphalocele and umbilical cord hernia and offer appropriate treatment. In consideration of possibly severe complications in the case of the torsion of a pedunculated accessory liver lobe, we strongly recommend primary removal once pre- or intraoperative identification has been made.

Midgut development in rat embryos using microcomputed tomography.

The development of the mammalian gut was first described more than a century ago. Since then, it has been believed that a series of highly orchestrated developmental processes occur before the intestine achieves its final formation. The key steps include the formation of the umbilicus, the so-called "physiological herniation" of the midgut into the umbilical cord, an intestinal "rotation", and the "return of the gut" into the abdominal cavity. However, this sequence of events is predominantly based on histological sections of dissected embryos, a 2D technique with methodological limitations. For a better understanding of spatial relationships in the embryo, we utilized microcomputed tomography (µCT), a nondestructive 3D imaging method. Here, we show the detailed processes and mechanisms of intestinal development in rat embryos, including the development of the umbilicus, the formation of loops inside the umbilical coelom, and the subsequent shift of these loops into the abdominal cavity. Our 3D datasets of developing intestines will substantially advance the understanding of normal mammalian midgut embryology and offer new possibilities to reveal unknown mechanisms in the pathogenesis of congenital disorders.

High resolution three-dimensional imaging and measurement of lung, heart, liver, and diaphragmatic development in the fetal rat based on micro-computed tomography (micro-CT).

Understanding of normal fetal organ development is crucial for the evaluation of the pathogenesis of congenital anomalies. Various techniques have been used to generate imaging of fetal rat organogenesis, such as histological dissection with 3-dimensional reconstruction and scanning electron microscopy. However, these techniques did not imply quantitative measurements of developing organs (volumes, surface areas of organs). Furthermore, a partial or total destruction of the embryos prior to analysis was inevitable. Recently, micro-computed tomography (micro-CT) has been established as a novel tool to investigate embryonic development in non-dissected embryos of rodents. In this study, we used the micro-CT technique to generate 4D datasets of rat embryos aged between embryonic day 15-22 and newborns. Lungs, hearts, diaphragms, and livers were digitally segmented in order to measure organ volumes and analyze organ development as well as generate high-resolution 3D images. These data provide objective values compiling a 4D atlas of pulmonary, cardiac, diaphragmatic, and hepatic development in the fetal rat.

SLC20A1 Is Involved in Urinary Tract and Urorectal Development.

Previous studies in developing Xenopus and zebrafish reported that the phosphate transporter slc20a1a is expressed in pronephric kidneys. The recent identification of SLC20A1 as a monoallelic candidate gene for cloacal exstrophy further suggests its involvement in the urinary tract and urorectal development. However, little is known of the functional role of SLC20A1 in urinary tract development. Here, we investigated this using morpholino oligonucleotide knockdown of the zebrafish ortholog slc20a1a. This caused kidney cysts and malformations of the cloaca. Moreover, in morphants we demonstrated dysfunctional voiding and hindgut opening defects mimicking imperforate anus in human cloacal exstrophy. Furthermore, we performed immunohistochemistry of an unaffected 6-week-old human embryo and detected SLC20A1 in the urinary tract and the abdominal midline, structures implicated in the pathogenesis of cloacal exstrophy. Additionally, we resequenced SLC20A1 in 690 individuals with bladder exstrophy-epispadias complex (BEEC) including 84 individuals with cloacal exstrophy. We identified two additional monoallelic de novo variants. One was identified in a case-parent trio with classic bladder exstrophy, and one additional novel de novo variant was detected in an affected mother who transmitted this variant to her affected son. To study the potential cellular impact of SLC20A1 variants, we expressed them in HEK293 cells. Here, phosphate transport was not compromised, suggesting that it is not a disease mechanism. However, there was a tendency for lower levels of cleaved caspase-3, perhaps implicating apoptosis pathways in the disease. Our results suggest SLC20A1 is involved in urinary tract and urorectal development and implicate SLC20A1 as a disease-gene for BEEC.

Regenerative capacity of the enteric nervous system: is immaturity defining the point of no return?

BACKGROUND: Intestinal obstruction in newborns is associated with intestinal motility disorders after surgery. Alterations in the enteric nervous system (ENS) might cause abnormal peristalsis, which may then result in intestinal motility disorders. We aimed to quantify alterations in the myenteric plexus after a ligation and to test if these alterations were reversible. METHODS: Small intestines of chicken embryos were ligated in ovo at embryonic day (ED) 11 for either 4 d (ED 11-15) or 8 d (ED 11-19). Both treated groups and control group were sacrificed and intestinal segments examined by means of both light and electron microscopy. RESULTS: The number of proximal myenteric ganglia increased (ED 19, 30.7 ± 3.16 versus 23.1 ± 2.03; P < 0.001) in the 8-d ligature group but had values similar to the control group in the 4-d ligature group. The size distribution was skewed toward small ganglia in the 8-d ligature group (ED 19, 83.71 ± 11.60% versus 3.88 ± 4.74% in the control group; P < 0.001) but comparable with the control group in the 4-d ligature group. Subcellular alterations in the 4-d ligature group were reversible. CONCLUSIONS: The pathologic alterations in the ENS were fully reversible in the 4-d ligature group. This reversibility might be linked to the degree of immaturity of the ENS.

Occurrence and colocalization of surfactant proteins A, B, C and D in the developing and adult rat brain.

BACKGROUND: Surfactant proteins (SP's) have been described as inherent proteins of the human central nervous system (CNS). Their distribution pattern in brain tissue and altered cerebrospinal fluid (CSF) - concentrations in different CNS pathologies are indicative of their immunological and rheological importance. The aim of this study has been to investigate when - compared to the lungs - SP's are expressed in the developing rat brain and which functional components in the CNS participate in their production. MATERIAL AND METHODS: Brain and lung tissue from embryonal (days 10, 12, 14, 16, 17 and 20), newborn, and adult rats were harvested and investigated for expression of SP-A, SP-B, SP-C and SP-D using immunofluorescence microscopy in order to identify and compare the time points of their occurence in the respective tissue. To better identify the location of SP expression in the rat brain, SP's were colocalized with use of an astrocyte marker (GFAP), a neuronal marker (NeuN), an endothelial marker (CD31) and an axonal marker (NF). RESULTS AND CONCLUSION: SP-A and SP-C are expressed in the CNS of rats during early embryonic age whereas SP-B and SP-D are first present in the adult rat brain. All SP's are expressed in cells adjacent to CSF spaces, probably influencing and maintaining physiological CSF flow. SP's A and C are abundant at the site of the blood brain barrier (BBB).

Reversible small bowel obstruction in the chicken foetus.

BACKGROUND: Ligation of the embryonic gut is an established technique to induce intestinal obstruction and subsequently intestinal atresia in chicken embryos. In this study, we modified this established chicken model of prenatal intestinal obstruction to describe (1) the kinetics of morphological changes, (2) to test if removal of the ligature in ovo is possible in later embryonic development and (3) to describe morphological adaptations following removal of the ligature. MATERIALS AND METHODS: On embryonic day (ED) 11, small intestines of chick embryos were ligated micro surgically in ovo. In Group 1 (n = 80) gut was harvested proximal and distal to the ligation on ED 12-19. In Group 2 (n = 20) the induced obstruction was released on day 15 and gut was harvested on ED 16-19. Acetyl choline esterase staining was used as to assess resulting morphological changes. RESULTS: A marked intestinal dilatation of the proximal segment can be seen 4 days after the operation (ED 15). The dilatation increased in severity until ED 19 and intestinal atresia could be observed after ED 16. In the dilated proximal segments, signs of disturbed enteric nervous system morphology were obvious. In contrast to this, release of the obstruction on ED 15 in Group 2 resulted in almost normal gut morphology at ED 19. CONCLUSION: Our model not only allows the description of morphological changes caused by an induced obstruction on ED 11 but also-more important - of morphological signs of adaptation following the release of the obstruction on ED 15.

A chicken embryo model for the study of umbilical and supraumbilical body wall malformations.

UNLABELLED: BACKGROUND/PURPOSE; The embryology of ventral body wall malformations is only partially understood, although their incidence is relatively common. As only few experimental data exist on the development of those defects, the aim of our study was to compare the teratogenic effect of trypan blue (TB) and suramin (SA) in their capability to induce umbilical and supraumbilical abdominal wall malformations in a chicken egg model. MATERIALS AND METHODS: A total of 255 fertilized chicken eggs were incubated at 38 °C and 75% relative humidity. Embryos were treated in ovo on incubation day 2.5 (Hamburger/Hamilton (HH) stage 13). The eggshell was windowed, and solutions of TB or SA were injected into the coelomic cavity at the region of the umbilicus. The window was closed and the embryos reincubated until examination on day 8 (HH 34). RESULTS: A total of 60 embryos survived in each group. The largest number of embryos presented with defects in the umbilical and supraumbilical region (25% in the SA group and 40% in the TB group). A combination of both defects (thoracoabdominoschisis) was seen in 20% of the TB and 8.3% of the SA groups, respectively. Associated anomalies found in both groups were head and eye defects, abnormal pelvic configurations, leg deformities, and mild forms of cloacal exstrophies. CONCLUSIONS: TB and SA have both a high potential to induce umbilical and supraumbilical ventral body wall malformations in chicken embryos. This novel animal model might help to establish a more profound understanding of the developmental steps in ventral body wall formation and the embryology for its malformations.

Embryology of the early foregut.

In embryology, no agreement exists how the early foregut differentiates into the respiratory tract and the intestinal tract. In particular, the formation of the early lung anlage as well as the process of separation of trachea and esophagus remains unclear. This process is explained in a rather schematic way and aims more to explain pathologic findings, whereas true embryologic investigations are extremely rare in this field. Here, scanning electron microscopy of the normal foregut development illustrates the steps, which finally leads to the development of larynx and trachea on the one hand, and pharynx and esophagus on the other hand. This study was performed in chicken embryos in accordance to the developmental stages described. As the main results from these illustrations show, we found no evidence for lateral foregut ridges inside the undivided foregut chamber and no fusion of lateral foregut components to form a trachea-esophageal septum.

Embryology of the midgut.

In most textbooks of embryology and pediatric surgery, the puzzling spectrum of midgut "malrotations" is explained by an "impaired" process of rotation of the midgut. However, this "process of rotation" is explained in a rather schematic way and aims more to explain pathologic findings whereas detailed embryologic investigations are still rare in this field. Good animal models which would allow the comparison of normal and abnormal midgut development are missing. In this paper we describe the development of the midgut in form of an atlas. Scanning electron microscopy is used in rat embryos to illustrate the crucial embryologic processes of midgut development. The main result shown in these illustrations is that clear signs of a process of rotation are missing.

Embryology of the hindgut.

Normal and abnormal development of the hindgut is still in debate. Normal development of the hindgut critically depends on the cloacal membrane. In this study, scanning electron microscopy of staged rat embryos between the gestational days 10-15 was performed to show the normal development of the hindgut and the abnormal development in Danforth's short tail (SD) mice. Our studies in normal and abnormal development indicate that the embryonic cloaca never passes through a stage that is similar to any form of anorectal malformation in neonates, including the so-called "cloacas" in females. To explain the abnormal development in anorectal malformations, further studies are mandatory.

The embryology of the diaphragm.

Despite the progress in prenatal diagnosis and intervention as well as postnatal therapeutic strategies, congenital diaphragmatic hernia (CDH) is still associated with a meaningful mortality because of the induced pulmonary hypoplasia. An essential key in understanding the pathogenesis of CDH is the underlying embryology, which has been neglected during the last decades. Likewise, the development of the normal diaphragm is still poorly understood. Obsolescent perceptions, mainly formed from histologic sections, are still propagated. In this review we present an atlas of scanning electron microscopy images that depict the normal and defective development of the diaphragm in the nitrofen rat model for CDH. Our findings suggest that the normal diaphragm mainly develops from the posthepatic mesenchymal plate. If the development of the posthepatic mesenchymal plate is impaired, a diaphragmatic defect occurs.

Embryology of the testicular descent.

Numerous researchers studied the morphology of the testicular descent, including the possible function of the gubernaculum. However, a clear illustration of this process is still missing. The aim of this paper was to illustrate the embryology of the testicular descent in the rat by scanning electron microscopy. In a first phase of the intra-abdominal testicular descent, the testis moves actively from the lower pole of the kidney towards the bladder neck. In a second inguinal phase the testis enters groin and moves in the developing processus vaginalis peritonei caused by the disappearance of the bulb of the gubernaculums testis.

Embryology of the distal urethra and external genitals.

Faulty ventral openings of the urethra constitute a broad spectrum of malformations that are subsumed under the term "hypospadia." The normal development of the urethra and the genitals critically depends on the following events: (a) formation of the external genitalia, (b) fate of the cloacal membrane, and (c) formation of the distal urethra. The purpose of this study was to demonstrate these events using microsurgical techniques and scanning electron microscopy in staged rat embryos.

Embryology of anorectal malformations.

Today, the normal and abnormal development of the hindgut is still a matter of speculation. However, as the result of recent studies in appropriate animal models, most embryologic events that finally lead to abnormal hindgut development are better known than in the past: (1) the process of maldevelopment starts in early embryonic stages; (2) the cloacal membrane is always too short in its dorsal part, thus, the dorsal cloaca is missing; and (3) as a result, the hindgut remains attached to the sinus urogenitalis, forming the recto-urethral fistula. In the past, an impaired process of septation was believed to be the main cause of abnormal hindgut development. In contrast to this, our results indicate that the development of the septum is more passive than active. Furthermore, the results of our studies in normal and abnormal development indicate that (1) the embryonic cloaca never passes through a stage that is similar to any form of anorectal malformation in neonates, including the so-called "cloacas" in female embryos, and (2) to explain abnormal development, studies in abnormal embryos are mandatory.

The testicular descent in the rat: a scanning electron microscopic study.

PURPOSE: Numerous researchers studied the morphology of testicular descent including the possible function of gubernaculum. However, a clear illustration of this process is still missing. The aim of this study was to illustrate testicular descent using scanning electron microscopy (SEM) in a rat model. METHODS: The abdomen of rat fetuses between gestational day (E) 15 and E 22 and newborns at postnatal day (D) 0 and D 1.5 was opened by microsurgery. Standard preparation for SEM was carried out. The position of the testis and gubernaculum testis was documented. RESULTS: The gubernaculum was obvious in male rat embryos at E 17.5. In a first phase (E 16-E 21) the testis moved from cranio-lateral and dorsal to caudo-medial and ventral, while clear signs of an active role of the gubernaculum were missing. In a second phase (E 22-D 1.5) the processus vaginalis peritonei (PVP) developed, while the conus of the gubernaculum disappeared, after which, the testis moved out of the abdominal cavity and entered the PVP. CONCLUSION: In our study, we could not specify the role of gubernaculum for testicular descent. However, our data showed that the testis lay intraperitoneal throughout the descensus testis.