bluuebunny/crossref_metadata_2025 huggingface dataset:
Longitudinal organization of the anterior neural plate and neural tube
Authors: Shimamura Kenji , Hartigan Dennis J. , Martinez Salvador , Puelles Luis , Rubenstein John L. R.
Published: April 2021

Over the last century, several morphological models of forebrain organization have been proposed that hypothesize alternative topological solutions for the relationships of the histogenic primordia. Central to all of these models are their definitions of the longitudinal axis and the longitudinal organization of the neural plate and neural tube. To understand the longitudinal organization of the anterior brain, we have sought to identify molecular properties that are continuous along the entire longitudinal axis of the embryonic CNS. In this essay, we describe studies of the expression of several genes in the mouse between 7.5 (presomite stage) and 10.5 days post coitum (dpc) that provide evidence for the trajectory of the anteriorposterior axis and the longitudinal organization of the anterior CNS.

Specifically, we report that the expression of noggin, sonic hedgehog and Nkx-2.2 define longitudinal columns of cells that are present along the entire CNS axis. Within the forebrain, the expression of these genes, as well as that of Nkx-2.1 and BF-1, are in distinct longitudinal regions in the neural plate and tube. We demonstrate that the earliest longitudinal axon pathways of the forebrain are spatially correlated with the longitudinal domain defined by Nkx-2.2. Finally, expression of the former genes, and Otx-1 and Emx-2, suggests that the cephalic neural plate is organized into molecularly distinct domains delimited by longitudinal and transverse borders; these results provide a foundation for defining the mechanisms that pattern the neural plate.
The role of yan in mediating the choice between cell division and differentiation
Authors: Rogge Ronald , Green Patricia J. , Urano Jun , Horn-Saban Shirley , Mlodzik Marek , Shilo Ben-Zion , Hartenstein Volker , Banerjee Utpal
Published: April 2021

An allele of the yan locus was isolated as an enhancer of the Ellipse mutation of the Drosophila epidermal growth factor receptor (Egfr) gene. This yan allele is an embryonic lethal and also fails to complement the lethality of anterior open (aop) mutations. Phenotypic and complementation analysis revealed that aop is allelic to yan and genetically the lethal alleles act as null mutations for the yan gene. Analysis of the lethal alleles in the embryo and in mitotic clones showed that loss of yan function causes cells to overproliferate in the dorsal neuroectoderm of the embryo and in the developing eye disc. Our studies suggest that the role of yan is defined by the developmental context of the cells in which it functions. An important role of this gene is in allowing a cell to choose between cell division and differentiation. The relationship of the Egfr and Notch pathways to this devel- opmental role of yan is discussed.
Expression of a truncated FGF receptor results in defective lens development in transgenic mice
Authors: Robinson Michael L. , MacMillan-Crow L. A. , Thompson John A. , Overbeek Paul A.
Published: April 2021

Members of the fibroblast growth factor (FGF) family are thought to initiate biological responses through the activation of cell surface receptors which must dimerize to transmit an intracellular signal. Mammalian lens epithelial cells respond to exogenous extracellular FGF, either in tissue culture or in transgenic mice, by initiating fiber cell differentiation. The role of FGF signalling in normal lens development was evaluated by lens-specific synthesis of a kinase-deficient FGF receptor type I (FGFR1) in transgenic mice. This truncated FGF receptor is thought to act as a dominant negative protein by heterodimerization with endogenous FGF receptors. The presence of transgenic mRNA in the lens was confirmed by in situ hybridization and by polymerase chain reaction amplification of reverse transcribed lens RNA (RT-PCR). The presence of transgenic protein was determined by Western blotting with antibodies to an extracellular domain of FGFR1. Three of four transgenic families expressing the truncated FGF receptor exhibited lens defects ranging from cataracts to severe microphthalmia. While the microphthalmic lenses displayed a normal pattern of differentiation-specific crystallin expression, the lens epithelial cells were reduced in number and the lens fiber cells displayed characteristics consistent with the induction of apoptosis. Our results support the view that FGF receptor signalling plays an essential role in normal lens biology.
Evolution of neuroblast identity: seven-up and prospero expression reveal homologous and divergent neuroblast fates in Drosophila and Schistocerca
Authors: Broadus Julie , Doe Chris Q.
Published: April 2021

In the Drosophila CNS, early neuroblast formation and fate are controlled by the pair-rule class of segmentation genes. The distantly related Schistocerca (grasshopper) embryo has a similar arrangement of neuroblasts, despite lack of known pair-rule gene function. Does divergent pair-rule gene function lead to different neuroblast identities, or can different patterning mechanisms produce homologous neuroblasts? We use four molecular markers to compare Drosophila and Schistocerca neuroblast identity: seven-up, prospero, engrailed, and fushi-tarazu/Dax. In both insects some early-forming neuroblasts share key features of neuroblast identity (position, time of formation, and temporally accurate gene expression); thus, different patterning mechanisms can generate similar neuroblast fates. In contrast, several later-forming neuroblasts show speciesspecific differences in position and/or gene expression; these neuroblast identities seem to have diverged, suggesting that evolution of the insect central nervous system can occur through changes in embryonic neuroblast identity.
Expression of truncated Sek-1 receptor tyrosine kinase disrupts the segmental restriction of gene expression in the Xenopus and zebrafish hindbrain
Authors: Xu Qiling , Alldus Graham , Holder Nigel , Wilkinson David G.
Published: April 2021

During development of the vertebrate hindbrain regulatory gene expression is confined to precise segmental domains. Studies of cell lineage and gene expression suggest that establishment of these domains may involve a dynamic regulation of cell identity and restriction of cell movement between segments. We have taken a dominant negative approach to interfere with the function of Sek-1, a member of the Eph-related receptor tyrosine kinase family expressed in rhombomeres r3 and r5. In Xenopus and zebrafish embryos expressing truncated Sek-1, lacking kinase sequences, expression of r3/r5 markers occurs in adjacent even-numbered rhombomeres, in domains contiguous with r3 or r5. This disruption is rescued by fulllength Sek-1, indicating a requirement for the kinase domain in the segmental restriction of gene expression. These data suggest that Sek-1, perhaps with other Ephrelated receptors, is required for interactions that regulate the segmental identity or movement of cells.
The gene virilizer is required for female-specific splicing controlled by Sxl, the master gene for sexual development in Drosophila
Authors: Hilfiker Andres , Amrein Hubert , Dübendorfer Andreas , Schneiter Roger , Nöthiger Rolf
Published: April 2021

The gene virilizer (vir) is needed for dosage compensation and sex determination in females and for an unknown vital function in both sexes. In genetic mosaics, XX somatic cells mutant for vir differentiate male structures. One allele, vir2f, is lethal for XX, but not for XY animals. This femalespecific lethality can be rescued by constitutive expression of Sxl or by mutations in msl (male-specific lethal) genes. Rescued animals develop as strongly masculinized intersexes or pseudomales. They have male-specifically spliced mRNA of tra, and when rescued by msl, also of Sxl. Our data indicate that vir is a positive regulator of femalespecific splicing of Sxl and of tra pre-mRNA.
Evolutionary conservation of a cell fate specification gene: the Hydra achaete-scute homolog has proneural activity in Drosophila
Authors: Grens Ann , Mason Elizabeth , Marsh J. Lawrence , Bode Hans R.
Published: April 2021

Members of the Achaete-scute family of basic helix-loophelix transcription factors are involved in cell fate specification in vertebrates and invertebrates. We have isolated and characterized a cnidarian achaete-scute homolog, CnASH, from Hydra vulgaris, a representative of an evolutionarily ancient branch of metazoans. There is a single achaete-scute gene in Hydra, and the bHLH domain of the predicted gene product shares a high degree of amino acid sequence similarity with those of vertebrate and Drosophila Achaete-scute proteins. In Hydra, CnASH is expressed in a subset of the interstitial cells as well as differentiation intermediates of the nematocyte pathways. In vitro translated CnASH protein can form heterodimers with the Drosophila bHLH protein Daughterless, and these dimers bind to consensus Achaete-scute DNA binding sites in a sequence-specific manner. Ectopic expression of CnASH in wild-type late third instar Drosophila larvae and early pupae leads to the formation of ectopic sensory organs, mimicking the effect of ectopic expression of the endogenous achaete-scute genes. Expression of CnASH in flies that are achaete and scute double mutants gives partial rescue of the mutant phenotype, comparable to the degree of rescue obtained by ectopic expression of the Drosophila genes. These results indicate that the achaete-scute type of bHLH genes for cell fate specification, as well as their mode of action, arose early and have been conserved during metazoan evolution.
Pax-2 controls multiple steps of urogenital development
Authors: Torres Miguel , Gómez-Pardo Emilia , Dressler Gregory R. , Gruss Peter
Published: April 2021

Urogenital system development in mammals requires the coordinated differentiation of two distinct tissues, the ductal epithelium and the nephrogenic mesenchyme, both derived from the intermediate mesoderm of the early embryo. The former give rise to the genital tracts, ureters and kidney collecting duct system, whereas mesenchymal components undergo epithelial transformation to form nephrons in both the mesonephric (embryonic) and metanephric (definitive) kidney. Pax-2 is a transcriptional regulator of the paired-box family and is widely expressed during the development of both ductal and mesenchymal components of the urogenital system. We report here that Pax-2 homozygous mutant newborn mice lack kidneys, ureters and genital tracts. We attribute these defects to dysgenesis of both ductal and mesenchymal components of the developing urogenital system. The Wolffian and Müllerian ducts, precursors of male and female genital tracts, respectively, develop only partially and degenerate during embryogenesis. The ureters, inducers of the metanephros are absent and therefore kidney development does not take place. Mesenchyme of the nephrogenic cord fails to undergo epithelial transformation and is not able to form tubules in the mesonephros. In addition, we show that the expression of specific markers for each of these components is de-regulated in Pax-2 mutants. These data show that Pax-2 is required for multiple steps during the differentiation of intermediate mesoderm. In addition, Pax-2 mouse mutants provide an animal model for human hereditary kidney diseases.
Cooperative interactions between extracellular matrix, integrins and parathyroid hormone-related peptide regulate parietal endoderm differentiation in mouse embryos
Authors: Behrendtsen Ole , Alexander Caroline M. , Werb Zena
Published: April 2021

The outgrowth of parietal endoderm (PE) cells from precursor endodermal cells is one of the first differentiation events that occur in mouse embryos. We have analyzed the molecular determinants of this process by placing isolated inner cell masses (ICMs) on defined extracellular matrix substrata in microdrop cultures. Differentiation and outgrowth of PE required a fibronectin substratum. Laminin supported the adhesion and outgrowth of visceral endoderm (VE) and actively suppressed the differentiation of PE in mixtures of fibronectin and laminin. Collagen type IV, gelatin, vitronectin or entactin supported little or no endodermal outgrowth. Trophectoderm (TE) cells have been implied to be important in PE induction in vivo. We found that recombination of ICMs in culture with TE cells, or with medium conditioned by TE cells, greatly increased the differentiation of PE. TE cells stimulated PE outgrowth on substrata other than fibronectin. One cytokine secreted by trophoblast and endodermal cells, parathyroid hormone-related peptide (PTHrP), was critical for outgrowth on any substratum. A function-perturbing antibody to PTHrP reduced the number of PE cells, whereas the addition of PTHrP increased that number. Furthermore, addition of PTHrP changed the substratum requirements for outgrowth, making laminin, vitronectin and low concentrations of fibronectin permissive for PE outgrowth. Immunostaining with anti-integrin antibodies showed that fully differentiated PE cells outgrowing on fibronectin expressed α5, α6 and αVβ3 integrins. However, analysis of outgrowths in the presence of function-perturbing antibodies to α5, α6 and αVβ3 integrins showed that these integrins directed PE outgrowth only on fibronectin, laminin and vitronectin substrata, respectively. We have shown that there is a cooperative interplay of extracellular matrix, integrins and PTHrP that modulates PE outgrowth.
headcase, an imaginal specific gene required for adult morphogenesis in Drosophila melanogaster
Authors: Weaver Thomas A. , White Robert A. H.
Published: April 2021

The majority of adult organs of a holometabolic insect like Drosophila melanogaster are derived from specific imaginal cells. These cells differ from their larval equivalents in many important cellular characteristics, ranging from the nature of the cell cycle to the timing and pattern of cellular differentiation. Here we describe the cellular, molecular and genetic characterization of a gene, headcase (hdc), which is required for imaginal cell development. hdc is the first gene to be described which is specifically expressed in all imaginal cells; this has allowed us to identify many imaginal primordia in the embryo and follow their mor- phogenesis throughout embryonic and larval development. The Hdc protein is an extremely basic (pI 9.6) cytoplasmic protein with no obvious sequence similarities or conserved motifs. Interestingly, the spatial-temporal pattern of hdc expression prefigures imaginal cell re-entry into the mitotic cell cycle and persists until the final cell divisions. hdc null alleles have been isolated and found to cause pupal lethality, with dead pharate adults exhibiting defects in the differentiation of many adult tissues, most notably in head development. Ectopic expression of hdc, provided by a hdc- minigene, rescues the pupal lethality. Imaginal disc mor- phology in null mutants appears normal, therefore loss of hdc expression does not affect imaginal cell growth, but instead interferes with the ability of the imaginal primordia to differentiate properly during pupal development, sug- gesting that hdc may be involved in hormonal responsive- ness during metamorphosis.
patched overexpression alters wing disc size and pattern: transcriptional and post-transcriptional effects on hedgehog targets
Authors: Johnson Ronald L. , Grenier Jennifer K. , Scott Matthew P.
Published: April 2021

The membrane protein, Patched, plays a critical role in patterning embryonic and imaginal tissues in Drosophila. patched constitutively inactivates the transcription of target genes such as wingless, decapentaplegic, and patched itself. The secreted protein, Hedgehog, induces transcription of target genes by opposing the Patched signaling pathway. Using the Gal4 UAS system we have overexpressed patched in wing imaginal discs and found that high Patched levels, expressed in either normal or ectopic patterns, result in loss of wing vein patterning in both compartments centering at the anterior/posterior border. In addition, patched inhibits the formation of the mechanosensory neurons, the campaniform sensilla, in the wing blade. The patched wing vein phenotype is modulated by mutations in hedgehog and cubitus interruptus (ci). Patched overexpression inhibits transcription of patched and decapentaplegic and post-transcriptionally decreases the amount of Ci protein at the anterior/posterior boundary. In hedgehogMrt wing discs, which express ectopic hedgehog, Ci levels are correspondingly elevated, suggesting that hedgehog relieves patched repression of Ci accumulation. Protein kinase A also regulates Ci; protein kinase A mutant clones in the anterior compartment have increased levels of Ci protein. Thus patched influences wing disc patterning by decreasing Ci protein levels and inactivating hedgehog target genes in the anterior compartment.
Temporal and spatial regulation of H19 imprinting in normal and uniparental mouse embryos
Authors: Sasaki Hiroyuki , Ferguson-Smith Anne C. , Shum Alisa S. W. , Barton Sheila C. , Surani M. Azim
Published: April 2021

The mouse H19 gene is imprinted so that the paternal copy is both methylated and repressed during fetal development. However, the CpG-rich promoter region encompassing the transcription start is not methylated in sperm; this region must therefore become methylated postzygotically. We first examined the timing of DNA methylation of this region and the corresponding expression of H19. Both parental copies are initially undermethylated in blastocysts and the paternal copy then becomes fully methylated in the embryo around implantation; this methylation is more protracted in the extraembryonic lineages, especially in the trophoblast. By contrast to the lineage-dependent methylation, we observed exclusive expression of the maternal copy in preimplantation embryos and in all the lineages of early postimplantation embryos although variability may exist in cultured embryos. This indicates that methylation of the CpG-rich promoter is not a prerequisite for the paternal repression. We then examined whether methylation and expression occurs appropriately in the absence of a maternal or a paternal genome. Both H19 copies in androgenetic embryos are fully methylated while they are unmethylated in parthenogenetic embryos. This correlates with the lack of expression in androgenetic embryos but expression in parthenogenetic embryos. However, the androgenetic trophoblast was exceptional as it shows reduced methylation and expresses H19. These results suggest that promoter methylation is not the primary inactivation mechanism but is a stabilizing factor. Differential methylation in the more upstream region, which is established in the gametes, is a likely candidate for the gametic signal and may directly control H19 activity.
Induction of avian cardiac myogenesis by anterior endoderm
Authors: Schultheiss Thomas M. , Xydas Steve , Lassar Andrew B.
Published: April 2021

An experimental system was devised to study the mecha-nisms by which cells become committed to the cardiac myocyte lineage during avian development. Chick tissues from outside the fate map of the heart (in the posterior primitive streak {PPS} of a Hamburger & Hamilton stage 4 embryo) were combined with potential inducing tissues from quail embryos and cultured in vitro. Species-specific RT-PCR was employed to detect the appearance of the cardiac muscle markers chick Nkx-2.5 (cNkx-2.5), cardiac troponin C and ventricular myosin heavy chain in the chick responder tissues. Using this procedure, we found that stage 4-5 anterior lateral (AL) endoderm and anterior central (AC) mesendoderm, but not AL mesoderm or posterior lateral mesendoderm, induced cells of the PPS to differentiate as cardiac myocytes. Induction of cardiogen-esis was accompanied by a marked decrease in the expression of ρ-globin, implying that PPS cells were being induced by anterior endoderm to become cardiac myocytes instead of blood-forming tissue. These results suggest that anterior endoderm contains signaling molecules that can induce cardiac myocyte specification of early primitive streak cells.

One of the cardiac muscle markers induced by anterior endoderm, cNkx-2.5, is here described for the first time. cNkx-2.5 is a chick homeobox-containing gene that shares extensive sequence similarity with the Drosophila gene tinman, which is required for Drosophila heart formation. The mesodermal component of cNkx-2.5 expression from stage 5 onward, as determined by in situ hybridization, is strikingly in accord with the fate map of the avian heart. By the time the myocardium and endocardium form distinct layers, cNkx-2.5 is found only in the myocardium. cNkx-2.5 thus appears to be the earliest described marker of avian mesoderm fated to give rise to cardiac muscle.
Gene expression, polarising activity and skeletal patterning in reaggregated hind limb mesenchyme
Authors: Hardy Adrian , Richardson Michael K. , Francis-West Philippa H. , Rodriguez Concepción , Izpisúa-Belmonte Juan-Carlos , Duprez Delphine , Wolpert Lewis
Published: April 2021

The developing chick limb has two major signalling centres; the apical ectodermal ridge maintains expression of several important genes and outgrowth of the limb, and the polarising region specifies the pattern of skeletal elements along the anteroposterior axis. We have used reaggregated leg grafts (mesenchyme dissociated into single cells, placed in an ectodermal jacket and grafted to a host) to study patterning in a system where the developmental axes are severely disrupted. Reaggregates from different regions of leg mesenchyme developed corre-spondingly different digits, giving a system in which skeletal phenotype could be compared with the expression of genes thought to be important in patterning.

We found that posterior third and whole leg reaggregates gave rise to different digits, yet expressed the same combination of HoxD, Bmp-2 and shh genes throughout their development. Anterior thirds initially only express the 3′ end of the HoxD cluster but activate the more 5′ members of the cluster sequentially over a period of 48 hours, a period during which Bmp-2 is activated but no shh or Fgf-4 expression could be detected. Our results suggest that there are two independent mechanisms for activating the HoxD complex, one polarising region-dependent and one independent, and that shh expression may not be necessary to maintain outgrowth and patterning once a ridge has been established.
Early even-skipped stripes act as morphogenetic gradients at the single cell level to establish engrailed expression
Authors: Fujioka Miki , Jaynes James B. , Goto Tadaatsu
Published: April 2021

even-skipped (eve) has been proposed to set up parasegment borders at the anterior edge of each of its seven stripes by providing a sharp expression boundary, where engrailed is activated on one side and wingless on the other. By expressing bell-shaped early eve stripes without the sharp boundary provided by narrow, late stripes, we find that the early gradient is sufficient for generating stable parasegment borders. Based on several lines of evidence, we propose that the anterior portion of each early stripe has morphogenic activity, repressing different target genes at different concentrations. These distinct repression thresholds serve to both limit and subdivide a narrow zone of paired expression. Within this zone, single cell rows express either engrailed, where runt and sloppy-paired are repressed, or wingless, where they are not. While the early eve gradient is sufficient to establish parasegmental borders without refined, late expression, late eve expression has a role in augmenting this boundary to provide for strong, continuous stripes of engrailed expression. In addition, we show that the early eve gradient is sufficient, at its posterior edge, for subdividing the ftz domain into engrailed expressing and non-expressing cells.
The homeobox gene repo is required for the differentiation and maintenance of glia function in the embryonic nervous system of Drosophila melanogaster
Authors: Halter Daniel A. , Urban Joachim , Rickert Christof , Ner Sarbjit S. , Ito Kei , Travers Andrew A. , Technau Gerhard M.
Published: April 2021

We describe the cloning, expression and phenotypic characterisation of repo, a gene from Drosophila melanogaster that is essential for the differentiation and maintenance of glia function. It is not, however, required for the initial determination of glial cells. In the embryo, the gene, which encodes a homeodomain protein, is expressed exclusively in all developing glia and closely related cells in both the central and peripheral nervous systems. The only observed exceptions in the CNS are the midline glia derived from the mesectoderm and two of three segmental nerve root glial cells. Using a polyclonal antibody we traced the spatial and temporal pattern of the protein expression in detail.

Embryos homozygous for null alleles of the protein exhibit late developmental defects in the nervous system, including a reduction in the number of glial cells, disrupted fasciculation of axons, and the inhibition of ventral nerve cord condensation. The expression of an early glial-specific marker is unaffected in such homozygotes. By contrast, the expression of late glial-specific markers is either substantially reduced or absent. The specificity of expression is also observed in the locust Schistocerca gregaria and is thus evolutionarily conserved.
Hox genes and the evolution of vertebrate axial morphology
Authors: Burke Ann C. , Nelson Craig E. , Morgan Bruce A. , Tabin Cliff
Published: April 2021

A common form of evolutionary variation between vertebrate taxa is the different numbers of segments that contribute to various regions of the anterior-posterior axis; cervical vertebrae, thoracic vertebrae, etc. The term ‘transposition’ is used to describe this phenomenon. Genetic experiments with homeotic genes in mice have demonstrated that Hox genes are in part responsible for the specification of segmental identity along the anterior-posterior axis, and it has been proposed that an axial Hox code determines the morphology of individual vertebrae (Kessel, M. and Gruss, P. (1990) Science 249, 347-379). This paper presents a comparative study of the developmental patterns of homeobox gene expression and developmental morphology between animals that have homologous regulatory genes but different morphologies. The axia expression boundaries of 23 Hox genes were examined in the paraxial mesoderm of chick, and 16 in mouse embryos by in situ hybridization and immunolocalization techniques. Hox gene anterior expression boundaries were found to be transposed in concert with morphological boundaries. This data contributes a mechanistic level to the assumed homology of these regions in vertebrates. The recognition of mechanistic homology supports the historical homology of basic patterning mechanisms between all organisms that share these genes.
Neural induction and regionalisation by different subpopulations of cells in Hensen’s node
Authors: Storey Kate G. , Selleck Mark A. J. , Stern Claudio D.
Published: April 2021

Cell lineage analysis has revealed that the amniote organizer, Hensen’s node, is subdivided into distinct regions, each containing a characteristic subpopulation of cells with defined fates. Here, we address the question of whether the inducing and regionalising ability of Hensen’s node is associated with a specific subpopulation. Quail explants from Hensen’s node are grafted into an extraem-bryonic site in a host chick embryo allowing host- and donor-derived cells to be distinguished. Cell-type- and region-specific markers are used to assess the fates of the mesodermal and neural cells that develop.

We find that neural inducing ability is localised in the epiblast layer and the mesendoderm (deep portion) of the medial sector of the node. The deep portion of the posterolateral part of the node does not have neural inducing ability. Neural induction also correlates with the presence of particular prospective cell types in our grafts: chordamesoderm (notochord/head process), definitive (gut) endoderm or neural tissue. However, only grafts that include the epiblast layer of the node induce neural tissue expressing a complete range of anteroposterior characteristics, although prospective prechordal plate cells may also play a role in specification of the forebrain.
Pair-rule expression of the Drosophila fushi tarazu gene: a nuclear receptor response element mediates the opposing regulatory effects of runt and hairy
Authors: Tsai Chihcheng , Gergen Peter
Published: April 2021

The segmentation genes runt and hairy are required for the proper transcriptional regulation of the pair-rule gene fushi tarazu during the blastoderm stage of Drosophila embryogenesis. The expression of different fushi tarazu reporter genes was examined in runt and hairy mutant embryos, as well as in runt over-expressing embryos in order to identify DNA elements responsible for mediating these regulatory effects. The results indicated that runt and hairy act through a common 32 base-pair element. This element, designated as fDE1, contains a binding site for a small family of orphan nuclear receptor proteins that are uniformly expressed in blastoderm embryos. The pair-rule expression of reporter gene constructs containing multi-merized fDE1 elements depends on activation by runt and repression by hairy. Examination of reporter genes with mutated fDE1 elements provided further evidence that this element mediates both transcriptional activation and repression. Genetic experiments indicated that the opposing effects of runt and hairy were not due solely to cross-regulatory interactions between these two genes and that fDE1-dependent expression is regulated by factors in addition to runt and hairy.
nubbin encodes a POU-domain protein required for proximal-distal patterning in the Drosophila wing
Authors: Ng Medard , Diaz-Benjumea Fernando J. , Cohen Stephen M.
Published: April 2021

The nubbin gene is required for normal growth and patterning of the wing in Drosophila. We report here that nubbin encodes a member of the POU family of transcription factors. Regulatory mutants which selectively remove nubbin expression from wing imaginal discs lead to loss of wing structures. Although nubbin is expressed throughout the wing primordium, analysis of genetic mosaics suggests a localized requirement for nubbin activity in the wing hinge. These observations suggest the existence of a novel proximal-distal growth control center in the wing hinge, which is required in addition to the well characterized anterior-posterior and dorsal-ventral compartment boundary organizing centers.
Embryonic activation of the myoD gene is regulated by a highly conserved distal control element
Authors: Goldhamer David J. , Brunk Brian P. , Faerman Alexander , King Ayala , Shani Moshe , Emerson Charles P.
Published: April 2021

MyoD belongs to a small family of basic helix-loop-helix transcription factors implicated in skeletal muscle lineage determination and differentiation. Previously, we identified a transcriptional enhancer that regulates the embryonic expression of the human myoD gene. This enhancer had been localized to a 4 kb fragment located 18 to 22 kb upstream of the myoD transcriptional start site. We now present a molecular characterization of this enhancer. Transgenic and transfection analyses localize the myoD enhancer to a core sequence of 258 bp. In transgenic mice, this enhancer directs expression of a lacZ reporter gene to skeletal muscle compartments in a spatiotemporal pattern indistinguishable from the normal myoD expression domain, and distinct from expression patterns reported for the other myogenic factors. In contrast to the myoD promoter, the myoD enhancer shows striking conservation between humans and mice both in its sequence and its distal position. Furthermore, a myoD enhancer/heterologous promoter construct exhibits muscle-specific expression in transgenic mice, demonstrating that the myoD promoter is dispensable for myoD activation. With the exception of E-boxes, the myoD enhancer has no apparent sequence similarity with regulatory regions of other characterized muscle-specific structural or regulatory genes. Mutation of these E-boxes, however, does not affect the pattern of lacZ transgene expression, suggesting that myoD activation in the embryo is E-box-independent. DNase I protection assays reveal multiple nuclear protein binding sites in the core enhancer, although none are strictly muscle-specific. Interestingly, extracts from myoblasts and fibroblasts yield identical protection profiles, indicating a similar complement of enhancerbinding factors in muscle and this non-muscle cell type. However, a clear difference exists between myoblasts and cells (and other non-muscle cell types) in the chromatin structure of the chromosomal myoD core enhancer, suggesting that the myoD enhancer is repressed by epigenetic mechanisms in cells. These data indicate that myoD activation is regulated at multiple levels by mechanisms that are distinct from those controlling other characterized muscle-specific genes.
Postaxial polydactyly in forelimbs of CRABP-II mutant mice
Authors: Fawcett Diana , Pasceri Peter , Fraser Robert , Colbert Melissa , Rossant Janet , Giguère Vincent
Published: April 2021

The cytoplasmic retinoic acid (RA)-binding protein CRABP-II is expressed widely throughout early morphogenesis in mouse embryo, but its expression becomes more restricted as organogenesis progresses. CRABP-II expression remains strong in the developing limb bud suggesting a role for this protein in limb patterning. Here, we show that the CRABP-II promoter can direct expression of a lacZ transgene in a specific posterior domain during limb bud development. In order to investigate in more detail the role played by CRABP-II in RA signal transduction, we have also generated mice homozygous for a null mutation of this gene. CRABPII-/- mice are viable and fertile but show a developmental defect of the forelimb, specifically an additional, postaxial digit. This digit is generally, but not exclusively, limited to a single forepaw of an individual animal. The penetrance of the phenotype varies according to the genetic background, occurring most frequently on the inbred 129Sv background (50%), less frequently on the C57Bl/6 background (30%) and rarely on the outbred CD1 background (10%). This developmental abnormality implies a role for CRABP-II in normal patterning of the limb.
Striatal precursors adopt cortical identities in response to local cues
Authors: Fishell Gord
Published: April 2021

One of the early steps in the regionalization of the CNS is the subdivision of the forebrain into dorsal and basal telen-cephalic ventricular zones. These ventricular zones give rise to the cortex and striatum respectively, in the mature brain. Previous work suggests that while neural precursors are able to move within both the dorsal cortical and basal striatal ventricular zones, they are unable to cross the boundary area between them. To determine if the regional identities of the cells in these ventricular zones are restricted, cells from the basal striatal ventricular zone were either transplanted back into their original environ-ment or into the dorsally adjacent cortical ventricular zone. Use of in vitro explants of mouse telencephalon demon-strated that striatal precursors are able to integrate het-erotopically within 12 hours of being placed onto the surface of cortical ventricular zone. To examine whether heterotopically placed neural precursors have phenotypes appropriate to their host or donor environment, in vivo transplants in rats were performed. Striatal ventricular zone cells transplanted to a striatal environment adopt morphologies and axonal projections characteristic of striatal cells. In contrast, striatal ventricular zone cells transplanted in vivo to a cortical environment acquired morphologies and axonal projections specific to cortex. These findings suggest that within forebrain, position-specific cues play an instructive role in determining critical aspects of regional phenotype.
A role for HGF/SF in neural induction and its expression in Hensen’s node during gastrulation
Authors: Streit Andrea , Stern Claudio D. , Théry Clotilde , Ireland Grenham W. , Aparicio Samuel , Sharpe Melanie J. , Gherardi Ermanno
Published: April 2021

It was previously shown (Roberts, C., Platt, N., Streit, A., Schachner, M. and Stern, C. D. (1991) Development 112, 959-970) that grafts of Hensen’s node into chick embryos enhance and maintain expression of the L5 carbohydrate in neighbouring epiblast cells, and that antibodies against L5 inhibit neural induction by such a graft. We now show that L5 is initially widely expressed in the epiblast, but as neural induction proceeds it gradually becomes confined to and up-regulated in the early neural plate. L5 can therefore be considered as a marker for cells that are competent to respond to neural induction.

We also show that Hepatocyte Growth Factor/Scatter Factor (HGF/SF) promotes the expression of L5 by extraembryonic epiblast in collagen gels after overnight culture. Explants cultured for several days in the presence of HGF/SF, as well as explants of prospective neural plate, can differentiate into cells with neuronal morphology expressing neuronal markers. To investigate whether HGF/SF is expressed in the chick embryo at appropriate stages of development, we produced specific cDNA probes and used them for in situ hybridization. We find that at the primitive streak stage, HGF/SF is expressed specifically in Hensen’s node. We therefore propose that HGF/SF plays a role during the early steps of neural induction, perhaps by inducing or maintaining the competence of the epiblast to respond to neural inducing signals.
A dominant inhibitory version of the small GTP-binding protein Rac disrupts cytoskeletal structures and inhibits developmental cell shape changes in Drosophila
Authors: Harden Nicholas , Loh Hui Yong , Chia William , Lim Louis
Published: April 2021

The Rho subfamily of Ras-related small GTP-binding proteins is involved in regulation of the cytoskeleton. The cytoskeletal changes induced by two members of this subfamily, Rho and Rac, in response to growth factor stim-ulation, have dramatic effects on cell morphology. We are interested in using Drosophila as a system for studying how such effects participate in development. We have identified two Drosophila genes, DRacA and DRacB, encoding proteins with homology to mammalian Rac1 and Rac2. We have made transgenic flies bearing dominant inhibitory (N17DRacA), and wild-type versions of the DRacA cDNA under control of an Hsp70 promoter. Expression of the N17DRacA transgene during embryonic development causes a high frequency of defects in dorsal closure which are due to disruption of cell shape changes in the lateral epidermis. Embryonic expression of N17DRacA also affects germband retraction and head involution. The epidermal cell shape defects caused by expression of N17DRacA are accompanied by disruption of a localized accumulation of actin and myosin thought to be driving epidermal cell shape change. Thus the Rho subfamily may be generating localized changes in the cytoskeleton during Drosophila development in a similar fashion to that seen in mammalian and yeast cells. The Rho subfamily is likely to be partic-ipating in a wide range of developmental processes in Drosophila through its regulation of the cytoskeleton.
A conserved enhancer of the human and murine Hoxa-7 gene specifies the anterior boundary of expression during embryonal development
Authors: Knittel Thomas , Kessel Michael , Kim Myoung Hee , Gruss Peter
Published: April 2021

The murine homeobox-containing gene Hoxa-7 is expressed in restricted patterns during embryogenesis and plays an important role in the control of region-specific differentiation. Previous studies have shown that separate elements specify lineage restriction and expression boundaries of Hoxa-7. In particular 3.6 kb of 5′ flanking sequences were sufficient to establish an anterior boundary of Hoxa-7 gene expression. To identify the minimal regulatory element specifying the anterior boundary of expression, transgenic mice were generated carrying chimeric constructs with deletions of 5′ flanking sequences fused to a thymidine kinase minimal promoter/E. coli lacZ reporter construct. By deletion analysis, a 470 bp long control element (AX 470) located 1.6 kb upstream of the transcription start site was identified that directed expression of the β-galactosidase protein in a pattern reflecting the anterior boundary of expression of the endogenous Hoxa-7 gene. This element was active in either orientation and conferred regionspecific expression to unrelated promoters, thereby behaving like an enhancer element. In contrast, transgenic mice carrying further 5′ and 3′ deletions of the 470 bp long element did not exhibit an anterior boundary of Hoxa-7 expression. Based on these results the minimal control element (AX 470) specifying the anterior boundary of Hox expression was designated as Hoxa-7 enhancer.

Furthermore, 3 kb of the human HOXA7 upstream region were sequenced and compared to its mouse homologue in order to identify conserved regions. Sequence comparison revealed motifs that were strongly conserved between both species. The human homologue of the mouse Hoxa-7 enhancer was 70% identical at the nucleotide level and was also capable of directing an anterior boundary in transgenic mice.

Using transgenic lines a detailed analysis of the Hoxa-7 enhancer-directed expression during embryogenesis was performed. lacZ expression was first detected in the allantois at day 7.5 p.c. and in mesoderm and ectoderm at day 8.5 of gestation. Between gestational ages E8.5 to E12.5 β-gal expression was observed in the somites, spinal cord, spinal ganglia and paraxial mesoderm as well as in mesenchymal layers of the kidney. A distinct anterior limit of expression was noted in transgenic lines at level C4 (neural tube) and C5 (spinal ganglia).

Our deletion experiments defined a minimal enhancer element specifying the anterior boundary of Hox gene expression in early and late phases of development. Further studies aim at characterizing the trans-acting factors that mediate the spatial and temporal expression of Hox genes in the developing embryo.
Vascular endothelial cell lineage-specific promoter in transgenic mice
Authors: Schlaeger Thorsten M. , Qin Ying , Fujiwara Yuko , Magram Jeanne , Sato Thomas N.
Published: April 2021

Vascular endothelial cells play essential roles in the function and development of the cardiovascular system. However, due to the lack of lineage-specific markers suitable for molecular and biochemical analyses, very little is known about the molecular mechanisms that regulate endothelial cell differentiation. We report the first vascular endothelial cell lineage-specific (including angioblastic precursor cells) 1.2 kb promoter in transgenic mice. Moreover, deletion analysis of this promoter region in transgenic embryos revealed multiple elements that are required for the maximum endothelial cell lineage-specific expression. This is a powerful molecular tool that will enable us to identify factors and cellular signals essential for the establishment of vascular endothelial cell lineage. It will also allow us to deliver genes specifically into this cell type in vivo to test specifically molecules that have been implicated in cardiovascular development. Furthermore, we have established embryonic stem (ES) cells from the blastocysts of the transgenic mouse that carry the 1.2 kb promoter-LacZ reporter transgene. These ES cells were able to differentiate in vitro to form cystic embryoid bodies (CEB) that contain endothelial cells determined by PECAM immunohistochemistry. However, these in vitro differentiated endothelial cells did not express the LacZ reporter gene. This indicates the lack of factors and/or cellular interactions which are required to induce the expression of the reporter gene mediated by this 1.2 kb promoter in this in vitro differentiation system. Thus this system will allow us to screen for the putative inducers that exist in vivo but not in vitro. These putative inducers are presumably important for in vivo differentiation of vascular endothelial cells.
SpZ12-1, a negative regulator required for spatial control of the territory-specific CyIIIa gene in the sea urchin embryo
Authors: Wang David G.-W. , Kirchhamer Carmen V. , Britten Roy J. , Davidson Eric H.
Published: April 2021

The CyIIIa cytoskeletal actin gene of the sea urchin Strongylocentrotus purpuratus is activated in late cleavage and expressed exclusively in the aboral ectoderm territory of the embryo. Previous gene transfer studies defined a 2.3 kb cis-regulatory region that is necessary and sufficient for correct temporal and spatial expression of a CyIIIa•CAT fusion gene. In this paper, a negative regulatory element within this region was identified that is required for repression of the CyIIIa gene in skeletogenic mesenchyme cells. The repression mediated by this regulatory element takes place after initial territorial specification. A cDNA clone encoding a DNA-binding protein with twelve Zn fingers (SpZ12-1) was isolated by probing an expression library with this cis-element. Deletion analysis of the SpZ12-1 protein confirmed that a DNA-binding domain is located within the Zn finger region. SpZ12-1 is the only DNAbinding protein in embryo nuclear extract that interacts with the specific cis-target sites required for repression of CyIIIa•CAT in skeletogenic mesenchyme and is likely to be the trans factor that mediates this repression.
The limbic system-associated membrane protein (LAMP) selectively mediates interactions with specific central neuron populations
Authors: Zhukareva Victoria , Levitt Pat
Published: April 2021

The limbic system-associated membrane protein (LAMP) is a 64–68×103Mr glycoprotein that is expressed by subsets of neurons that are functionally interconnected. LAMP exhibits characteristics that are indicative of a developmentally significant protein, such as an early and restricted pattern of expression and the ability to mediate specific fiber-target interactions. A potential, selective adhesive mechanism by which LAMP may regulate the formation of specific circuits is investigated in the present experiments. LAMP is readily released from intact membranes by phosphatidyl inositol-specific phospholipase C. Purified, native LAMP, isolated by PI-PLC digestion and immunoaffinity chromatography, is capable of mediating fluorescent Covasphere aggregation via homophilic binding. To test the ability of LAMP to selectively facilitate substrate adhesion and growth of neurons from LAMP-positive, in contrast to LAMP-negative regions of the developing brain, purified LAMP was dotted onto nitrocellulose-coated dishes and test cells plated. Limbic neurons from perirhinal cortex bind specifically to substrate-bound LAMP within 4 hours, forming small cell aggregates with short neuritic processes that continue to grow through a 48 hour period of monitoring. Preincubation of cells with antiLAMP has a modest effect on cell binding but significantly reduces initiation of process growth. Non-limbic neurons from somatosensory cortex and olfactory bulb fail to bind or extend processes on the LAMP substrate to any significant extent. All cell populations bind equally well and form neurites on poly-D-lysine and laminin. The present results provide direct evidence that LAMP can specifically facilitate interactions with select neurons in the CNS during development. The data support the concept that patterned expression of unique cell adhesion molecules in functionally related regions of the mammalian brain can regulate circuit formation.