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Developmental Biochemistry of Cottonseed Embryogenesis and Germination : XIII. REGULATION OF BIOSYNTHESIS OF PRINCIPAL STORAGE PROTEINS.

The synthesis of the principal cottonseed storage proteins during embryogenesis has been followed by analyses of stained protein gels and of fluorographs of protein synthesized in vivo and from purified RNA in vitro in the wheat germ system. The kinetics of in vivo labeling as well as immunochemical cross-reactivity indicate that the 52- and 48-kilodalton mature storage protein sets are derived from 70- and 67-kilodalton precursor protein sets that are abundant proteins in embryonic cotyledons and disappear in late embryogenesis. Identification of the initial translation products of the storage protein mRNA has not been clearly established although products of apparent molecular weights of 69,000 and 60,000 are the likely storage protein precursors.Storage protein synthesis falls off markedly in late embryogenesis simultaneously with the loss of a superabundant class of mRNAs (shown by cDNA:RNA reassociation) that are presumed to be those for the storage proteins. The synthesis of these proteins ceases abruptly when immature embryos are removed from the boll and allowed to germinate precociously or when this precocious germination is prevented by incubation in abscisic acid. Thus, abscisic acid is not implicated in the expression of the storage protein genes.A scheme involving co-translational processing into vesicles, glycosylation, and slow in situ cleavage to produce the mature storage proteins is proposed.

Journal Article↗

Interruption of Somatic Embryogenesis in Daucus carota L. by 5-Bromodeoxyuridine.

Embryogenic Daucus carota L. cells grown in 9 micromolar 2.4-dichlorophenoxyacetic acid are resistant to greater than 5 micromolar 5-bromodeoxyuridine (BrdU). In contrast, 5 micromolar BrdU strongly inhibits somatic embryogenesis within 24 hours after transfer of cells to an auxin-free medium. DNA synthesis rates in control and BrdU-treated cultures are rapid and similar; however, the DNA content does not reach levels as great in the presence of BrdU as in control cultures. BrdU substitutes for thymidine in the DNA in 28% of the available sites 48 hours after auxin removal. Following DNA repair, somatic embryogenesis resumes. BrdU DNA incorporation leads to somatic embryogenesis inhibition and provides an alternative to auxin treatment for the interruption of carrot cell culture differentiation.

Journal Article↗

Soybean Seed Protein Genes Are Regulated Spatially during Embryogenesis.

We used in situ hybridization to investigate Kunitz trypsin inhibitor gene expression programs at the cell level in soybean embryos and in transformed tobacco seeds. The major Kunitz trypsin inhibitor mRNA, designated as KTi3, is first detectable in a specific globular stage embryo region, and then becomes localized within the axis of heart, cotyledon, and maturation stage embryos. By contrast, a related Kunitz trypsin inhibitor mRNA class, designated as KTi1/2, is not detectable during early embryogenesis. Nor is the KTi1/2 mRNA detectable in the axis at later developmental stages. Outer perimeter cells of each cotyledon accumulate both KTi1/2 and KTi3 mRNAs early in maturation. These mRNAs accumulate progressively from the outside to inside of each cotyledon in a "wave-like" pattern as embryogenesis proceeds. A similar KTi3 mRNA localization pattern is observed in soybean somatic embryos and in transformed tobacco seeds. An unrelated mRNA, encoding [beta]-conglycinin storage protein, also accumulates in a wave-like pattern during soybean embryogenesis. Our results indicate that cell-specific differences in seed protein gene expression programs are established early in development, and that seed protein mRNAs accumulate in a precise cellular pattern during seed maturation. We also show that seed protein gene expression patterns are conserved at the cell level in embryos of distantly related plants, and that these patterns are established in the absence of non-embryonic tissues.

Journal Article↗

Laser capture microdissection for the analysis of gene expression during embryogenesis of Arabidopsis.

It is during embryogenesis that the body plan of the developing plant is established. Analysis of gene expression during embryogenesis has been limited due to the technical difficulty of accessing the developing embryo. Here we demonstrate that laser capture microdissection can be applied to the analysis of embryogenesis. We show how this technique can be used in concert with DNA microarray for the large-scale analysis of gene expression in apical and basal domains of the globular-stage and heart-stage embryo, respectively, when critical events of polarity, symmetry and biochemical differentiation are established. This high resolution spatial analysis shows that up to approximately 65% of the genome is expressed in the developing embryo, and that differential expression of a number of gene classes can be detected. We discuss the validity of this approach for the functional analysis of both published and previously uncharacterized essential genes.

Arabidopsis↗

Ci-Rga, a gene encoding an MtN3/saliva family transmembrane protein, is essential for tissue differentiation during embryogenesis of the ascidian Ciona intestinalis.

A novel gene (Ci-Rga) essential for tissue differentiation during embryogenesis of the ascidian Ciona intestinalis is reported here. This gene was identified through functional screening of Ciona genes required for development by translational inhibition experiments with morpholino antisense oligonucleotides. The deduced protein of Ci-Rga contains two copies of a domain with unknown function called the MtN3/saliva domain. Phylogenetic analysis showed that Ci-Rga belongs to the MtN3/saliva family of genes conserved among metazoans and plants, and is an ortholog of mouse Rga (Recombination-activating gene 1 gene activation). During Ciona embryogenesis, both maternal and zygotic transcripts of Ci-Rga were expressed. Translational inhibition of Ci-Rga with specific morpholino resulted in abnormal embryos in which the cleavage pattern became atypical and expression of marker genes for each of the six major tissues, namely the endoderm, muscle, mesenchyme, notochord, neural tissue, and epidermis, was lost or suppressed at the tailbud stage. Although differentiation of all the six major tissues was affected by Ci-Rga knock-down, the degree of abnormalities and the timing of appearance of abnormalities were different among tissues. Expression analysis of developmentally important genes involved in the fate specification, such as Ci-Bra, Ci-Twist-like1a, Ci-Otx, Ci-Fgf9/16/20, Ci-Lhx3, Ci-FoxD, and Ci-Tbx6b, showed that an initial step of the fate specification of notochord, mesenchyme, and neural tissue, but not of endoderm or muscle, is impaired in the knock-down embryo. These results showed that Ci-Rga is a multifunctional gene essential for tissue differentiation during embryogenesis, and is primarily required for the fate specification of notochord, mesenchyme, and neural tissue, and provide some insights into the function of this little-known group of genes.

Amino Acid Sequence↗

Congenital malformations in offspring of diabetic women treated with oral hypoglycaemic agents during embryogenesis.

A markedly increased risk (50%) of congenital malformations in the offspring of women treated with oral hypoglycaemic agents during the first trimester has recently been reported. With this background, the medical records of a consecutive sample of 25 pregnant Type 2 diabetic women treated with oral hypoglycaemic agents during embryogenesis between 1966 and 1991 in the diabetic service of a university hospital, were studied retrospectively. None of the infants had major congenital malformations disclosed in the neonatal period (0%, 97.5% confidence interval 0.0-13.7%), but one minor congenital malformation was found (4.0%, 95% confidence interval 0.1-20.3%). Although this study, due to the limited number of pregnancies examined, does not exclude an association between treatment with oral hypoglycaemic agents at the time of embryogenesis and major congenital malformations in the offspring, the previously reported association was not confirmed. Thus we find no obvious indication for therapeutic abortions in patients who have accidentally been treated with oral hypoglycaemic agents during embryogenesis. On the contrary it seems reasonable to reassure these women with respect to their risk of having a malformed baby, stop the treatment with oral hypoglycaemic agents and initiate insulin treatment.

Abnormalities, Drug-Induced↗

Cold shock domain family members YB-1 and MSY4 share essential functions during murine embryogenesis.

Three cold shock domain (CSD) family members (YB-1, MSY2, and MSY4) exist in vertebrate species ranging from frogs to humans. YB-1 is expressed throughout embryogenesis and is ubiquitously expressed in adult animals; it protects cells from senescence during periods of proliferative stress. YB-1-deficient embryos die unexpectedly late in embryogenesis (embryonic day 18.5 [E18.5] to postnatal day 1) with a runting phenotype. We have now determined that MSY4, but not MSY2, is also expressed during embryogenesis; its abundance declines substantially from E9.5 to E17.5 and is undetectable on postnatal day 1(adult mice express MSY4 in testes only). Whole-mount analysis revealed similar patterns of YB-1 and MSY4 RNA expression in E11.5 embryos. To determine whether MSY4 delays the death of YB-1-deficient embryos, we created and analyzed MSY4-deficient mice and then generated YB-1 and MSY4 double-knockout embryos. MSY4 is dispensable for normal development and survival, but the testes of adult mice have excessive spermatocyte apoptosis and seminiferous tubule degeneration. Embryos doubly deficient for YB-1 and MSY4 are severely runted and die much earlier (E8.5 to E11.5) than YB-1-deficient embryos, suggesting that MSY4 indeed shares critical cellular functions with YB-1 in the embryonic tissues where they are coexpressed.

Animals↗

Somatic Embryogenesis, Rhizogenesis, and Morphinan Alkaloids Production in Two Species of Opium Poppy.

A study of somatic embryogenesis and rhizogenesis and their influence on production of morphinan alkaloids on two species of opium poppy is presented. We identified the ratios of auxin and cytokinin that caused somatic embryogenesis and rhizogenesis in hypocotyl and cotyledons of Papaver somniferum album and Papaver orientale splendidissimum. The hypocotyls and cotyledons both show somatic embryogenesis in Papaver somniferum album whereas only the cotyledons were embryogenic in Papaver orientale splendidissimum. For rhizogenesis, the most important response is on the cotyledons and leaves in these two species. Histology showed characteristic stages of somatic embryo: Globular, cotyledonous, and heart cotyledonary. High performance liquid chromatography analysis showed that the roots of both species synthesized codeine, thebaine, and papaverine. Morphine was only detected in aerial parts of Papaver somniferum album. Codeine and thebaine were detected in the rhizogenous but no embryonic callus. These results suggest that root organogenesis is causally related to alkaloid biosynthesis.

Journal Article↗

A novel role for vitamin K1 in a tyrosine phosphorylation cascade during chick embryogenesis.

The development of the embryo is dependent upon a highly coordinated repertoire of cell division, differentiation, and migration. Protein-tyrosine phosphorylation plays a pivotal role in the regulation of these processes. Vitamin K-dependent gamma-carboxylated proteins have been identified as ligands for a unique family (Tyro 3 and 7) of receptor tyrosine kinases (RTKs) with transforming ability. The involvement of vitamin K metabolism and function in two well characterized birth defects, warfarin embryopathy and vitamin K epoxide reductase deficiency, suggests that developmental signals from K-dependent pathways may be required for normal embryogenesis. Using a chick embryogenesis model, we now demonstrate the existence of a vitamin K1-dependent protein-tyrosine phosphorylation cascade involving c-Eyk, a member of the Tyro 12 family, and key intracellular proteins, including focal adhesion kinase (pp125FAK), paxillin, and pp60src. This cascade is sensitive to alteration in levels or metabolism of vitamin K1. These findings provide a major clue as to why, in the mammalian (and human) fetus, the K-dependent proteins are maintained in an undercarboxylated state, even to the point of placing the newborn at hemorrhagic risk. The precise regulation of vitamin K1-dependent regulatory pathways would appear to be critical for orderly embryogenesis.

Amino Acid Sequence↗

Expression of histidine decarboxylase and cellular histamine-like immunoreactivity in rat embryogenesis.

In this study we investigated the developmental expression of histidine decarboxylase (HDC) mRNA and the distribution of histamine-immunoreactive (histamine-ir) cells in the rat embryonic tissues. We applied Northern blot analysis, in situ hybridization with synthetic oligonucleotide probes complementary to the rat HDC cDNA, and indirect histamine immunocytochemistry. Northern blot analysis revealed the appearance of a major (2.6 KB) HDC mRNA species in liver on embryonic Day 14. Its hybridization level peaked on Day E18, when two minor (1.6 and 3.5 KB) mRNA species were also present. During the periparturition period, a rapid decrease in HDC RNA was apparent, as the 2.6 KB mRNA species was expressed at a low level on postnatal Day P1. The embryonic liver expressed HDC on days E14-E20. On days E18 and E20, the periosteum and the epiphyseal growth plates of the endochondrally ossificating bones, and some striated muscle cells, showed hybridization signal for HDC. Histamine immunoreactivity was detected in many epithelial and neuronal cell types during embryogenesis. An intense histamine immunoreaction appeared first in essentially all cells of the liver parenchyma on day E12. This parenchymal histamine immunoreactivity disappeared by birth, after which this immunofluorescence in liver was restricted to a few scattered mast cells until adulthood. Some neurons in the peripheral sensory, sympathetic and cranial nerve ganglia were histamine-immunoreactive from day E16 to birth. In addition, many immunoreactive nerve fibers were detected in the gastrointestinal muscularis externa, mesentery, salivary glands, kidney, lung, and muscle tissue. We conclude that during rat embryogenesis histamine is produced and stored transiently by cells in liver, developing bone, and a few striated muscle cells, in addition to previously reported neurons in rat brain. Many peripheral neurons, epithelial cells, and mast cells display histamine immunoreactivity during rat embryogenesis but are devoid of detectable HDC mRNA with the current method. It remains possible that histamine is formed by another enzyme or is taken up from the extracellular space. The results support the concept that a significant proportion of histamine is formed and stored by embryonic cells other than mast cells.

Animals↗

Genetic chimerism of Vitis vinifera cv. Chardonnay 96 is maintained through organogenesis but not somatic embryogenesis.

BACKGROUND: Grapevine can be a periclinal chimera plant which is composed at least of two distinct cell layers (L1, L2). When the cell layers of this plant are separated by passage through somatic embryogenesis, regenerated plants could show distinct DNA profiles and a novel phenotype which proved different from that of the parent plant. RESULTS: Genetically Chardonnay clone 96 is a periclinal chimera plant in which is L1 and L2 cell layers are distinct. Plants obtained via organogenesis through meristematic bulks are shown to be composed of both cell layers. However, plants regenerated through somatic embryogenesis starting from anthers or nodal explants are composed only of L1 cells. These somaclones do not show phenotypic differences to the parental clone up to three years after regeneration. Interestingly, the only somaclone showing an atypical phenotype (asymmetric leave) shows a genotypic modification. CONCLUSION: These results suggest that the phenotype of Chardonnay 96 does not result from an interaction between the two distinct cell layers L1 and L2. If phenotype conformity is further confirmed, somatic embryogenesis will result in true-to-type somaclones of Chardonnay 96 and would be well suitable for gene transfer.

Chimera↗

Increase of mitochondrial DNA content and transcripts in early bovine embryogenesis associated with upregulation of mtTFA and NRF1 transcription factors.

BACKGROUND: Recent work has shown that mitochondrial biogenesis and mitochondrial functions are critical determinants of embryonic development. However, the expression of the factors controlling mitochondrial biogenesis in early embryogenesis has received little attention so far. METHODS: We used real-time quantitative PCR to quantify mitochondrial DNA (mtDNA) in bovine oocytes and in various stages of in vitro produced embryos. To investigate the molecular mechanisms responsible for the replication and the transcriptional activation of mtDNA, we quantified the mRNA corresponding to the mtDNA-encoded cytochrome oxidase 1 (COX1), and two nuclear-encoded factors, i.e. the Nuclear Respiratory Factor 1 (NRF1), and the nuclear-encoded Mitochondrial Transcription Factor A (mtTFA). RESULTS: Unlike findings reported in mouse embryos, the mtDNA content was not constant during early bovine embryogenesis. We found a sharp, 60% decrease in mtDNA content between the 2-cell and the 4/8-cell stages. COX1 mRNA was constant until the morula stage after which it increased dramatically. mtTFA mRNA was undetectable in oocytes and remained so until the 8/16-cell stage; it began to appear only at the morula stage, suggesting de novo synthesis. In contrast, NRF1 mRNA was detectable in oocytes and the quantity remained constant until the morula stage. CONCLUSION: Our results revealed a reduction of mtDNA content in early bovine embryos suggesting an active process of mitochondrial DNA degradation. In addition, de novo mtTFA expression associated with mitochondrial biogenesis activation and high levels of NRF1 mRNA from the oocyte stage onwards argue for the essential function of these factors during the first steps of bovine embryogenesis.

Animals↗

Deiodinase activity is present in Xenopus laevis during early embryogenesis.

Thyroid hormones orchestrate amphibian metamorphosis. The type 2 and type 3 deiodinases make vital contributions to this process by controlling levels of the thyroid hormones T(4) and T(3) available to different tissues. Because the tadpole thyroid gland is not functional until stage NF44, it has been widely assumed that thyroid signaling is absent during amphibian early development, thyroid hormone only becoming a major regulator during premetamorphic stages. Similarly, in mammals, thyroid function is known to be essential to neuronal development, especially during the perinatal stages, but again little is known about early stages of development. Here we demonstrate that key elements of thyroid hormone signaling are present during early development of Xenopus. In particular, we find functional thyroid hormone-activating deiodinases and significant levels of their substrates, T(4) and T(3), during early embryogenesis. Furthermore, we have further characterized a recently identified deiodinase in amphibians, homologous to mammalian type 1 deiodinase (D1). This enzyme is expressed in marked, spatially defined patterns during embryogenesis. The patterns of expression of type 1 deiodinase are distinct from those of type 2 and type 3 deiodinases. Deiodinase expression is found in neurogenic areas from stage NF30 onward, both in the central and peripheral nervous systems. We conclude that both activating and inactivating deiodinases show dynamic patterns of expression during early embryogenesis in amphibians, particularly in neurogenic areas. These findings suggest that thyroid hormone signaling is a key component of early neuronal development in vertebrates.

Animals↗

Expression of messenger ribonucleic acids encoding the inhibin/activin system during mid- and late-gestation rat embryogenesis.

We recently demonstrated that inhibin/activin alpha-, beta A-, and beta B-subunit messenger RNAs (mRNAs) are localized in a variety of embryonic rat tissues from 12-20 days post coitum (pc) and reported localizations consistent with possible growth effects of activin during rat embryogenesis. In the present study, we examined the tissue-specific distribution of mRNAs encoding all known players of the inhibin/activin system. In situ hybridization with radiolabeled RNA probes specific for mouse activin receptors (ActRII and ActRIIB), rat follistatin, and rat inhibin/activin subunits was used to examine the spatiotemporal expression of these molecules in adjacent sections of postimplantation rat embryos (8-20 days pc) as well as in midgestation placenta and uterine tissues (8-12 days pc). With the exception of the dorsal root ganglion and salivary gland, alpha- and beta B-subunit mRNAs were found exclusively in reproductive tissues (brain, pituitary, and/or gonads). beta A-Subunit mRNA signal was observed in the brain and gonads as well as in a variety of other tissues during embryogenesis. ActRII mRNA was found exclusively in neuronal tissue from 14 days pc until birth. ActRIIB mRNA was also found in brain, spinal cord, and ganglion, but usually appeared earlier in development than the ActRII message. ActRIIB message was also expressed in a number of other tissues, in some cases along with beta A-subunit mRNA. In these tissues, ActRIIB expression was confined to epithelial and endothelial cell types. Follistatin message was observed in all tissues (except the heart and vessels) localizing beta A-subunit and/or ActRIIB but not in the same cell type. Outside the embryo, beta A-subunit mRNA was localized in the decidua capsularis during midgestation, whereas ActRIIB message was found in placenta as early as 9 days pc. Expression of follistatin message was apparent in decidua from 8-11 days pc, then disappeared from this tissue and was abundant in myometrium at 12 days pc. These data suggest that: 1) inhibin and activin regulate aspects of the fetal reproductive system, whereas activin A may regulate the growth and differentiation of many embryonic tissues; 2) ActRII and ActRIIB serve different roles during development of the rat embryo; and 3) follistatin is in a position to modulate the effects of activin during postimplantation rat embryogenesis.

Activins↗

Split cord malformation: Part I: A unified theory of embryogenesis for double spinal cord malformations.

Much confusion still exists concerning the pathological definitions and clinical significance of double spinal cord malformations. Traditional terms used to describe the two main forms of these rare malformations, diastematomyelia and diplomyelia, add to the confusion by their inconsistent usage, ambiguities, and implications of their dissimilar embryogenesis. Based on the detailed radiographic and surgical findings of 39 cases of double cord malformations and the autopsy data on two other cases, this study endorses a new classification for double cord malformations and proposes a unified theory of embryogenesis for all their variant forms and features. The new classification recommends the term split cord malformation (SCM) for all double spinal cords. A Type I SCM consists of two hemicords, each contained within its own dural tube and separated by a dura-sheathed rigid osseocartilaginous median septum. A Type II SCM consists of two hemicords housed in a single dural tube separated by a nonrigid, fibrous median septum. These two essential features necessary for typing, the state of the dural tube and the nature of the median septum, do not ever overlap between the two main forms and can always be demonstrated by imaging studies so that accurate preoperative typing is always possible. All other associated structures in SCM such as paramedian nerve roots, myelomeningoceles manqué, and centromedian vascular structures frequently do overlap between types and are not reliable typing criteria. The unified theory of embryogenesis proposes that all variant types of SCMs have a common embryogenetic mechanism. Basic to this mechanism is the formation of adhesions between ecto- and endoderm, leading to an accessory neurenteric canal around which condenses an endomesenchymal tract that bisects the developing notochord and causes formation of two hemineural plates. The altered state of the emerging split neural tube and the subsequent ontogenetic fates of the constituent components of the endomesenchymal tract ultimately determine the configuration and orientation of the hemicords, the nature of the median septum, the coexistence of various vascular, lipomatous, neural, and fibrous oddities within the median cleft, the high association with open myelodysplastic and cutaneous lesions, and the seemingly unlikely relationship with fore and midgut anomalies. The multiple facets of this theory are presented in increasing complexity against the background of known embryological facts and theories; the validity of each facet is tested by comparing structures and phenomena predicted by the facet with actual radiographic, surgical, and histopathological findings of these 41 cases of SCM.

Adolescent↗

Beta 3 tubulin expression characterizes the differentiating mesodermal germ layer during Drosophila embryogenesis.

During embryogenesis, the beta 3 tubulin gene of Drosophila is transcribed predominantly in the mesoderm. We have raised antibodies specific to the C-terminal domain of the beta 3 tubulin and analysed by immunostaining the distribution of this tubulin isotype during Drosophila embryogenesis. The protein is first detectable in the cephalic mesoderm at maximal germband extension. Shortly afterwards, beta 3 tubulin is expressed in single cells at identical positions of the thoracic and abdominal segments. We suggest that these cells represent muscle pioneer cells of Drosophila. During later embryonic development the somatic musclature, visceral musculature, dorsal vessel and macrophages contain beta 3 tubulin. In dorsalizing mutants dorsal, snail and twist, which do not form a ventral furrow during gastrulation, beta 3 expression is greatly reduced but not completely abolished. Our analysis shows that beta 3 tubulin immunostaining characterizes the differentiation of mesodermal derivatives during embryogenesis.

Animals↗

The regulation of the cell cycle during Drosophila embryogenesis: the transition to polyteny.

The process of polytenization plays a crucial role in Drosophila development, and most of the larval tissues are polytene. By analyzing the pattern of DNA replication in embryos pulse-labeled with BrdU, we show that many larval tissues undergo a transition to begin becoming polytene late in embryogenesis. Our results demonstrate that in these larval tissues polyteny results from a modified cell cycle, the endo cell cycle, in which there is only an S (synthesis) phase and a G (gap) phase. A key regulator of the mitotic cell cycle, the product of the string gene (the Drosophila homologue of cdc25), is not required for the endo cell cycle. The developmental regulation of the endo cell cycle is striking in that tissue-specific domains undergo polytene DNA replication in a dynamic pattern at defined times in embryogenesis. During subsequent rounds of the endo cell cycle in late embryogenesis and first instar larval development, the domains are subdivided and the temporal control is not as rigid. The length of the G phase varies among different tissues. By quantifying DNA content, we show that during the early polytene S phases the genome is not fully duplicated.

Animals↗

Expression of the two mannose 6-phosphate receptors is spatially and temporally different during mouse embryogenesis.

Mammalian cells express two mannose 6-phosphate receptors, MPR46 and MPR300, both of which mediate the targeting of lysosomal enzymes to lysosomes. Additionally the receptors mediate the secretion (MPR46) and the endocytosis (MPR300) of lysosomal enzymes and the binding of IGFII (MPR300). We have analyzed the distribution of MPR46 and MPR300 transcripts during mouse embryogenesis by in situ hybridization. Up to day 15.5 of embryonic development we found a non-overlapping distribution of the transcripts for the two receptors. High expression of MPR46 was observed at sites of hemopoiesis and in the thymus while MPR300 was highly expressed in the cardiovascular system. Late in embryogenesis (day 17.5) a wide variety of tissues expressed the receptors, but still the expression pattern was almost non-overlapping. This unexpected complementary expression pattern points to specific functions of the two mannose 6-phosphate receptors during mouse embryogenesis.

Animals↗