bluuebunny/crossref_metadata_2025 huggingface dataset:
Altered rhombomere-specific gene expression and hyoid bone differentiation in the mouse segmentation mutant, kreisler (kr)
Authors: Frohman Michael A. , Martin Gail R. , Cordes Sabine P. , Halamek Louis P. , Barsh Gregory S.
Published: April 2021

Rhombomeres appear transiently in the vertebrate hindbrain shortly after neurulation and are thought to represent embryologic compartments in which the expression of different combinations of genes leads to segment-specific differentiation of the developing hind-brain, the cranial ganglia, and the branchial arches. To determine the extent to which gene expression is related to the formation of visible rhombomere boundaries, we have examined, by in situ hybridization, the expression of five rhombomere-specific genes in mouse embryos homozygous for the kreisler (kr) mutation, in which rhombomeres 4-7 are replaced by a smooth morpho-logically unsegmented neural tube.

Using molecular probes specific for Hoxb-1 (Hox-2.9), Hoxb-3 (Hox-2.7), Hoxb-4 (Hox-2.6), Krox-20, or Fgf-3 (Int-2), we found that the kr mutation affects the expression of all the genes we examined, but, surprisingly, the altered patterns of expression are not restricted to that portion of the mutant hindbrain which is morphologically abnormal. Rostral expression boundaries of Hoxb-3 and Hoxb-4 are displaced from their normal positions at r4/5 and r6/7 to the approximate positions of r3/4 and r4/5, respectively. The expression domains of Krox-20 and Fgf-3 are also displaced in a rostral direction and the intensity of Fgf-3 hybridization is greatly reduced. The expression domain of Hoxb-1 is affected differently from the other genes in kr/kr embryos; its rostral boundary at r3/4 is intact but the caudal boundary is displaced from its normal location at r4/5 to the approximate position of r5/6. Because boundaries of gene expression for Hoxb-1 and Hoxb-4 are found in a region of the kr/kr hindbrain that lacks visible rhombomeres, establishment of regional identity, as reflected by differential gene expression, does not require overt segmentation.

To investigate whether the altered patterns of gene expression we observed in the kr/kr embryonic hind-brain are associated with morphologic changes in the adult, we examined neural crest-derived tissues of the second and third branchial arches, which normally arise from rhombomeres 4 and 6, respectively. We found that the hyoid bone in kr/kr animals exhibited an accessory process on the greater horn (a third arch structure) most easily explained by ectopic development of a second arch structure (the hyoid lesser horn) in an area normally derived from the third arch.
Development of Drosophila wing sensory neurons in mutants with missing or modified cell surface molecules
Authors: Whitlock Kathleen E.
Published: April 2021

The neurons of the sensory receptors on the wing of Drosophila melanogaster have highly characteristic axon projections in the central nervous system (CNS). The morphology of these projections was studied in flies bearing mutations that affect cell surface molecules thought to be important in axon guidance. The animals used were mutant for the fasciclinI (fasI), fasciclinII (fasII), fasciclinIII (fasIII) and neurally altered carbohy - drate (nac) genes. Axon populations were visualized by staining with DiI and light-reacting the dye with diaminobenzidine to yield permanent preparations.

The fasI, fasII and fasIII mutants as well as the nac mutant display altered axonal trajectories in the CNS. One phenotype seen in fasII mutants and in animals mutant for both fasI and fasIII was extra branching within the axon projection pattern. A second phenotype observed was a reduction or complete loss of one of the tracts, apparently due to the axons shifting to a neigh-boring tract. This was seen in the most extreme form in nac mutants and to a lesser degree in fasIII mutants.

To determine if the mutations discussed here affected axon guidance, wing discs were analyzed using the anti- body 22C10 to label sensory neurons in the wing during metamorphosis. Both misrouting of axons and the appearance of ectopic neurons in the wing were observed. In the fasI;fasIII, the fasII and the nac mutants, there was misrouting of sensory axons in the developing wing. In addition, the fasII and nac mutants displayed ectopic sensory neurons in the wing. This implies that the cell surface molecules missing (fasci - clins) or modified (by the nac gene product), in these mutants may play a role in both neurogenesis and axon guidance.
Mesodermal cell interactions in the sea urchin embryo: properties of skeletogenic secondary mesenchyme cells
Authors: Ettensohn Charles A. , Ruffins Seth W.
Published: April 2021

An interaction between the two principal populations of mesodermal cells in the sea urchin embryo, primary and secondary mesenchyme cells (PMCs and SMCs, respectively), regulates SMC fates and the process of skeleto-genesis. In the undisturbed embryo, skeletal elements are produced exclusively by PMCs. Certain SMCs also have the ability to express a skeletogenic phenotype; however, signals transmitted by the PMCs direct these cells into alternative developmental pathways. In this study, a combination of fluorescent cell-labeling meth-ods, embryo microsurgery and cell-specific molecular markers have been used to study the lineage, numbers, normal fate(s) and developmental potential of the skeletogenic SMCs. Previous fate-mapping studies have shown that SMCs are derived from the veg2 layer of blastomeres of the 64-cell-stage embryo and from the small micromeres. By specifically labeling the small micromeres with 5-bromodeoxyuridine, we demonstrate that descendants of these cells do not participate in skeletogenesis in PMC-depleted larvae, even though they are the closest lineal relatives of PMCs. Skeleto-genic SMCs are therefore derived exclusively from the veg2 blastomeres. Because the SMCs are a heteroge-neous population of cells, we have sought to gain information concerning the normal fate(s) of skeletogenic SMCs by determining whether specific cell types are reduced or absent in PMC( ) larvae. Of the four known SMC derivatives: pigment cells, blastocoelar (basal) cells, muscle cells and coelomic pouch cells, only pig-ment cells show a major reduction (>50%) in number following SMC skeletogenesis. We therefore propose that the PMC-derived signal regulates a developmental switch, directing SMCs to adopt a pigment cell pheno-type instead of a default (skeletogenic) fate. Ablation of SMCs at the late gastrula stage does not result in the recruitment of any additional skeletogenic cells, demon-strating that, by this stage, the number of SMCs with skeletogenic potential is restricted to 60-70 cells. Previ-ous studies showed that during their switch to a skele-togenic fate, SMCs alter their migratory behavior and cell surface properties. In this study, we demonstrate that during conversion, SMCs become insensitive to the PMC-derived signal, while at the same time they acquire PMC-specific signaling properties.
Developmental regulation of transcription of a novel prespore-specific gene (Dp87) in Dictyostelium discoideum
Authors: Ozaki Toshinori , Nakao Hajime , Orii Hidefumi , Morio Takahiro , Takeuchi Ikuo , Tasaka Masao
Published: April 2021

The Dp87 is a novel prespore specific gene of Dictyostelium discoideum which has a long open reading frame of 555 amino acids. The entire amino acid sequence had low but significant homology to the spore coat proteins, SP96 and SP70, of this organism. When a chimeric gene, containing a 1380 bp of the 5 upstream region of this gene fused with CAT gene, as reporter, was introduced into cells of this organism, it was expressed only in prespore cells of the slug. Transformation experiments, using chimeric genes, containing a series of 5 deletions of the upstream region, showed that 447 bp to 357 bp is an important cis-acting regulatory region for transcription. A nuclear factor(s) that specifically bind to this cis-acting region were detected from slug cell nuclei. Transformation experiments using a chimeric gene consisting of the 5 region between 666 bp and +149 bp of this gene, a β-galactosidase reporter and an actin 8 terminator, showed that the reporter gene was expressed as early as in aggregation streams, indicating that Dp87 become transcribed a few hours earlier than the other prespore-specific genes so far reported. This was confirmed by northern hybridization detected using an image plate analyzer. The fact that cells expressing Dp87 appeared at random in aggregation streams gives solid support to the idea that position-independent differentiation of prespore and pre-stalk cells, followed by their sorting, brings about pattern formation in this organism.
The Drosophila Ras2 and Rop gene pair: a dual homology with a yeast Ras-like gene and a suppressor of its loss-of-function phenotype
Authors: Salzberg Adi , Cohen Noa , Halachmi Naomi , Kimchie Ziva , Lev Zeev
Published: April 2021

The promoter of the Drosophila melanogaster Ras2 gene is bidirectional, regulating an additional gene oriented in the opposite polarity. The two divergently transcribed genes are only 93 bases apart and deletion analysis proved that common cis-acting elements within this promoter region are required for the transcriptional activity of both genes. We cloned the gene paired with Ras2 in the bidirectional promoter and isolated cDNAs corresponding to its mRNA. The Ras opposite (Rop) gene encodes for a 68 103Mr protein which shares sequence homology with the members of a novel Saccharomyces cerevisiae gene family, including the SLY1, SEC1 and VPS33 (SLP1) genes, all of which are involved in vesicle trafficking among yeast cellular compartments. A highly conserved motif in this family is also found in β-COP, a coat protein isolated from rat Golgi-bound non-clathrin vesicles. Thus, the Rop protein may be a component of one of the vesicle trafficking pathways in Drosophila cells. The Rop gene expression during embryogenesis is restricted to the central nervous system (CNS) and the garland cells, a small group of nephrocytes that takes up waste materials from the haemolymph by endocytosis. Ras2 is also expressed in the embryonic garland cells. In postembryonic stages, the two genes are co-expressed in the larval salivary glands and the central nervous system, and in the adult CNS and reproductive systems. Interestingly, the S. cerevisiae SLY1-20 allele is a suppressor of the loss of the YPT1 gene, a ras-like gene implicated in vesicle translocation, suggesting that the two genes may inter-act with one another. Since Sec1p and β-COP may also interact with small GTP-binding proteins of the ras superfamily, it is conceivable that the Rop and Ras2 gene products are not just co-expressed in common tissues, but may also functionally interact with one another in these tissues.
Control of imaginal cell development by the escargot gene of Drosophila
Authors: Hayashi Shigeo , Hirose Susumu , Metcalfe Tony , Shirras Alan D.
Published: April 2021

Mutations in the escargot (esg) locus, which codes for a zinc-finger-containing protein with similarity to the product of the snail gene, cause a variety of defects in adult structures such as loss of abdominal cuticle and malformation of the wings and legs. esg RNA is expressed in wing, haltere, leg and genital imaginal discs and in abdominal histoblast nests in the embryo. Expression in imaginal tissues is also found in third instar larvae. In esg mutant larvae, normally diploid abdominal histoblasts replicate their DNA without cell division and become similar in appearance to the polytene larval epidermal cells. A similar phenotype was also found in imaginal discs of larvae mutant for both esg and the Drosophila raf gene. These results suggest that one of the normal functions of esg may be the maintenance of diploidy in imaginal cells.
Identification and characterization of a gene activated by the deformed homeoprotein
Authors: Mahaffey James W. , Jones Denise F. , Hickel Jo Ann , Griswold Charles M.
Published: April 2021

In Drosophila, the homeotic genes encode transcription factors which control segment identity during embryogenesis by specifying the appropriate set of ‘target’ genes necessary to produce the individual segmental characteristics. Though we know much about the homeotic genes and the proteins they encode, we know little of their targets. Here we identify and characterize one such target gene, a gene activated by the product of the homeotic gene Deformed. DNA binding assays and expression of reporter gene constructs indicate that activation of this gene requires a direct interaction between the Deformed protein and an upstream enhancer element at this target gene. However, although Deformed is required to activate this gene in cells of the maxillary segment, ectopically expressed Deformed does not activate the gene in other regions of the embryo. We conclude from this and other observations that additional factors may be required to activate the target gene, and, therefore, Deformed may participate in either a combinatorial or hierarchical activation signal in the maxillary cells. This newly identified gene encodes a novel protein of unknown function, though proteins with similar amino acid composition have been found. The pattern of transcript accumulation during embryogenesis indicates that this gene may be regulated by other homeoproteins in addition to Deformed.
Analysis of the gooseberry locus in Drosophila embryos: gooseberry determines the cuticular pattern and activates gooseberry neuro
Authors: Gutjahr Thomas , Patel Nipam H. , Li Xuelin , Goodman Corey S. , Noll Markus
Published: April 2021

The segment-polarity class of segmentation genes in Drosophila are primarily involved in the specification of sub-segmental units. In addition, some of the segmentpolarity genes have been shown to specify cell fates within the central nervous system. One of these loci, gooseberry, consists of two divergently transcribed genes, gooseberry and gooseberry neuro, which share a paired box as well as a paired-type homeobox. Here, the expression patterns of the two gooseberry gene products are described in detail. The gooseberry protein appears in a characteristic segment-polarity pattern of stripes at gastrulation and persists until head involution. It is initially restricted to the ectodermal and neuroectodermal germ layer, but is later detected in mesodermal and neuronal cells as well. The gooseberry neuro protein first appears during germ band extension in cells of the central nervous system and also, much later, in epidermal stripes and in a small number of muscle cells. P-elementmediated transformation with the gooseberry gene has been used to demonstrate that gooseberry transactivates gooseberry neuro and is sufficient to rescue the gooseberry cuticular phenotype in the absence of gooseberry neuro.
Isolation of the murine cyclin B2 cDNA and characterization of the lineage and temporal specificity of expression of the B1 and B2 cyclins during oogenesis, spermatogenesis and early embryogenesis
Authors: Chapman Deborah L. , Wolgemuth Debra J.
Published: April 2021

A cDNA encoding the murine cyclin B2 (cycB2) was isolated from an adult mouse testis cDNA library as part of studies designed to identify cyclins involved in murine germ cell development. This cycB2 cDNA was then used to examine the pattern of cycB2 expression during male and female germ cell development and in early embryogenesis, and to compare this expression with the previously characterized expression of cycB1. A single 1.7 kb cycB2 transcript was detected by northern blot hybridization analysis of total RNA isolated from midgestation embryos and various adult tissues. Northern blot and in situ hybridization analyses revealed that cycB2 expression in the testis was most abundant in the germ cells, specifically in pachytene spermatocytes. This is in contrast to the highest levels of expression of cycB1 being present in early spermatids. In situ analysis of the ovary revealed cycB2 transcripts in both germ cells and somatic cells, specifically in the oocytes and granulosa cells of growing and mature follicles. The pattern of cycB1 and cycB2 expression in ovulated and fertilized eggs was also examined. While the steady state level of cycB1 and cycB2 signal remained constant in oocytes and ovulated eggs, signal of both appeared to decrease following fertilization. In addition, both cycB1 and cycB2 transcripts were detected in the cells of the inner cell mass and the trophectoderm of the blastocyst. These results demonstrate that there are lineageand developmental-specific differences in the pattern of the B cyclins in mammalian germ cells, in contrast to the coexpression of B cyclins in the early conceptus.
Isolated sequences from the linked Myf-5 and MRF4 genes drive distinct patterns of muscle-specific expression in transgenic mice
Authors: Patapoutian Ardem , Miner Jeffrey H. , Lyons Gary E. , Wold Barbara
Published: April 2021

In developing mouse embryos, MyoD family regulatory genes are expressed specifically in muscle precursors and mature myofibers. This pattern, taken together with the well-established ability of MyoD family members to convert a variety of cell types to skeletal muscle, suggests a significant role for these genes in regulating skeletal myogenesis. The possibility that expression of these genes may be causally associated with segregation of the myogenic lineage from other mesodermal derivatives, or with the subsequent maintenance of muscle phenotypes at later times, raises the issue of how MyoD family genes are themselves regulated during development. In this work, we have initiated studies to identify DNA sequences that govern Myf-5 and MRF4 (herculin, myf-6) transcription. Myf-5 is the first of the MyoD family to be expressed in the developing mouse embryo, while MRF4 is the most abundantly expressed myogenic factor in postnatal animals. In spite of their strikingly divergent patterns of expression, Myf-5 and MRF4 are tightly linked in the mouse genome; their translational start codons are only 8.5 kilobases apart. Here, the 5′ flanking regions of the mouse Myf-5 and MRF4 genes were separately linked to a bacterial β-galactosidase (lacZ) gene, and these constructs were each used to produce several lines of transgenic mice. Transgene expression was monitored by X-gal staining of whole embryos and by in situ hybridization of embryo sections. For the Myf-5/lacZ lines, the most intense transgene expression was in the visceral arches and their craniofacial muscle derivatives, beginning at day 8.75 post coitum (p.c.). This correlates with endogenous Myf-5 expression in visceral arches. However, while Myf-5 is also expressed in somites starting at day 8 p.c., transgene expression in the trunk is not observed until day 12 p.c. Thus, the Myf-5/lacZ construct responds to early Myf-5 activators in the visceral arches but not in the somites, suggesting that myogenic determination in the nonsomitic head mesoderm may be under separate control from that of the somitic trunk mesoderm. MRF4/lacZ lines displayed an entirely different pattern from Myf-5. Transgene expression appeared in muscles starting at day 16.5 p.c. and became increasingly prominent at later times. However, an early wave of myotomal expression that is characteristic of the endogenous MRF4 was not recapitulated by the transgene.
Two distinct populations of prestalk cells within the tip of the migratory Dictyostelium slug with differing fates at culmination
Authors: Early Anne E. , Gaskell Michael J. , Traynor David , Williams Jeffrey G.
Published: April 2021

The ecmA gene of Dictyostelium encodes an extracellular matrix protein and is selectively expressed in prestalk cells. We show that its promoter contains discrete elements that direct expression in different subpopulations of prestalk cells. Prestalk(pst)A cells occupy the front half of the prestalk region. Expression in pstA cells requires DNA sequences close to the cap site of the gene and a separate, upstream region that acts in combination with the gene proximal sequences. PstO cells are situated in the rear half of the prestalk region and at least two separate and redundant promoter regions direct expression within them. All constructs that are expressed in pstO cells are also expressed in anteriorlike cells (ALCs); cells that resemble prestalk cells but which are scattered throughout the prespore region.

This observation suggests that pstO cells and ALCs may be very similar in their properties. If development occurs under conditions in which a migratory slug is not formed, there is an ordered movement of cells into the stalk tube. PstA cells enter the stalk tube first, followed by a proportion of the pstO cells. The remainder of the pstO cells contribute to the upper cup, an ALC-derived subpopulation of prestalk cells which is located at the apex of the spore head. After prolonged slug migration, a discrete pstO zone appears not to be maintained and, at culmination, pstO cells are found scattered throughout the stalk.
Targeted gene expression as a means of altering cell fates and generating dominant phenotypes
Authors: Brand Andrea H. , Perrimon Norbert
Published: April 2021

We have designed a system for targeted gene expression that allows the selective activation of any cloned gene in a wide variety of tissueand cell-specific patterns. The gene encoding the yeast transcriptional activator GAL4 is inserted randomly into the Drosophila genome to drive GAL4 expression from one of a diverse array of genomic enhancers. It is then possible to introduce a gene containing GAL4 binding sites within its promoter, to activate it in those cells where GAL4 is expressed, and to observe the effect of this directed misexpression on development. We have used GAL4-directed transcription to expand the domain of embryonic expression of the homeobox protein even-skipped. We show that even-skipped represses wingless and transforms cells that would normally secrete naked cuticle into denticle secreting cells. The GAL4 system can thus be used to study regulatory interactions during embryonic development. In adults, targeted expression can be used to generate dominant phenotypes for use in genetic screens. We have directed expression of an activated form of the Dras2 protein, resulting in dominant eye and wing defects that can be used in screens to identify other members of the Dras2 signal transduction pathway.
In vitro stalk cell differentiation in wild-type and ‘slugger’ mutants of Dictyostelium discoideum
Authors: Inouye Kei , Gross Julian
Published: April 2021

In ‘slugger’ mutants of Dictyostelium discoideum, aggregates of cells remain for an abnormally long time in the migratory phase under conditions where wild-type aggregates form fruiting bodies. In the present work, we have examined the relationship between the defect in fruiting body formation in these mutants and their ability to form mature stalk cells. We dissociated anterior cells from slugs of the mutants and their parents and tested their ability to form stalk cells when incubated at low density in the presence of (1) the stalk cell morphogen Differentiation Inducing Factor-1 (DIF1) together with cyclic AMP, or (2) 8-Br-cAMP, which is believed to penetrate cell membrane and activate cAMPdependent protein kinase (PKA).

Most of the mutants were markedly defective in forming stalk cells in response to DIF-1 plus cAMP, confirming a close relationship between fruiting body formation and stalk cell maturation. On the other hand, many of these same mutants formed stalk cells efficiently in response to 8-Br-cAMP. This supports evidence for an essential role of PKA in stalk cell maturation and fruiting body formation. It also indicates that many of the mutants owe their slugger phenotype to defects in functions required for optimal adenylyl cyclase activity.
The role of the monopteros gene in organising the basal body region of the Arabidopsis embryo
Authors: Berleth Thomas , Jürgens Gerd
Published: April 2021

The monopteros (mp) gene contributes to apical-basal pattern formation in the Arabidopsis embryo. mp mutant seedlings lack basal body structures such as hypocotyl, radicle and root meristem, and this pattern deletion has been traced back to alterations in the octant-stage embryo. Cells of the embryo proper and the uppermost cell of the suspensor fail to establish division patterns that would normally generate the basal body structures. The resulting absence of a morphological axis seems to be responsible for another phenotypic trait of mp seedlings, variable positioning of cotyledons. This relationship is suggested by weak mp seedling phenotypes in which the presence of a short hypocotyl is correlated with normal arrangement of cotyledons. Root formation has been induced in mp seedlings grown in tissue culture. This result supports the notion that the mp gene is required for organising the basal body region, rather than for making the root, in the developing embryo.
Histological studies on the development of retinotectal projections from nasoventral quarter-eyes in Xenopus laevis
Authors: Degen N. , Brändle K. , Peter L.
Published: April 2021

In Xenopus larvae, the size and location of the retinotectal projection of nasoventral quarter-eyes was analyzed in early stages (43–47), midlarval stages (50 and 53) and metamorphic stages (56 and 60), by labelling the optic nerve with the cobalt-lysine complex or with horseradish-peroxidase (HRP). For direct comparison, both fragment and normal eye projections were determined simultaneously in the same specimen in brain whole mounts. During early stages (up to stage 47), the projection fields of normal eyes and quarter-eyes are confined to the rostral part of the tectum. The extension of the projection in rostrocaudal direction of eye fragments does not differ from that of normal eyes. During later development up to metamorphosis, normal eyes expand their projection over the newly formed tectal surface in a caudal direction, whereas the fiber terminations of nasoventral quarter-eyes still remain in the rostral part of the tectum. Quantitative studies revealed that there is no difference in the size of both halves of the tectum. At least for quarter-eyes, however, a strict correlation between eye size and extension of the contralateral projection field could be established. According to our results, it is unlikely that during development local tectal markers are involved in determining the location of the projection field and the retinotopic ordering of the optic fibers. Instead we suggest that the optic fibers separate in accordance with their retinal specificity.
Molecular characterization of the lethal of scute genetic function
Authors: Martín-Bermudo D. María , González Francisco , Domínguez María , Rodríjuez Isabel , Ruiz-Gômez Mar , Romani Susana , Modolell Juan , Jiménez Fernando
Published: April 2021

The lethal of scute (l’sc) genetic function, which plays an essential role in the early development of the central nervous system of the Drosophila embryo, is localized within the achaete-scute complex (AS-C). Several lines of evidence have suggested that the AS-C T3 transcription unit corresponds to the l’sc function. We demonstrate that short fragments of DNA, containing the T3 transcribed region and a few kilobases of flanking sequences, rescue, albeit partially, the lethality and neural phenotype of l’sc deletions. Still, the complex wild-type pattern of expression of T3 is not reproduced by the transduced genes. This depends on cis-control elements scattered within the entire AS-C DNA and intermingled with regulatory elements specific for other AS-C transcription units. These elements are necessary for the initial activation of T3 in the neuroectoderm, probably mediated by axis-patterning genes. The presence of a cluster of E-boxes, upstream of the T3 transcribed region, suggests another level of control of T3 expression by basic-helix-loop-helix proteins, among them its own gene product.
Segmental migration of the hindbrain neural crest does not arise from its segmental generation
Authors: Sechrist J. , Serbedzija G. N. , Scherson T. , Fraser S. E. , Bronner-Fraser M.
Published: April 2021

The proposed pathways of chick cranial neural crest migration and their relationship to the rhombomeres of the hindbrain have been somewhat controversial, with differing results emerging from grafting and DiI-labelling analyses. To resolve this discrepancy, we have examined cranial neural crest migratory pathways using the combination of neurofilament immunocytochemistry, which recognizes early hindbrain neural crest cells, and labelling with the vital dye, DiI. Neurofilament-positive cells with the appearance of premigratory and early-migrating neural crest cells were noted at all axial levels of the hindbrain. At slightly later stages, neural crest cell migration in this region appeared segmented, with no neural crest cells obvious in the mesenchyme lateral to rhombomere 3 (r3) and between the neural tube and the otic vesicle lateral to r5. Focal injections of DiI at the levels of r3 and r5 demonstrated that both of these rhombomeres generated neural crest cells. The segmental distribution of neural crest cells resulted from the DiI-labelled cells that originated in r3 and r5 deviating rostrally or caudally and failing to enter the adjacent preotic mesoderm or otic vesicle region. The observation that neural crest cells originating from r3 and r5 avoided specific neighboring domains raises the intriguing possibility that, as in the trunk, extrinsic factors play a major role in the axial patterning of the cranial neural crest and the neural crest-derived peripheral nervous system.
Analyses of PS integrin functions during Drosophila development
Authors: Zusman Susan , Grinblat Yevgenya , Yee Gene , Kafatos Fotis C. , Hynes Richard O.
Published: April 2021

The Drosophila position-specific (PS) antigens are homologues of the vertebrate integrins, a family of trans-membrane proteins that function in cell-matrix and cell-cell adhesion. The common β subunit of PS integrins (PSβ) is encoded by the lethal(l)myospheroid gene (mys) and is required during wing, eye and muscle development. By expressing PSβ protein at defined developmental periods, we have shown that PS integrins are required throughout pupation, but not earlier, for normal development of wings. In contrast, the key requirement for PS integrins in eye development occurs only in the late pupa. Furthermore, PS integrins are apparently not required for the differentiation of the ommatidial cells; only for their organization. These results are consistent with roles for PS integrins in the interactions between the wing epithelia during the two phases of pupal wing expansion and in maintaining the attachment of a fully formed fenestrated membrane to the basement membrane of the retina.

We have also examined the functional significance of alternative splicing of the transcript of the mys gene using P element-mediated transformation to introduce transgenes producing only one of the two spliced forms of PSβ. We find that either form is sufficient to rescue postem-bryonic mys phenotypes in the wing, eye and muscle but that both of the two splice forms are necessary to rescue the mys embryonic defects. This result indicates a requirement for the alternative splicing of mys during embryogenesis. The location of the alternative exons suggests that the two forms of the PSβ integrin subunit may interact with alternative α subunits and/or ligands.
A common precursor for glia and neurons in the embryonic CNS of Drosophila gives rise to segment-specific lineage variants
Authors: Udolph Gerald , Prokop Andreas , Bossing Torsten , Technau Gerhard M.
Published: April 2021

The nervous system consists of two classes of cells, neurons and glia, which differ in morphology and function. They derive from precursors located in the neurogenic region of the ectoderm. In this study, we present the complete embryonic lineage of a neuroectodermal precursor in Drosophila that gives rise to neurons as well as glia in the abdominal CNS. This lineage is conserved among different Drosophila species. We show that neuronal and glial cell types in this clone derive from one segregating precursor, previously described as NB1-1. Thus, in addition to neuroblasts and glioblasts, there exists a third class of CNS precursors in Drosophila, which we call neuroglioblasts. We further show that the NB 1-1 lineage exhibits characteristic segment-specific differences on the cellular level.
Large-scale normal cell death in the developing rat kidney and its reduction by epidermal growth factor
Authors: Coles Harriet S. R. , Burne Julia F. , Raff Martin C.
Published: April 2021

Although normal cell death is known to occur in many developing vertebrate organs, it has not been thought to play an important part in the development of the mammalian kidney. We show here that normal cell death is found in both the nephrogenic region and medullary papilla of the developing rat kidney and, in each of these areas, it follows a distinct developmental time course. As many as 3% of the cells in these areas have a typical apoptotic morphology and the dead cells seem to be cleared rapidly (within 1-2 hours) by phagocytosis by neighbouring parenchymal cells. These values are similar to those in vertebrate neural tissues where 50% or more of the cells die during normal development, suggesting that large-scale death is a normal feature of kidney development. We also show that in vivo treatment with epidermal growth factor inhibits cell death in the developing kidney, consistent with the possibility that the cells normally die because they lack sufficient survival factors. Our findings suggest that the extent of normal cell death in developing animals is still greatly underestimated and they raise the possibility that many of these cell deaths may reflect limiting amounts of survival factors.
EGF abrogation-induced fusilli-form dysmorphogenesis of Meckel’s cartilage during embryonic mouse mandibular morphogenesis in vitro
Authors: Shum Lillian , Sakakura Yasunori , Bringas Pablo , Luo Wen , Snead Malcolm L. , Mayo Mark , Crohin Constant , Millar Sharon , Werb Zena , Buckley Sue , Hall Fredrick L. , Warburton David , Slavkin Harold C.
Published: April 2021

Mutations associated with genes of the EGF superfamily are implicated in facial malformations arising from abnormal development of the first branchial arch. EGF and EGF receptor (EGFr) transcripts are expressed in the mouse embryonic first branchial arch and derivatives from E9 through E15. EGF transcripts are localized to ectomesenchymal cells associated with precartilage, cartilage, bone and tooth-forming cells. EGF and EGFr proteins co-localize to the same cells suggesting an autocrine regulation. To test whether EGF effects the timing and positional information required for Meckel’s cartilage (MC) and tooth development, we cultured E10 mandibular explants in serumless, chemically defined medium with either antisense or sense EGF oligodeoxynucleotides. Antisense inhibition of EGF expression produces bilaterally symmetrical Fusilli form dysmorphogenesis of MC and decreases tooth bud size; these effects are reversed by the addition of exogenous EGF to the culture medium. Tyrphostin RG 50864, which inhibits EGF receptor kinase activity, inhibits EGF stimulation of tyrosine phosphorylation in a concentration-dependent manner and severely retards mandibular development yet increases tooth size. These findings support the hypothesis that endogenous EGF and EGF-like proteins provide signalling to regulate the size and shape both of cartilage and tooth formation during craniofacial morphogenesis.
Expression of relB transcripts during lymphoid organ development: specific expression in dendritic antigen-presenting cells
Authors: Carrasco Daniel , Ryseck Rolf-Peter , Bravo Rodrigo
Published: April 2021

We have studied the expression of the relB gene during mouse development using in situ hybridization and immunocytochemical analysis. The results show that the expression of the relB gene is highly restricted to a sub-population of cells that colonize the lymphoid tissues and that appear very late during the process of hemapoietic diversification. RNA transcripts of relB are very low or undetectable in early and late embryos. Low relB expression is observed in the thymus at late stages of embryogenesis but rapidly increases after birth. In adult lymphoid tissues, relB is detected in the medullary region of the thymus, the periarterial lymphatic sheaths of the spleen, and the deep cortex of the lymph nodes, which correspond to the regions where T cells of mature phenotype and interdigitating dendritic cells are present. Using double immunofluorescent labeling of thymic cell suspensions, we have identified the interdigitating dendritic cells as the target of RelB expression. These cells are part of a system of antigen-presenting cells that function in the induction of several immune responses, such as, tolerance, sensitization of MHC-restricted T cells, rejection of organ transplants and formation of T-dependent antibodies. Our observations indicate that RelB may play a particular role in the signal transduction pathways that regulate dendritic cell differentiation and its cellular responses.
Cortical cytoplasm, which induces dorsal axis formation in Xenopus, is inactivated by UV irradiation of the oocyte
Authors: Holowacz Tamara , Elinson Richard P.
Published: April 2021

Localized maternal determinants control the formation of dorsal axial structures in Xenopus embryos. To examine the spatial distribution of dorsal determinants, we injected cytoplasm from various regions of the egg and 16-cell embryo into the ventral vegetal cells of a 16-cell recipient embryo. Cortical cytoplasm from the egg vegetal surface induced the formation of a secondary dorsal axis in 53% of recipients. In contrast, animal cortical, equatorial cortical and vegetal deep cytoplasm never induced secondary axis formation. We also compared the axis-inducing ability of animal versus vegetal dorsal cortical cytoplasm from 16-cell embryos. Significantly more dorsalizing activity was found in vegetal dorsal cytoplasm compared to animal dorsal cytoplasm at this stage.

Previous work has shown that UV irradiation of the vegetal surface of either prophase I oocytes, or fertilized eggs, leads to the development of embryos that lack dorsal structures. Egg vegetal cortical cytoplasm was capable of restoring the dorsal axis of 16-cell recipient embryos derived from UV-irradiated oocytes or fertil-ized eggs. We also tested the axis inducing ability of cytoplasm obtained when UV-irradiated oocytes and eggs were treated as donors of cytoplasm. While vegetal cortical cytoplasm from UV-irradiated fertilized eggs retains its dorsalizing activity, cytoplasm obtained from eggs, UV irradiated as oocytes, does not.

The egg vegetal cortex provides a suitable source for the isolation of maternal dorsal determinants. In addition, since UV irradiation of the oocyte vegetal surface destroys the dorsalizing activity of transferred cytoplasm, UV can be used to further restrict possible candidates for such determinants.
FGF-4 regulates expression of Evx-1 in the developing mouse limb
Authors: Niswander Lee , Martin Gail R.
Published: April 2021

We describe here the temporal and spatial pattern of expression of Evx-1, a murine homolog of the Drosophila even-skipped gene, in the developing limb bud. Evx-1 RNA is first detected in distal limb (progress zone) mes-enchyme shortly after the formation of the apical ecto-dermal ridge. The level of Evx-1 RNA increases during the next 24 hours of development, and then decreases in the subsequent 24 hours, such that by the time the ridge regresses Evx-1 RNA is undetectable. At all these stages, Evx-1 RNA is localized primarily to the posterior distal mesenchyme, in the region immediately underlying that portion of the ridge in which the Fgf-4 gene is expressed. Using an in vitro culture system, we show that the ridge is required for both the induction and maintenance of Evx-1 expression in the distal mesenchyme. We also demonstrate that in the absence of the ridge, FGF-4, as well as other FGF proteins, can induce Evx-1 expression in the limb bud. However, this effect appears to be indirect, since it can be blocked by an inhibitor of protein synthesis. Additional studies demonstrate that the effect of FGF-4 on Evx-1 expression is modulated by BMP-2. These data serve to identify Evx-1 as a downstream gene in the FGF signal transduction pathway in the limb.
Heterogeneity in the expression of fibroblast growth factor receptors during limb regeneration in newts (Notophthalmus viridescens)
Authors: Poulin Matthew L. , Patrie Kevin M. , Botelho Mary Jane , Tassava Roy A. , Chiu Ing-Ming
Published: April 2021

Two closely related fibroblast growth factor receptors, FGFR1 and FGFR2, have been cloned from a newt (Notophthalmus viridescens) limb blastema cDNA library. Sequence analysis revealed that we have isolated both the bek and KGFR variants of FGFR2. These two variants differ only in the second half of the last of their three Ig-like domains. The expression patterns of FGFR1 and FGFR2 during limb regeneration have been determined by in situ hybridization. During the preblastema stages of regeneration, FGFR2 expression is observed in the basal layer of the wound epithelium and in the cells of the periosteum. As regeneration progresses to the blastema stages, FGFR2 expression continues to be observed in the basal layer of the wound epithelium with additional hybridization seen in the blastema mesenchyme closely associated with the bisected bones. From the early bud to the mid-bud blastema stage, FGFR1 expression is observed throughout the blastema mesenchyme but, unlike FGFR2, is distinctly absent from the wound epithelium. In the differentiation stages of regeneration, the mesenchymal expression of FGFR2 becomes restricted to the cells of the condensing cartilage and later to the perichondrium. During these later stages of regeneration, the wound epithelium hybridization to the FGFR2 probe is no longer observed. The expression patterns of these receptors suggest that FGFR1 and FGFR2 have distinct roles in limb regeneration, despite their sharing a number of the FGF ligands. Further investigation regarding the potential sources of the FGF ligands will help establish the role that FGFs and FGFRs play in limb regeneration.
CLAVATA1, a regulator of meristem and flower development in Arabidopsis
Authors: Clark Steven E. , Running Mark P. , Meyerowitz Elliot M.
Published: April 2021

We have investigated the effects on plant development of mutations in the Arabidopsis thaliana CLAVATA1 gene. In clavata1 plants, vegetative, inflorescence and floral meristems are all enlarged relative to wild type. The apical meristem can fasciate in the more severe mutant alleles, and this fasciation can occur prior to the transition to flowering. Flowers of clavata1 plants can have increased numbers of organs in all four whorls, and can also have additional whorls not present in wild-type flowers. Double mutant combinations of clavata1 with agamous, apetala2, apetala3 and pistillata indicate that CLAVATA1 controls the underlying floral meristem structure upon which these homeotic genes act. Double mutant combinations of clavata1 with apetala1 and leafy indicate CLAVATA1 plays a role in establishing and maintaining floral meristem identity, in addition to its role in controlling meristem size. In support of this, RNA expression patterns of AGAMOUS and APETALA1 are altered in clavata1 flowers.
Ciliary neurotrophic factor maintains the pluripotentiality of embryonic stem cells
Authors: Conover Joanne C. , Ip Nancy Y. , Poueymirou William T. , Bates Brian , Goldfarb Mitchell P. , DeChiara Thomas M. , Yancopoulos George D.
Published: April 2021

Ciliary neurotrophic factor was discovered based on its ability to support the survival of ciliary neurons, and is now known to act on a variety of neuronal and glial populations. Two distant relatives of ciliary neurotrophic factor, leukemia inhibitory factor and oncostatin M, mimic ciliary neurotrophic factor with respect to its actions on cells of the nervous system. In contrast to ciliary neurotrophic factor, leukemia inhibitory factor and oncostatin M also display a broad array of actions on cells outside of the nervous system. The overlapping activities of leukemia inhibitory factor, oncostatin M and ciliary neurotrophic factor can be attributed to shared receptor components. The specificity of ciliary neurotrophic factor for cells of the nervous system results from the restricted expression of the alpha component of the ciliary neurotrophic factor receptor complex, which is required to convert a functional leukemia inhibitory factor/oncostatin M receptor complex into a ciliary neurotrophic factor receptor complex. The recent observation that the alpha component of the ciliary neurotrophic factor receptor complex is expressed by very early neuronal precursors suggested that ciliary neurotrophic factor may act on even earlier precursors, particularly on cells previously thought to be targets for leukemia inhibitory factor action. Here we show the first example of ciliary neurotrophic factor responsiveness in cells residing outside of the nervous system by demonstrating that embryonic stem cells express a functional ciliary neurotrophic factor receptor complex, and that ciliary neurotrophic factor is similar to leukemia inhibitory factor in its ability to maintain the pluripotentiality of these cells.
Complex fiber-type-specific expression of fast skeletal muscle troponin I gene constructs in transgenic mice
Authors: Hallauer Patricia L. , Bradshaw Holly L. , Hastings Kenneth E. M.
Published: April 2021

We analyzed, in transgenic mice, the cellular expression pattern of the quail fast skeletal muscle troponin I (TnIfast) gene and of a chimeric reporter construct in which quail TnIfast DNA sequences drive expression of E. coli beta-galactosidase ( -gal). Both constructs were actively expressed in skeletal muscle and specifically in fast, as opposed to slow, muscle fibers. Unexpectedly, both constructs showed a marked differential expression among the adult fast fiber subtypes according to the pattern IIB>IIX>IIA. This expression pattern was consistent in multiple lines and differed from the endogenous mouse TnIfast pattern, which shows approximately equal expression in all fast fibers. These observations indicate that distinct regulatory mechanisms contribute to high-level expression of TnIfast in the various fast fiber subtypes and suggest that the outwardly simple pattern of equal expression in all fast fiber types shown by the endogenous mouse TnIfast gene is based on an intricate system of counterbalancing mechanisms. The adult expression pattern of the TnIfast/β-gal construct emerged in a two-stage developmental process. Differential expression in fast versus slow fibers was evident in neonatal animals, although expression in fast fibers was relatively weak and homogeneous. During the first two weeks of postnatal life, expression in maturing IIB fibers was greatly increased whereas that in IIA /IIX fibers remained weak, giving rise to marked differential expression among fast fiber types. Thus at least two serially acting (pre- and post-natal) fiber-type-specific regulatory mechanisms contribute to high-level gene expression in adult fast muscle fibers. Unexpected similarities between TnIfast transgene expression and that of the myosin heavy chain gene family (which includes differentially expressed IIB-, IIX- and IIA-specific members) suggest that similar mechanisms may regulate adult fast muscle gene expression in a variety of unrelated muscle gene families.