bluuebunny/crossref_metadata_2025 huggingface dataset:
Spatially regulated SpEts4 transcription factor activity along the sea urchin embryo animal-vegetal axis
Authors: Wei Zheng , Angerer Lynne M. , Angerer Robert C.
Published: April 2021

Because the transcription of the SpHE gene is regulated cell-autonomously and asymmetrically along the maternally determined animal-vegetal axis of the very early sea urchin embryo, its regulators provide an excellent entry point for investigating the mechanism(s) that establishes this initial polarity. Previous studies support a model in which spatial regulation of SpHE transcription relies on multiple nonvegetal positive transcription factor activities (Wei, Z., Angerer, L. M. and Angerer, R. C. (1997) Dev. Biol. 187, 71-78) and a yeast one-hybrid screen has identified one, SpEts4, which binds with high specificity to a cis element in the SpHE regulatory region and confers positive activation of SpHE promoter transgenes (Wei, Z., Angerer, R. C. and Angerer, L. M. (1999) Mol. Cell. Biol. 19, 1271-1278). Here we demonstrate that SpEts4 can bind to the regulatory region of the endogenous SpHE gene because a dominant repressor, created by fusing SpEts4 DNA binding and Drosophila engrailed repression domains, suppresses its transcription. The pattern of expression of the SpEts4 gene is consistent with a role in regulating SpHE transcription in the nonvegetal region of the embryo during late cleavage/early blastula stages. Although maternal transcripts are uniformly distributed in the egg and early cleaving embryo, they rapidly turn over and are replaced by zygotic transcripts that accumulate in a pattern congruent with SpHE transcription. In addition, in vivo functional tests show that the SpEts4 cis element confers nonvegetal transcription of a β-galactosidase reporter gene containing the SpHE basal promoter, and provide strong evidence that the activity of this transcription factor is an integral component of the nonvegetal transcriptional regulatory apparatus, which is proximal to, or part of, the mechanism that establishes the animal-vegetal axis of the sea urchin embryo.
GoosecoidandMix.1repressBrachyuryexpression and are required for head formation inXenopus
Authors: Latinkić B. V. , Smith J. C.
Published: April 2021

The Xenopus homologue of Brachyury, Xbra, is expressed in the presumptive mesoderm of the early gastrula. Induction of Xbra in animal pole tissue by activin occurs only in a narrow window of activin concentrations; if the level of inducer is too high, or too low, the gene is not expressed. Previously, we have suggested that the suppression of Xbra by high concentrations of activin is due to the action of genes such as goosecoid and Mix.1. Here, we examine the roles played by goosecoid and Mix.1 during normal development, first in the control of Xbra expression and then in the formation of the mesendoderm. Consistent with the model outlined above, inhibition of the function of either gene product leads to transient ectopic expression of Xbra. Such embryos later develop dorsoanterior defects and, in the case of interference with Mix.1, additional defects in heart and gut formation. Goosecoid, a transcriptional repressor, appears to act directly on transcription of Xbra. In contrast, Mix.1, which functions as a transcriptional activator, may act on Xbra indirectly, in part through activation of goosecoid.
A novel ontogenetic pathway in hybrid embryos between species with different modes of development
Authors: Raff Elizabeth C. , Popodi Ellen M. , Sly Belinda J. , Turner F. Rudolf , Villinski Jeffrey T. , Raff Rudolf A.
Published: April 2021

To investigate the bases for evolutionary changes in developmental mode, we fertilized eggs of a direct-developing sea urchin, Heliocidaris erythrogramma, with sperm from a closely related species, H. tuberculata, that undergoes indirect development via a feeding larva. The resulting hybrids completed development to form juvenile adult sea urchins. Hybrids exhibited restoration of feeding larval structures and paternal gene expression that have been lost in the evolution of the direct-developing maternal species. However, the developmental outcome of the hybrids was not a simple reversion to the paternal pluteus larval form. An unexpected result was that the ontogeny of the hybrids was distinct from either parental species. Early hybrid larvae exhibited a novel morphology similar to that of the dipleurula-type larva typical of other classes of echinoderms and considered to represent the ancestral echinoderm larval form. In the hybrid developmental program, therefore, both recent and ancient ancestral features were restored. That is, the hybrids exhibited features of the pluteus larval form that is present in both the paternal species and in the immediate common ancestor of the two species, but they also exhibited general developmental features of very distantly related echinoderms. Thus in the hybrids, the interaction of two genomes that normally encode two disparate developmental modes produces a novel but harmonious ontongeny.
Identification and characterization of hydra metalloproteinase 2 (HMP2): a meprin-like astacin metalloproteinase that functions in foot morphogenesis
Authors: Yan Li , Fei Kaiyin , Zhang Jinsong , Dexter Sharon , Sarras Michael P.
Published: April 2021

Several members of the newly emerging astacin metalloproteinase family have been shown to function in a variety of biological events, including cell differentiation and morphogenesis during both embryonic development and adult tissue differentiation. We have characterized a new astacin proteinase, hydra metalloproteinase 2 (HMP2) from the Cnidarian, Hydra vulgaris. HMP2 is translated from a single mRNA of 1.7 kb that contains a 1488 bp open reading frame encoding a putative protein product of 496 amino acids. The overall structure of HMP2 most closely resembles that of meprins, a subgroup of astacin metalloproteinases. The presence of a transient signal peptide and a putative prosequence indicates that HMP2 is a secreted protein that requires post-translational processing. The mature HMP2 starts with an astacin proteinase domain that contains a zinc binding motif characteristic of the astacin family. Its COOH terminus is composed of two potential protein-protein interaction domains: an “MAM” domain (named after meprins, A-5 protein and receptor protein tyrosine phosphatase μ) that is only present in meprin-like astacin proteinases; and a unique C-terminal domain (TH domain) that is also present in another hydra metalloproteinase, HMP1, in Podocoryne metalloproteinase 1 (PMP1) of jellyfish and in toxins of sea anemone. The spatial expression pattern of HMP2 was determined by both mRNA whole-mount in situ hybridization and immunofluorescence studies. Both morphological techniques indicated that HMP2 is expressed only by the cells in the endodermal layer of the body column of hydra. While the highest level of HMP2 mRNA expression was observed at the junction between the body column and the foot process, immunofluorescence studies indicated that HMP2 protein was present as far apically as the base of the tentacles. In situ analysis also indicated expression of HMP2 during regeneration of the foot process. To test whether the higher levels of HMP2 mRNA expression at the basal pole related to processes underlying foot morphogenesis, antisense studies were conducted. Using a specialized technique named localized electroporation (LEP), antisense constructs to HMP2 were locally introduced into the endodermal layer of cells at the basal pole of polyps and foot regeneration was initiated and monitored. Treatment with antisense to HMP2 inhibited foot regeneration as compared to mismatch and sense controls. These functional studies in combination with the fact that HMP2 protein was expressed not only at the junction between the body column and the foot process, but also as far apically as the base of the tentacles, suggest that this meprin-class metalloproteinase may be multifunctional in hydra.
Formation of the avian primitive streak from spatially restricted blastoderm: evidence for polarized cell division in the elongating streak
Authors: Wei Yan , Mikawa Takashi
Published: April 2021

Gastrulation in the amniote begins with the formation of a primitive streak through which precursors of definitive mesoderm and endoderm ingress and migrate to their embryonic destinations. This organizing center for amniote gastrulation is induced by signal(s) from the posterior margin of the blastodisc. The mode of action of these inductive signal(s) remains unresolved, since various origins and developmental pathways of the primitive streak have been proposed. In the present study, the fate of chicken blastodermal cells was traced for the first time in ovo from prestreak stages XI-XII through HH stage 3, when the primitive streak is initially established and prior to the migration of mesoderm. Using replication-defective retrovirus-mediated gene transfer and vital dye labeling, precursor cells of the stage 3 primitive streak were mapped predominantly to a specific region where the embryonic midline crosses the posterior margin of the epiblast. No significant contribution to the early primitive streak was seen from the anterolateral epiblast. Instead, the precursor cells generated daughter cells that underwent a polarized cell division oriented perpendicular to the anteroposterior embryonic axis. The resulting daughter cell population was arranged in a longitudinal array extending the complete length of the primitive streak. Furthermore, expression of cVg1, a posterior margin-derived signal, at the anterior marginal zone induced adjacent epiblast cells, but not those lateral to or distant from the signal, to form an ectopic primitive streak. The cVg1-induced epiblast cells also exhibited polarized cell divisions during ectopic primitive streak formation. These results suggest that blastoderm cells located immediately anterior to the posterior marginal zone, which secretes an inductive signal, undergo spatially directed cytokineses during early primitive streak formation.
Caenorhabditis elegansTwist plays an essential role in non-striated muscle development
Authors: Corsi Ann K. , Kostas Stephen A. , Fire Andrew , Krause Michael
Published: April 2021

The basic helix-loop-helix (bHLH) transcription factor Twist plays a role in mesodermal development in both invertebrates and vertebrates. In an effort to understand the role of the unique Caenorhabditis elegans Twist homolog, hlh-8, we analyzed mesodermal development in animals with a deletion in the hlh-8 locus. This deletion was predicted to represent a null allele because the HLH domain is missing and the reading frame for the protein is disrupted. Animals lacking CeTwist function were constipated and egg-laying defective. Both of these defects were rescued in transgenic mutant animals expressing wild-type hlh-8. Observing a series of mesoderm-specific markers allowed us to characterize the loss of hlh-8 function more thoroughly. Our results demonstrate that CeTwist performs an essential role in the proper development of a subset of mesodermal tissues in C. elegans. We found that CeTwist was required for the formation of three out of the four non-striated enteric muscles born in the embryo. In contrast, CeTwist was not required for the formation of the embryonically derived striated muscles. Most of the post-embryonic mesoderm develops from a single lineage. CeTwist was necessary for appropriate patterning in this lineage and was required for expression of two downstream target genes, but was not required for the expression of myosin, a marker of differentiation. Our results suggest that mesodermal patterning by Twist is an evolutionarily conserved function.
OOC-3, a novel putative transmembrane protein required for establishment of cortical domains and spindle orientation in the P1 blastomere of C. elegans embryos
Authors: Pichler Silke , Gönczy Pierre , Schnabel Heinke , Pozniakowski Andrei , Ashford Anthony , Schnabel Ralf , Hyman Anthony A.
Published: April 2021

Asymmetric cell divisions require the establishment of an axis of polarity, which is subsequently communicated to downstream events. During the asymmetric cell division of the P1 blastomere in C. elegans, establishment of polarity depends on the establishment of anterior and posterior cortical domains, defined by the localization of the PAR proteins, followed by the orientation of the mitotic spindle along the previously established axis of polarity. To identify genes required for these events, we have screened a collection of maternal-effect lethal mutations on chromosome II of C. elegans. We have identified a mutation in one gene, ooc-3, with mis-oriented division axes at the two-cell stage. Here we describe the phenotypic and molecular characterization of ooc-3. ooc-3 is required for the correct localization of PAR-2 and PAR-3 cortical domains after the first cell division. OOC-3 is a novel putative transmembrane protein, which localizes to a reticular membrane compartment, probably the endoplasmic reticulum, that spans the whole cytoplasm and is enriched on the nuclear envelope and cell-cell boundaries. Our results show that ooc-3 is required to form the cortical domains essential for polarity after cell division.
Regulation of proliferation and differentiation in spermatogonial stem cells: the role of c-kit and its ligand SCF
Authors: Ohta Hiroshi , Yomogida Kentaro , Dohmae Kayoko , Nishimune Yoshitake
Published: April 2021

To study self-renewal and differentiation of spermatogonial stem cells, we have transplanted undifferentiated testicular germ cells of the GFP transgenic mice into seminiferous tubules of mutant mice with male sterility, such as those dysfunctioned at Steel (Sl) locus encoding the c-kit ligand or Dominant white spotting (W) locus encoding the receptor c-kit. In the seminiferous tubules of Sl/Sld or Sl17H/Sl17H mice, transplanted donor germ cells proliferated and formed colonies of undifferentiated c-kit (−) spermatogonia, but were unable to differentiate further. However, these undifferentiated but proliferating spermatogonia, retransplanted into Sl (+) seminiferous tubules of W mutant, resumed differentiation, indicating that the transplanted donor germ cells contained spermatogonial stem cells and that stimulation of c-kit receptor by its ligand was necessary for maintenance of differentiated type A spermatogonia but not for proliferation of undifferentiated type A spermatogonia. Furthermore, we have demonstrated that their transplantation efficiency in the seminiferous tubules of Sl17H/Sl17H mice depended upon the stem cell niche on the basement membrane of the recipient seminiferous tubules and was increased by elimination of the endogenous spermatogonia of mutant mice from the niche by treating them with busulfan.
Pax6 defines the di-mesencephalic boundary by repressing En1 and Pax2
Authors: Matsunaga Eiji , Araki Isato , Nakamura Harukazu
Published: April 2021

Transcriptional factors and signaling molecules are responsible for regionalization of the central nervous system. In the early stage of neural development, Pax6 is expressed in the prosencephalon, while En1 and Pax2 are expressed in the mesencephalon. Here, we misexpressed Pax6 in the mesencephalon to elucidate the mechanism of the di-mesencephalic boundary formation. Histological analysis, expression patterns of diencephalic marker genes, and fiber trajectory of the posterior commissure indicated that Pax6 misexpression caused a caudal shift of the dimesencephalic boundary. Pax6 repressed En1, Pax2 and other tectum (mesencephalon)-related genes such as En2, Pax5, Pax7, but induced Tcf4, a diencephalon marker gene. To know how Pax6 represses En1 and Pax2, we ectopically expressed a dominant-active or negative form of Pax6. The dominant-active form of Pax6 showed a similar but more severe phenotype than Pax6, while the dominant-negative form showed an opposite phenotype, suggesting that Pax6 acts as a transcriptional activator. Thus Pax6 may repress tectum-related genes by activating an intervening repressor. The results of misexpression experiments, together with normal expression patterns of Pax6, En1 and Pax2, suggest that repressive interaction between Pax6 and En1/Pax2 defines the di-mesencephalic boundary.
Sox2 regulatory sequences direct expression of a β-geo transgene to telencephalic neural stem cells and precursors of the mouse embryo, revealing regionalization of gene expression in CNS stem cells
Authors: Zappone Massimo V. , Galli Rossella , Catena Raffaella , Meani Natalia , Biasi Silvia De , Mattei Elisabetta , Tiveron Cecilia , Vescovi Angelo L. , Lovell-Badge Robin , Ottolenghi Sergio , Nicolis Silvia K.
Published: April 2021

Sox2 is one of the earliest known transcription factors expressed in the developing neural tube. Although it is expressed throughout the early neuroepithelium, we show that its later expression must depend on the activity of more than one regionally restricted enhancer element. Thus, by using transgenic assays and by homologous recombination-mediated deletion, we identify a region upstream of Sox2 (−5.7 to −3.3 kb) which can not only drive expression of a β-geo transgene to the developing dorsal telencephalon, but which is required to do so in the context of the endogenous gene. The critical enhancer can be further delimited to an 800 bp fragment of DNA surrounding a nuclease hypersensitive site within this region, as this is sufficient to confer telencephalic expression to a 3.3 kb fragment including the Sox2 promoter, which is otherwise inactive in the CNS.

Expression of the 5.7 kb Sox2β-geo transgene localizes to the neural plate and later to the telencephalic ventricular zone. We show, by in vitro clonogenic assays, that transgene-expressing (and thus G418-resistant) ventricular zone cells include cells displaying functional properties of stem cells, i.e. self-renewal and multipotentiality. We further show that the majority of telencephalic stem cells express the transgene, and this expression is largely maintained over two months in culture (more than 40 cell divisions) in the absence of G418 selective pressure. In contrast, stem cells grown in parallel from the spinal cord never express the transgene, and die in G418. Expression of endogenous telencephalic genes was similarly observed in long-term cultures derived from the dorsal telencephalon, but not in spinal cord-derived cultures.

Thus, neural stem cells of the midgestation embryo are endowed with region-specific gene expression (at least with respect to some networks of transcription factors, such as that driving telencephalic expression of the Sox2 transgene), which can be inherited through multiple divisions outside the embryonic environment.
Regulation of central neuron synaptic targeting by the Drosophila POU protein, Acj6
Authors: Certel Sarah J. , Clyne Peter J. , Carlson John R. , Johnson Wayne A.
Published: April 2021

Mutations in the Drosophila class IV POU domain gene, abnormal chemosensory jump 6 (acj6), have previously been shown to cause physiological deficits in odor sensitivity. However, loss of Acj6 function also has a severe detrimental effect upon coordinated larval and adult movement that cannot be explained by the simple loss in odorant detection. In addition to olfactory sensory neurons, Acj6 is expressed in a distinct subset of postmitotic interneurons in the central nervous system from late embryonic to adult stages. In the larval and adult brain, Acj6 is highly expressed in central brain, optic and antennal lobe neurons. Loss of Acj6 function in larval optic lobe neurons results in disorganized retinal axon targeting and synapse selection. Furthermore, the lamina neurons themselves exhibit disorganized synaptic arbors in the medulla of acj6 mutant pupal brains, suggesting that Acj6 may play a role in regulating synaptic connections or structure. To further test this hypothesis, we misexpressed two Acj6 isoforms in motor neurons where they are not normally found. The two Acj6 isoforms are produced from alternatively spliced acj6 transcripts, resulting in significant structural differences in the amino-terminal POU IV box. Acj6 misexpression caused marked alterations at the neuromuscular junction, with contrasting effects upon nerve terminal branching and synapse formation associated with specific Acj6 isoforms. Our results suggest that the class IV POU domain factor, Acj6, may play an important role in regulating synaptic target selection by central neurons and that the amino-terminal POU IV box is important for regulation of Acj6 activity.
Glial cell fate specification modulated by the bHLH gene Hes5in mouse retina
Authors: Hojo Masato , Ohtsuka Toshiyuki , Hashimoto Nobuo , Gradwohl Gérald , Guillemot François , Kageyama Ryoichiro
Published: April 2021

Neurons and glial cells differentiate from common precursors. Whereas the gene glial cells missing (gcm) determines the glial fate in Drosophila, current data about the expression patterns suggest that, in mammals, gcm homologues are unlikely to regulate gliogenesis. Here, we found that, in mouse retina, the bHLH gene Hes5 was specifically expressed by differentiating Müller glial cells and that misexpression of Hes5 with recombinant retrovirus significantly increased the population of glial cells at the expense of neurons. Conversely, Hes5-deficient retina showed 30-40% decrease of Müller glial cell number without affecting cell survival. These results indicate that Hes5 modulates glial cell fate specification in mouse retina.
Querkopf, a MYST family histone acetyltransferase, is required for normal cerebral cortex development
Authors: Thomas Tim , Voss Anne K. , Chowdhury Kamal , Gruss Peter
Published: April 2021

In order to find, and mutate, novel genes required for regulation of neurogenesis in the cerebral cortex, we performed a genetic screen in mice. As the result of this screen, we created a new mouse mutant, querkopf. The querkopf mutation is due to an insertion into a MYST family histone acetyltransferase gene. Mice homozygous for the querkopf mutation have craniofacial abnormalities, fail to thrive in the postnatal period and have defects in central nervous system development. The defects in central nervous system development are particularly prominent in the cerebral cortex, which is disproportionally smaller than in wild-type mice. A large reduction in the size of the cortical plate was already apparent during embryogenesis. Homozygous mice show a lack of large pyramidal cells in layer V of the cortex, which is reflected in a reduction in the number of Otx1-positive neurons in this layer during postnatal development. Homozygous mice also show a reduction in the number of GAD67-positive interneurons throughout the cortex. Our results suggest that Querkopf is an essential component of a genetic cascade regulating cell differentiation in the cortex, probably acting in a multiprotein complex regulating chromatin structure during transcription.
Ace/Fgf8 is required for forebrain commissure formation and patterning of the telencephalon
Authors: Shanmugalingam Shantha , Houart Corinne , Picker Alexander , Reifers Frank , Macdonald Rachel , Barth Anukampa , Griffin Kevin , Brand Michael , Wilson Stephen W.
Published: April 2021

Fibroblast growth factors (Fgfs) form a large family of secreted signalling proteins that have a wide variety of roles during embryonic development. Within the central nervous system (CNS) Fgf8 is implicated in patterning neural tissue adjacent to the midbrain-hindbrain boundary. However, the roles of Fgfs in CNS tissue rostral to the midbrain are less clear. Here we examine the patterning of the forebrain in zebrafish embryos that lack functional Fgf8/Ace. We find that Ace is required for the development of midline structures in the forebrain. In the absence of Ace activity, midline cells fail to adopt their normal morphology and exhibit altered patterns of gene expression. This disruption to midline tissue leads to severe commissural axon pathway defects, including misprojections from the eye to ectopic ipsilateral and contralateral targets. Ace is also required for the differentiation of the basal telencephalon and several populations of putative telencephalic neurons but not for overall regional patterning of forebrain derivatives. Finally, we show that ace expression co-localises with anterior neural plate cells that have previously been shown to have forebrain patterning activity. Removal of these cells leads to a failure in induction of ace expression indicating that loss of Ace activity may contribute to the phenotypes observed when anterior neural plate cells are ablated. However, as ace mutant neural plate cells still retain at least some inductive activity, then other signals must also be produced by the anterior margin of the neural plate.
RGS proteins inhibit Xwnt-8 signaling in Xenopus embryonic development
Authors: Wu Chunlai , Zeng Qingyi , Blumer Kendall J. , Muslin Anthony J.
Published: April 2021

RGS family members are GTPase activating proteins (GAPs) that antagonize signaling by heterotrimeric G proteins. Injection of Xenopus embryos with RNA encoding rat RGS4 (rRGS4), a GAP for Gi and Gq, resulted in shortened trunks and decreased skeletal muscle. This phenotype is nearly identical to the effect of injection of either frzb or dominant negative Xwnt-8. Injection of human RGS2, which selectively deactivates Gq, had similar effects. rRGS4 inhibited the ability of early Xwnt-8 but not Xdsh misexpression to cause axis duplication. This effect is distinct from axin family members that contain RGS-like domains but act downstream of Xdsh. We identified two Xenopus RGS4 homologs, one of which, Xrgs4a, was expressed as a Spemann organizer component. Injection of Xenopus embryos with Xrgs4a also resulted in shortened trunks and decreased skeletal muscle. These results suggest that RGS proteins modulate Xwnt-8 signaling by attenuating the function of a G protein.
Mice lacking the transcriptional corepressor TIF1β are defective in early postimplantation development
Authors: Cammas Florence , Mark Manuel , Dollé Pascal , Dierich Andrée , Chambon Pierre , Losson Régine
Published: April 2021

TIF1β, a member of the transcriptional intermediary factor 1 family, has been reported to function as a corepressor for the large class of KRAB domain-containing zinc finger proteins of the Krüppel type. To address the biological function of TIF1β, we have generated TIF1β-deficient mice by gene disruption. TIF1β protein was detected in wild-type but not TIF1β−/− blastocysts. Homozygous mutant embryos, which developed normally until the blastocyst stage and underwent uterine implantation, were arrested in their development at the early egg-cylinder stage at about embryonic day (E) 5.5 and were completely resorbed by E8.5. Taken together, these results provide genetic evidence that TIF1β is a developmental regulatory protein that exerts function(s) essential for early postimplantation development.
Regulation of retinoic acid signaling during lung morphogenesis
Authors: Malpel Sarah , Mendelsohn Cathy , Cardoso Wellington V.
Published: April 2021

Little is known about how retinoic acid (RA) synthesis, utilization and metabolism are regulated in the embryonic lung and how these activities relate to lung pattern formation. Here we report that early lung bud formation and subsequent branching morphogenesis are characterized by distinct stages of RA signaling. At the onset of lung development RA signaling is ubiquitously activated in primary buds, as shown by expression of the major RA-synthesizing enzyme, RALDH-2 and activation of a RARE-lacZ transgene. Nevertheless, further airway branching appears to require downregulation of RA pathways by decreased synthesis, increased RA degradation in the epithelium via P450RAI-mediated metabolism, and inhibition of RA signaling in the mesenchyme by COUPTF-II expression. These mechanisms controlling local RA signaling may be critical for normal branching, since we show that manipulating RA levels in vitro to maintain RA signaling activated as in the initial stage, leads to an immature lung phenotype characterized by failure to form typical distal buds. We show that this phenotype likely results from RA interfering with the establishment of a distal signaling center, altering levels and distribution of Fgf10 and Bmp4, genes that are essential for distal lung formation. Furthermore, RA upregulates P450RAI expression, suggesting the presence of feedback mechanisms controlling RA availability. Our study illustrates the importance of regional mechanisms that control RA availability and utilization for correct expression of pattern regulators and normal morphogenesis during lung development.
Imprint switching for non-random X-chromosome inactivation during mouse oocyte growth
Authors: Tada Takashi , Obata Yayoi , Tada Masako , Goto Yuji , Nakatsuji Norio , Tan Seong-Seng , Kono Tomohiro , Takagi Nobuo
Published: April 2021

In mammals, X-chromosome inactivation occurs in all female cells, leaving only a single active X chromosome. This serves to equalise the dosage of X-linked genes in male and female cells. In the mouse, the paternally derived X chromosome (XP) is imprinted and preferentially inactivated in the extraembryonic tissues whereas in the embryonic tissues inactivation is random. To investigate how XP is chosen as an inactivated X chromosome in the extraembryonic cells, we have produced experimental embryos by serial nuclear transplantation from non-growing (ng) oocytes and fully grown (fg) oocytes, in which the X chromosomes are marked with (1) an X-linked lacZ reporter gene to assay X-chromosome activity, or (2) the Rb(X.9)6H translocation as a cytogenetic marker for studying replication timing. In the extraembryonic tissues of these ng/fg embryos, the maternal X chromosome (XM) derived from the ng oocyte was preferentially inactivated whereas that from the fg oocyte remained active. However, in the embryonic tissues, X inactivation was random. This suggests that (1) a maternal imprint is set on the XM during oocyte growth, (2) the maternal imprint serves to render the XM resistant to inactivation in the extraembryonic tissues and (3) the XM derived from an ng oocyte resembles a normal XP.
Notch signaling regulates the pattern of auditory hair cell differentiation in mammals
Authors: Zine Azel , Water Thomas R. Van De , Ribaupierre Francois de
Published: April 2021

The development of the mammalian cochlea is an example of patterning in the peripheral nervous system. Sensory hair cells and supporting cells in the cochlea differentiate via regional and cell fate specification. The Notch signaling components shows both distinct and overlapping expression patterns of Notch1 receptor and its ligands Jagged1 (Jag1) and Jagged2 (Jag2) in the developing auditory epithelium of the rat. On embryonic day 16 (E16), many precursor cells within the Kölliker’s organ immunostained for the presence of both Notch1 and Jag1, while the area of hair cell precursors did not express either Notch1 and Jag1. During initial events of hair cell differentiation between E18 and birth, Notch1 and Jag1 expression predominated in supporting cells and Jag2 in nascent hair cells. Early after birth, Jag2 expression decreased in hair cells while the pattern of Notch1 expression now included both supporting cells and hair cells. We show that the normal pattern of hair cell differentiation is disrupted by alteration of Notch signaling. A decrease of either Notch1 or Jag1 expression by antisense oligonucleotides in cultures of the developing sensory epithelium resulted in an increase in the number of hair cells. Our data suggest that the Notch1 signaling pathway is involved in a complex interplay between the consequences of different ligand-Notch1 combinations during cochlear morphogenesis and the phases of hair cell differentiation
Expression of neurogenin3 reveals an islet cell precursor population in the pancreas
Authors: Schwitzgebel Valérie M. , Scheel David W. , Conners Jennifer R. , Kalamaras Julie , Lee Jacqueline E. , Anderson David J. , Sussel Lori , Johnson Jeffrey D. , German Michael S.
Published: April 2021

Differentiation of early gut endoderm cells into the endocrine cells forming the pancreatic islets of Langerhans depends on a cascade of gene activation events controlled by transcription factors including the basic helix-loop-helix (bHLH) proteins. To delineate this cascade, we began by establishing the position of neurogenin3, a bHLH factor found in the pancreas during fetal development. We detect neurogenin3 immunoreactivity transiently in scattered ductal cells in the fetal mouse pancreas, peaking at embryonic day 15.5. Although not detected in cells expressing islet hormones or the islet transcription factors Isl1, Brn4, Pax6 or PDX1, neurogenin3 is detected along with early islet differentiation factors Nkx6.1 and Nkx2.2, establishing that it is expressed in immature cells in the islet lineage. Analysis of transcription factor-deficient mice demonstrates that neurogenin3 expression is not dependent on neuroD1/BETA2, Mash1, Nkx2.2, Nkx6.1, or Pax6. Furthermore, early expression of neurogenin3 under control of the Pdx1 promoter is alone sufficient to drive early and ectopic differentiation of islet cells, a capability shared by the pancreatic bHLH factor, neuroD1/BETA2, but not by the muscle bHLH factor, MyoD. However, the islet cells produced in these transgenic experiments are overwhelmingly α cells, suggesting that factors other than the bHLH factors are required to deviate from a default α cell fate. These data support a model in which neurogenin3 acts upstream of other islet differentiation factors, initiating the differentiation of endocrine cells, but switching off prior to final differentiation. The ability to uniquely identify islet cell precursors by neurogenin3 expression allows us to determine the position of other islet transcription factors in the differentiation cascade and to propose a map for the islet cell differentiation pathway.
dackel acts in the ectoderm of the zebrafish pectoral fin bud to maintain AER signaling
Authors: Grandel Heiner , Draper Bruce W. , Schulte-Merker Stefan
Published: April 2021

Classical embryological studies have implied the existence of an apical ectodermal maintenance factor (AEMF) that sustains signaling from the apical ectodermal ridge (AER) during vertebrate limb development. Recent evidence suggests that AEMF activity is composed of different signals involving both a sonic hedgehog (Shh) signal and a fibroblast growth factor 10 (Fgf10) signal from the mesenchyme. In this study we show that the product of the dackel (dak) gene is one of the components that acts in the epidermis of the zebrafish pectoral fin bud to maintain signaling from the apical fold, which is homologous to the AER of tetrapods. dak acts synergistically with Shh to induce fgf4 and fgf8 expression but independently of Shh in promoting apical fold morphogenesis. The failure of dak mutant fin buds to progress from the initial fin induction phase to the autonomous outgrowth phase causes loss of both AER and Shh activity, and subsequently results in a proximodistal truncation of the fin, similar to the result obtained by ridge ablation experiments in the chicken. Further analysis of the dak mutant phenotype indicates that the activity of the transcription factor engrailed 1 (En1) in the ventral non-ridge ectoderm also depends on a maintenance signal probably provided by the ridge. This result uncovers a new interaction between the AER and the dorsoventral organizer in the zebrafish pectoral fin bud.
Hes6 acts in a positive feedback loop with the neurogenins to promote neuronal differentiation
Authors: Koyano-Nakagawa Naoko , Kim Jaesang , Anderson David , Kintner Chris
Published: April 2021

During the development of the vertebrate nervous system, neurogenesis is promoted by proneural bHLH proteins such as the neurogenins, which act as potent transcriptional activators of neuronal differentiation genes. The pattern by which these proteins promote neuronal differentiation is thought to be governed by inhibitors, including a class of transcriptional repressors called the WRPW-bHLH proteins, which are similar to Drosophila proteins encoded by hairy and genes in the enhancer of split complex (E-(SPL)-C). Here, we describe the isolation and characterization of Hes6, which encodes a novel WRPW-bHLH protein expressed during neurogenesis in mouse and Xenopus embryos. We show that Hes6 expression follows that of neurogenins but precedes that of the neuronal differentiation genes. We provide several lines of evidence to show that Hes6 expression occurs in developing neurons and is induced by the proneural bHLH proteins but not by the Notch pathway. When ectopically expressed in Xenopus embryos, Hes6 promotes neurogenesis. The properties of Hes6 distinguish it from other members of the WRPW-bHLH family in vertebrates, and suggest that it acts in a positive-feedback loop with the proneural bHLH proteins to promote neuronal differentiation.
The fate of cells in the tailbud of Xenopus laevis
Authors: Davis Robert L. , Kirschner Marc W.
Published: April 2021

The vertebrate tailbud and trunk form very similar tissues. It has been a controversial question for decades whether cell determination in the developing tail proceeds as part of early axial development or whether it proceeds by a different mechanism. To examine this question more closely, we have used photoactivation of fluorescence to mark small neighborhoods of cells in the developing tailbud of Xenopus laevis. We show that, in one region of the tailbud, very small groups of adjacent cells can contribute progeny to the neural tube, notochord and somitic muscle, as well as other identified cell types within a single embryo. Groups averaging three adjacent cells at a later stage can contribute progeny with a similar distribution. Our data suggest that the tailbud contains multipotent cells that make very late germ-layer decisions.
Phosphorylation of Bicoid on MAP-kinase sites: contribution to its interaction with the torso pathway
Authors: Janody Florence , Sturny Rachel , Catala Françoise , Desplan Claude , Dostatni Nathalie
Published: April 2021

The Torso signal transduction pathway exhibits two opposite effects on the activity of the Bicoid (Bcd) morphogen: (i) Bcd function is repressed by Torso (Tor) at the anterior pole of the embryo leading to a retraction of the expression of many Bcd targets from the most anterior region of the embryo, where the Tor tyrosine kinase receptor is activated, and (ii) Bcd function is strengthened by Tor in a broader anterior region, as indicated by a shift of the posterior border of Bcd targets towards the anterior pole in embryos deprived from Tor activity. Anterior repression of Bcd targets was not observed in embryos lacking maternal contribution of D-sor, which acts downstream of Tor and encodes a MAP-kinase kinase. This indicates that the Ras signalling cascade is directly involved in this process, although the known transcriptional effectors of the Tor pathway, tll and hkb, are not (Ronchi, E., Treisman, J., Dostatni, N., Struhl, G. and Desplan, C. (1993) Cell 74, 347-355). Bcd is a good in vitro substrate for phosphorylation by MAP-kinase and phosphorylation of the protein occur in vivo on MAP-kinase sites. In the presence of a Bcd mutant that could no longer be phosphorylated by MAP-kinase, expression of Bcd targets remained repressed by Tor at the pole while strengthening of Bcd activity was reduced. These experiments indicate that phosphorylation of Bcd by MAP-kinase is likely to be required for the Tor pathway to induce its full positive effect on Bcd. This suggests that Tor signalling acts at a distance from the anterior pole by direct modification of the diffusing Bcd morphogen.
Glia dictate pioneer axon trajectories in the Drosophila embryonic CNS
Authors: Hidalgo Alicia , Booth Gwendolen E.
Published: April 2021

Whereas considerable progress has been made in understanding the molecular mechanisms of axon guidance across the midline, it is still unclear how the axonal trajectories of longitudinal pioneer neurons, which never cross the midline, are established. Here we show that longitudinal glia of the embryonic Drosophila CNS direct formation of pioneer axon pathways. By ablation and analysis of glial cells missing mutants, we demonstrate that glia are required for two kinds of processes. Firstly, glia are required for growth cone guidance, although this requirement is not absolute. We show that the route of extending growth cones is rich in neuronal cell bodies and glia, and also in long processes from both these cell types. Interactions between neurons, glia and their long processes orient extending growth cones. Secondly, glia direct the fasciculation and defasciculation of axons, which pattern the pioneer pathways. Together these events are essential for the selective fasciculation of follower axons along the longitudinal pathways.
Positive and negative interactions of GDNF, NTN and ART in developing sensory neuron subpopulations, and their collaboration with neurotrophins
Authors: Baudet Christel , Mikaels Åsa , Westphal Heiner , Johansen Jens , Johansen Teit E. , Ernfors Patrik
Published: April 2021

Glial cell line-derived neurotrophic factor (GDNF), neurturin (NTN) and neublastin/artemin (ART) are distant members of the transforming growth factor β; family, and have been shown to elicit neurotrophic effects upon several classes of peripheral and central neurons. Limited information from in vitro and expression studies has also substantiated a role for GDNF family ligands in mammalian somatosensory neuron development. Here, we show that although dorsal root ganglion (DRG) sensory neurons express GDNF family receptors embryonically, they do not survive in response to their ligands. The regulation of survival emerges postnatally for all GDNF family ligands. GDNF and NTN support distinct subpopulations that can be separated with respect to their expression of GDNF family receptors, whereas ART supports neurons in populations that are also responsive to GDNF or NTN. Sensory neurons that coexpress GDNF family receptors are medium sized, whereas small-caliber nociceptive cells preferentially express a single receptor. In contrast to brain-derived neurotrophic factor (BDNF)-dependent neurons, embryonic nerve growth factor (NGF)-dependent nociceptive neurons switch dependency to GDNF, NTN and ART postnatally. Neurons that survive in the presence of neurotrophin 3 (NT3) or neurotrophin 4 (NT4), including proprioceptive afferents, Merkel end organs and D-hair afferents, are also supported by GDNF family ligands neonatally, although at postnatal stages they lose their dependency on GDNF and NTN. At late postnatal stages, ART prevents survival elicited by GDNF and NTN. These data provide new insights on the roles of GDNF family ligands in sensory neuron development.
Gli2 functions in FGF signaling during antero-posterior patterning
Authors: Brewster R. , Mullor J. L. , Altaba A. Ruiz i
Published: April 2021

Patterning along the anteroposterior (A-P) axis involves the interplay of secreted and transcription factors that specify cell fates in the mesoderm and neuroectoderm. While FGF and homeodomain proteins have been shown to play different roles in posterior specification, the network coordinating their effects remains elusive. Here we have analyzed the function of Gli zinc-finger proteins in mesodermal A-P patterning. We find that Gli2 is sufficient to induce ventroposterior development, functioning in the FGF-brachyury regulatory loop. Gli2 directly induces brachyury, a gene required and sufficient for mesodermal development, and Gli2 is in turn induced by FGF signaling. Moreover, the homeobox gene Xhox3, a critical determinant of posterior development, is also directly regulated by Gli2. Gli3, but not Gli1, has an activity similar to that of Gli2 and is expressed in ventroposterior mesoderm after Gli2. These findings uncover a novel function of Gli proteins, previously only known to mediate hedgehog signals, in the maintenance and patterning of the embryonic mesoderm. More generally, our results suggest a molecular basis for an integration of FGF and hedgehog inputs in Gli-expressing cells that respond to these signals.
Attractive and repulsive interactions between female and male gametophytes in Arabidopsis pollen tube guidance
Authors: Shimizu Kentaro K. , Okada Kiyotaka
Published: April 2021

Sexual reproduction in plants, unlike that of animals, requires the action of multicellular haploid gametophytes. The male gametophyte (pollen tube) is guided to a female gametophyte through diploid sporophytic cells in the pistil. While interactions between the pollen tube and diploid cells have been described, little is known about the intercellular recognition systems between the pollen tube and the female gametophyte. In particular, the mechanisms that enable only one pollen tube to interact with each female gametophyte, thereby preventing polysperm, are not understood. We isolated female gametophyte mutants named magatama (maa) from Arabidopsis thaliana by screening for siliques containing half the normal number of mature seeds. In maa1 and maa3 mutants, in which the development of the female gametophyte was delayed, pollen tube guidance was affected. Pollen tubes were directed to mutant female gametophytes, but they lost their way just before entering the micropyle and elongated in random directions. Moreover, the mutant female gametophytes attracted two pollen tubes at a high frequency. To explain the interaction between gametophytes, we propose a monogamy model in which a female gametophyte emits two attractants and prevents polyspermy. This prevention process by the female gametophyte could increase a plant’s inclusive fitness by facilitating the fertilization of sibling female gametophytes. In addition, repulsion between pollen tubes might help prevent polyspermy. The reproductive isolations observed in interspecific crosses in Brassicaceae are also consistent with the monogamy model.
BMP4 rescues a non-cell-autonomous function ofMsx1in tooth development
Authors: Bei Marianna , Kratochwil Klaus , Maas Richard L.
Published: April 2021

The development of many organs depends on sequential epithelial-mesenchymal interactions, and the developing tooth germ provides a powerful model for elucidating the nature of these inductive tissue interactions. In Msx1-deficient mice, tooth development arrests at the bud stage when Msx1 is required for the expression of Bmp4 and Fgf3 in the dental mesenchyme (Bei, M. and Maas, R. (1998) Development 125, 4325-4333). To define the tissue requirements for Msx1 function, we performed tissue recombinations between wild-type and Msx1 mutant dental epithelium and mesenchyme. We show that through the E14.5 cap stage of tooth development, Msx1 is required in the dental mesenchyme for tooth formation. After the cap stage, however, tooth development becomes Msx1 independent, although our experiments identify a further late function of Msx1 in odontoblast and dental pulp survival. These results suggest that prior to the cap stage, the dental epithelium receives an Msx1-dependent signal from the dental mesenchyme that is necessary for tooth formation.

To further test this hypothesis, Msx1 mutant tooth germs were first cultured with either BMP4 or with various FGFs for two days in vitro and then grown under the kidney capsule of syngeneic mice to permit completion of organogenesis and terminal differentiation. Previously, using an in vitro culture system, we showed that BMP4 stimulated the growth of Msx1 mutant dental epithelium (Chen, Y., Bei, M. Woo, I., Satokata, I. and Maas, R. (1996). Development 122, 3035-3044). Using the more powerful kidney capsule grafting procedure, we now show that when added to explanted Msx1-deficient tooth germs prior to grafting, BMP4 rescues Msx1 mutant tooth germs all the way to definitive stages of enamel and dentin formation. Collectively, these results establish a transient functional requirement for Msx1 in the dental mesenchyme that is almost fully supplied by BMP4 alone, and not by FGFs. In addition, they formally prove the postulated downstream relationship of BMP4 with respect to Msx1, establish the non-cell-autonomous nature of Msx1 during odontogenesis, and disclose an additional late survival function for Msx1 in odontoblasts and dental pulp.