pinecone/core-2020-05-10-deduplication huggingface dataset:
Identification of Endogenous Retinoids, Enzymes, Binding Proteins, and Receptors during Early Postimplantation Development in Mouse: Important Role of Retinal Dehydrogenase Type 2 in Synthesis of All-trans-Retinoic Acid

AbstractSpecific combinations of nuclear retinoid receptors acting as ligand-inducible transcription factors mediate the essential role of retinoids in embryonic development. Whereas some data exist on the expression of these receptors during early postimplantation development in mouse, little is known about the enzymes controlling the production of active ligands for the retinoid receptors. Furthermore, at early stages of mouse development virtually no data are available on the presence of endogenous retinoids. In the present study we have used a recently developed high-performance liquid chromatographic (HPLC) technique to identify endogenous retinoids in mouse embryos down to the egg cylinder stage. All-trans-retinoic acid, a ligand for the retinoic acid receptors, was detected in embryos dissected as early as 7.5 dpc (i.e., a combination of midstreak until late allantoic bud stage embryos). At these stages, we detected mRNA coding for all the retinoid receptors, retinoid binding proteins, and two enzymes able to convert retinol to retinal (retinol dehydrogenase 5 (RDH5) and alcohol dehydrogenase 4 (ADH4)). We also detected retinal dehydrogenase type 2 (RALDH2), an enzyme capable of oxidising the final step in the all-trans-retinoic acid synthesis. In egg cylinder stage mouse embryos no all-trans-retinoic acid was detected. However, at this stage its precursor all-trans-retinal was present. In accordance with these HPLC observations, RDH5 and ADH4 were expressed, but no transcripts coding for enzymes that oxidise retinal to retinoic acid. Therefore, our results suggest that RALDH2 is a key regulator in initiating retinoic acid synthesis sometime between the mid-primitive streak stage and the late allantoic bud stage in mouse embryos
Targeted Misexpression of Constitutively Active BMP Receptor-IB Causes Bifurcation, Duplication, and Posterior Transformation of Digit in Mouse Limb

AbstractMembers of bone morphogenetic proteins (BMPs) play important roles in many aspects of vertebrate embryogenesis. In developing limbs, BMPs have been implicated in control of anterior–posterior patterning, outgrowth, chondrogenesis, and apoptosis. These diverse roles of BMPs in limb development are apparently mediated by different BMP receptors (BMPR). To identify the developmental processes in mouse limb possibly contributed by BMP receptor-IB (BMPR-IB), we generated transgenic mice misexpressing a constitutively active Bmpr-IB (caBmpr-IB). The transgene driven by the mouse Hoxb-6 promoter was ectopically expressed in the posterior mesenchyme of the forelimb bud, the lateral plate mesoderm, and the whole mesenchyme of the hindlimb bud. While the forelimbs appeared normal, the transgenic hindlimbs exhibited several phenotypes, including bifurcation, preaxial polydactyly, and posterior transformation of the anterior digit. However, the size of bones in the transgenic limbs seemed unaltered. Defects in sternum and ribs were also found. The bifurcation in the transgenic hindlimb occurred early in the limb development (E10.5) and was associated with extensive cell death in the mesenchyme and occasionally in the apical ectodermal ridge (AER). Sonic hedgehog (Shh) and Patched (Ptc) expression appeared unaffected in the transgenic limb buds, suggesting that the BMPR-IB mediated signaling pathway is downstream from Shh. However, ectopic Fgf4 expression was found in the anterior AER, which may account for the duplication of the anterior digit. An ectopic expression of Gremlin found in the transgenic limb bud would be responsible for the ectopic Fgf4 expression. The observations that Hoxd-12 and Hoxd-13 expression patterns were extended anteriorly provide a molecular basis for the posterior transformation of the anterior digit. Together these results suggest that BMPR-IB is the endogenous receptor to mediate the role of BMPs in anterior–posterior patterning and apoptosis in mouse developing limb. In addition, BMPR-IB may represent a critical component in the Shh/FGF4 feedback loop by regulating Gremlin expression
The Lens Organizes the Anterior Segment: Specification of Neural Crest Cell Differentiation in the Avian Eye

AbstractDuring the development of the anterior segment of the eye, neural crest mesenchyme cells migrate between the lens and the corneal epithelium. These cells contribute to the structures lining the anterior chamber: the corneal endothelium and stroma, iris stroma, and trabecular meshwork. In the present study, removal of the lens or replacement of the lens with a cellulose bead led to the formation a disorganized aggregate of mesenchymal cells beneath the corneal epithelium. No recognizable corneal endothelium, corneal stroma, iris stroma, or anterior chamber was found in these eyes. When the lens was replaced immediately after removal, a disorganized mass of mesenchymal cells again formed beneath the corneal epithelium. However, 2 days after surgery, the corneal endothelium and the anterior chamber formed adjacent to the lens. When the lens was removed and replaced such that only a portion of its anterior epithelial cells faced the cornea, mesenchyme cells adjacent to the lens epithelium differentiated into corneal endothelium. Mesenchyme cells adjacent to lens fibers did not form an endothelial layer. The cell adhesion molecule, N-cadherin, is expressed by corneal endothelial cells. When the lens was removed the mesenchyme cells that accumulated beneath the corneal epithelium did not express N-cadherin. Replacement of the lens immediately after removal led to the formation of an endothelial layer that expressed N-cadherin. Implantation of lens epithelia from older embryos showed that the lens epithelium maintained the ability to support the expression of N-cadherin and the formation of the corneal endothelium until E15. This ability was lost by E18. These studies provide evidence that N-cadherin expression and the formation of the corneal endothelium are regulated by signals from the lens. N-cadherin may be important for the mesenchymal-to-epithelial transformation that accompanies the formation of the corneal endothelium
Analysis of Gene Expressions during Xenopus Forelimb Regeneration

AbstractXenopus laevis can regenerate an amputated limb completely at early limb bud stages, but the metamorphosed froglet gradually loses this capacity and can regenerate only a spike-like structure. We show that the spike formation in a Xenopus froglet is nerve dependent as is limb regeneration in urodeles, since denervation concomitant with amputation is sufficient to inhibit the initiation of blastema formation and fgf8 expression in the epidermis. Furthermore, in order to determine the cause of the reduction in regenerative capacity, we examined the expression patterns of several key genes for limb patterning during the spike-like structure formation, and we compared them with those in developing and regenerating limb buds that produce a complete limb structure. We cloned Xenopus HoxA13, a marker of the prospective autopodium region, and the expression pattern suggested that the spike-like structure in froglets is accompanied by elongation and patterning along the proximodistal (PD) axis. On the other hand, shh expression was not detected in the froglet blastema, which expresses fgf8 and msx1. Thus, although the wound epidermis probably induces outgrowth of the froglet blastema, the polarizing activity that organizes the anteroposterior (AP) axis formation is likely to be absent there. Our results demonstrate that the lost region in froglet limbs is regenerated along the PD axis and that the failure of organization of the AP pattern gives rise to a spike-like incomplete structure in the froglet, suggesting a relationship between regenerative capacity and AP patterning. These findings lead us to conclude that the spike formation in postometamorphic Xenopus limbs is epimorphic regeneration
The zebrafish floating head mutant demonstrates podocytes play an important role in directing glomerular differentiation

In zebrafish, the pronepheric glumerulus occupies a middle position underneath the notochord and is vascularized through angiogenic capillary ingrowth from the dorsal aorta. The midline mutants floating head (flh), sonic you (syu), and you-too (yot) provide the opportunity to study glnzuerolur differentiation in the absence of the notochord and vascularization from the dorsal aorta. In flh, syn, and yot mutants, glomeruli differentiate at ectopic lateral positions within the embryo and contain morphologically identifiable podocyte and endothelial cell types. In the absence of the dorsal aorta, endothelia from an alternate source are recruited by podocytes during glomerular vascularization to make functional glomeruli. Our results suggest that middle signals are required for proper glomerular morphogenesis but not for the differentiation of podocytes. Podocytes apper to play an important role in directing celluar recruitment events leading to glomerular differentiation. Furthermore, we find defects in sclerotomal development that correlate with defects in glomerular morphogenesis suggesting a possible link between the formation of these embryonic stuctures
Antagonizing Activity of Chick Grg4 against Tectum-Organizing Activity

AbstractAlar plate of chick mesencephalon differentiates into the optic tectum. It has been shown that factors expressed in the mes–metencephalic boundary induce the tectum and give positional specificity. Chick Grg4 is expressed at first in the anterior neural fold. The expression localizes from the posterior diencephalon to the mesencephalon by stage 10. To investigate the function of Grg4 in mesencephalic development, Grg4 overexpression was carried out by in ovo electroporation. After Grg4 overexpression, expression of En-2, Pax5, Fgf8, and EphrinA2 was repressed, and Pax6 was upregulated in the mesencephalic region. Grg4 overexpression caused the morphological change; mesencephalic swelling became smaller and the di–mesencephalic boundary shifted posteriorly, that is, the anterior limit of tectum shifted posteriorly. Importantly, cotransfection of Grg4 with Pax5 canceled the tectum-inducing activity of Pax5. These results suggest that Grg4 works as an antagonist against tectum-organizing activity. It was also shown that transfected N-terminal domains of Grg4 induced En-2 expression. Since N-terminal domains were transported to the nucleus in the neuroepithelium, they could act as dominant negative for endogenous Grg4. These results indicate that Grg4 has repressing activity against the organizing molecules and suggest that Grg4 plays important roles in formation of anterior tectal boundary and polarity
Medial Edge Epithelial Cell Fate during Palatal Fusion

AbstractTo explain the disappearance of medial edge epithelial (MEE) cells during palatal fusion, programmed cell death, epithelial–mesenchymal transformation, and migration of these cells to the oral and nasal epithelia have been proposed. However, MEE cell death has not always been accepted as a mechanism involved in midline epithelial seam disappearance. Similarly, labeling of MEE cells with vital lipophilic markers has not led to a clear conclusion as to whether MEE cells migrate, transform into mesenchyme, or both. To clarify these controversies, we first utilized TUNEL techniques to detect apoptosis in mouse palates at the fusion stage and concomitantly analyzed the presence of macrophages by immunochemistry and confocal microscopy. Second, we in vitro infected the MEE with the replication-defective helper-free retroviral vector CXL, which carries the Escherichia coli lacZ gene, and analyzed β-galactosidase activity in cells after fusion to follow their fate. Our results demonstrate that MEE cells die and transform into mesenchyme during palatal fusion and that dead cells are phagocytosed by macrophages. In addition, we have investigated the effects of the absence of transforming growth factor β3 (TGF-β3) during palatal fusion. Using environmental scanning electron microscopy and TUNEL labeling we compared the MEE of the clefted TGF-β3 null and wild-type mice. We show that MEE cell death in TGF-β3 null palates is greatly reduced at the time of fusion, revealing that TGF-β3 has an important role as an inducer of apoptosis during palatal fusion. Likewise, the bulging cells observed on the MEE surface of wild-type mice prior to palatal shelf contact are very rare in the TGF-β3 null mutants. We hypothesize that these protruding cells are critical for palatal adhesion, being morphological evidence of increased cell motility/migration
Bromodeoxyuridine Specifically Labels the Regenerative Stem Cells of Planarians

AbstractThe singular regenerative abilities of planarians require a population of stem cells known as neoblasts. In response to wounding, or during the course of cell turnover, neoblasts are signaled to divide and/or differentiate, thereby replacing lost cell types. The study of these pluripotent stem cells and their role in planarian regeneration has been severely hampered by the reported inability of planarians to incorporate exogenous DNA precursors; thus, very little is known about the mechanisms that control proliferation and differentiation of this stem cell population within the planarian. Here we show that planarians are, in fact, capable of incorporating the thymidine analogue bromodeoxyuridine (BrdU), allowing neoblasts to be labeled specifically during the S phase of the cell cycle. We have used BrdU labeling to study the distribution of neoblasts in the intact animal, as well as to directly demonstrate the migration and differentiation of neoblasts. We have examined the proposal that a subset of neoblasts is arrested in the G2 phase of the cell cycle by double-labeling with BrdU and a mitosis-specific marker; we find that the median length of G2 (∼6 h) is sufficient to account for the initial mitotic burst observed after feeding or amputation. Continuous BrdU-labeling experiments also suggest that there is not a large, slow-cycling population of neoblasts in the intact animal. The ability to label specifically the regenerative stem cells, combined with the recently described use of double-stranded RNA to inhibit gene expression in the planarian, should serve to reignite interest in the flatworm as an experimental model for studying the problems of metazoan regeneration and the control of stem cell proliferation
Primary Neuronal Differentiation in Xenopus Embryos Is Linked to the β3 Subunit of the Sodium Pump

AbstractIn amphibian embryos, activation of additional sodium pumps in neural plate cell membranes ensures that neural plate-derived neurons differentiate subsequently in the neural tube. When the sodium pump is inhibited during the mid-neural fold stages, neuronal differentiation fails. The effect is irreversible. We find that these events operate through the Na pump β3 subunit. When neural plate-specific Na pumps are activated, transcripts for β3 decline precipitately during the mid-neural fold stages, first in the neural plate and then in the dorsal mesoderm. As the neural tube closes, β3 returns, specifically in motor neurons and interneurons. Inhibition of the Na pump with the cardiac glycoside strophanthidin prevents the normal fall in β3 during neurulation: β3 is maintained in the neural plate until the neural tube closes, but lost from the dorsal mesoderm. Complete elimination of β3 transcripts from dorsal structures then occurs. Inhibiting the Na pump does not induce cell death (assessed by TUNEL staining) in the nervous system. Transcripts for X-Delta, NeuroD, and GSK3β are not affected by inhibition of the Na pump. Xotch and N-tubulin transcripts fall to very low levels and Xotch disappears permanently from the nervous system. When β3 transcript expression is maintained throughout neurulation, by over expression of injected mRNAs, Xotch is eliminated from the neural tube and somites and switches to the ectoderm
Brachyury (T) Expression in Embryos of a Larvacean Urochordate, Oikopleura dioica, and the Ancestral Role of T

AbstractThe Brachyury, or T, gene is required for notochord development in animals occupying all three chordate subphyla and probably also had this role in the last common ancestor of the chordate lineages. In two chordate subphyla (vertebrates and cephalochordates), T is also expressed during gastrulation in involuting endodermal and mesodermal cells, and in vertebrates at least, this expression domain is required for proper development. In the basally diverging chordate subphylum Urochordata, animals in the class Ascidiacea do not employ T during gastrulation in endodermal or nonaxial mesodermal cells, and it has been suggested that nonnotochordal roles for T were acquired in the cephalochordate–vertebrate lineage after it split with Urochordata. To test this hypothesis, we cloned T from Oikopleura dioica, a member of the urochordate class Appendicularia (or Larvacea), which diverged basally in the subphylum. Investigation of the expression pattern in developing Oikopleura embryos showed early expression in presumptive notochord precursor cells, in the notochord, and in parts of the developing gut and cells of the endodermal strand. We conclude that the ancestral role of T likely included expression in the developing gut and became necessary in chordates for construction of the notochord
Regulatory Mutations of the Drosophila Sox Gene Dichaete Reveal New Functions in Embryonic Brain and Hindgut Development

AbstractSox domain proteins encompass a conserved family of transcriptional regulators that are implicated in a variety of developmental processes in eukaryotes from worm to man. The Dichaete gene of Drosophila encodes a group B Sox protein related to mammalian Sox1, -2, and -3 and, like these proteins, it is widely and dynamically expressed throughout embryogenesis. In order to unravel new Dichaete functions, we characterized the organization of the Dichaete gene using a combination of regulatory mutant alleles and reporter gene constructs. Dichaete expression is tightly controlled during embryonic development by a complex of regulatory elements distributed over 25 kb downstream and 3 kb upstream of the transcription unit. A series of regulatory alleles which affect tissue-specific domains of Dichaete were used to demonstrate that Dichaete has functions in addition to those during segmentation and midline development previously described. First, Dichaete has functions in the developing brain. A specific group of neural cells in the tritocerebrum fails to develop correctly in the absence of Dichaete, as revealed by reduced expression of labial, zfh-2, wingless, and engrailed. Second, Dichaete is required for the correct differentiation of the hindgut. The Dichaete requirement in hindgut morphogenesis is, in part, via regulation of dpp, since ectopically supplied dpp can rescue Dichaete phenotypes in the hindgut. Taken together, there are now four distinct in vivo functions described for Dichaete that can be used as models for context-dependent comparative studies of Sox function
Composition, Synthesis, and Assembly of the Embryonic Chick Retinal Basal Lamina

AbstractTo study the biology of basal laminae in the developing nervous system the protein composition of the embryonic retinal basal lamina was investigated, the site of synthesis of its proteins in the eye was determined, and basal lamina assembly was studied in vivo in two assay systems. Laminin, nidogen, agrin, collagen IV, and XVIII are major constituents of the retinal basal lamina. However, only agrin is synthesized by the retina, whereas the other matrix constituents originate from cells of the ciliary body, the lens, or the optic disc. The synthesis from extraretinal tissues infers that the retinal basal lamina proteins must be shed from their tissues of origin into the vitreous body and from there bind to receptor proteins provided by the retinal neuroepithelium. The fact that all proteins typical for the retinal basal lamina are abundant in the vitreous body and a new basal lamina is only formed when the vitreous body was directly adjacent to the retina is consistent with the contention of the vitreous body having a function in retinal basal lamina formation. Basal lamina assembly was also studied after disrupting the retinal basal lamina by intraocular injection of collagenase. The basal lamina regenerated after chasing the collagenase with Matrigel, which served as a collagenase inhibitor. The basal lamina was reconstituted within 6 h. However, the regenerated basal lamina was located deeper in the retina than normal by reconstituting along the retracted neuroepithelial endfeet demonstrating that these endfeet are the preferred site of basal lamina assembly
The Ecdysone Regulatory Pathway Controls Wing Morphogenesis and Integrin Expression during Drosophila Metamorphosis

AbstractDrosophila imaginal discs are specified and patterned during embryonic and larval development, resulting in each cell acquiring a specific fate in the adult fly. Morphogenesis and differentiation of imaginal tissues, however, does not occur until metamorphosis, when pulses of the steroid hormone ecdysone direct these complex morphogenetic responses. In this paper, we focus on the role of ecdysone in regulating adult wing development during metamorphosis. We show that mutations in the EcR ecdysone receptor gene and crooked legs (crol), an ecdysone-inducible gene that encodes a family of zinc finger proteins, cause similar defects in wing morphogenesis and cell adhesion, indicating a role for ecdysone in these morphogenetic responses. We also show that crol and EcR mutations interact with mutations in genes encoding integrin subunits—a family of αβ heterodimeric cell surface receptors that mediate cell adhesion in many organisms. α-Integrin transcription is regulated by ecdysone in cultured larval organs and some changes in the temporal patterns of integrin expression correlate with the ecdysone titer profile during metamorphosis. Transcription of α- and β-integrin subunits is also altered in crol and EcR mutants, indicating that integrin expression is dependent upon crol and EcR function. Finally, we describe a new hypomorphic mutation in EcR which indicates that different EcR isoforms can direct the development of adult appendages. This study provides evidence that ecdysone controls wing morphogenesis and cell adhesion by regulating integrin expression during metamorphosis. We also propose that ecdysone modulation of integrin expression might be widely used to control multiple aspects of adult development
Hox C Cluster Genes Are Dispensable for Overall Body Plan of Mouse Embryonic Development

AbstractHox genes encode transcription factors which provide positional information during morphogenesis along the body axis; genetic interaction among Hox genes is thought to be necessary for correct pattern formation. One of the most curious features of the 39 vertebrate Hox genes is that they form four clusters each composed of several genes paralogous between clusters. This raises the question are all four clusters necessary for the development of vertebrates and, if so, what is the function of each cluster? To provide an answer to this question, we prepared Hox C cluster null mice utilizing the Cre-loxP system. Hox C cluster null mice, lacking all nine Hox C genes, die at the time of birth; however, the mutant pups develop to this stage with minor transformations. This development shows that the Hox C genes are dispensable for the overall body plan of mouse embryogenesis. Furthermore, transformations observed in the skeletal system of the Hox C cluster null mice are milder than those observed in the Hoxc-9 null mice, providing further evidence that at least some genes within a cluster exhibit interaction functions with each other