Carnegie Stages Research Articles

Growth and asymmetry of the human liver during the embryonic period.

Previous studies had suggested that the development of asymmetry in the human liver could occur as a consequence of the earlier developing asymmetry of the heart and that the preferential right-sidedness of hepatic volume was secondary to favored venous return on the right. To examine this question we reconstructed the liver from serial histologic sections of human embryos in the Carnegie Embryological Collection of stages 11 through 23. Photomicrographs of embryo sections were projected, traced into the digitizing pad on an image analyzer microcomputer, and the liver volume and relative volumes to right and left of the median plane of the body calculated. The 38 embryos studied represent each Carnegie stage, sectioned in transverse, frontal, or sagittal (except stage 11) planes. The liver appears in stage 11 and grows to over 90 mm3 at stage 23, as the embryo enlarges from 3 to 30 mm crown-rump length (CRL). A significantly greater proportion of the liver was found on the right of the median plane in all embryos from stage 11 onward (p less than 0.001). Volume in mm3 is given by the semilogarithmic regression formulas volume = 1.49 x 10(-6)e0.826 stage, or volume = 0.0177 e0.333 CRL. Insignificant variation from the average proportion of 57.8% (SE +/- 0.8%, range 51-70%) on the right of the median plane was present throughout the embryonic period. The observation that the liver is right-sided from its first appearance is not well explained by properties of the vascular pattern. The explanation for this intrinsic hepatic asymmetry remains obscure.

Development of the choroid plexus anlage and supraependymal structures in the fourth ventricular roof plate of human embryos: scanning electron microscopic observations.

The developing anlage of the choroid plexus and supraependymal structures in the fourth ventricular roof plates of nine normal human embryos ranging from Carnegie stages 14 to 19 were investigated with scanning electron microscopy. In the human embryos at stage 18, the first semimacroscopic choroidal anlage developed in the form of bilateral evaginations that ran dorsomedially and caudally from the bilateral corners of the rhombencephalon. The anlage became evident with even smaller and parallel ridges in the embryo at stage 19. Embryos at earlier stages exhibited surface membrane modifications such as convexity, microvilli, cilia, and spherical protrusions at the middle one-third of the rhombencephalon, which corresponded to the future choroidal anlage region. Two morphologically different groups of supraependymal cells (SE cells) were elucidated throughout the stages examined. Type 1 SE cells has spindle or tear-drop-like bodies, frequently with one or more long cytoplasmic processes. Type 2 SE cells were globular, with numerous fine pseudopodial processes. Type 1 SE cells were distributed mainly at the future choroidal anlage regions or on the anlage itself and were less frequently located at the rostral end of the roof. We found no general pattern in the distribution of type 2 SE cells. Supraependymal fibers (SE fibers) were seen as fine processes that were distributed similarly to type 1 SE cells and extended transversely for a long distance.

High prevalence of defective human embryos at the early postimplantation period.

Thirty-seven cases of human embryos at the early postimplantation period were procured after induced abortion and examined histologically. Their developmental stages ranged from Carnegie stages 6 to 11, and their standard ages ranged from 14 to 24 days after fertilization. Five cases (13.5%) were grossly abnormal, and seven (18.9%) were degenerating partially or in toto. Gross abnormalities included distorted embryonic disc, disorganized neural groove or tube, and neural tube dysraphism. The high prevalence rate of defective embryos at the early postimplantation period supports the clinical finding that a substantial proportion of human conceptions are eliminated from an early stage of pregnancy, often without the knowledge of the mother. The fate of undifferentiated pathological embryos is uncertain and remains to be determined.

Fourth ventricular floor in human embryos: scanning electron microscopic observations.

The ultrastructural surface features of the normal fourth ventricular floor of seven human embryos ranging from Carnegie stage 14 to stage 19 (crown-rump length: 7.6-16.2 mm) were examined by using scanning electron microscopy (SEM). Low-power SEM views showed the median sulcus, sulcus limitans, and neuromeres, transient structures characteristic of the earlier embryonic period. High-power SEM observation revealed supraependymal cells (SE cells) and supraependymal fibers (SE fibers) which exhibited a characteristic localization, as well as generalized surface-membrane modifications such as microvilli and cilia. SE cells could be classified into two major groups. The type 1 SE cells seem to possess neuronal functions, as deduced from morphological similarities to their counterparts in adults and the specialized distribution closely related to neuromeres. The type 2 SE cell morphologically resembled the phagocytic SE cell described in related literature. SE fibers ran a course either rostrocaudally in the median sulcus or mediolaterally on the neuromeres, most frequently near the interneuromeric cleft; they made contact with type 1 SE cells and ependymal surface modifications and then penetrated the ependymal layer.

Axiale Mesenchymstrukturen in der Anlage der Halswirbelsäule des Menschen

The anlage of the cervical vertebral column of a human embryo has been investigated (9 mm CRL, Carnegie stage 16). The nuclear density in the axial mesenchyme increases rhythmically from cranial to caudal. This phenomenon superimposes a metameric pattern on the blastema. Furthermore, cell formations are shaped by the orientation of the mesenchymal cells. The name ‘formationes quasi distensae’ is proposed for this system. The anlage of the atlas shows a distinct mesenchymal anlage of a vertebral body. The conclusion is drawn that the dens axis is predominantly formed out of the anlage of the atlas body. The opinion that man does not show an anlage of the atlas body can no longer be sustained.

Rotation of the junction of the outflow tract and great arteries in the embryonic human heart.

The factors which give rise to the normal relationship between the great arteries and their respective ventricles are unknown. The developmental anatomy of this region was studied by using frontal, sagittal, or transverse serial histologic sections of 17 normal human embryos of Carnegie stages 15-19 from the Carnegie Embryological Collection. Distances and angles between major anatomic landmarks were determined by using computer reconstructions of the serially sectioned embryos, three-dimensional analytic geometry, and Euclidean distance formulas. The findings show that between stages 15 and 19 there is a marked rotation of the axis of the semilunar valves: frontal 121 degrees counterclockwise, sagittal 196 degrees counterclockwise, and transverse 240 degrees clockwise. Simultaneously the great arteries lengthen at a faster rate than the rest of the heart; and there is also an increase in the caliber and wall thickness of the great arteries. These results suggest that the changing rate of growth between the great arteries and the heart is necessary to align the great arteries, the semilunar valves, and the muscular outflow tract septum appropriately with respect to the interventricular septum. Reductions in the rate of growth of the great arteries relative to the heart could, by causing changes in the rotation of great arteries and outflow tract septum, have a role in the pathogenesis of cardiovascular malformations such as tetralogy of Fallot and transposition of the great arteries.

Development of aortic and mitral valve continuity in the human embryonic heart.

The anatomic relationship of the aortic and mitral valves is a useful landmark in assessing congenital heart malformations. The atrioventricular and semilunar valve regions originate in widely separated parts of the early embryonic heart tube, and the process by which the normal fibrous continuity between the aortic and mitral valves is acquired has not been clearly defined. The development of the aortic and mitral valve relationship was studied in normal human embryos in the Carnegie Embryological Collection, and specimens of Carnegie stages 13, 15, 17, 19, and 23, prepared as serial histologic sections cut in the sagittal plane, were selected for reconstruction. In stage 13, the atrioventricular valve area is separated from the semilunar valve area by the large bend between the atrioventricular and outflow-tract components of the single lumen heart tube created by the left interventricular sulcus. In stages 15 and 17, the aortic valve rotates into a position near the atrioventricular valves with development of four chambers and a double circulation. In stage 19, there is fusion of aortic and mitral endocardial cushion material along the endocardial surface of the interventricular flange, and this relationship is maintained in subsequent stages. Determination of three-dimensional Cartesian coordinates of the midpoints of valve positions shows that, while there is growth of intervalvular distances up to stage 17, the aortic to mitral distance is essentially unchanged thereafter. During the period studied, the left ventricle increases in length over threefold. The relative lack of growth in the saddle-shaped fold between the atrioventricular and outflow tract components of the heart, contrasting with the rapid growth of the outwardly convex components of most of the atrial and ventricular walls, may be attributed to the different mechanical properties of the two configurations. It is postulated that the pathogenesis of congenital heart malformations, which characteristically have failure of development of aortic and mitral valve continuity, may involve abnormalities of rotation of the aortic region or malpositioning of the fold in the heart tube.

Development of the atrioventricular valve region in the human embryo

Severe cardiac malformations may involve the atrioventricular valve region, but the sequence of embryonic development of this important area has been little studied. In particular, the basis of atrioventricular muscular discontinuity, except at the conduction system, has remained unexplained. To examine this question, serial histologic sections of human embryos from the Carnegie Embryological Collection and from the Hopkins Pathology Collection were studied and six embryos were reconstructed. The atrioventricular sulcus can be identified in Carnegie stage 10 as an indentation or crease on the right side separating the heart tube from the umbilical vein. By stage 12 the sulcus has deepened and rotated anteriorly as the atria appear and the heart tube elongates rapidly within the confining pericardial space. Selective accumulation of cardiac jelly on the endocardial aspect of the constriction of the heart tube produced by the atrioventricular sulcus is pronounced by stage 14. By stage 16 the separation of the atrioventricular orifice into right and left components is well advanced, and by stage 18 the septation of the atria and ventricles is largely complete. The muscular connection between the atria and the ventricles becomes interrupted around most of the artioventricular sulcus, except for the His bundle, during the latter part of the embryonic period. The topography of the original sulcus assumes a catenoidal or saddle-shaped configuration, i.e., convex in one plane and concave in the perpendicular plane. The tension and pressure relationships in such a structure would favor cardiac jelly accumulation and the eventual disintegration of lines of myocyte connections passing across the groove. The preservation of the His bundle connection is explained by the failure of the sulcus to completely encircle the heart.

Development of .GAMMA.-enolase immunoreactive cells in the nervous system of human embryos.

The development of γ-enolase immunoreactive cells in five human embryos (Carnegie stages: 13-16 and 21; crown-rump length: 6.3-23.4mm) was investigated by immunohistochemistry, using the basal plate of the neural tube, dorsal root ganglion at the cervical level, trigeminal and vestibulocochlear ganglia, and para-esophageal and para-tracheal ganglia.In the basal plate of the stage 13 embryo, not only the motor neurons but also the anterior root was immunoreactive at the level of the cervical spinal cord. Some of these reactive neurons were in the rhombencephalon. The motor neurons in the basal plate of mesencephalon and thoracic spinal cord of the stage 14 embryo were immunoreactive, while those of the lumbar spinal cord were immunoreactive in the stage 15 embryo. In the dorsal root ganglion, trigeminal and vestibulocochlear ganglia, and para-esophageal and para-tracheal ganglia, immunoreactive cells were observed first in the stage 15 embryo. In the stage 21 embryo, immunoreactivity was observed around the peripheral respiratory tract.As the appearance of γ-enolase in neurons is related to functional and morphological maturation, our study will provide descriptive information for the evaluation of neuronal development in early human embryos.

Cerebral dysraphia (future anencephaly) in a human twin embryo at stage 13.

Cerebral dysraphia was studied histologically and by graphic reconstruction in a twin at stage 13, and comparisons were made with the normal (discordant) twin. The normal, bidirectional closure of the rostral neuropore was investigated in several embryos, from which it was concluded that the situs neuroporicus is represented by the future commissural plate rather than by the (adult) lamina terminalis. In the abnormal twin the neural tube was open over part of the midbrain and forebrain, although the situs neuroporicus was closed. The experimental production of anencephaly by Giroud and co-workers was reviewed, and comparisons between embryonic staging systems in the rat, mouse, and human were made. Three corresponding phases are found in the human: 1) cerebral dysraphia, occurring before or during Carnegie stage 11 (approximately 23-25 days); 2) exposure of a highly developing and well-differentiated brain during the remainder of the embryonic period; and 3) degeneration of the exposed brain throughout the fetal period, resulting in anencephaly. Hence the abnormal twin described here is believed to represent a precursor of typical anencephaly, and is the earliest example of purely cerebral dysraphia so far recorded.

Structural organization of the human cerebral cortex prior to the appearance of the cortical plate.

The early development and the structural organization of the human cerebral cortex, prior to the appearance of the cortical plate (Carnegie stage 22, ca. 54 days), was studied in two embryos: 43 (stage 18) and 50 day old (stage 20), respectively. It has been shown that the human cerebral cortex begins its ontogenetic development around the sixth rather than around the eighth week of gestation as it has been previously assumed. The human cerebral cortex starts to develop soon after the cerebral vesicles have been formed (stage 15) and a primitive internal capsule has been established (stage 17, ca. 41 days). By stage 18 of human development fibres from this primitive internal capsule have reached and probably have penetrated into the developing cerebral vesicle, through its more superficial zone. Fibres from this primitive internal capsule have been traced backward through the ventral thalamus to the mesencephalic tegmentum. The possible existence of primitive ascending fibres from the mid-brain which terminate in the superficial zone of the developing cerebral cortex (tegmento-thalamostriato-cortical tract) is suggested. The arrival of these primitive corticipetal fibres establishes in the outer zone of the cerebral cortex a primordial plexiform lamina or an external white matter. Horizontal-bipolar cells (embryonic Cajal-Retzius neurons) begin to differentiate by stage 18 of human development (43 days in our case). By stage 20 (50 days in our case), the primordial plexiform lamina is well established, extends throughout the entire surface of the developing cerebral cortex, and is considered to be functionally active. It is, by this age, a superficial, 40 micrometers thick, complex fibrillar neuronal organization composed of numerous horizontal corticipetal fibres (demonstrable with silver methods), horizontal-bipolar Cajal-Retzius neurons and a few other, less defined, cellular elements. This primordial plexiform lamina is considered to represent a primitive "premammalian" cortical organization. The next event in cortical ontogenesis is the appearance of the cortical plate or the mammalian neocortical grey at stage 22 (ca. 54 days). Migrating neuroblasts attracted toward the preexisting primordial plexiform lamina and guided by glial fibres start to accumulate within it.(ABSTRACT TRUNCATED AT 400 WORDS)

Role of the vagus nerve and its recurrent laryngeal branch in the development of the human ductus arteriosus.

The reason that the normal ductus arteriosus has a muscular media, contrasting with the elastic lamellar structure of the adjacent great arteries, is unknown. We examined the hypothesis that the anatomic relationship of the ductus arteriosus to the vagus and recurrent laryngeal nerves during early development might be of importance in influencing ductal morphology. Normal human embryos from the Carnegie Embryological Collection and embryos and fetuses from the Hopkins Pathology Collection were studied microscopically, by reconstructions made from serial histologic sections, or by gross dissection. At Carnegie stage 16 the recurrent laryngeal nerves pass medially from the vagus nerve to the laryngeal area and are caudal to the bilaterally symmetric sixth aortic arches. By stage 18 the right sixth aortic arch has disappeared and the left sixth aortic arch is in a more caudal position relative to the larynx. The left vagus nerve and its recurrent laryngeal branch form a sling supporting the distal (or ductus arteriosus component) of the left sixth aortic arch. In subsequent development there is greater relative separation of the larynx and ductus arteriosus. The media of the ductus arteriosus beneath the supporting nerves is thinner and has less elastic fiber formation than the elastic lamellar media of the adjacent aortic arches. The study shows that the vagus and recurrent laryngeal nerves are in a position to provide mechanical support to the ductus arteriosus during its development and that the morphology of the media of the supported ductus arteriosus differs from that of the adjacent unsupported aortic arches. It is suggested that this local mechanical support may be the reason that the normal ductus arteriosus differentiates as a muscular artery and is therefore able to obliterate its lumen in postnatal life. Without such support the ductal media could develop the abundant elastic fibers characteristic of the normal unsupported aorta and pulmonary trunk and become an abnormal, persistently patent ductus arteriosus.

The growth of the human brain during the embryonic period proper. 1. Linear axes.

Linear axes of the brain were measured in 143 human embryos from Carnegie stages 11 to 23 (3 1/2-8 postovulatory weeks). The embryos ranged from 3 to 30 mm in C.-R. length. Both Born reconstructions and serial sections of the central nervous system were used. The brain axes included were the fronto-occipital diameter, bitemporal diameter, and length and width of both the mesencephalon and cerebellum. A least squares line was fitted to the set of data points corresponding to each brain axis measured, and a t test verified that a linear model was an appropriate representation of the data. Based on these linear measurements it can be concluded that for forebrain grows more rapidly than the rest of the brain at the onset of tubular brain enlargement. Furthermore, as seen by comparing growth along two dimensions, the forebrain and midbrain grow at the same rate, whereas the cerebellum grows at different rates along the length and height axes. In addition, the cerebellum begins to grow later than the rostral part of the brain. Covariance analysis of the data points of the embryonic brain axes with data points of identical brain axes of the fetus showed that the measurements from the embryonic and fetal brain axes cannot be represented by a single regression line.

The estimated age of staged human embryos and early fetuses

The appearance of anatomic features during embryogenesis relative to menstrual history is not well established. The 621 staged human embryos and fetuses in the Carnegie Embryological Collection were examined, and morphologic features were recorded in 494 specimens in good condition, from 382 intrauterine, 49 ectopic, and 55 pregnancies of other or unknown source. There were menstrual data on 276 specimens, and for 10 the date of a single coitus was known. The embryo develops to 1 mm at the twenty-eighth menstrual day, after which its crown-rump length increases by 0.7 mm per day. Coital ages and postovulatory ages estimated from menstrual data correlated significantly (p < 0.001) with crown-rump length and Carnegie stage, but estimated postovulatory age was highly variable (standard deviation, 10.5 days). Morphologic features appeared within a narrow interval (standard deviation, 0.0 to 1.5 Carnegie stages). Normal human embryogenesis is a stereotyped sequence with little statistical variation, but menstrual data in individual cases may be unreliable in dating this sequence.

The early development of the nervous system in staged insectivore and primate embryos.

The early development of the nervous system was studied in stage embryos of hemicentetes semispinosus, Microcebus murinus, Alouatta seniculus, Cebus appella, Cebus albifrons, macaca mulatta, and Homo sapiens. The specimens were assigned to Carnegie stages 11-13. Serial transverse sections were examined and graphic reconstructions were prepared. The early development of the neural tube is basically similar in all the species investigated but differences in detail are noticeable. The mesencephalic flexure serves in all cases as a landmark for malpighi's tripartite subdivision of the brain. The nonhuman embryos seem to show a little more variation than the human in the closure of the neuropores in relation to somitic count. With the exception of the later-appearing terminal-vomeronasal component, all major portions of the neural crest as classified by O'Rahilly ('65) are represented in both the nonhuman and the human embryos studied. No crest is present at the level of rhombomere 1, nor at rhombomere 3 except in the platyrrhines and some human embryos, nor at rhombomere 5 except in certain human specimens. An indication of the division of the trigeminal ganglion into its primary divisions is rare at stage 11 (C. apella), may be visible at stage 12 (Alouatta, macaca, Homo), and is definite (in Homo) at stage 13. Ganglionic contributions from head ectoderm (epipharyngeal placodes), as previously described in the human and some other vertebrate embryos, were sought and found in Cebus apella. In both nonhuman and human, a tendency is noted whereby the rostral limit of the occipitospinal crest, high at stage 11, seems to descend relatively at stage 12, and ascend again at stage 13 (at least in the human) to become associated with the appearance of the accessory and hypoglossal nerves. In general, the motor components of the nerves are identifiable before the sensory elements, and, in the present study, nerve fibers were first observed in the human at stage 13 in some of the cranial nerves and in the ventral roots of the spinal nerves.

Development of the interpeduncular nucleus in the midbrain of rhesus monkey and human

The development of the interpeduncular nucleus (IPN) in primates was studied in rhesus monkey with 3H-thymidine autoradiographic, Nissl and Golgi methods and in humans in histological preparations from embryos and fetuses of different ages. Autoradiographic analysis demonstrated that the neurons of the monkey IPN underwent their final cell division between postconception day 36 (E36) and E42, which corresponds to Stages 17 through 21 of Hendrickx and Sawyer. Autoradiograms of monkeys sacrificed at various short intervals following exposure to a pulse of 3H-thymidine showed that IPN neurons were generated in the proximity of the ventricular surface near the confluence of the 3rd ventricle and cerebral aqueduct, migrated ventrally along the midline and then spread laterally after reaching the ventral midbrain, where IPN was first recognized at E45 (Stage 23). The distribution of successively generated neurons in autoradiograms revealed caudal to rostal and lateral to medial spatiotemporal gradients. Differentiation of IPN neuronal size and development of Nissl substance began in rhesus monkey only after postmitotic cells had reached their destination and seemed to be pronounced mainly through E104. However, growth of the dendrites and elaboration of their side branches as seen in Golgi impregnations progressed gradually from E81 to birth (E165) and perhaps even later. Analysis of histological preparations of a series of human embryos and fetuses was used to derive similar information indirectly, since the autoradiographic method cannot be applied to man. It was found that IPN neurons in human probably underwent their final division between Carnegie Stage 17 and 21. Similarly, as in monkey, postmitotic cells in human IPN displayed an inverted fountain pattern of cellular migration. IPN could first be delineated at Stage 23. There was evidence for both caudal to rostral and lateral to medial spatiotemporal gradients in the human, as in the monkey. Thus, in monkey and human, all IPN neurons are generated within the first quarter of intrauterine life and there is remarkable similarity in the timing, tempo and pattern of IPN neuronal differentiation in both species, indicating the validity of using nonhuman primates as an experimental model for understanding the development of this structure in man.

An analysis of the age and size of 483 human embryos.

The crown-rump length of 483 fixed human embryos of Carnegie stages 6-23 was analyzed and median and predicted mean lengths were calculated. The results were compared with those of other series and confirmed that early human growth rates are different from those of macaques with which human embryo growth has previously been compared. The study indicated that it is possible to predict: 1. the median size of an embryo of given Streeter horizon or Carnegie stage; 2. the age of a fresh embryo, or one of undisputed Streeter staging, by comparison with mean figures of other authorities; and 3. the corrected age of an embryo of known length or known Streeter staging or both in terms of postovulation age. Since teratogens may reduce the embryonic growth rate this information is relevant in the analysis of teratogenic factors in human development.

Cardiovascular malformations found in 1286 externally normal human embryos alive in utero

AbstractThe heart and great vessels of 1286 human embryos were microdissected and examined for malformations. Most of the embryos were obtained by induced abortion conducted for socioeconomic reasons and the remainder by hysterectomy performed because of some minor disturbance. Only embryos that were alive in utero, at Carnegie stages 18–23, and showed no external anomalies were included. A microdissection procedure that enables precise examination of the heart was used. Criteria for diagnosis of such defects as ventricular septal defect, stenosis, and coarctation were established. Twenty‐seven (2.1%) embryos with congenital cardiovascular malformation (CCM) were found. The frequency was higher than that (0.46–1.17%) reported for newborns. The defects included most of the comparatively common types of CCM, and it was confirmed that the major types of CCM are manifested early in life. It was also noted that certain severe types of anomaly, reported to be scarce in infants, are relatively common in embryos.

Pathogenesis of preaxial polydactyly of the hand in human embryos

Hand plates with preaxial polydactyly from 13 human embryos of the Carnegie stages 17-23 were examined macro- and microscopically. Morphological features in early pathogenesis of preaxial polydactyly are (1) an abnormal extension and a delayed involution of the apical ectodermal ridge on the preaxial border of the hand plate in stages 17 and 18, (2) a precocious development of an interdigital notch between the duplicated thumbs in stages 17 and 18, and (3) bifurcation of the distal part of the first digital ray in stage 19. A disorder of the interaction between limb ectoderm and mesoderm is considered to be the pathogenetic event.

Normal development of early human embryos: observation of 90 specimens at Carnegie stages 7 to 13.

Abstract1. A systematic analysis of the relation between 2 parameters of developmental stage — clinically assessed embryonic age and growth — was made for 90 human embryos at Carnegie stages 7–13 from healthy pregnancies. 2. The data showed remarkable individual variation in the relation between age and developmental stage of embryos at stages 11–13. It appears that the mean age for these stages in our specimens was a few days greater than the corresponding age in the currently cited standards, although a definite conclusion must await further studies. 3. The relation between body length and developmental stages in our embryos was not different from the corresponding standards presented by previous investigators. The relation of the number of somites to developmental stage and greatest length in 13 embryos at stages 9–11 was approximately in accord with that reported for several corresponding embryos by other investigators. 4. The oldest age for embryos at each stage in our data may have practical use in denying a causal relation between the exposure of pregnant women to an exogenous agent at such a stage and the subsequent appearance of specific malformations in their progeny.