bluuebunny/crossref_metadata_2025 huggingface dataset:
Synergy betweenHoxa1andHoxb1: the relationship between arch patterning and the generation of cranial neural crest
Authors: Gavalas Anthony , Trainor Paul , Ariza-McNaughton Linda , Krumlauf Robb
Published: April 2021

Hoxa1 and Hoxb1 have overlapping synergistic roles in patterning the hindbrain and cranial neural crest cells. The combination of an ectoderm-specific regulatory mutation in the Hoxb1 locus and the Hoxa1 mutant genetic background results in an ectoderm-specific double mutation, leaving the other germ layers impaired only in Hoxa1 function. This has allowed us to examine neural crest and arch patterning defects that originate exclusively from the neuroepithelium as a result of the simultaneous loss of Hoxa1 and Hoxb1 in this tissue. Using molecular and lineage analysis in this double mutant background we demonstrate that presumptive rhombomere 4, the major site of origin of the second pharyngeal arch neural crest, is reduced in size and has lost the ability to generate neural crest cells. Grafting experiments using wild-type cells in cultured normal or double mutant mouse embryos demonstrate that this is a cell-autonomous defect, suggesting that the formation or generation of cranial neural crest has been uncoupled from segmental identity in these mutants. Furthermore, we show that loss of the second arch neural crest population does not have any adverse consequences on early patterning of the second arch. Signalling molecules are expressed correctly and pharyngeal pouch and epibranchial placode formation are unaffected. There are no signs of excessive cell death or loss of proliferation in the epithelium of the second arch, suggesting that the neural crest cells are not the source of any indispensable mitogenic or survival signals. These results illustrate that Hox genes are not only necessary for proper axial specification of the neural crest but that they also play a vital role in the generation of this population itself. Furthermore, they demonstrate that early patterning of the separate components of the pharyngeal arches can proceed independently of neural crest cell migration.
mshspecifies dorsal cell fate in theDrosophilawing
Authors: Milán Marco , Weihe Ulrich , Tiong Stanley , Bender Welcome , Cohen Stephen M.
Published: April 2021

Drosophila limbs develop from imaginal discs that are subdivided into compartments. Dorsal-ventral subdivision of the wing imaginal disc depends on apterous activity in dorsal cells. Apterous protein is expressed in dorsal cells and is responsible for (1) induction of a signaling center along the dorsal-ventral compartment boundary (2) establishment of a lineage restriction boundary between compartments and (3) specification of dorsal cell fate. Here, we report that the homeobox gene msh (muscle segment homeobox) acts downstream of apterous to confer dorsal identity in wing development.
Thelin-11LIM homeobox gene specifies olfactory and chemosensory neuron fates inC. elegans
Authors: Sarafi-Reinach Trina R. , Melkman Tali , Hobert Oliver , Sengupta Piali
Published: April 2021

Chemosensory neuron diversity in C. elegans arises from the action of transcription factors that specify different aspects of sensory neuron fate. In the AWB and AWA olfactory neurons, the LIM homeobox gene lim-4 and the nuclear hormone receptor gene odr-7 are required to confer AWB and AWA-specific characteristics respectively, and to repress an AWC olfactory neuron-like default fate. Here, we show that AWA neuron fate is also regulated by a member of the LIM homeobox gene family, lin-11. lin-11 regulates AWA olfactory neuron differentiation by initiating expression of odr-7, which then autoregulates to maintain expression. lin-11 also regulates the fate of the ASG chemosensory neurons, which are the lineal sisters of the AWA neurons. We show that lin-11 is expressed dynamically in the AWA and ASG neurons, and that misexpression of lin-11 is sufficient to promote an ASG, but not an AWA fate, in a subset of neuron types. Our results suggest that differential temporal regulation of lin-11, presumably together with its interaction with asymmetrically segregated factors, results in the generation of the distinct AWA and ASG sensory neuron types. We propose that a LIM code may be an important contributor to the generation of functional diversity in a subset of olfactory and chemosensory neurons in C. elegans.
Tumorhead, aXenopusgene product that inhibits neural differentiation through regulation of proliferation
Authors: Wu Chuan Fen , Nakamura Hisashi , Chan Agnes Pui-Yee , Zhou Yi-Hong , Cao Tongyu , Kuang Jian , Gong Siew-Ging , He Guangan , Etkin Laurence D.
Published: April 2021

Tumorhead (TH) is a novel maternal gene product from Xenopus laevis containing several basic domains and a weak coiled-coil. Overexpression of wild-type TH resulted in increased proliferation of neural plate cells, causing expansion of the neural field followed by neural tube and craniofacial abnormalities. Overexpressed TH protein repressed neural differentiation and neural crest markers, but did not inhibit the neural inducers, pan-neural markers or mesodermal markers. Loss of function by injection of anti-TH antibody inhibited cell proliferation. Our data are consistent with a model in which tumorhead functions in regulating differentiation of the neural tissues but not neural induction or determination through its effect on cell proliferation.
Developmentally regulated cell cycle dependence of swelling-activated anion channel activity in the mouse embryo
Authors: Kolajova Marika , Hammer Mary-Anne , Collins Jennifer L. , Baltz Jay M.
Published: April 2021

Anion channels activated by increased cell volume are a nearly ubiquitous mechanism of cell volume regulation, including in early preimplantation mouse embryos. Here, we show that the swelling-activated anion current (ICl,swell) in early mouse embryos is cell-cycle dependent, and also that this dependence is developmentally regulated. ICl,swell is present both in first meiotic prophase (germinal vesicle stage) mouse oocytes and in unfertilized mature oocytes in second meiotic metaphase, and it persists after fertilization though the 1-cell and 2-cell stages. ICl,swell was found to remain unchanged during metaphase at the end of the 1-cell stage. However, ICl,swell decreased during prophase and became nearly undetectable upon entry into metaphase at the end of the 2-cell stage. Entry into prophase/metaphase was required for the decrease in ICl,swell at the end of the 2-cell stage, since it persisted indefinitely in 2-cell embryos arrested in late G2. There is considerable evidence that the channel underlying ICl,swell is not only permeable to inorganic anions, but to organic osmolytes as well. We found a similar pattern of cell cycle and developmental dependence in the 1-cell and 2-cell stages for the swelling-induced increase in permeability to the organic osmolyte glycine. Thus, entry into metaphase deactivates ICl,swell in embryos, but only after developmental progression through the 2-cell stage.
Sphingosine-1-phosphate lyase has a central role in the development of Dictyostelium discoideum
Authors: Li Guochun , Foote Christopher , Alexander Stephen , Alexander Hannah
Published: April 2021

Sphingosine-1-phosphate, a product of sphingomyelin degradation, is an important element of signal transduction pathways that regulate cell proliferation and cell death. We have demonstrated additional roles for sphingosine-1-phosphate in growth and multicellular development. The specific disruption in Dictyostelium discoideum of the sphingosine-1-phosphate lyase gene, which encodes the enzyme that catalyzes sphingosine-1-phosphate degradation, results in a mutant strain with aberrant morphogenesis, as well as an increase in viability during stationary phase. The absence of sphingosine-1-phosphate lyase affects multiple stages throughout development, including the cytoskeletal architecture of aggregating cells, the ability to form migrating slugs, and the control of cell type-specific gene expression and terminal spore differentiation. This pleiotropic effect, which is due to the loss of sphingosine-1-phosphate lyase, establishes sphingolipids as pivotal regulatory molecules in a wide range of processes in multicellular development.
four-jointedinteracts withdachs, abelsonandenabledand feeds back onto theNotchpathway to affect growth and segmentation in theDrosophilaleg
Authors: Buckles Gerri R. , Rauskolb Cordelia , Villano John Lee , Katz Flora N.
Published: April 2021

The molecular basis of segmentation and regional growth during morphogenesis of Drosophila legs is poorly understood. We show that four-jointed is not only required for these processes, but also can direct ectopic growth and joint initiation when its normal pattern of expression is disturbed. These effects are non-autonomous, consistent with our demonstration of both transmembrane and secreted forms of the protein in vivo. The similarities between four-jointed and Notch phenotypes led us to further investigate the relationships between these pathways. Surprisingly, we find that although four-jointed expression is regulated downstream of Notch activation, four-jointed can induce expression of the Notch ligands, Serrate and Delta, and may thereby participate in a feedback loop with the Notch signaling pathway. We also show that four-jointed interacts with abelson, enabled and dachs, which leads us to suggest that one target of four-jointed signaling is the actin cytoskeleton. Thus, four-jointed may bridge the gap between the signals that direct morphogenesis and those that carry it out.
A role for frizzled 3 in neural crest development
Authors: Deardorff Matthew A. , Tan Change , Saint-Jeannet Jean-Pierre , Klein Peter S.
Published: April 2021

Wnts are a large family of secreted molecules implicated in numerous developmental processes. Frizzled proteins are likely receptors for Wnts and are required for Wnt signaling in invertebrates. A large number of vertebrate frizzled genes have also been identified, but their roles in mediating specific responses to endogenous Wnts have not been well defined. Using a functional assay in Xenopus, we have performed a large screen to identify potential interactions between Wnts and frizzleds. We find that signaling by Xwnt1, but not other Wnts, can be specifically enhanced by frizzled 3 (Xfz3). As both Xfz3 and Xwnt1 are highly localized to dorsal neural tissues that give rise to neural crest, we examined whether Xfz3 mediates Xwnt1 signaling in the formation of neural crest. Xfz3 specifically induces neural crest in ectodermal explants and in embryos, similar to Xwnt1, and at lower levels of expression, synergizes with Xwnt1 in neural crest induction. Furthermore, loss of Xfz3 function, either by depletion with a Xfz3-directed morpholino antisense oligonucleotide or by expression of an inhibitory form of Xfz3 (Nfz3), prevents Xwnt1-dependent neural crest induction in ectodermal explants and blocks neural crest formation in whole embryos. These results show that Xfz3 is required for Xwnt1 signaling in the formation of the neural crest in the developing vertebrate embryo.
Kermit, a frizzled interacting protein, regulates frizzled 3 signaling in neural crest development
Authors: Tan Change , Deardorff Matthew A. , Saint-Jeannet Jean-Pierre , Yang Jing , Arzoumanian Arpine , Klein Peter S.
Published: April 2021

Wnts are a family of secreted glycoproteins that are important for multiple steps in early development. Accumulating evidence suggests that frizzled genes encode receptors for Wnts. However, the mechanism through which frizzleds transduce a signal and the immediate downstream components that convey that signal are unclear. We have identified a new protein, Kermit, that interacts specifically with the C-terminus of Xenopus frizzled-3 (Xfz3). Kermit is a 331 amino acid protein with a central PDZ domain. Kermit mRNA is expressed throughout Xenopus development and is localized to neural tissue in a pattern that overlaps Xfz3 expression temporally and spatially. Co-expression of Xfz3 and Kermit results in a dramatic translocation of Kermit to the plasma membrane. Inhibition of Kermit function with morpholino antisense oligonucleotides directed against the 5′ untranslated region of Kermit mRNA blocks neural crest induction by Xfz3, and this is rescued by co-injection of mRNA encoding the Kermit open reading frame. These observations suggest that Kermit is required for Wnt/frizzled signaling in neural crest development. To the best of our knowledge, Kermit is the first protein identified that interacts directly with the cytoplasmic portion of frizzleds to modulate their signaling activity.
Downregulation of the tissue-specific transcription factor Fork head byBroad-Complexmediates a stage-specific hormone response
Authors: Renault Nathalie , King-Jones Kirst , Lehmann Michael
Published: April 2021

Drosophila development is coordinated by pulses of the steroid hormone 20-hydroxyecdysone (20E). During metamorphosis, the 20E-inducible Broad-Complex (BR-C) gene plays a key role in the genetic hierarchies that transduce the hormone signal, being required for the destruction of larval tissues and numerous aspects of adult development. Most of the known BR-C target genes, including the salivary gland secretion protein (Sgs) genes, are terminal differentiation genes that are thought to be directly regulated by BR-C-encoded transcription factors. Here, we show that repression of Sgs expression is indirectly controlled by the BR-C through transcriptional down-regulation of fork head, a tissue-specific gene that plays a central role in salivary gland development and is required for Sgs expression. Our results demonstrate that integration of a tissue-specific regulatory gene into a 20E-controlled genetic hierarchy provides a mechanism for hormonal repression. Furthermore, they suggest that the BR-C is placed at a different position within the 20E-controlled hierarchies than previously assumed, and that at least part of its pleiotropic functions are mediated by tissue-specific regulators.
GDNF and neurturin are target-derived factors essential for cranial parasympathetic neuron development
Authors: Hashino Eri , Shero Marlene , Junghans Dirk , Rohrer Hermann , Milbrandt Jeffrey , Johnson Eugene M.
Published: April 2021

During development, parasympathetic ciliary ganglion neurons arise from the neural crest and establish synaptic contacts on smooth and striate muscle in the eye. The factors that promote the ciliary ganglion pioneer axons to grow toward their targets have yet to be determined. Here, we show that glial cell line-derived neurotrophic factor (GDNF) and neurturin (NRTN) constitute target-derived factors for developing ciliary ganglion neurons. Both GDNF and NRTN are secreted from eye muscle located in the target and trajectory pathway of ciliary ganglion pioneer axons during the period of target innervation. After this period, however, the synthesis of GDNF declines markedly, while that of NRTN is maintained throughout the cell death period. Furthermore, both in vitro and in vivo function-blocking of GDNF at early embryonic ages almost entirely suppresses ciliary axon outgrowth. These results demonstrate that target-derived GDNF is necessary for ciliary ganglion neurons to innervate ciliary muscle in the eye. Since the down-regulation of GDNF in the eye is accompanied by down-regulation of GFRα1 and Ret, but not of GFRα2, in innervating ciliary ganglion neurons, the results also suggest that target-derived GDNF regulates the expression of its high-affinity coreceptors.
Facultative heterochromatization in parahaploid male mealybugs: involvement of a heterochromatin-associated protein
Authors: Bongiorni Silvia , Mazzuoli Milena , Masci Stefania , Prantera Giorgio
Published: April 2021

The behavior of chromosomes during development of the mealybug Planococcus citri provides one of the most dramatic examples of facultative heterochromatization. In male embryos, the entire haploid paternal chromosome set becomes heterochromatic at mid-cleavage. Male mealybugs are thus functionally haploid, owing to heterochromatization (parahaploidy). To understand the mechanisms underlying facultative heterochromatization in male mealybugs, we have investigated the possible involvement of an HP-1-like protein in this process. HP-1 is a conserved, nonhistone chromosomal protein with a proposed role in heterochromatinization in other species. It was first identified in Drosophila melanogaster as a protein enriched in the constitutive heterochromatin of polytene chromosome. Using a monoclonal antibody raised against the Drosophila HP-1 in immunoblot and immunocytological experiments, we provide evidence for the presence of an HP-1-like in Planococcus citri males and females. In males, the HP-1-like protein is preferentially associated with the male-specific heterochromatin. In the developing male embryos, its appearance precedes the onset of heterochromatization. In females, the HP-1-like protein displays a scattered but reproducible localization pattern along chromosomes. The results indicate a role for an HP-1-like protein in the facultative heterochromatization process.
The nematode even-skipped homolog vab-7 regulates gonad and vulva position in Pristionchus pacificus
Authors: Jungblut Benno , Sommer Ralf J.
Published: April 2021

In free-living nematodes, developmental processes like the formation of the vulva, can be studied at a cellular level. Cell lineage and ablation studies have been carried out in various nematode species and multiple changes in vulval patterning have been identified. In Pristionchus pacificus, vulva formation differs from Caenorhabditis elegans with respect to several autonomous and conditional aspects of cell fate specification. To understand the molecular basis of these evolutionary changes, we have performed a genetic analysis of vulva formation in P. pacificus. Here, we describe two mutants where the vulva is shifted posteriorly, affecting which precursor cells will form vulval tissue in P. pacificus. Mutant animals show a concomitant posterior displacement of the gonadal anchor cell, indicating that the gonad and the vulva are affected in a similar way. We show that mutations in the even-skipped homolog of nematodes, vab-7, cause these posterior displacements. In addition, cell ablation studies in the vab-7 mutant indicate that the altered position of the gonad not only changes the cell fate pattern but also the developmental competence of vulval precursor cells. Investigation of Cel-vab-7 mutant animals showed a similar but weaker vulva defective phenotype to the one described for Ppa-vab-7.
Asymmetric Prospero localization is required to generate mixed neuronal/glial lineages in theDrosophilaCNS
Authors: Freeman Marc R. , Doe Chris Q.
Published: April 2021

In many organisms, single neural stem cells can generate both neurons and glia. How are these different cell types produced from a common precursor? In Drosophila, glial cells missing (gcm) is necessary and sufficient to induce glial development in the CNS. gcm mRNA has been reported to be asymmetrically localized to daughter cells during precursor cell division, allowing the daughter cell to produce glia while precursor cell generates neurons. We show that (1) gcm mRNA is uniformly distributed during precursor cell divisions; (2) the Prospero transcription factor is asymmetrically localized into the glial-producing daughter cell; (3) Prospero is required to upregulate gcm expression and induce glial development; and (4) mislocalization of Prospero to the precursor cell leads to ectopic gcm expression and the production of extra glia. We propose a novel model for the separation of glia and neuron fates in mixed lineages in which the asymmetric localization of Prospero results in upregulation of gcm expression and initiation of glial development in only precursor daughter cells.
Fgf signalling through MAPK cascade is required for development of the subpallial telencephalon in zebrafish embryos
Authors: Shinya Minori , Koshida Sumito , Sawada Atsushi , Kuroiwa Atsushi , Takeda Hiroyuki
Published: April 2021

The telencephalon is formed in the most anterior part of the central nervous system (CNS) and is organised into ventral subpallial and dorsal pallial domains. In mice, it has been demonstrated that Fgf signalling has an important role in induction and patterning of the telencephalon. However, the precise role of Fgf signalling is still unclear, owing to overlapping functions of Fgf family genes. To address this, we have examined, in zebrafish embryos, the activation of Ras/mitogen-activated protein kinase (MAPK), one of the major downstream targets of Fgf signalling. Immunohistochemical analysis reveals that an extracellular signal-regulated kinase (ERK), a vertebrate MAPK is activated in the anterior neural boundary (ANB) of the developing CNS at early segmentation stages. Experiments with Fgf inhibitors reveal that ERK activation at this stage is totally dependent on Fgf signalling. Interestingly, a substantial amount of ERK activation is observed in ace mutants in which fgf8 gene is mutated. We then examine the function of Fgf signalling in telencephalic development by use of several inhibitors to Fgf signalling cascade, including dominant-negative forms of Ras (RasN17) and the Fgf receptor (Fgfr), and a chemical inhibitor of Fgfr, SU5402. In treated embryos, the induction of telencephalic territory normally proceeded but the development of the subpallial telencephalon was suppressed, indicating that Fgf signalling is required for the regionalisation within the telencephalon. Finally, antisense experiments with morpholino-modified oligonucleotides suggest that zebrafish fgf3, which is also expressed in the ANB, co-operates with fgf8 in subpallial development.
spiel ohne grenzen/pou2is required during establishment of the zebrafish midbrain-hindbrain boundary organizer
Authors: Belting Heinz-Georg , Hauptmann Giselbert , Meyer Dirk , Abdelilah-Seyfried Salim , Chitnis Ajay , Eschbach Cathrin , Söll Iris , Thisse Christine , Thisse Bernard , Artinger Kristin B. , Lunde Karen , Driever Wolfgang
Published: April 2021

The vertebrate midbrain-hindbrain boundary (MHB) organizes patterning and neuronal differentiation in the midbrain and anterior hindbrain. Formation of this organizing center involves multiple steps, including positioning of the MHB within the neural plate, establishment of the organizer and maintenance of its regional identity and signaling activities. Juxtaposition of the Otx2 and Gbx2 expression domains positions the MHB. How the positional information is translated into activation of Pax2, Wnt1 and Fgf8 expression during MHB establishment remains unclear. In zebrafish spiel ohne grenzen (spg) mutants, the MHB is not established, neither isthmus nor cerebellum form, the midbrain is reduced in size and patterning abnormalities develop within the hindbrain. In spg mutants, despite apparently normal expression of otx2, gbx1 and fgf8 during late gastrula stages, the initial expression of pax2.1, wnt1 and eng2, as well as later expression of fgf8 in the MHB primordium are reduced. We show that spg mutants have lesions in pou2, which encodes a POU-domain transcription factor. Maternal pou2 transcripts are distributed evenly in the blastula, and zygotic expression domains include the midbrain and hindbrain primordia during late gastrulation. Microinjection of pou2 mRNA can rescue pax2.1 and wnt1 expression in the MHB of spg/pou2 mutants without inducing ectopic expression. This indicates an essential but permissive role for pou2 during MHB establishment. pou2 is expressed normally in noi/pax2.1 and ace/fgf8 zebrafish mutants, which also form no MHB. Thus, expression of pou2 does not depend on fgf8 and pax2.1. Our data suggest that pou2 is required for the establishment of the normal expression domains of wnt1 and pax2.1 in the MHB primordium.
Unique functions of Sonic hedgehog signaling during external genitalia development
Authors: Haraguchi Ryuma , Mo Rong , Hui Chi-chung , Motoyama Jun , Makino Shigeru , Shiroishi Toshihiko , Gaffield William , Yamada Gen
Published: April 2021

Coordinated growth and differentiation of external genitalia generates a proximodistally elongated structure suitable for copulation and efficient fertilization. The differentiation of external genitalia incorporates a unique process, i.e. the formation of the urethral plate and the urethral tube. Despite significant progress in molecular embryology, few attempts have been made to elucidate the molecular developmental processes for external genitalia. The sonic hedgehog (Shh) gene and its signaling genes have been found to be dynamically expressed during murine external genitalia development. Functional analysis by organ culture revealed that Shh could regulate mesenchymally expressed genes, patched 1 (Ptch1), bone morphogenetic protein 4 (Bmp4), Hoxd13 and fibroblast growth factor 10 (Fgf10), in the anlage: the genital tubercle (GT). Activities of Shh for both GT outgrowth and differentiation were also demonstrated. Shh–/– mice displayed complete GT agenesis, which is compatible with such observations. Furthermore, the regulation of apoptosis during GT formation was revealed for the first time. Increased cell death and reduced cell proliferation of the Shh–/– mice GT were shown. A search for alterations of Shh downstream gene expression identified a dramatic shift of Bmp4 gene expression from the mesenchyme to the epithelium of the Shh mutant before GT outgrowth. Regulation of mesenchymal Fgf10 gene expression by the epithelial Shh was indicated during late GT development. These results suggest a dual mode of Shh function, first by the regulation of initiating GT outgrowth, and second, by subsequent GT differentiation.
Increase of cGMP, cADP-ribose and inositol 1,4,5-trisphosphate preceding Ca2+ transients in fertilization of sea urchin eggs
Authors: Kuroda Ritsu , Kontani Kenji , Kanda Yasunari , Katada Toshiaki , Nakano Takashi , Satoh Yu-ichi , Suzuki Norio , Kuroda Hideyo
Published: April 2021

Transient increases, or oscillations, of cytoplasmic free Ca2+ concentration, [Ca2+]i, occur during fertilization of animal egg cells. In sea urchin eggs, the increased Ca2+ is derived from intracellular stores, but the principal signaling and release system involved has not yet been agreed upon. Possible candidates are the inositol 1,4,5-trisphosphate receptor/channel (IP3R) and the ryanodine receptor/channel (RyR) which is activated by cGMP or cyclic ADP-ribose (cADPR). Thus, it seemed that direct measurements of the likely second messenger candidates during sea urchin fertilization would be essential to an understanding of the Ca2+ signaling pathway. We therefore measured the cGMP, cADPR and inositol 1,4,5-trisphosphate (IP3) contents of sea urchin eggs during the early stages of fertilization and compared these with the [Ca2+]i rise in the presence or absence of an inhibitor against soluble guanylate cyclase. We obtained three major experimental results: (1) cytosolic cGMP levels began to rise first, followed by cADPR and IP3 levels, all almost doubling before the explosive increase of [Ca2+]i; (2) most of the rise in IP3 occurred after the Ca2+ peak; IP3 production could also be induced by the artificial elevation of [Ca2+]i, suggesting the large increase in IP3 is a consequence, rather than a cause, of the Ca2+ transient; (3) the measured increase in cGMP was produced by the soluble guanylate cyclase of eggs, and inhibition of soluble guanylate cyclase of eggs diminished the production of both cADPR and IP3 and the [Ca2+]i increase without the delay of Ca2+ transients. Taken together, these results suggest that the RyR pathway involving cGMP and cADPR is not solely responsible for the initiating event, but contributes to the Ca2+ transients by stimulating IP3 production during fertilization of sea urchin eggs.
BMPR-IA signaling is required for the formation of the apical ectodermal ridge and dorsal-ventral patterning of the limb
Authors: Ahn Kyung , Mishina Yuji , Hanks Mark C. , Behringer Richard R. , Crenshaw E. Bryan
Published: April 2021

We demonstrate that signaling via the bone morphogenetic protein receptor IA (BMPR-IA) is required to establish two of the three cardinal axes of the limb: the proximal-distal axis and the dorsal-ventral axis. We generated a conditional knockout of the gene encoding BMPR-IA (Bmpr) that disrupted BMP signaling in the limb ectoderm. In the most severely affected embryos, this conditional mutation resulted in gross malformations of the limbs with complete agenesis of the hindlimbs. The proximal-distal axis is specified by the apical ectodermal ridge (AER), which forms from limb ectoderm at the distal tip of the embryonic limb bud. Analyses of the expression of molecular markers, such as Fgf8, demonstrate that formation of the AER was disrupted in the Bmpr mutants. Along the dorsal/ventral axis, loss of engrailed 1 (En1) expression in the non-ridge ectoderm of the mutants resulted in a dorsal transformation of the ventral limb structures. The expression pattern of Bmp4 and Bmp7 suggest that these growth factors play an instructive role in specifying dorsoventral pattern in the limb. This study demonstrates that BMPR-IA signaling plays a crucial role in AER formation and in the establishment of the dorsal/ventral patterning during limb development.
BMP controls proximodistal outgrowth, via induction of the apical ectodermal ridge, and dorsoventral patterning in the vertebrate limb
Authors: Pizette Sandrine , Abate-Shen Cory , Niswander Lee
Published: April 2021

Dorsoventral (DV) patterning of the vertebrate limb requires the function of the transcription factor Engrailed 1 (EN1) in the ventral ectoderm. EN1 restricts, to the dorsal half of the limb, the expression of the two genes known to specify dorsal pattern. Limb growth along the proximodistal (PD) axis is controlled by the apical ectodermal ridge (AER), a specialized epithelium that forms at the distal junction between dorsal and ventral ectoderm. Using retroviral-mediated misexpression of the bone morphogenetic protein (BMP) antagonist Noggin or an activated form of the BMP receptor in the chick limb, we demonstrate that BMP plays a key role in both DV patterning and AER induction. Thus, the DV and PD axes are linked by a common signal. Loss and gain of BMP function experiments show that BMP signaling is both necessary and sufficient to regulate EN1 expression, and consequently DV patterning. Our results also indicate that BMPs are required during induction of the AER. Manipulation of BMP signaling results in either disruptions in the endogenous AER, leading to absent or severely truncated limbs or the formation of ectopic AERs that can direct outgrowth. Moreover, BMP controls the expression of the MSX transcription factors, and our results suggest that MSX acts downstream of BMP in AER induction. We propose that the BMP signal bifurcates at the level of EN1 and MSX to mediate differentially DV patterning and AER induction, respectively.
The establishment of segmentation in theDrosophilaleg
Authors: Rauskolb Cordelia
Published: April 2021

Segmentation is a developmental mechanism that subdivides a tissue into repeating functional units, which can then be further elaborated upon during development. In contrast to embryonic segmentation, Drosophila leg segmentation occurs in a tissue that is rapidly growing in size and thus segmentation must be coordinated with tissue growth. I demonstrate that segmentation of the Drosophila leg, as assayed by expression of the key regulators of segmentation, the Notch ligands and fringe, occurs progressively and I define the sequence in which the initial segmental subdivisions arise. I further demonstrate that the proximal-distal patterning genes homothorax and dachshund are positively required, while Distal-less is unexpectedly negatively required, to establish the segmental pattern of Notch ligand and fringe expression. Two Serrate enhancers that respond to regulation by dachshund are also identified. Together, these studies provide evidence that distinct combinations of the proximal-distal patterning genes independently regulate each segmental ring of Notch ligand and fringe expression and that this regulation occurs through distinct enhancers. These studies thus provide a molecular framework for understanding how segmentation during tissue growth is accomplished.
Clonal analysis of differentiating embryonic stem cells reveals a hematopoietic progenitor with primitive erythroid and adult lymphoid-myeloid potential
Authors: Perlingeiro Rita C. R. , Kyba Michael , Daley George Q.
Published: April 2021

Embryonic stem (ES) cells differentiate into multiple hematopoietic lineages during embryoid body formation in vitro, but to date, an ES-derived hematopoietic stem cell has not been identified and subjected to clonal analysis in a manner comparable with hematopoietic stem cells from adult bone marrow. As the chronic myeloid leukemia-associated BCR/ABL oncogene endows the adult hematopoietic stem cell with clonal dominance without inhibiting pluripotent lymphoid and myeloid differentiation, we have used BCR/ABL as a tool to enable engraftment and clonal analysis. We show that embryoid body-derived hematopoietic progenitors expressing BCR/ABL maintain a primitive hematopoietic blast stage of differentiation and generate only primitive erythroid cell types in vitro. These cells can be cloned, and when injected into irradiated adult mice, they differentiate into multiple myeloid cell types as well as T and B lymphocytes. While the injected cells express embryonic (β-H1) globin, donor-derived erythroid cells in the recipient express only adult (β-major) globin, suggesting that these cells undergo globin gene switching and developmental maturation in vivo. These data demonstrate that an embryonic hematopoietic stem cell arises in vitro during ES cell differentiation that constitutes a common progenitor for embryonic erythroid and definitive lymphoid-myeloid hematopoiesis.
The Snail protein family regulates neuroblast expression ofinscuteableandstring, genes involved in asymmetry and cell division inDrosophila
Authors: Ashraf Shovon I. , Ip Y. Tony
Published: April 2021

Delaminated neuroblasts in Drosophila function as stem cells during embryonic central nervous system development. They go through repeated asymmetric divisions to generate multiple ganglion mother cells, which divide only once more to produce postmitotic neurons. Snail, a zinc-finger transcriptional repressor, is a pan-neural protein, based on its extensive expression in neuroblasts. Previous results have demonstrated that Snail and related proteins, Worniu and Escargot, have redundant and essential functions in the nervous system. We show that the Snail family of proteins control central nervous system development by regulating genes involved in asymmetry and cell division of neuroblasts. In mutant embryos that have the three genes deleted, the expression of inscuteable is significantly lowered, while the expression of other genes that participate in asymmetric division, including miranda, staufen and prospero, appears normal. The deletion mutants also have much reduced expression of string, suggesting that a key component that drives neuroblast cell division is abnormal. Consistent with the gene expression defects, the mutant embryos lose the asymmetric localization of prospero RNA in neuroblasts and lose the staining of Prospero protein that is normally present in ganglion mother cells. Simultaneous expression of inscuteable and string in the snail family deletion mutant efficiently restores Prospero expression in ganglion mother cells, demonstrating that the two genes are key targets of Snail in neuroblasts. Mutation of the dCtBP co-repressor interaction motifs in the Snail protein leads to reduction of the Snail function in central nervous system. These results suggest that the Snail family of proteins control both asymmetry and cell division of neuroblasts by activating, probably indirectly, the expression of inscuteable and string.
The functions ofpannierduringDrosophilaembryogenesis
Authors: Herranz Hector , Morata Ginés
Published: April 2021

The pannier (pnr) gene of Drosophila encodes a zinc-finger transcription factor of the GATA family and is involved in several developmental processes during embryonic and imaginal development. We report some novel aspects of the regulation and function of pnr during embryogenesis. Previous work has shown that pnr is activated by decapentaplegic (dpp) in early development, but we find that after stage 10, the roles are reversed and pnr becomes an upstream regulator of dpp. This function of pnr is necessary for the activation of the Dpp pathway in the epidermal cells implicated in dorsal closure and is not mediated by the JNK pathway, which is also necessary for Dpp activity in these cells. In addition, we show that pnr behaves as a selector-like gene in generating morphological diversity in the dorsoventral body axis. It is responsible for maintaining a subdivision of the dorsal half of the embryo into two distinct, dorsomedial and dorsolateral, regions, and also specifies the identity of the dorsomedial region. These results, together with prior work on its function in adults, suggest that pnr is a major factor in the genetic subdivision of the body of Drosophila.
Mutations inLIKE HETEROCHROMATIN PROTEIN 1affect flowering time and plant architecture inArabidopsis
Authors: Gaudin Valérie , Libault Marc , Pouteau Sylvie , Juul Trine , Zhao Gengchun , Lefebvre Delphine , Grandjean Olivier
Published: April 2021

In plants, recent studies have demonstrated links between the regulation of developmental processes and chromatin dynamics and organisation. Analysis of new mutations affecting overall plant architecture, leaf development and flowering time in Arabidopsis has allowed us to clone and characterise LHP1, the Drosophila heterochromatin protein 1 (HP1) homologue. LHP1 has the chromo and chromo shadow domains central to the function of animal proteins. Yeast two hybrid studies and in planta deletion experiments suggest similar modes of action in plants and animals via homodimer formation. In vivo localisation experiments revealed a specific subnuclear protein distribution in foci throughout the nucleus. Our data suggest that LHP1 may act as a main regulator of gene expression in plants, through formation of heterochromatin-like repressive complexes, to control developmental pathways involved in organ and cell size, and the vegetative to reproductive phase transition.
Role ofC. elegans lin-40MTA in vulval fate specification and morphogenesis
Authors: Chen Zhe , Han Min
Published: April 2021

Vulval differentiation in Caenorhabditis elegans involves several fundamental cellular events, including cell fusion, division and migration. We have characterized the role of the lin-40 (also known as egr-1) gene in these cellular processes. LIN-40 is homologous to the metastasis-associated factor 1 (MTA1) in mammals, which has been identified as a component of the nucleosome remodeling and histone deacetylation (NuRD) complex that functions as a transcriptional co-repressor. We show here that lin-40 negatively regulates vulval fate specification at least partly by promoting cell fusion between the vulval precursor cells and the hypodermal syncytium at an early larval stage. This inhibitory function of lin-40 might be carried out by downregulating lin-39 Hox expression. We also show that lin-40 is specifically required for cell divisions along the transverse orientation during vulval morphogenesis.
FGF-induced lens cell proliferation and differentiation is dependent on MAPK (ERK1/2) signalling
Authors: Lovicu Frank J. , McAvoy John W.
Published: April 2021

Members of the fibroblast growth factor (FGF) family induce lens epithelial cells to undergo cell division and differentiate into fibres; a low dose of FGF can stimulate cell proliferation (but not fibre differentiation), whereas higher doses of FGF are required to induce fibre differentiation. To determine if these cellular events are regulated by the same signalling pathways, we examined the role of mitogen-activated protein kinase (MAPK) signalling in FGF-induced lens cell proliferation and differentiation. We show that FGF induced a dose-dependent activation of extracellular regulated kinase 1/2 (ERK1/2) as early as 15 minutes in culture, with a high (differentiating) dose of FGF stimulating a greater level of ERK phosphorylation than a lower (proliferating) dose. Subsequent blocking experiments using UO126 (a specific inhibitor of ERK activation) showed that activation of ERK is required for FGF-induced lens cell proliferation and fibre differentiation. Interestingly, inhibition of ERK signalling can block the morphological changes associated with FGF-induced lens fibre differentiation; however, it cannot block the synthesis of some of the molecular differentiation markers, namely, β-crystallin. These findings are consistent with the in vivo distribution of the phosphorylated (active) forms of ERK1/2 in the lens. Taken together, our data indicate that different levels of ERK signalling may be important for the regulation of lens cell proliferation and early morphological events associated with fibre differentiation; however, multiple signalling pathways are likely to be required for the process of lens fibre differentiation and maturation.
Hedgehog signaling is required for pituitary gland development
Authors: Treier Mathias , O’Connell Shawn , Gleiberman Anatoli , Price Jenny , Szeto Daniel P. , Burgess Rob , Chuang Pao-Tien , McMahon Andrew P. , Rosenfeld Michael G.
Published: April 2021

Pituitary gland development serves as an excellent model system in which to study the emergence of distinct cell types from a common primordium in mammalian organogenesis. We have investigated the role of the morphogen Sonic hedgehog (SHH) in outgrowth and differentiation of the pituitary gland using loss-and gain-of-function studies in transgenic mice. Shh is expressed throughout the ventral diencephalon and the oral ectoderm, but its expression is subsequently absent from the nascent Rathke’s pouch as soon as it becomes morphologically visible, creating a Shh boundary within the oral epithelium. We used oral ectoderm/Rathke’s pouch-specific 5′ regulatory sequences (Pitx1HS) from the bicoid related pituitary homeobox gene (Pitx1) to target overexpression of the Hedgehog inhibitor Hip (Huntingtin interacting protein) to block Hedgehog signaling, finding that SHH is required for proliferation of the pituitary gland. In addition, we provide evidence that Hedgehog signaling, acting at the Shh boundary within the oral ectoderm, may exert a role in differentiation of ventral cell types (gonadotropes and thyrotropes) by inducing Bmp2 expression in Rathke’s pouch, which subsequently regulates expression of ventral transcription factors, particularly Gata2. Furthermore, our data suggest that Hedgehog signaling, together with FGF8/10 signaling, synergizes to regulate expression of the LIM homeobox gene Lhx3, which has been proved to be essential for initial pituitary gland formation. Thus, SHH appears to exert effects on both proliferation and cell-type determination in pituitary gland development.
Neuronal differences prefigure somatotopy in the zebrafish lateral line
Authors: Gompel Nicolas , Dambly-Chaudière Christine , Ghysen Alain
Published: April 2021

The central projection of the fish lateral line displays somatotopic ordering. In order to know when and how this ordering is established, we have labelled single sensory neurones and followed the growth of their neurites. We show that the neuromast cells and the corresponding neurones are not related by a fixed lineage, and also that somatotopic differences between anterior and posterior line neurones, and among neurones of the posterior line,are present before innervation of the sense organs. We propose that the position of the central projection defines the peripheral position that the neurone will innervate.
Otx2 is required for visceral endoderm movement and for the restriction of posterior signals in the epiblast of the mouse embryo
Authors: Perea-Gomez Aitana , Lawson Kirstie A. , Rhinn Muriel , Zakin Lise , Brûlet Philippe , Mazan Sylvie , Ang Siew-Lan
Published: April 2021

Genetic and embryological experiments have demonstrated an essential role for the visceral endoderm in the formation of the forebrain; however, the precise molecular and cellular mechanisms of this requirement are poorly understood. We have performed lineage tracing in combination with molecular marker studies to follow morphogenetic movements and cell fates before and during gastrulation in embryos mutant for the homeobox gene Otx2. Our results show, first, that Otx2 is not required for proliferation of the visceral endoderm, but is essential for anteriorly directed morphogenetic movement. Second, molecules that are normally expressed in the anterior visceral endoderm, such as Lefty1 and Mdkk1, are not expressed in Otx2 mutants. These secreted proteins have been reported to antagonise, respectively, the activities of Nodal and Wnt signals, which have a role in regulating primitive streak formation. The visceral endoderm defects of the Otx2 mutants are associated with abnormal expression of primitive streak markers in the epiblast, suggesting that anterior epiblast cells acquire primitive streak characteristics. Taken together, our data support a model whereby Otx2 functions in the anterior visceral endoderm to influence the ability of the adjacent epiblast cells to differentiate into anterior neurectoderm, indirectly, by preventing them from coming under the influence of posterior signals that regulate primitive streak formation.
lost-a-fin encodes a type I BMP receptor, Alk8, acting maternally and zygotically in dorsoventral pattern formation
Authors: Mintzer Keith A. , Lee Michelle A. , Runke Greg , Trout Jamie , Whitman Malcolm , Mullins Mary C.
Published: April 2021

TGFβ signaling pathways of the bone morphogenetic protein (BMP) subclass are essential for dorsoventral pattern formation of both vertebrate and invertebrate embryos. Here we determine by chromosomal mapping, linkage analysis, cDNA sequencing and mRNA rescue that the dorsalized zebrafish mutant lost-a-fin (laf) is defective in the gene activin receptor-like kinase 8 (alk8), which encodes a novel type I TGFβ receptor. The alk8 mRNA is expressed both maternally and zygotically. Embyros that lack zygotic, but retain maternal Laf/Alk8 activity, display a weak dorsalization restricted to the tail and die by 3 days postfertilization. We rescued the laf dorsalized mutant phenotype by alk8 mRNA injection and generated homozygous laf/alk8 mothers to investigate the maternal role of Laf/Alk8 activity. Adult fish lacking Laf/Alk8 activity are fertile, exhibit a growth defect and are significantly smaller than their siblings. Embryos derived from homozygous females, which lack both maternal and zygotic Laf/Alk8 activity, display a strongly dorsalized mutant phenotype, no longer limited to the tail. These mutant embryos lack almost all gastrula ventral cell fates, with a concomitant expansion of dorsal cell types. During later stages, most of the somitic mesoderm and neural tissue circumscribe the dorsoventral axis of the embryo. Zygotic laf/alk8 mutants can be rescued by overexpression of the BMP signal transducer Smad5, but not the Bmp2b or Bmp7 ligands, consistent with the Laf/Alk8 receptor acting within a BMP signaling pathway, downstream of a Bmp2b/Bmp7 signal. Antibodies specific for the phosphorylated, activated form of Smad1/5, show that BMP signaling is nearly absent in gastrula lacking both maternal and zygotic Laf/Alk8 activity, providing further evidence that Laf/Alk8 transduces a BMP signal. In total, our work strongly supports the role of Laf/Alk8 as a type I BMP receptor required for the specification of ventral cell fates.