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Teratoma Formation and Genomic Profiling Using Multi-Omics Approaches.

Teratoma formation is the gold standard assay for evaluating the developmental pluripotency of human and mouse embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Following subcutaneous injection into immunodeficient mice, pluripotent stem cells spontaneously differentiate into derivatives representing all three embryonic germ layers-ectoderm, mesoderm, and endoderm. Beyond serving as a functional assay for pluripotency, teratomas provide a unique three-dimensional model system for studying early human development and lineage specification in vivo. This chapter describes comprehensive protocols for teratoma formation in immunodeficient mice, tissue processing for multiple downstream genomic applications, and multi-omics profiling approaches. We detail methods for embryonic stem cell culture, teratoma generation via subcutaneous injection, tissue dissection and processing for chromatin immunoprecipitation followed by sequencing (ChIP-Seq), RNA sequencing (RNA-Seq), single-cell multiome profiling combining chromatin accessibility (ATAC-Seq) and gene expression (scRNA-Seq), and histological analysis using hematoxylin and eosin (H&E) staining. Additionally, we provide bioinformatics workflows for analyzing the resulting genomic datasets to characterize the epigenetic and transcriptional landscapes of teratoma-derived tissues. These methods enable comprehensive molecular characterization of developmental processes and provide valuable resources for stem cell biologists studying pluripotency, differentiation, and early embryonic development.

Teratoma

Ecological determinants in microbial colonization of the murine gastrointestinal tract: adherence of Torulopsis pintolopesii to epithelial surfaces.

Torulopsis pintolopesii is a yeast indigenous to the gastrointestinal tracts of conventional mice and rats from many colonies. In such natively colonized animals, the organism forms layers on the surface of the epithelium in the secreting portion of the stomach and can be cultured from all areas of the gastrointestinal tract. When given in water or food to germfree mice or specific pathogen-free mice possessing an indigenous microbiota free of yeast, T. pintolopesii also can be cultured from all areas of the tract at population levels ranging from 10(5) to 10(8) cells per g (wet weight). Likewise, as in its native hosts, the organism forms layers on gastric surfaces in the associated animals. The layers form on the secreting surface in both the specific pathogen-free and monoassociated ex-germfree mice. In the latter animal, however, a layer of yeast also forms on the nonsecreting gastric surface. In tests of its capacity to adhere to gastrointestinal surfaces in vitro, the organism adheres to epithelia from all areas of the mouse tract. These findings support an hypothesis that the capacity of T. pintolopesii to adhere to epithelial surfaces may be only one determinant influencing it to form layers on the gastric secreting surface in its native hosts.

Animals

Suppression of alpha-amylase gene expression by antisense oligodeoxynucleotide in barley cultured aleurone layers.

Antisense oligodeoxynucleotides (ODNs) have been applied to regulate gene expression using cell-free media or animal cells. Here we demonstrate the specific inhibition of barley alpha-amylase gene expression by synthetic antisense ODNs. In a cell free system using wheat-germ extracts, 5 microM of a 20-mer antisense ODN prevented the synthesis of the polypeptide corresponding to the predetermined length of alpha-amylase translated in vitro, whereas there was no effect on other protein synthesis. Furthermore, in cultured aleurone cells, alpha-amylase activity was efficiently decreased by addition of ODNs. At the concentrations higher than 5 microM, antisense ODN inhibited alpha-amylase gene expression almost completely. These results imply that ODN could transport into the cultured aleurone cells crossing the cell membrane, and regulate specific gene expression. This simple model system could be applicable not only for the analysis of the alpha-amylase multigene family in barley but also for studying functions of cryptic genes in higher plant.

Base Sequence

Localization of alpha-galactomannan and of wheat germ agglutinin receptors in Schizosaccharomyces pombe.

The location of galactomannan on the surface of Schizosaccharomyces pombe cells was reexamined by scanning electron microscopy by an indirect but specific method using gold markers. The polysaccharide was found on the cell surface and at the end beginning to grow but not on the wall established by division. Galactomannan was also localized on S. pombe thin sections by transmission electron microscopy using the same method. The polysaccharide was found deposited in two layers in the cell wall, i.e. at the periphery of the wall and near the plasmalemma. The septum was also marked but mainly near the plasmalemma. These results indicated that the polysaccharide is elaborated onto the outside of the wall during extension but not during septum formation. When thin sections of S. pombe were marked with gold granules labeled with wheat germ agglutinin, marking was found in vacuoles but not in the cell wall. This confirmed that S. pombe cell wall is devoid of chitin.

Ascomycota

Multiple gap junction genes are utilized during rat skin and hair development.

The expression of four different gap junction gene products (alpha 1, beta 1, beta 2, and beta 3) has been analysed during rat skin development and the hair growth cycle. Both alpha 1 (Cx43) and beta 2 (Cx26) connexins were coexpressed in the undifferentiated epidermis. A specific, developmentally regulated elimination of beta 2 expression was observed in the periderm at E16. Coinciding with the differentiation of the epidermis, differential expression of alpha 1 and beta 2 connexins was observed in the newly formed epidermal layers. alpha 1 connexin was expressed in the basal and spinous layers, while beta 2 was confined to the differentiated spinous and granular layers. Large gap junctions were present in the basal layer, while small gap junctions, associated with many desmosomes, were typical for the differentiated layers. Although the distribution pattern for alpha 1 and beta 2 expression remained the same in the neonatal and postnatal epidermis, the RNA and protein levels decreased markedly following birth. Hair follicle development was marked by expression of alpha 1 connexin in hair germs at E16. Following beta 2 detection at E20, the expression increased for both alpha 1 and beta 2 in developing follicles. A cell-type-specific expression was detected in the outer root sheath, in the matrix, in the matrix-derived cells (inner root sheath, cortex and medulla) and in the dermal papilla. In addition, alpha 1 was specifically expressed in the arrector pili muscle, while sebocytes expressed both alpha 1 and beta 3 (Cx31) connexin. beta 1 connexin (Cx32) was not detected at any stage analysed. The results indicate that multiple gap junction genes contribute to epidermal and follicular morphogenesis. Moreover, based on the utilization of gap junctions in all living cells of the surface epidermis, it appears that the epidermis may behave as a large communication compartment that may be coupled functionally to epidermal appendages (hair follicles and sebaceous glands) via gap junctional pathways.

Animals

In vitro development of secondary blastodiscs from dispersed blastoderm cells of Gallus domesticus.

A study of the in vitro growth of embryonic structures from dispersed blastoderm cells is reported. The specific type of blastoderm cells with the capability of growing on the coverslip developed into the discoidal embryonic structures resembling those of avian species. The glass surface was apparently an initiator of differentiation into at least three types of cells specifically distributed in the blastodisc. Groups of structures formed were evaluated at 6, 12, 24 and 36 hours to study the developmental pattern in vitro and to estimate the number of cells per structure. Microscopic examination of the area pellucida revealed that all three basic germ layers were established after 24 hours of incubation in vitro. The 36 hour stage was represented by bulky growth of mesodermal-like cells and changes in hypoblast layer where some of the cells degenerated and some were transformed to mesenchymal spindle-like cells.

Animals

Investigation of the effect of hydroxyurea on the cell cycle and the development of necrosis in the embryonic CNS of mice.

On day 10 of gestation pregnant mice (strain NMRI) were given an intravenous injection of 500 mg/kg Hydroxyurea (HU). Simultaneously either 5 muCi/g or 10 muCi/g 3H-thymidine (3H-Tdr; specific activity 5Ci/mmol) was administered to the animals. At various times after treatment embryos and electron microscopy. Two hours after administration of HU condensations of the chromatin structure could be detected electron microscopically in some cells. Thirty minutes later the nucleolus became smaller and denser, the cytoplasm shrank, and the cell organelles moved closer together. Three hours after application of the drug break-down of the involved cells set in. As in the autoradiograms about 98% of the counted necroses were labelled, and since labelled thymidine is almost exclusively incorporated during the S-phase, it can be stated the HU influences only metabolic processes which take place during the S-phase. From the morphological findings it can be concluded that in the case of the S-phase-specific metabolic pathway, which is influenced by HU, we are primarily dealing with DNA synthesis.

Animals

[Effect of neuropharmacologic preparations and colchicine on morphogenetic processes in embryonal amphibian cells].

The effect of antagonists of biogenous amines (antitransmitters--AT) and colchicine on rapid morphogenetic processes in the explants of embryonic ectoderm with underlying mesoderm cut from the lateral region of Rana temporaria embryos at the late neurula-early taibud stages was studied. The normal morphogenesis of the explants consists of two successive phases: phase of contact polarization and phase of cell movement into the fragment. The high concentrations of AT inhibited completely the morphogenesis of explants but somewhat lower concentrations inhibited the second phase of morphogenesis and not only did not prevent the cell polarization but even assisted its propagation over all the fragment. The inhibiting effect of AT was relieved by 5-hydroxytryptamine which per se stimulated the morphogenesis of explants. Thus, AT exert a specific inhibition of the motility of embryonic cells but do not prevent the contact interactions responsible for cell polarization within every layer.

Amphetamines

[Action of microbial flora of the digestive tract on the metabolism of bile acids in the rat (author's transl)].

An isotopic balance is established in rats receiving a regular feed intake of [4-14-C]cholesterol so that various chemical species of bile acids have the same specific activity. This property is used to study bile acids distribution in the rat liver, digestive tract and fecal excretion. Bile acids are separated by thin-layer chromatography, radioactivity is determined by liquid scintillation, and the mass by 3-hydroxysteroid dehydrogenase action. The resulting comparative study made between the germ-free rat (axenic rat) and the rat exposed to microbes ("holoxenic" or conventional rat) receiving a semi-synthetetic feed, shows the influence excercised on the metabolism by the microbial flora of the digestive tract. This study confirms that the axenic rat compared to its holoxenic homologue has a higher bile acids pool and a lower fecal excretion. At all levels of the digestive tract (small intestine and the whole caecum and large intestine), probably as well as in the liver, the total amount of bile acids which is observed in the axenic rat is about twice the amount observed in the holoxenic rat, but fecal excretion is decreased by 20%. Values obtained by this method are higher than those previously observed by other authors using gas-liquid chromatography or [14-C]cholic acid isotopic dilution. This study also confirms that cholic and beta-muricholic acids are the main bile acids in the axenic rat and in addition establishes that in this animal bile acids composition is complex and varies from the small intestine to the feces. Besides cholic, alpha- and beta-muricholic, chenodeoxycholic and ursodeoxycholic acids, unidentified chemical species constitute 21% of the whole in the feces. Comparing the compositions observed in axenic and holoxenic rats in this experiment, it could not be determined if the relative activity of the two pathways of bile acid biosynthesis is deeply or only slightly changed by the presence of microbial flora. This is because of a large fraction of unknown composants in the feces of the axenic rat and the extreme complexity in the feces of the holoxenic rat.

Animals

Reactivity of lipopolysaccharides from various salmonella SR and R chemotypes Ra-Re mutants with concanavalin A.

Lipopolysaccharides from different R mutants of Salmonella minnesota and Salmonella typhimurium belonging to chemotypes Ra to Re, as well as from three SR mutants of Salmonella typhimurium were selected for a study of their precipitability with Concanavalin A. Predictions as to the outcome of the reaction could be made since both the chemical structure of the Salmonella R lipopolysaccharides and structural requirements for a positive reaction with Concanavalin A are well established. Precipitation studies in the immuno-electrophoretic assay and in the microcapillary test were carried out with alkali-treated lipopolysaccharides as untreated lipopolysaccharide is too highly aggregated to allow a sufficient migration in agarose layers. Lipopolysaccharides of all mutants--except the SR mutants--were obtained by the phenol/chloroform/petroleum ether method in order to avoid contaminations by glucans or glycogen which are known to occur in phenol/water extracted lipopolysaccharides and which would lead to erroneous results. Additional precipitation studies were carried out with two other lectins of different polysaccharide specificity: Wheat Germ Agglutinin and Soybean Agglutinin. As expected, lipopolysaccharides of chemotypes Ra, Rb1, and RcP- mutants reacted strongly with Concanavalin A, whereas no reaction was demonstrable with lipopolysaccharides of chemotypes Rb2, Rb3, Rd and Re mutants. The lipopolysaccharide of an RcP+ mutant unexpectedly failed to precipitate unless it was dephosphorylated with HF. This artificially prepared RcP-lipopolysaccharide showed a strong reaction, thus demonstrating that negative charges in the direct neighborhood of reactive sugar units as in RcP+ LPS may prevent precipitation with Concanavalin A. No reactivity demonstrable by precipitation could be obtained using either Wheat Germ Agglutinin or Soybean Agglutinin with alkali-treated lipopolysaccharide even of those chemotypes which had the supposedly reactive sugar in a terminal position, such as N-acetyl-D-glucosamine in Ra mutants (Wheat Germ Agglutinin) or D-galactose in Rb2 or Rb3 mutants (Soybean Agglutinin).

Acetylgalactosamine

Fine needle aspiration of ovarian masses. II. Correlative cytologic and histologic study of nonneoplastic cysts and noncelomic epithelial neoplasms.

A wide variety of neoplasms and nonneoplastic cysts involves the ovary. Seventy-seven ovarian masses were aspirated using fine needles. They included 32 nonneoplastic cysts of various types, 29 celomic epithelial neoplasms, 10 germ cell neoplasms and 6 other neoplasms. The study was performed in order to elucidate the cytologic features of aspirates from these lesions and correlate them with the histologic features. Material aspirated from malignant neoplasms resulted in abundantly cellular specimens while aspirates from benign neoplasms and nonneoplastic cysts showed only scanty cells. Follicular cysts and corpus luteum cysts showed similar cell types but with various degrees of cellularity and incidence of cell types. Cysts lined by a single layer of cuboidal cells, such as paraovarian and paratubal cysts, were very similar cytologically. The three types of germ cell neoplasms encountered showed features which were diagnostic of each entity. It was difficult to discriminate between some of the types of the sex cord and mesenchymal neoplasms. Aspiration cytology can achieve a satisfactory classification of ovarian masses. It has a specific role to play especially in young patients, in whom preservation of ovarian function is advisable.

Biopsy, Needle

Ultrastructural localization of wheat germ agglutinin-binding sites on surfaces of chick embryo cells during early differentiation.

The objective of this work was to examine changes in a surface component of cells from the chick embryo during morphogenetic migrations of gastrulation. Two electron microscope techniques were used to localize cell-bound wheat germ agglutinin (WGA), a lectin which specifically binds N-acetyl glucosamine residues. One technique involved conjugation of peroxidase to WGA before reaction with the cells; the other technique used glucose oxidase to mark WGA which was already cell-bound. In both cases, binding was revealed using diaminobenzidine. Before formation of the primitive streak, all surfaces of the two-layered embryo bound WGA. After migration of cells through the streak, to form the three-layered embryo, not all cell surfaces bound WGA equally. Epiblast cells generally bound WGA lateral to the primitive streak but not during passage through the streak. Mesenchyme cells, after passage through the streak, bound WGA increasingly as they migrated away from the streak. A WGA-binding matrix was observed in the vicinity of the mesenchyme cells and on the dorsal surface of the endoblast. The ventral surface of the endoblast bound the lectin very poorly. In some instances, a peroxidase reaction product was consistently seen on certain surfaces which was not removable by addition of the simple hapten N-acetyl glucosamine. In these cases, the density of the deposit was lessened by use of diacetyl chitobiose as a hapten. This result, together with the reduction of reaction product following certain hyaluronidase treatments, suggests that WGA may be binding to hyaluronic acid as well as membrane glycoproteins.

Animals

Stage- and cell-specific expression of cyclic adenosine 3',5'-monophosphate-dependent protein kinases in rat seminiferous epithelium.

Expression of mRNAs in the rat testis encoding cyclic AMP (cAMP)-dependent protein kinases (PKAs) was studied. A microdissection method was used to isolate 10 pools of seminiferous tubules representing various stages of the cycle of the seminiferous epithelium in combination with Northern blots and in situ hybridization. The results showed a differential expression of the four isoforms of the regulatory subunits (PKA-R) at various stages of the cycle. RI alpha mRNA was detected at approximately the same levels at all stages while expression of RI beta mRNA was low at stages XIII-III, started to increase at stages IV-V, and reached a maximum at stages VIII-XI. The level of RII alpha mRNA was low at stages II-VI, increased markedly at stage VIIa,b, and reached maximal levels at stages VIIc,d and VIII, followed by a reduced expression at later stages, RII beta mRNA levels increased significantly at stage VI with maximal levels at stages VII and VIII. In situ hybridization of sections from the adult rat testis revealed RI alpha mRNA in the layers of pachytene spermatocytes and round spermatids of all stages. RI beta mRNA was detected over late pachytene spermatocytes and round spermatids of stages VII-XIII. RII alpha mRNA was seen in the layers of round spermatids of stages VII-VIII and elongating spermatids of later stages while RII beta mRNA was detected only in the round spermatid region of stages VII-VIII and in some tubules of stages I-VI. These data show that mRNAs encoding PKA-R are expressed in a stage-specific manner in differentiating male germ cells with different patterns of expression for each subunit; this suggests specific roles for these protein kinases at different times of spermatogenesis.

Animals

Topographical organization of the nigrotectal projection in rat: evidence for segregated channels.

Recent evidence suggests that projections from the superior colliculus to the brainstem in rat are organized into a series of anatomically segregated output channels. To understand how collicular function may be modified by the basal ganglia it is important to know whether particular output modules of the superior colliculus can be selectively influenced by input from substantia nigra. The purpose of the present study was, therefore, to examine in more detail topography within the nigrotectal system in the rat. Small injections (10-50 nl) of a 1% solution of wheatgerm agglutinin conjugated with horseradish peroxidase were made at different locations within substantia nigra and surrounding structures. A discontinuous puff-like pattern of anterogradely transported label was found in medial and caudal parts of the ipsilateral intermediate layers of the superior colliculus. In contrast, the rostrolateral enlargement of the intermediate layers contained a greater density of more evenly distributed terminal label. Injection sites associated with this dense pattern of laterally located label were concentrated in lateral pars reticulata, while the puff-like pattern was produced by injections into ventromedial pars reticulata. Retrograde tracing experiments with the fluorescent dyes True Blue and Fast Blue revealed that injections involving the rostrolateral intermediate layers were consistently associated with a restricted column of labelled cells in the dorsolateral part of ipsilateral pars reticulata. Comparable injections into medial and caudal regions of the superior colliculus produced retrograde labelling in ventral and medial parts of the rostral two-thirds of pars reticulata. Both anterograde and retrograde tracing data indicated that contralateral nigrotectal projections arise from cells located in ventral and medial pars reticulata. The present results suggest that the main ipsilateral projection from substantia nigra pars reticulata to the superior colliculus comprises two main components characterized by regionally segregated populations of output cells and spatially separated zones of termination. Of particular interest is the apparent close alignment between terminal zones of the nigrotectal channels and previously defined populations of crossed descending output cells in the superior colliculus. Thus, the rostrolateral intermediate layers contain a concentration of terminals specifically from dorsolateral pars reticulata and output cells which project to the contralateral caudal medulla and spinal cord. Conversely, the medial and caudal intermediate layers receive terminals from ventral and medial pars reticulata and contain cells which project specifically to contralateral regions of the paramedian pontine and medullary reticular formation.(ABSTRACT TRUNCATED AT 400 WORDS)

Amidines

A novel lamina lucida component of epithelial and endothelial basement membranes detected by LH39 monoclonal antibody.

The murine monoclonal antibody, LH39 was characterized in this study and appeared to bind to a novel basement membrane epitope. This antigen was expressed in the epithelial basement membrane of human tissue derived from all three germ cell layers and in basement membranes surrounding small blood vessels within the stroma of all organs examined. LH39 antigen could be first detected in fetal skin at the dermo-epidermal junction at 7 weeks estimated gestational age but was not present in the dermal vasculature until 16 weeks. When tested against tissue from a range of lower mammalian species, LH39 antigen appeared to be primate-specific. The epithelial basement membrane zone in organotypical cultures, where there is de novo synthesis of basement membrane components, contained abundant LH39 antigen in contrast to other basement membrane components, type IV collagen, laminin, and type VII collagen. Ultrastructural localization of LH39 epitope, using immunogold electron microscopy on unfixed freshly frozen tissue, was to the lamina lucida. No cross-reactivity could be detected between LH39 and laminin, fibronectin, and collagens I, III, IV, and V using the ELISA assay. In vitro studies with a range of proteolytic enzymes suggested that the antigen was non-collagenous in nature. LH39 precipitated a polypeptide with a molecular weight of 185 kD from extracts of metabolically labelled cultured keratinocytes, and polypeptides of 185 and 200 kD from the culture medium. The tissue distribution of LH39 antigen suggested that it may be an epitope within anchoring filaments. Potential applications of this antibody include the study of benign and malignant human vascular disorders, diseases and tumours associated with angiogenesis, epithelial neoplasms, and conditions of tissue regeneration and repair, such as wound healing.

Adult

Identification of basic fibroblast growth factor sensitivity and receptor and ligand expression in human colon tumor cell lines.

Basic fibroblast growth factor (bFGF) has been shown to be mitogenic to many different eukaryotic cell lines of mesodermal and neuroectodermal origin. Addition of exogenous bFGF to the chemically defined media of five characterized human colon tumor cell lines, cultured in the absence of epidermal growth factor (EGF), resulted in stimulation of growth from 24% to 146% in four of five cell lines, as measured by a colorimetric MTT assay. A positive dose-response relationship was observed when colon cells were treated with bFGF concentrations from 1 pM to 1 nM. bFGF showed a cumulative effect with EGF in stimulating the proliferation of colon tumor cells. The growth-inhibitory effect of exogenous transforming growth factor-beta (TGF-beta) on these cells was abolished by bFGF. When colon tumor cells were examined on immunoblots with a fibroblast growth factor (FGF) receptor-specific antibody, bands were detected at apparent molecular weights of 131 and 145 kDa. Conditioned media and cell lysates from the same human colon tumor cell lines were immunoprecipitated with a bFGF-specific antibody. An immunoreactive band was detected that comigrated with authentic human recombinant bFGF (16 kDa). Furthermore, preabsorption of anti-bFGF antibody with authentic ligand blocked immunodetection of the 16 kDa band on immunoblots. Documentation of a bFGF response, receptor, and ligand expression in human colon tumor cell lines is novel, and may represent a more widespread role for FGF that extends to epithelial cells and tumors of endodermal germ layer origin. The expression of both ligand and receptors by these cells indicates that bFGF could be involved in their growth regulation at the autocrine level.

Adenocarcinoma

Binding of plant lectins to mycoplasma cells and membranes.

The binding of iodinated wheat germ agglutinin, Ricinus communis agglutinin, and concanavalin A to mycoplasma cells and membranes was examined. All mycoplasmas studied specifically bound concanavalin A or R. communis agglutinin and, to a lesser degree, wheat germ agglutinin. The binding of lectins to whole cells was similar to that recorded for membranes, suggesting that significant binding only occurred on the outer surface of the mycoplasma membrane. Proteolysis of the membrane almost always increased the capacity to bind lectins, which indicates that additional carbohydrate groups on the mycoplasma membrane are masked by a protein layer or protein complexes on the membrane. The observation that carbohydrates are apparently exposed on the surface of mycoplasma membranes should stimulate more concentrated study on the isolation and chemical characterization of these substances since it is quite likely that they are responsible for a variety of reactions between mycoplasmas and host cells.

Acholeplasma

The laminar distribution of macaque tectobulbar and tectospinal neurons.

The superior colliculus exerts its most direct influence over orienting movements, and saccades in particular, via its descending projections to the brain stem and spinal cord. However, while there is detailed physiological data concerning the generation of saccade-related activity in the primate superior colliculus, there is relatively little data on the detailed connectivity of this structure in primates. Consequently, retrograde transport techniques were utilized to determine the locations of the cells of origin of these descending pathways in macaque monkeys. Tectal cells that projected to the ipsilateral pontine reticular formation were mainly found in the deep gray layer and occasionally in the intermediate gray layer. Tectal cells that projected to the contralateral pontine reticular formation were predominantly located in the intermediate gray layer. The contralaterally projecting population could be subdivided into two groups. The cells in upper sublamina of the intermediate gray layer project primarily to the saccade-related regions of the paramedian reticular formation. Cells in the lower sublamina project primarily to more lateral regions of the pontine reticular formation and to the spinal cord. We conclude that the primate colliculus is provided with at least three descending output channels, which are likely to differ in their connections and functions. Specifically, it seems likely that the lower portion of the intermediate gray layer may be specialized to subserve combined head and eye orienting movements, while the upper sublamina subserves saccades.

Animals