bluuebunny/crossref_metadata_2025 huggingface dataset:
A new Drosophila homeobox gene, bsh, is expressed in a subset of brain cells during embryogenesis
Authors: Jones Bradley , McGinnis William
Published: April 2021

Homeobox genes have been shown to control the determination of positional, tissue and cellular identity during the development of the fruitfly Drosophila melanogaster. Because genes involved in the determination of internal structures derived from neural, mesodermal and endodermal tissues may have been overlooked in conventional genetic screens, we undertook the identification of new homeobox genes expressed in these internal tissues. Here we describe the characterization of one of these new Drosophila homeobox genes, called brain-specific-homeobox (bsh). In embryos, bsh is expressed exclusively in the brain. bsh protein accumu-lates in approximately 30 cells in each brain hemisphere. One of these bsh expressing cells is closely associated with the terminus of the larval visual nerve (Bolwig’s nerve). While deletions of chromosomal interval containing the bsh gene show no dramatic changes in embryonic brain morphology, the expression pattern of the bsh gene suggests that it may play a highly specialized role in the determination and function of cell type in the Drosophila brain.
An activity gradient of decapentaplegic is necessary for the specification of dorsal pattern elements in the Drosophila embryo
Authors: Wharton Kristi A. , Ray Robert P. , Gelbart William M.
Published: April 2021

decapentaplegic (dpp) is a zygotically expressed gene encoding a TGF-β-related ligand that is necessary for dorsal-ventral patterning in the Drosophila embryo. We show here that dpp is an integral part of a gradient that specifies many different cell fates via intercellular signalling. There is a graded requirement for dpp activity in the early embryo: high levels of dpp activity specify the amnioserosa, while progressively lower levels specify dorsal and lateral ectoderm. This potential for dpp to specify cell fate is highly dosage sensitive. In the wildtype embryo, increasing the gene dosage of dpp can shift cell fates along the dorsal-ventral axis. Furthermore, in mutant embryos, in which only a subset of the dorsalventral pattern elements are represented, increasing the gene dosage of dpp can specifically transform those pattern elements into more dorsal ones. We present evidence that the zygotic dpp gradient and the maternal dorsal gradient specify distinct, non-overlapping domains of the dorsal-ventral pattern.
Cortical development associated with conjugation of Paramecium
Authors: Romero M. Rosario , Torres Antonio
Published: April 2021

The cortical cytoskeleton of Paramecium is character-ized by a complex, polarized and asymmetrical organi-zation. In order to analyse the cortical development of Paramecium tetraurelia during the sexual process of con-jugation, different antibodies have been used to follow the development of five cortical components: basal bodies, ciliary rootlets, outer lattice, epiplasm and cytospindle. This study demonstrates that the cortex of Paramecium undergoes an elaborate development process following conjugation. Some of the cortical structures that are not renewed at any other moment of the cell cycle, are resorbed after conjugation and replaced by other newly formed ones. The reorganiza-tion of the cortical components occurs according to dif-ferent morphogenetic waves that spread over the cell surface. The oral system and the preoral suture line act as morphogenetic epicentres.
Isoform-specific immunological detection of newt retinoic acid receptor δ1 in normal and regenerating limbs
Authors: Hill David S. , Ragsdale Clifton W. , Brockes Jeremy P.
Published: April 2021

Retinoic acid (RA) exerts a variety of effects on the regenerating urodele limb including positional respecification of the blastema. The major RA receptor expressed in the newt limb and blastema is the 1 isoform and, in order to detect 1 in this context, we have made five affinity-purified antibodies against fusion proteins and peptides from non-overlapping regions of the molecule. These antibodies have been evaluated by reaction with transfected COS-7 cells, newt limb cells in culture and newt limb tissue sections. The most informative antibodies were RP6, directed against N-terminal region A sequence, and RP8, directed against C-terminal sequence. In western blots of blastemal extracts, 1 protein was detected as two major bands of immunoreactivity at positions consistent with the employment of two candidate methionine initiators identified by cDNA sequencing. Staining of adult limb sections with RP6 and RP8 showed reactivity in half of the nuclei in epidermal and mesenchymal tissues, a heterogeneity that was observed with adjacent nuclei in muscle fibres. In the regenerating limb, nuclei in the blastemal mesenchyme and wound epidermis were strongly reactive, although no axial variation in expression was detected.
A functionally specialized α-tubulin is required for oocyte meiosis and cleavage mitoses in Drosophila
Authors: Matthews Kathleen A. , Rees Dianne , Kaufman Thomas C.
Published: April 2021

Three α-tubulin proteins contribute to microtubules during oogenesis and early embryogenesis in Drosophila melanogaster: αTUB84B, αTUB84D, and αTUB67C. αTUB67C is unique in two respects. It is a structurally divergent α-tubulin, sharing only 67% amino acid identity with the generic isotypes αTUB84B and αTUB84D, and its expression is exclusively maternal. The genetic analysis of the Tub67C gene described here demon strates that αTUB67C is required for nuclear division in the oocyte and early embryo. Both meiosis and cleavage-stage mitoses are severely affected by mutations that result in a substantial decrease in the ratio of αTUB67C/ αTUB84B+ αTUB84D. A large increase in this ratio, achieved by increasing the gene dosage of Tub67C, has little or no effect on meiosis, but severely disrupts mitotic spindle function. Thus, both classes of α-tubulin isotype present in the mature oocyte, αTUB67C and αTUB84B/84D, are essential for normal spindle function in early Drosophila development. These α-tubulins provide the first example of tubulin isotypes known to be coexpressed in wild-type animals whose encoded variation is required for the normal function of a microtubule array.
Aggregation chimeras demonstrate that the primary defect responsible for aganglionic megacolon in lethal spotted mice is not neuroblast autonomous
Authors: Kapur Raj P. , Yost Cynthia , Palmiter Richard D.
Published: April 2021

The lethal spotted (ls) mouse has been used as a model for the human disorder Hirschsprung’s disease, because as in the latter condition, ls/ls homozygotes are born without ganglion cells in their terminal colons and, without surgical intervention, die early as a consequence of intestinal obstruction. Previous studies have led to the conclusion that hereditary aganglionosis in ls/ls mice occurs because neural crest-derived enteric neuroblasts fail to colonize the distal large intestine during embryogenesis, perhaps due to a primary defect in non-neuroblastic mesenchyme rather than migrating neuroblasts themselves. In this investigation, the latter issue was addressed directly, in vivo, by comparing the distributions of ls/ls and wild-type neurons in aggregation chimeras. Expression of a transgene, D H-nlacZ, in enteric neurons derived from the vagal neural crest, was used as a marker for ls/ls enteric neurons in chimeric mice. In these animals, when greater than 20% of the cells were wild-type, the ls/ls phenotype was rescued; such mice were neither spotted nor aganglionic. In addition, these ‘rescued’ mice had mixtures of ls/ls and wildtype neurons throughout their gastrointestinal systems including distal rectum. In contrast, mice with smaller relative numbers of wild-type cells exhibited the classic ls/ls phenotype. The aganglionic terminal bowel of the latter mice contained neither ls/ls nor wild-type neurons. These results confirm that the primary defect in ls/ls embryos is not autonomous to enteric neuroblasts, but instead exists in the non-neuroblastic mesenchyme of the large intestine.
Regulation of development and differentiation by the extracellular matrix
Authors: Adams Josephine C. , Watt Fiona M.
Published: April 2021

Introduction

Evidence that cell-ECM interactions regulate

differentiation and development

Developmental mutations

Experimental perturbation of embryonic development

Cell and organ cultures

Regulatory mechanisms

ECM diversity

Interactions of growth factors with the ECM

ECM receptors

Non-integrin receptors

Integrins

Signal transduction

Cytoskeleton

Second messengers

‘ECM-response elements’

Conclusion
Regional repression of a Drosophila POU box gene in the endoderm involves inductive interactions between germ layers
Authors: Affolter Markus , Walldorf Uwe , Kloter Urs , Schier Alexander F. , Gehring Walter J.
Published: April 2021

An induction process occurring between the mesodermal and the endodermal germ layers has recently been described in the regulation of the Drosophila homeotic gene labial (lab). We report here that proper spatial regulation of the Drosophila POU box gene pdm-1 products also involves interaction between these two germ layers. pdm-1 transcripts are initially present in both the anterior and the posterior endodermal midgut primordia. Upon fusion of the two primordia, transcripts disappear from two regions in the endoderm, a central domain and an anterior domain. The anterior repression domain of pdm-1 is independent of the expression of known homeotic genes and genes encoding secreted signalling molecules in the visceral mesoderm, both for its positioning and its repression. Repression in the central domain requires both the homeotic gene Ultrabithorax (Ubx) and the decapentaplegic (dpp) gene, which encodes a secreted protein. Both of these genes are also required for lab induction. However, the analysis of pdm-1 expression in various mutant backgrounds indicates that the regulation of lab and pdm-1 across germ layers is controlled by different genetic cascades. Our study indicates that dpp is not the signal that dictates central pdm-1 repression across germ layers and suggests that in the same midgut region, different signalling pathways result in the differential activation or repression of potential transcription factors.
Cross regulation of decapentaplegic and Ultrabithorax transcription in the embryonic visceral mesoderm of Drosophila
Authors: Hursh Deborah A. , Padgett Richard W. , Gelbart William M.
Published: April 2021

The Drosophila decapentaplegic gene (dpp) encodes a TGF-β family member involved in signal transduction during embryonic midgut formation. The shortvein (shv) class of cis-regulatory dpp mutants disrupt expression in parasegments 4 and 7 (ps4 and ps7) of the embryonic visceral mesoderm (VM) surrounding the gut and cause abnormalities in gut morphogenesis. We demonstrate that cis-regulatory elements directing expression in ps4 and ps7 are separable and identify DNA fragments that generate ps4 and ps7 expression patterns using reporter gene constructs. dpp reporter gene expression in both ps4 and ps7 is autoregulated as it requires endogenous dpp+ activity. Reporter gene ps7 expression requires the wild-type action of Ultra bithorax (Ubx), and abdominal-A. Furthermore, the expression of certain Ubx reporter genes is coincident with dpp in the VM. Both the mis-expression of Ubx reporter genes in the developing gastric caecae at ps4 and its normal expression in ps7 are dependent upon endogenous dpp+ activity. We conclude that dpp both responds to and regulates Ubx in ps7 of the visceral mesoderm and that Ubx autoregulation within this tissue may be indirect as it requires more components than have previously been thought.
Analysis of genetic mosaics in developing and adult Drosophila tissues
Authors: Xu Tian , Rubin Gerald M.
Published: April 2021

We have constructed a series of strains to facilitate the generation and analysis of clones of genetically distinct cells in developing and adult tissues of Drosophila. Each of these strains carries an FRT element, the target for the yeast FLP recombinase, near the base of a major chromosome arm, as well as a gratuitous cellautonomous marker. Novel markers that carry epitope tags and that are localized to either the cell nucleus or cell membrane have been generated. As a demonstration of how these strains can be used to study a particular gene, we have analyzed the developmental role of the Drosophila EGF receptor homolog. Moreover, we have shown that these strains can be utilized to identify new mutations in mosaic animals in an efficient and unbiased way, thereby providing an unprecedented opportunity to perform systematic genetic screens for mutations affecting many biological processes.
Lithium perturbation and goosecoid expression identify a dorsal specification pathway in the pregastrula zebrafish
Authors: Stachel Scott E. , Grunwald David J. , Myers Paul Z.
Published: April 2021

The zebrafish dorsoventral axis can first be distin-guished at gastrulation, upon formation of the embry-onic shield, the site of the organizer. We have asked whether the shield is specified before gastrulation. First, we show that brief exposure of premidblastula embryos to lithium, which is known to shut down the phospho-inositol signaling pathway, produces excessive shield formation and extreme hyper-dorsal development. Second, we show that the zebrafish goosecoid homeobox gene is activated at or just after the midblastula in a localized domain of cells that subsequently populate the most anterior region of the incipient shield and axial hypoblast. goosecoid expression is elevated and radialized by early lithium treatment, suggesting that goosec -oid plays a role in establishing the organizer and shield. Our results demonstrate that the zebrafish dorsal axis is signaled by a pathway initiated in the cleavage-stage embryo. Furthermore, they provide novel insights into anterior morphogenesis.
Chimeric analysis of T (Brachyury) gene function
Authors: Wilson Valerie , Rashbass Penny , Beddington Rosa S. P.
Published: April 2021

To investigate T(Brachyury) gene function, a chimeric analysis of midgestation (9.5–11.5 days post coitum) embryos has been performed. Embryonic stem (ES) cell lines homozygous or heterozygous for the T gene have been introduced into wild-type host embryos by blasto-cyst injection, and the resulting chimeras scored for morphological abnormality and extent of colonization by T/T cells. As observed previously in earlier stage chimeras (Rashbass, P., Cooke, L. A., Herrmann, B. G. and Beddington, R. S. P. (1991) Nature 353, 348-350), 9.5-11.5 dpc T/T +/+ chimeras exhibit many morpho-logical features of intact T/T mutants. In addition, a dramatic bias of T/T cells towards caudal regions (such as tail and allantois) was observed in all chimeras tested. This is likely to result from accumulation of nascent T/T mesoderm cells with time near the primitive streak, possibly because of altered migration or adhesion proper-ties. T/+ cells colonized rostral regions efficiently, but a slight bias towards the distal end of the tail was still evident. No such bias was observed in control chimeras. The presence of T/T cells in the allantois resulted in its failure to form a correct placental connection and thus arrested later development. In contrast, chimeras in which T/T cells were present predominantly in the tail developed normally but exhibited severe tail abnormalities such as foreshortening, branching and haemorrhagic cavities. Moreover, in these embryos, much higher levels of chimerism were present in the distal end of the tail than in younger (9.5 dpc) embryos. Later in gestation, such abnormal tails probably degenerated, giving rise to neonates with absent or severely abnormal tails but no evidence of chimerism. In situ analysis of T expression in the tail reveals that normally T is expressed highly in the tailbud (the growing portion of the tail) during its elongation between 9.5 and 11.5 dpc. Thus, evidence both from chimeras and from T expression in the tail suggest that T may play a role in the correct deployment of cells emerging from the tailbud.
Isolation and characterisation of murine homologues of the Drosophila seven in absentia gene (sina)
Authors: Della Neil G. , Senior Paul V. , Bowtell David D. L.
Published: April 2021

The seven in absentia gene (sina) is required for formation of the R7 photoreceptor cell in the developing eye of Drosophila melanogaster. The sina protein contains a putative zinc finger domain and localises to the cell nucleus in Drosophila. We report here the identification of a family of genes in the mouse (designated Siah) with extensive sequence homology to Drosophila sina. The Siah genes fall into two main groups: Siah-1, which consists of four closely related members, two of which appear to be functional, and Siah-2, which contains a single functional member. The predicted Siah proteins show an unusually high degree of conservation with sina over the majority of their lengths, diverging significantly only at their amino terminal ends. The Siah-1 and Siah-2 genes are widely expressed at a low level in the embryo and adult. Analysis of Siah-2 by hybridisation histo-chemistry shows that it is expressed at a higher level in a restricted number of sites during development, including the olfactory epithelium, retina, forebrain and pro-liferating cartilage of developing bone. The striking degree of sequence homology observed between the Drosophila and murine genes implies strong conservation pressure on the Siah genes and suggests that they play a significant role in vertebrate development.
Ventral spinal cord inhibition of neurite outgrowth from embryonic rat dorsal root ganglia
Authors: Fitzgerald Maria , Kwiat Geoffrey C. , Middleton Jacqueta , Pini Adrian
Published: April 2021

Organotypic culture of embryonic rat lumbar spinal cord and dorsal root ganglia has been used to demonstrate an inhibitory effect of ventral spinal cord on neurite growth from dorsal root ganglion explants. When dorsal root ganglion explants from 14 – 15 day old embryos were cultured alone or in close proximity to a dorsal cord explant, the pattern of dorsal root ganglion neurite outgrowth was typically radial. However, when E14 – 15 dorsal root ganglion explants were cocultured for 22 – 24 hours in proximity to a ventral spinal cord explant from the same embryo, few, if any, dorsal root ganglion neurites grew in the direction of the ventral cord explant. This inhibitory effect appeared to be developmentally regulated; it was diminished or absent in cocultures prepared from 18 day old embryos. In contrast, in cocultures of dorsal cord and ventral cord explants from E14-15 embryos, dorsal cord neurites grew abundantly toward the ventral cord explant suggesting that the inhibition is not likely to be due to a nonspecific neurotoxic effect and that the activity responsible selectively inhibits dorsal root ganglion neurite outgrowth. We conclude that a diffusible, primary afferent inhibitory factor(s) produced by embryonic ventral horn may be responsible for the inhibition. Our results are discussed with respect to the possible involvement of inhibition in the normal development of primary afferent innervation of the spinal cord.
Mechanisms of dorsal-ventral axis determination in Drosophila embryos revealed by cytoplasmic transplantations
Authors: Roth Siegfried
Published: April 2021

The establishment of the dorsal-ventral pattern in Drosophila embryos depends on a signal transduction process: a putative extracellular ligand released into the perivitelline space surrounding the embryo binds to the Toll receptor. Toll activation triggers the formation of the nuclear gradient of dorsal protein, the morphogen of the dorsal-ventral axis. Here, I analyse the dorsal protein distribution and the expression of zygotic dorsal-ventral genes in Toll− embryos that have been injected with wild-type cytoplasm under a variety of different injection conditions. Injections into two positions within a single embryo lead to the formation of two dorsal-ventral patterns in one embryo, allowing the analysis of interactions between pattern-forming processes. The results of single and double injections suggest that the spatial information for the embryonic dorsal-ventral axis is largely derived from spatial cues present in the extraembryonic compartment, which restrict the release of the putative Toll ligand. They argue against a Toll-dependent pattern-formation process employing local self-enhancement and lateral inhibition to enhance a weak initial asymmetry. The putative Toll ligand appears to originate from a ventrally restricted zone which extends along the entire anterior-posterior axis. Ligand diffusion or its graded release are required to determine the slope of the nuclear dorsal protein gradi-ent. Both the Toll receptor and the putative ligand of Toll are in excess in wild-type embryos. Since spatial information for the embryonic dorsal-ventral axis is already present in the vitelline membrane or the perivitelline space, it is most likely generated during oogen-esis. Oogenic pattern formation is also responsible for the perpendicular orientation the dorsal-ventral axis maintains with respect to the anterior-posterior axis.
Analysis of Hox-4.5 and Hox-3.6 expression during newt limb regeneration: differential regulation of paralogous Hox genes suggest different roles for members of different Hox clusters
Authors: Simon Hans-Georg , Tabin Clifford J.
Published: April 2021

Adult urodele amphibians can regenerate their limbs and tail. Based on their roles in other developing sys-tems, Hox genes are strong candidates for genes that play a role in regulating pattern formation during regeneration. There are four homologous clusters of Hox genes in vertebrate genomes. We isolated cDNA clones of two newt homeobox genes from homologous positions within two Hox clusters; Hox-4.5 and Hox-3.6. We used RNase protection on nonamputated (normal) and regenerating newt appendages and tissue to com-pare their transcriptional patterns. Both genes show increased expression upon amputation with similar kinetics. Hox-4.5 and Hox-3.6 transcription is limited to the mesenchymal cells in the regenerates and is not found in the epithelial tissue. In addition to regenerat-ing appendages, both genes are transcriptionally active in adult kidney of the newt. Striking differences were found in the regulation of Hox-4.5 and Hox-3.6 when they were compared in unamputated limbs and in regenerating forelimbs versus regenerating hindlimbs.

Hox-4.5 is expressed in the blastema of regenerating fore- and hindlimbs, but Hox-4.5 transcripts are not detectable in normal limbs. In contrast, Hox-3.6 tran-scripts are found exclusively in posterior appendages, but are present in normal as well as regenerating hindlimbs and tails. Hox-4.5 is also expressed at a higher level in proximal (mid-humerus) regenerates than in distal ones (mid-radius). When we proximalized the positional memory of a distal blastema with retinoic acid, we find that the early expression level of Hox-4.5 is also proximalized. When the expression of these genes is compared to the expression of two previously reported newt Hox genes, a consistent pattern emerges, which can be interpreted in terms of differential roles for the dif-ferent Hox clusters in determining regenerative limb morphology.
Maintenance of ZPA signaling in cultured mouse limb bud cells
Authors: Anderson Rosalie , Landry Michael , Muneoka Ken
Published: April 2021

The positional signal localized to the posterior (zone of polarizing activity or ZPA) region of the vertebrate limb is transiently expressed during development and a decline in ZPA signaling is accelerated when posterior cells are dis-sociated and cultured in vitro. The evidence that cultured posterior cells display a precocious decline in ZPA signaling when compared to in vivo studies suggests that a factor present in the limb bud maintains or stabilizes ZPA sig-naling during limb outgrowth and that this maintenance factor is lost and/or exhausted in in vitro studies. We have developed a new culture technique, ‘microdissociation’, which preserves extracellular components that we have found to be necessary for ZPA signal maintenance. Our data suggest that the limb bud ectoderm produces a maintenance activity that becomes stored in the extracellular matrix where it acts on limb bud cells to stabilize the activity of the ZPA signal. Using our initial characterization of this maintenance activity, we have identified a growth factor, FGF-2 (bFGF), that can replace all of the ZPA signaling maintenance activity observed in microdissociate cultures. The existence of various members of the FGF family in the developing limb strongly argues a role for FGF in stabiliz-ing ZPA signaling in vivo.
Contractile protein gene expression in primary myotubes of embryonic mouse hindlimb muscles
Authors: Ontell Marcia , Ontell Martin P. , Sopper Maggie M. , Mallonga Rosa , Lyons Gary , Buckingham Margaret
Published: April 2021

The time course of contractile protein [actin, myosin heavy chain (MHC) and myosin light chain (MLC)] gene expression in the hindlimb muscles of the embryonic mouse (<15 days gestation) has been correlated with the expression of genes for the myogenic regulatory factors, myogenin and MyoD, and with morphogenetic events. At 14 days gestation, secondary myotubes are not yet present in crural muscles (M. Ontell and K. Kozeka (1984) Am. J. Anat. 171, 133-148; M. Ontell, D. Bourke and D. Hughes (1988)Am. J. Anat. 181, 267-278); there-fore, all transcripts for contractile proteins found in these muscles must be produced in primary myotubes. In situ hybridization, with 35S-labeled antisense cRNAs, demonstrates the versatility of primary myotubes in that transcripts for (1) α-cardiac and α-skeletal actin, (2) MHCembryonic, MHCperinatal and MHC /slow, and (3) MLC1A, MLC1F and MLC3F are detectable at 14 days gestation. While the general patterns of early activation of the cardiac genes and early activation of the genes for the developmental isoforms are preserved in both myotomal and limb muscles (D. Sassoon, I. Garner and M. Buckingham (1988)Development 104, 155-164 and G. E. Lyons, M. Ontell, R. Cox, D. Sassoon and M. Buckingham (1990)J. Cell Biol. 111, 1465-1476 for myotomal muscle), there are a number of differences in contractile protein gene expression. For example, in the myotome, when myosin light chain genes are initially transcribed, hybridization signal with probe for MLC1A mRNA is greater than that with probe for MLC1F tran-scripts, whereas the relative intensity of signal with these same probes is reversed in the hindlimb. The order in which myosin heavy chain genes are activated is also different, with MHCembryonic and MHCperinatal preced-ing the appearance of MHC β/slow transcripts in limb muscles, while MHCembryonic and MHC /slow appear simultaneously in the myotomes prior to MHCperinatal. In the myotome, an intense hybridization signal for α-cardiac and a weak signal for α-skeletal actin tran-scripts are detectable prior to myosin mRNAs, whereas in the limb α-cardiac actin transcripts accumulate with myosin transcripts before α-skeletal actin mRNA is detectable. These differences indicate that there is no single coordinate pattern of expression of contractile protein genes during initial formation of the muscles of the mouse. The fact that different myogenic factors are present initially in myotomes than are present initially in the hindlimb (cf. M. E. Buckingham (1992)Trends Genet. 8, 144-149) suggests that there may be subtle dif-ferences in the capacity of these factors to activate dif-ferent muscle genes. These results are discussed in the context of muscle cell lineages.
Acidic and basic FGF in ocular media and lens: implications for lens polarity and growth patterns
Authors: Schulz Mark W. , Chamberlain Coral G. , Iongh Robbert U. de , McAvoy John W.
Published: April 2021

We have shown previously that FGF induces lens epithelial cells in explant culture to proliferate, migrate and differentiate into fibre cells in a progressive concentration-dependent manner. In situ, these processes occur in a distinct anterior-posterior pattern in clearly defined regions of the lens. Thus anterior-posterior differences in the bio-availability of FGF in the lens environment may play a role in determining lens polarity and growth patterns. In this study, using heparin chromatography and western blotting (or ELISA), we established that both acidic and basic FGF are present in the aqueous and vitreous (the ocular media that bathe the anterior and posterior compartments of the lens, respectively). In addition, substantially more FGF was recovered from vitreous than from aqueous. Both forms of FGF were also detected in lens fibre cells and capsule. A truncated form of basic FGF (less than 20×103Mr) predominated in every case with traces of higher Mr forms in lens cells. For acidic FGF, the classical full-length form (about 20×103Mr) predominated in lens cells and a truncated form was found in vitreous. The capsule contained a higher Mr form. Using our explant system, we also tested the biological activity of ocular media and FGF fractions obtained from vitreous and lens cells. Vitreous but not aqueous contained fibre-differentiating activity. Furthermore, virtually all the fibre-differentiating activity of vitreous was shown to be FGF-associated, as follows: (a) this activity remained associated with FGF during fractionation of vitreous by heparin and MonoS chromatography and (b) the activity of the major FGF-containing fraction was blocked by antibodies to acidic and basic FGF. Posterior, but not anterior, capsule was shown to have mitogenic activity, which was neutralised by FGF antibodies and associated only with the cellular surface. These results support our hypothesis that FGF is involved in determining the behaviour of lens cells in situ. In particular, a key role for FGF in determining lens polarity and growth patterns is suggested by the anterior-posterior differences in the bio-availability of FGF in the ocular media and capsule.
Segmental repetition of neuronal phenotype sets in the chick embryo hindbrain
Authors: Clarke J. D. W. , Lumsden Andrew
Published: April 2021

The neurons within the segmented hindbrain of the early chick embryo have been mapped with the neuronal tracers HRP and fluorescent lysinated dextran. We have categorised neurons according to their axonal pathways and have then compared rhombomeres with respect to the number and class of neurons present. The results indicate that most rhombomeres are similar in that they contain the same set of basic neuronal types but differ in that particular neuronal types are more abundant in some rhombomeres than others. The data support the concept that the hindbrain develops according to ‘variations on a segmental theme’ rather than ‘each segment is unique’. Many of the cell types occupy distinct mediolateral domains that are probably established by both the differential migration of some neuronal classes and the spatial segregation of distinct precursors. The caudal rhombomeres 7 and 8 are exceptional in that they do not have the full set of basic neuronal types and also contain two additional medial cell types that are not present rostrally. The mechanisms that may generate the regional diversity apparent in the more mature hindbrain are discussed.
Occurrence of dorsal axis-inducing activity around the vegetal pole of an uncleaved Xenopus egg and displacement to the equatorial region by cortical rotation
Authors: Fujisue M. , Kobayakawa Y. , Yamana K.
Published: April 2021

Specification of the dorsoventral axis is a subject of great importance in amphibian embryogenesis. We have found that cytoplasm of the vegetal dorsal cells of a 16cell embryo of Xenopus laevis, when injected into the ventral vegetal cells of a recipient at the same stage, can induce formation of a second axis. In the present experiments, using the same assay procedure, we found that the cytoplasm around the vegetal pole of an egg before cortical rotation is also active in inducing a second axis, that the activity decreases throughout the second half of the cell cycle and appears in a presumptive dorsal equatorial region at the 2to 16-cell stages. This is the first demonstration of the localization of dorsal forming activity in any specific region of an egg. After UV irradiation, a treatment that is known to block cortical rotation and thereby inhibit axis specification, the activity remains near the vegetal pole beyond the first cell cycle and does not appear in an equatorial region, at least at the 16-cell stage. This suggests that cortical rotation or a related force is in some way involved in changes in distribution of the activity.

We also found that UV-irradiated 8-cell embryos can rescue dorsal development when they are cut into halves along the first cleavage plane. Histological examination revealed that the rescued embryos have a neural tube and notochord. In the half embryo, the animal and vegetal regions came into contact during wound healing, an event that enables the activity to localize in the new equator of an embryo. Therefore this rescue suggests that, if the activity is distributed only in the equatorial region, dorsal specification occurs. In fact, the dorsal side of the rescued embryos seems to correspond to the plane through which the embryos have been cut.

Based on our results, we propose (1) that a determinant that carries axis-inducing activity is first present around the vegetal pole, (2) that the determinant shifts from the vegetal pole to an equatorial region by or in close association with cortical rotation and (3) that occurrence of the determinant in the equatorial region is a prerequisite for axis specification.
Genomic organization and expression of the planarian homeobox genes Dth-1 and Dth-2
Authors: Garcia-Fernàndez Jordi , Baguñà Jaume , Saló Emili
Published: April 2021

We have characterized the genomic organization of Dth1 and Dth-2, planarian homeobox-containing genes, previously described at the cDNA level (J. Garcia-Fernàndez, J. Baguñà and E. Saló (1991), Proc. Natl. Acad. Sci. USA, 88, 7338-7342). Genomic analysis shows that Dth-1 and Dth-2 genes encode proteins of 533 and 363 amino acids respectively. The open reading frame of Dth-1 is interrupted by two large introns of 8 kb and 12 kb Dth-2 also shows two introns, but these are short (42 bp and 44 bp) and the second interrupts helix III at position 44-45, as is the case with other homeobox genes from such divergent animals as Drosophila, honeybee, C. elegans, ascidians, and mouse, which suggests an ancient evolutionary relationship between these genes.

The spatial distribution of transcripts in adult tissues, determined by in situ hybridization, demonstrates that Dth-1 is expressed at a high level in the gastrodermal cells, while Dth-2 is expressed in the peripheral parenchyma, at higher levels in the dorsal than the ventral regions. Their specific spatial distribution suggests a possible role for these homeobox genes in determination and/or differentiation of specific cell types. The expression pattern of both genes is more or less continuous, but in Dth-1 clustered discontinuous labelling in areas surrounding the gastrodermis may indicate a specific expression of this gene in groups of undifferentiated cells (neoblasts) already committed or determined to gastrodermal cell fates.

In situ hybridization analysis during early regeneration shows expression only in the postblastema (stump) differentiated areas while no expression has been detected in the undifferentiated blastema, indicating that neither gene has a role in pattern formation mechanisms known to occur at the early stages of regeneration (0–3 days). Hence, Dth-1 and Dth-2 are planarian homeobox genes presumably involved in specific cell or tissue determination and/or differentiation.
Drosophila hairy pair-rule gene regulates embryonic patterning outside its apparent stripe domains
Authors: Lardelli Michael , Ish-Horowicz David
Published: April 2021

The hairy (h) segmentation gene of Drosophila regulates segmental patterning of the early embryo, and is expressed in a set of anteroposterior stripes during the blastoderm stage. We have used a set of h gene deletions to study the h promoter and the developmental requirements for individual h stripes. The results confirm upstream regulation of h striping but indicate that expression in the anterodorsal head domain depends on sequences downstream of the two transcription initiation sites. Surprisingly, the two anterior-most h domains appear to be dispensable for head development and embryonic viability. One partial promoter deletion expresses ectopic h, leading to misexpression of other segmentation genes and embryonic pattern defects. We demonstrate that h affects patterning outside its apparent stripe domains, supporting a model in which primary pair-rule genes act as concentration-dependent transcriptional regulators, i.e. as local morphogens.
Proximal cis-acting elements cooperate to set Hoxb-7 (Hox-2.3) expression boundaries in transgenic mice
Authors: Vogels Ronald , Charité Jeroen , Graaff Wim de , Deschamps Jacqueline
Published: April 2021

The Hox genes have been proved to be instrumental in establishing the positional identity of cells along the embryonic anteroposterior (A–P) axis. Studying the regulation of these genes is a first step toward elucidating the molecular basis of regionalization during embryogenesis. We report here on the identification of cis-acting elements controlling the expression of Hoxb-7 (Hox-2.3). We show that elements driving A–P restricted gene expression are located within the 3.5 kb proximal upstream sequences of the Hoxb-7 gene. A deletion analysis provides evidence for at least three cis-acting control elements upstream from Hoxb-7, and for cooperative interactions between some of these elements in generating the A–P restricted transgenic pattern. One element, conferring by itself Hox-like expression boundaries to the transgene, has been studied in more detail and found to act in an orientation- and promoter-dependent manner. Together the 3.5 kb sequences proximal to Hoxb-7 mediate A–P restricted Hoxb-7/lacZ gene expression in a domain showing rostral boundaries more posterior than those of Hoxb-7. The evolution throughout embryogenesis of the expression pattern of a transgene carrying these sequences has been analysed and shown to mimick that of the endogenous gene, except for a slight delay in the initial expression. We conclude that the transgenes that we tested, spanning a total of 27 kb genomic sequences, do not reproduce all the features of the Hoxb-7 expression pattern. The differences in expression between Hoxb-7 and the transgenes may reveal an aspect of the Hox regulation for which either remote cis-acting control elements and/or gene clustering is required. Additional features that may have favoured maintenance of clustered organisation during evolution are partial overlap of transcription units with the regulatory regions of the neighbouring genes, and cis-regulatory interactions between multiple Hox genes: not only do cis-acting control elements of the Hoxb-7 gene map in the 3′ untranslated sequences of the Hoxb-8 (Hox-2.4) gene, but our experiments suggest that Hoxb-7 control sequences modulate expression of the Hoxb-8 gene as well.
Retroviral expression of FGF-2 (bFGF) affects patterning in chick limb bud
Authors: Riley Bruce B. , Savage Mary P. , Simandl B. Kay , Olwin Bradley B. , Fallon John F.
Published: April 2021

To investigate the role of fibroblast growth factor-2 (basic fibroblast growth factor) in chick limb development, we constructed a replication-defective spleen necrosis virus to ectopically express fibroblast growth factor-2 in stage 2022 chick limb bud. Because infecting cells in vivo proved to be inefficient, limb bud cells were dissociated, infected in vitro, and then grafted back into host limbs. This procedure caused duplications of anterior skeletal elements, including proximal humerus, distal radius, and digits 2 and 3. Eighty-nine percent of host wings receiving infected grafts at their anterior borders had duplications of one or more of these elements. The frequency of duplication declined dramatically when infected cells were grafted to progressively more posterior sites of host limb buds, and grafting to the posterior border had no effect at all. Several techniques were used to determine the role of infected tissue in forming skeletal duplications. First, staining with an fibroblast growth factor-2 specific monoclonal antibody showed higher than endogenous levels of fibroblast growth factor-2 expression associated with extra elements. Second, the host/donor composition of duplicated elements was determined by simultaneously infecting donor cells with viruses encoding fibroblast growth factor-2 or β-galactosidase; donor tissue was then visualized by X-gal staining. Patterns of ectopic fibroblast growth factor-2 expression and X-gal staining confirmed the presence of infected donor tissue near duplicated structures, but the duplicated skeletal elements themselves showed very little staining. Similar results were obtained in duplications caused by infected quail wing bud cells grafted to the chick wing bud. These observations suggest that fibroblast growth factor-2expressing donor tissue induced host tissue to form normally patterned extra elements. In support of this conclusion, implanting beads containing fibroblast growth factor-2 caused partial duplications of digit 2. These data provide the first direct evidence that fibroblast growth factor-2 plays a role in patterning in the limb bud.
Cell proliferation in the gastrulating chick embryo: a study using BrdU incorporation and PCNA localization
Authors: Sanders E. J. , Varedi M. , French A. S.
Published: April 2021

Cell proliferation in the gastrulating chick embryo was assessed using two independent techniques which mark cells in S phase of the mitotic cycle: nuclear incorporation of bromodeoxyuridine (BrdU) detected immunocytochemically and immunolocalization of proliferating cell nuclear antigen (PCNA). Computer-reconstructed maps were produced showing the distribution of labelled nuclei in the primitive streak and the cell layers. These distributions were also normalized to take into account regional differences in cell density across the embryo. Results from a 2 hour pulse of BrdU indicated that although cells at caudal levels of the primitive streak showed the highest incorporation, this region showed a similar proportion of labelled cells to the surrounding caudal regions of the epiblast and mesoderm when normalized for cell density. The entire caudal third of the embryo showed the highest proportion of cells in S phase. Cells of Hensen’s node showed a relatively low rate of incorporation and, although the chordamesoderm cells showed many labelled nuclei, this appeared to be a reflection of a high cell density in this region. Combining this result with results from a 4 hour pulse of BrdU permitted mapping of cell generation time across the entire embryo. Generation times ranged from a low value of approximately 2 hours at caudal levels of both the epiblast and mesoderm, to an upper value of approximately 10 hours in the rostral regions of the primitive streak, in the mid-lateral levels of the epiblast and in the chordamesoderm rostral to Hensen’s node.

Cells at caudal regions of the primitive streak showed a generation time of approximately 5 hours. Taking into account that cells are generally considered to be continuously moving through the primitive streak, we conclude that cell division, as judged by generation time, is greatly reduced during transit through this region, despite the presence there of cells in S phase and M phase. Immunocytochemical localization of PCNA-positive nuclei gave generally similar distributions to those obtained with BrdU incorporation, confirming that this endogenous molecule is a useful S-phase marker during early embryogenesis. Mid-levels and caudal levels of the primitive streak showed the highest numbers of positive nuclei, and the highest proportion of labelling after cell density was accounted for. As with BrdU incorporation, the highest proportions of PCNA-positive nuclei were found towards the caudal regions of the epiblast and mesoderm. These results suggest that the differential growth of the caudal region of the embryo at this time is a direct consequence of elevated levels of cell proliferation in this region. However, no evidence was obtained to indicate that the primitive streak cells, or the cells of the emerging mesoderm, are highly proliferative, and we therefore conclude that the morphogenesis of the early mesoderm does not rely on elevated rates of cell division.
trkC, a receptor for neurotrophin-3, is widely expressed in the developing nervous system and in non-neuronal tissues
Authors: Tessarollo Lino , Tsoulfas Pantelis , Martin-Zanca Dionisio , Gilbert Debra J. , Jenkins Nancy A. , Copeland Neal G. , Parada Luis F.
Published: April 2021

The Trk family of tyrosine kinases encodes receptors for nerve growth factor-related neurotrophins. Here we present a developmental expression study of trkC, which encodes a receptor for neurotrophin-3 (NT-3). Like the related genes, trk and trkB, trkC is expressed primarily in neural lineages although the pattern is complex and includes non-neuronal cells. Direct comparison with trk and trkB developmental expression patterns permits the following observations. (1) trkC is expressed in novel neural tissues where other Trk genes are silent. (2) Some tissues appear to coexpress trkB and trkC receptors in the embryo and in the adult. (3) trkC expression can be detected in the gastrulating embryo. These data provide insights into the role of Trk-family receptors and nerve growth factor-related neurotrophins during development and suggest that, in addition to regulating neuronal survival and differentiation, the neurotrophin/Trk receptor system may have broader physiological effects. Finally, interspecific mouse backcrosses have been used to map the location of each of the Trk genes on mouse chromosomes. Alignment with available chromosomal maps identify possible linkage between the Trk genes and known neurological mutations.
PDGF and its receptors in the developing rodent retina and optic nerve
Authors: Mudhar Hardeep S. , Pollock, Richard A. , Wang Chiayeng , Stiles Charles D. , Richardson William D.
Published: April 2021

We have used in situ hybridization to visualize cells in the developing rat retina and optic nerve that express mRNAs encoding the A and B chains of platelet-derived growth factor (PDGF-A and PDGF-B), and the alpha and beta subunits of the PDGF receptor (PDGFR αR and PDGF βR). We have also visualized PDGF-A protein in these tissues by immunohistochemistry. In the retina, PDGF-A mRNA is present in pigment epithelial cells, ganglion neurons and a subset of amacrine neurons. PDGF-A transcripts accumulate in ganglion neurons during target innervation and in amacrine neurons around the time of eye opening, suggesting that PDGFA expression in these cells may be regulated by targetderived signals or by electrical activity. In the mouse retina, PDGF-A immunoreactivity is present in the cell bodies, dendrites and proximal axons of ganglion neurons, and throughout the inner nuclear layer. PDGFR αR mRNA is expressed in the retina by astrocytes in the optic fibre layer and by a subset of cells in the inner nuclear layer that might be Müller glia or bipolar neurons. Taken together, our data suggest short-range paracrine interactions between PDGF-A and PDGFR, the ligand and its receptor being expressed in neighbouring layers of cells in the retina. In the optic nerve, PDGF-A immunoreactivity is present in astrocytes but apparently not in the retinal ganglion cell axons. PDGF αR+ cells in the optic nerve first appear near the optic chiasm and subsequently spread to the retinal end of the nerve; these PDGFαR+ cells are probably oligodendrocyte precursors (Pringle et al., 1992). RNA transcripts encoding PDGF-B and PDGFβR are expressed by cells of the hyaloid and mature vascular systems in the eye and optic nerve.