bluuebunny/crossref_metadata_2025 huggingface dataset:
The interaction of boar sperm proacrosin with its natural substrate, the zona pellucida, and with polysulfated polysaccharides
Authors: Urch U. A. , Patel H.
Published: April 2021

Boar sperm acrosin is an acrosomal protease with trypsin-like specificity, and it functions in fertilization by assisting sperm passage through the zona pellucida by limited hydrolysis of this extracellular matrix. In addition to a proteolytic active site domain, acrosin binds the zona pellucida at a separate binding domain that is lost during proacrosin autolysis. In this study, we quantitate the binding of proacrosin to the physiological substrate for acrosin, the zona pellucida, and to a nonsubstrate, the polysulfated polysaccharide fucoidan. Binding was analogous to sea urchin sperm bindin that binds egg jelly fucan and the vitelline envelope of sea urchin eggs. Proacrosin was found to bind to fucoidan and to the zona pellucida with binding affinities similar to bindin interaction with egg jelly fucan. These interactions were competitively inhibited by similar relative molecular mass polysulfated polymers. Since bindin and proacrosin have distinctly different amino acid sequences, their interaction with acidic sulfate esters demonstrates an example of convergent evolution wherein different macromolecules localized in analogous sperm compartments have the same biological function. From cDNA sequence analysis of proacrosin, this binding may be mediated through a consensus sequence for binding sulfated glycoconjugates. Proacrosin binding to the zona pellucida may serve as both a recognition or primary sperm receptor, as well as maintaining the sperm on the zona peUucida once the acrosome reaction has occurred.
Retarded nuclear migration in Drosophila embryos with aberrant F-actin reorganization caused by maternal mutations and by cytochalasin treatment
Authors: Hatanaka Ko , Okada Masukichi
Published: April 2021

Three X-linked mutations of Drosophila melanogaster, gs(l)N26, gs(l)N441 and paralog, had a common maternal-effect phenotype. Mutant embryos show reduced egg contraction that normally occurs at an early cleavage stage in wild-type embryos. In addition, the mutants exhibited retarded nuclear migration while synchronous nuclear divisions were unaffected. The retarded migration causes nuclei to remain in the anterior part of the embryo retaining their spherical distribution even in a late cleavage stage. This consequently results in an extreme delay in nuclear arrival in the posterior periplasm. A mutant phenocopy was induced in wild-type embryos that were treated with cytochalasin B or D at a very early cleavage stage. Remarkable differences were noticed in the organization of cortical F-actin between the mutants and the wild type throughout the cleavage stage: obvious F-actin aggregates were dispersed in the cortex of mutant embryos, in contrast to the wild type where the cortical F-actin layer was smooth and underlying F-actin aggregates were smaller than those in the mutants; the transition of the distribution pattern of F-actin in the yolk mass, from the centralized to the fragmented type, occurred later in the mutants than in wild type. The results suggest that these mutations affect the mechanism underlying establishment and transition of F-actin organization required for normal egg contraction and nuclear migration in the cleavage embryos.
The development of adult muscles in Drosophila: ablation of identified muscle precursor cells
Authors: Broadie Kendal S. , Bate Michael
Published: April 2021

A small subset of mesodermal cells continues to express twist in the late embryo of Drosophila. These cells are the precursors of adult muscles. Each late twist-expressing cell begins to divide early in the second larval instar and division continues throughout the second and third instars, resulting in a small clone of twist-expressing cells at puparium formation. Treatment with a DNA-synthesis inhibitor, hydroxyurea (HU), ablates these cells if applied during S-phase of their replication cycle. We ablated twist-expressing lineages in the larva and demonstrated that this results in the absence of subsets of muscles in the adult abdomen and leg. HU treatment during this larval period has no discernible effect on the adult epidermis or innervation. We conclude that the twist-expressing cells identified in the late embryo are the unique primordia of adult muscles.

Each primordium is fated to establish 6-10 adult muscle fibres, defined here as a ‘muscle fibre group’. Each primordium has a unique fate and, after ablation, is not replaced by neighbouring cells. This unique fate does not rest with a particular founder cell within the primordium but is specified at the primordium level: ablation of a subset of cells within a muscle primordium does not result in an ablation of the resulting muscle group or in a decrease in the number of fibres within that muscle group, but rather results in a uniform decrease in the number of nuclei/fibres throughout the entire muscle. Thus, the twist-expressing primordia in the abdomen appear to be fated to give rise to a particular muscle group but act as an equivalent precursor pool in the formation of that muscle group. Our results permit the conclusion that specific muscle groups in the adult leg arise from restricted pools of twist-expressing adepithelial cells in the larval imaginal disc in a similar fashion. We conclude that the fate restriction of myoblast pools in early development defines elements of the final adult muscle pattern. The fate restriction of myoblast cells may be a result of genetic determination to form a specified muscle group or, alternatively, reflect the spatial isolation of otherwise equivalent cells to form muscle-specific precursor pools.
Methylation levels of maternal and paternal genomes during preimplantation development
Authors: HOWLETT SARAH K. , REIK WOLF
Published: April 2021

The methylation status of three highly repeated sequences was studied in sperm, eggs and preimplantation embryos with different combinations of parental chromosomes. High levels of methylation of the IAP and MUP sequence families were found in sperm and in eggs, whereas the LI repeat was found to be highly methylated in sperm but only about 42 % methylated in eggs. To assess how the two parental genomes behaved during preimplantation development, normal, fertilised embryos were compared with parthenogenetic embryos where the chromosomes are exclusively of maternal origin. It was observed that the high levels of methylation at the IAP and MUP sequences were retained through early development, with the first signs of demethylation at the IAP sequences apparent on both parental chromosomes in the blastocyst. Methylation at the sperm-derived LI sequences dropped to about the same level as that of the egg-derived sequences by the late 2-cell stage, both then remain at this intermediate level until around the time of cavitation when levels fell to about 10 % in the blastocyst. High levels of DNA methylase were detected in germinal vesicle and metaphase II oocytes; these high levels were maintained in fertilised and parthenogenetic embryos through into the morula and then declined to be undetectable in the blastocyst. Our comparison of maternal and paternal genomes suggests that methylation levels at repeat sequences are remarkably similar at the time of fertilization or, as in the case of the LI sequences, they become so during the first few cell cycles. Hence, there do not appear to be global methylation differences between the genomes that are retained through preimplantation development. The marked loss of methylation from sperm-derived LI sequences during the first two cell cycles may be indicative of the re-modelling of paternal chromosomes in the egg.
Tissue interactions affecting the migration and differentiation of neural crest cells in the chick embryo
Authors: Stern C.D. , Artinger K.B. , Bronner-Fraser M.
Published: April 2021

A series of microsurgical operations was performed in chick embryos to study the factors that control the polarity, position and differentiation of the sympathetic and dorsal root ganglion cells developing from the neural crest. The neural tube, with or without the notochord, was rotated by 180 degrees dorsoventrally to cause the neural crest cells to emerge ventrally. In some embryos, the notochord was ablated, and in others a second notochord was implanted. Sympathetic differentiation was assessed by catecholamine fluorescence after aldehyde fixation. Neural crest cells emerging from an inverted neural tube migrate in a ventral-to-dorsal direction through the sclerotome, where they become segmented by being restricted to the rostral half of each sclerotome. Both motor axons and neural crest cells avoid the notochord and the extracellular matrix that surrounds it, but motor axons appear also to be attracted to the notochord until they reach its immediate vicinity. The dorsal root ganglia always form adjacent to the neural tube and their dorsoventral orientation follows the direction of migration of the neural crest cells. Differentiation of catecholaminergic cells only occurs near the aorta/mesonephros and in addition requires the proximity of either the ventral neural tube (floor plate/ventral root region) or the notochord. Prior migration of presumptive catecholaminergic cells through the sclerotome, however, is neither required nor sufficient for their adrenergic differentiation.
Rescue and regulation of proboscipedia: a homeotic gene of the Antennapedia Complex
Authors: Randazzo F.M. , Cribbs D.L. , Kaufman T.C.
Published: April 2021

The extraordinary positional conservation of the homeotic genes within the Antennapedia and the Bithorax Complexes (ANT-C and BX-C) in Drosophila melanogaster and the murine Hox and human HOX clusters of genes can be interpreted as a reflection of functional necessity. The homeotic gene proboscipedia (pb) resides within the ANT-C, and its sequence is related to that of Hox-1.5. We show that two independent pb minigene P-element insertion lines completely rescue the labial palp-to-first leg homeotic transformation caused by pb null mutations; thus, a homeotic gene of the ANT-C can properly carry out its homeotic function outside of the complex. Despite the complete rescue of the null, the minigene expresses pb protein in only a subset of pb's normal domains of expression. Therefore, the biological significance of the excluded expression pattern elements remains unclear except to note they appear unnecessary for specifying normal labial identity. Additionally, by using reporter gene constructs inserted into the Drosophila genome and by comparing pb-associated genomic sequences from two divergent species, we have located cis-acting regulatory elements required for pb expression in embryos and larvae.
The relationship of decapentaplegic and engrailed expression in Drosophila imaginal disks: do these genes mark the anterior-posterior compartment boundary?
Authors: Raftery L.A. , Sanicola M. , Blackman R.K. , Gelbart W.M.
Published: April 2021

Imaginal disks, the primordia of the adult appendages in Drosophila, are divided into anterior and posterior compartments. However, the developmental role of such compartments remains unclear. The expression of decapentaplegic (dpp), a pattern formation gene required for imaginal disk development, has the intriguing property of being expressed in a line at or near the boundary between these compartments. Here, we compare the distribution of dpp-driven reporter gene expression to the pattern of expression of the engrailed (en) gene, known to be required for the maintenance of the compartment boundary. Using confocal microscopy to obtain single cell resolution, we have determined that the majority of the en+ imaginal disk cells expressing the dpp-driven reporter genes about those cells expressing en, while a small percentage of dpp reporter gene expressing cells also express en. In posterior regions of en mutant disks, where compartmentalization is abnormal, we observe ectopic expression of the dpp-driven reporter genes. We conclude that the pattern of dpp expression in imaginal disks is delimited in part through the direct or indirect repression by engrailed. Our results lead us to question the widely held assumption that the anterior edge of en expression demarcates the A/P compartment boundary.
Sex chimaerism, fertility and sex determination in the mouse
Authors: Patek C. E. , Kerr J. B. , Gosden R. G. , Jones K. W. , Hardy K. , Muggleton-Harris A. L. , Handyside A. H. , Whittingham D. G. , Hooper M. L.
Published: April 2021

Adult intraspecific mouse chimaeras, derived by introducing male embryonal stem cells into unsexed host blastocysts, were examined to determine whether gonadal sex was correlated with the sex chromosome composition of particular cell lineages. The fertility of XX↔XY and XY↔XY male chimaeras was also compared. The distribution of XX and XY cells in 34 XX↔XY ovaries, testes and ovotestes was determined by in situ hybridisation using a Y-chromosome-specific probe. Both XX and XY cells were found in all gonadal somatic tissues but Sertoli cells were predominantly XY and granulosa cells predominantly XX. The sex chromosome composition of the tunica albuginea and testicular surface epithelium could not, in general, be fully resolved, owing to diminished hybridisation efficiency in these tissues, but the ovarian surface epithelium (which like the testicular surface epithelium derives from the coelomic epithelium) was predominantly XX. These findings show that the claim that Sertoli cells were exclusively XY, on which some previous models of gonadal sex determination were based, was incorrect, and indicate instead that in the mechanism of Sertoli cell determination there is a step in which XX cells can be recruited. However, it remains to be established whether the sex chromosome constitution of the coelomic epithelium lineage plays a causal role in gonadal sex determination.

Male chimaeras with XX↔XY testes were either sterile or less fertile than chimaeras with testes composed entirely of XY cells. This impaired fertility was associated with the loss of XY germ cells in atrophic seminiferous tubules. Since this progressive lesion was correlated with a high proportion of XX Leydig cells, we suggest that XX Leydig cells are functionally defective, and unable to support spermatogenesis.
N-CAM alterations in splotch neural tube defect mouse embryos
Authors: Moase Connie E. , Trasler Daphne G.
Published: April 2021

The splotch (Sp) mouse is a model for both neurulation defects and defects in neural crest cell (NCC) derivatives. Since neurulation and NCC emigration from the neural tube occur at similar times in development, we suggest that these two events share a mechanism that, if disrupted, leads to malformations in both developmental pathways. Previous studies have shown that the underlying defect in these mutants may involve a mechanism that alters cellular organization and communication. Cell adhesion molecules (CAMs) have been linked with such interactions and because some, including N-CAM, are involved in neural development, we were interested in their pattern of expression in the splotch mutant. Immunolocalization studies showed similar temporospatial distributions of N-CAM antibody in embryonic day 9 mutants and controls. However, mutant embryos had a much higher intensity of anti-N-CAM fluorescence compared to controls. Further characterization using immunoblot analysis revealed that Sp mutants have an altered N-CAM polypeptide profile. Two N-CAM isoforms (Mr 140K and 180K, K=103) are normally present at this time of development. However, extracts from Sp embryos display a heavier N-CAM species (Mr 200K), as well as an altered 140K isoform. Heterozygotes also exhibit a different N-CAM profile, displaying a band between 180K and 200K in addition to the normal 180K and 140K species. Microheterogeneity was also observed in mutant and heterozygous embryos carrying Spd, an allele of Sp. However, these differences were less dramatic than that of Sp. The Sp locus may be involved in post-translational modification of N-CAM. An aberration in N-CAM processing could be the primary target of the mutation that leads to the developmental abnormalities observed in this mouse mutant.
Developmental analysis of the retinoic acid-inducible RAR-βJ2 promoter in transgenic animals
Authors: Mendelsohn C. , Ruberte E. , Lemeur M. , Morriss-Kay G. , Chambon P.
Published: April 2021

Retinoic acid (RA) is a signalling molecule important for pattern formation during development. There are three known types of nuclear receptors for RA in mammals, RAR-α, RAR-β and RAR-γ, which transduce the RA signal by inducing or repressing the transcription of target genes. Here we describe the developmental expression pattern of the mouse RAR-β2 promoter. Independent lines of transgenic animals expressing RAR-β2 promoter sequences fused to the E. coil β- galactosidase gene were examined throughout the course of embryogenesis and found to exhibit reproducible and specific patterns of β-galactosidase expression in a majority of sites that have been shown previously to contain mRAR-β transcripts. In the limbs, mRAR-β2 promoter activity and mRAR-β transcripts were both excluded from precartilagenous condensations; interestingly, mRAR-β2 promoter activity was observed in the apical ectodermal ridge (AER) where mRAR-β transcripts could not be detected, while no mRAR-β2 promoter activity or mRAR-β transcripts were associated with the limb region that contains the zone of polarizing activity (ZPA). Analysis of the lacZ expression pattern in embryos from mothers treated with teratogenic doses of RA, indicated that mRAR-β2 promoter is selectively induced in a manner suggesting that overexpression of the mRAR-β2 isoform is involved in RA-generated malformations. The normal and induced expression pattern of the mRAR-β2 promoter suggests several possible roles for mRAR-β2 in development of the limbs, as an inhibitor of cartilage formation, in programmed cell death and in the formation of loose connective tissue.
The genes encoding the major 42S storage particle proteins are expressed in male and female germ cells of Xenopus laevis
Authors: Abdallah Bassima , Hourdry Jacques , Deschamps Stephane , Denis Herman , Mazabraud Andre
Published: April 2021

As components of the 42S storage particles (thesauri-somes), thesaurin a and thesaurin b are involved in the long-term storage of tRNA and 5S RNA in previtello-genic oocytes of Xenopus laevis. Thesaurin a and thesaurin b are among the most abundant proteins in previtellogenic oocytes. We show here that the mRNAs encoding thesaurin a and thesaurin b are present not only in previtellogenic oocytes but also in pre-meiotic germ cells (oogonia). These mRNAs can also be detected in spermatogonia and early spermatocytes, and are translated into protein in testis, as they are in ovary. We conclude that male germ cells mimic female germ cells in several aspects of gene activity related to RNA accumu-lation and metabolism.
Regulated expression and growth inhibitory effects of transforming growth factor-β isoforms in mouse mammary gland development
Authors: Robinson Stephen D. , Silberstein Gary B. , Roberts Anita B. , Flanders Kathleen C. , Daniel Charles W.
Published: April 2021

Transforming Growth Factor-βl (TGF-β1) was pre-viously shown to inhibit reversibly the growth of mouse mammary ducts when administered in vivo by miniature slow-release plastic implants. We now report a compara-tive analysis of three TGF-β isoforms with respect to gene expression and localization of protein products within the mouse mammary gland. Our studies revealed overlapping expression patterns of TGF-β 1, TGF-β 2 and TGF-β 3 within the epithelium of the actively-growing mammary end buds during branching morpho-genesis, as well as within the epithelium of growth-quiescent ducts. However, TGF-J83 was the only isoform detected in myoepithelial progenitor cells (cap cells) of the growing end buds and myoepithelial cells of the mature ducts. During pregnancy, TGF-β2 and TGF-β 3 transcripts increased to high levels, in contrast to TGF-β1 transcripts which were moderately abundant; TGF-β2 was significantly transcribed only during pregnancy. Molecular hybridization in situ revealed overlapping patterns of expression for the three TGF-β isoforms during alveolar morphogenesis, but showed that, in contrast to the patterns of TGF-β1 and TGF-β2 expression, TGF-β3 is expressed more heavily in ducts than in alveoli during pregnancy. Developing alveolar tissue and its associated ducts displayed striking TGF-β3 immunoreactivity which was greatly reduced during lactation. All three isoforms showed dramatically reduced expression in lactating tissue.

The biological effects of active, exogenous TGF-β 2 and TGF-β3 were tested with slow-release plastic implants. These isoforms, like TGF-β1, inhibited mam-mary ductal elongation in situ by causing the disappear-ance of the proliferating stem cell layer (cap cells) and rapid involution of ductal end buds. None of the isoforms were active in inhibiting alveolar morphogenesis. We conclude that under the limited conditions of these tests, the three mammalian isoforms are functionally equival-ent. However, striking differences in patterns of gene expression and in the distribution of immunoreactive peptides suggest that TGF-β isoforms may have distinct roles in mammary growth regulation, morphogenesis and functional differentiation.
Differential expression of R- and N-cadherin in neural and mesodermal tissues during early chicken development
Authors: Inuzuka Hiroyuki , Redies Christoph , Takeichi Masatoshi
Published: April 2021

R-cadherin is a newly identified member of the cadherin family of cell adhesion receptors. The expression of R-cadherin in early chicken embryos was studied using affinity-purified antibodies to this molecule, comparing it with that of N-cadherin. Inununoblot analysis of various organs of 10.5-day embryos showed that R-cadherin is most abundantly expressed in the retina and brain. Immunostaining of the cervical and thoracic regions of embryos revealed that R- and N-cadherin are expressed in all neural tissues. In the neural tube, R-cadherin appears at around stage 21, although N-cadherin expression begins at a much earlier stage. The distribution of R-cadherin in the neural tube differs from that of N-cadherin; for example, some regions of the tube express only R-cadherin, and other regions only N-cadherin. In the peripheral ganglia, these two cadherins are also expressed in different patterns which change during development. Some mesenchymal tissues including the notochord, the myotome, myotubes and perichondria also express these cadherins, again in different patterns. Thus, R- and N-cadherin are differentially expressed in all the tissues examined, and they may contribute to the spatial segregation of heterogeneous cells in a tissue.
Associations between transforming growth factor β1 RNA expression and epithelial – mesenchymal interactions during tooth morphogenesis
Authors: Vaahtokari Anne , Vainio Seppo , Thesleff Irma
Published: April 2021

We have studied the expression of transforming growth factor beta-1 (TGF-β1) RNA during mouse tooth development, using in situ hybridization and experimental tissue recombinations. Analysis of the serial sections revealed the appearance of local expression of TGF-β1 RNA in the dental epithelium at bud-staged teeth (13day embryos). Just before transition to the cap stage, TGF-β1 RNA expression rapidly increased in the epithelial bud, and it also extended to the condensed dental mesenchyme. At cap stage (14- and 15-day embryos), there was an intense expression of TGF-β1 RNA in the morphologically active cervical loops of the dental epithelium.

During early bell stage (16- and 17-day embryos), TGF-β1 RNA expression was detected in the inner enamel epithelium where it subsequently almost disappeared (18-day embryos). After birth TGF-β1 transcripts transiently appeared in these cells when they were differentiating into ameloblasts (1-day mice). The transcripts were lost from the ameloblasts when they became secretory (4-day mice), but the expression continued in ameloblasts in enamel-free areas. Transient expression of TGF-β1 RNA was also detected in epithelial stratum intermedium cells at the time of ameloblast differentiation. In the mesenchyme, TGF-β1 RNA was not detected during bell stage until it appeared in differentiated odontoblasts (18-day embryos). The secretory odontoblasts continued to express TGF-β1 RNA at all stages studied including the odontoblasts of incisor roots.

Analysis of the distribution of bromodeoxyuridine (BrdU) incorporation indicated apparent correlations between TGF-β1 RNA expression and cell proliferation at the bud and cap stages but not at later stages of tooth development. Tissue recombination experiments of bud-staged (13-day embryos) dental and non-dental tissues showed that tooth epithelium, when cultured together with tooth mesenchyme, expressed TGF-β1 RNA. When the tooth epithelium was combined with non-dental jaw mesenchyme, TGF-β1 transcripts were not expressed. However, TGF-β1 RNA expression was seen in oral epithelium cultured with dental mesenchyme, while no expression of TGF-β1 transcripts was seen in the oral epithelium during normal development. Thus, TGF-β1 RNA expression seems to be regulated by epithelial– mesenchymal interactions.
Macromere cell fates during sea urchin development
Authors: Cameron R. Andrew , Fraser Scott E. , Britten Roy J. , Davidson Eric H.
Published: April 2021

This paper examines the cell lineage relationships and cell fates in embryos of the sea urchin Strongylocentrotus purpuratus leading to the various cell types derived from the definitive vegetal plate territory or the veg2 tier of cells. These cell types are gut, pigment cells, basal cells and coelomic pouches. They are cell types that constitute embryonic structures through cellular migration or rearrangement unlike the relatively non-motile ectoderm cell types. For this analysis, we use previous knowledge of lineage to assign macromeres to one of four types: VOM, the oral macromere; VAM, the aboral macro-mere, right and left VLM, the lateral macromeres. Each of the four macromeres contributes progeny to all of the cell types that descend from the definitive vegetal plate. Thus in the gut each macromere contributes to the esophagus, stomach and intestine, and the stripe of labeled cells descendant from a macromere reflects the re-arrangement of cells that occurs during archenteron elongation. Pigment cell contributions exhibit no consistent pattern among the four macromeres, and are haphazardly distributed throughout the ectoderm. Gut and pigment cell contributions are thus radially symmetrical. In contrast, the VOM blastomere contributes to both of the coelomic pouches while the other three macromeres contribute to only one or the other pouch. The total of the macromere contribution amounts to 60 % of the cells constituting the coelomic pouches.
Target sequences for hunchback in a control region conferring Ultrabithorax expression boundaries
Authors: Zhang Cheng-Cai , Müller Jürg , Hoch Michael , Jäckle Herbert , Bienz Mariann
Published: April 2021

Boundaries of Ultrabithorax expression are mediated by long-range repression acting through the PBX or ABX control region. We show here that either of these control regions confers an early band of β-galactosidase expression which is restricted along the anteroposterior axis of the blastoderm embryo. This band is succeeded by a stripe pattern with very similar anteroposterior limits. Dissection of the PBX control region demonstrates that the two patterns are conferred by distinct cis-regulatory sequences contained within separate PBX subfragments. We find several binding sites for hunchback protein within both PBX subfragments. Zygotic hunchback function is required to prevent ectopic PBX expression. Moreover, the PBX pattern is completely suppressed in embryos containing uniformly distributed maternal hunchback protein. Our results strongly suggest that hunchback protein directly binds to the PBX control region and acts as a repressor to specify the boundary positions of the PBX pattern.
Selective expression of an acetylcholine receptor–lacZ transgene in synaptic nuclei of adult muscle fibers
Authors: Sanes Joshua R. , Johnson Yvette R. , Kotzbauer Paul T. , Mudd Jacqueline , Hanley Theresa , Marttnou Jean-Claude , Merlie John P.
Published: April 2021

Acetylcholine receptors (AChRs) are highly concentrated in the postsynaptic membrane at the neuromuscular junction. To investigate mechanisms that lead to the formation or maintenance of this synaptic specialization, we generated transgenic mice in which regulatory elements from the AChR α- or ϵ-subunit genes are linked to a gene for a reporter protein that is targeted to the nucleus (nlacZ). Both transgenes were selectively expressed and developmentally regulated in muscle; nuclei in both extrafusal (ordinary) and intrafusal (spindle) muscle fibers were labeled. Within individual muscle fibers from є-nlacZ mice, nuclei near synaptic sites were nlacZ-positive, whereas extrasynaptic nuclei were nlacZ-negative. In contrast, nlacZ was expressed in both synaptic and extrasynaptic nuclei when under the control of regulatory elements from the AChR α-subunit gene; however, synaptic nuclei were somewhat more intensely stained than extrasynaptic nuclei in a minority of muscle fibers from these mice. Together, our results provide direct evidence for molecular differences between synaptic and extrasynaptic nuclei within a single cytoplasm, and suggest that the motor nerve regulates synapse formation by selectively affecting transcription in synaptic nuclei.
Expression of the zebrafish paired box gene pax[zf-b] during early neurogenesis
Authors: Krauss Stefan , Johansen Terje , Korzh Vladimir , Fjose Anders
Published: April 2021

The paired box-containing (pax) gene family encodes a group of putative transcription factors differentially expressed during embryonic development. In this study, we describe the cloning and expression of a zebrafish gene pax[zf-b], which most probably is a direct homologue to the mouse Pax2 gene. The putative protein encoded by pax[zf-b] contains a paired box, an octapeptide, but no homeobox. However, a region of homology to the N-terminal half of paired-type homeoboxes is detected C-terminal to the pax[zf-b] paired domain. In zebrafish embryos, pax[zf-b] transcripts are first seen during the formation of the neural keel. At 9–10 h of development, two laterally located transverse stripes of cells expressing the gene appear in the rostral 1/3 of the embryo. The two areas sub

sequently move towards the midline and form the posterior portion of the midbrain. In the following stages of development, at 10–12 h, transcripts are detected in the otic placode, the Wolffian duct including the nephritic primordium and in the optic stalk. At a later time point, beginning at 14 –15 h, single cells along the spinal cord, presumably interneurons, start to express the gene. The characteristic expression pattern of pax[zf-b] in the neural tube suggests an involvement of this gene in the regionalization of the midbrain as well as in the specification of neuronal cell fates at early embryonic stages.
Segmental origin and migration of neural crest cells in the hindbrain region of the chick embryo
Authors: Lumsden Andrew , Sprawson Nicola , Graham Anthony
Published: April 2021

A vital dye analysis of cranial neural crest migration in the chick embryo has provided a positional fate map of greater resolution than has been possible using labelled graft techniques. Focal injections of the fluorescent membrane probe DiI were made into the cranial neural folds at stages between 3 and 16 somites. Groups of neuroepithelial cells, including the premigratory neural crest, were labelled by the vital dye. Analysis of whole-mount embryos after 1–2 days further development, using conventional and intensified video fluorescence microscopy, revealed the pathways of crest cells migrating from mesencephalic and rhombencephalic levels of the neuraxis into the subjacent branchial region. The patterns of crest emergence and emigration correlate with the segmented disposition of the rhomben-cephalon. Branchial arches 1, 2 and 3 are filled by crest cells migrating from rhombomeres 2, 4 and 6 respectively, in register with the cranial nerve entry/exit points in these segments. The three streams of ventrally migrating cells are separated by alternating regions, rhombomeres 3 and 5, which release no crest cells. Rostrally, rhombomere 1 and the caudal mesencephalon also contribute crest to the first arch, primarily to its upper (maxillary) component. Both r3 and r5 are associated with enhanced levels of cell death amongst cells of the dorsal midline, suggesting that crest may form at these levels but is then eliminated. Organisation of the branchial region is thus related by the dynamic process of neural crest immigration to the intrinsic mechanisms that segment the neuraxis.
In vitro development of murine T cells from prethymic and preliver embryonic yolk sac hematopoietic stem cells
Authors: Liu Chih-Pin , Auerbach Robert
Published: April 2021

Mature T cells are derived from prethymic stem cells, which arise at one or more extrathymic sites and enter and differentiate in the thymus. The nature of these prethymic stem cells is a critical factor for the formation of the T-cell repertoire. Although the bone marrow of adult mice can provide such stem cells, their origin during murine embryogenesis is still undetermined. Among potential sites for these progenitor cells are the fetal liver and the embryonic yolk sac. Our studies focus on the yolk sac, both because the yolk sac appears earlier than any other proposed site, and because the mammalian yolk sac is the first site of hematopoiesis. Although it has been shown that the yolk sac in midgestation contains stem cells that can enter the thymic rudiment and differentiate toward T-cell lineage, our aim was to analyze the developmental potential of cells in the yolk sac from earlier stages, prior to the formation of the liver and any other internal organ. We show here that the yolk sac from 8- and 9-day embryos (2–9 and 13–19 somites, respectively) can reconstitute alymphoid congenie fetal thymuses and acquire mature T-cell-specific characteristics. Specifically, thymocytes derived from the early embryonic yolk sac can progress to the expression of mature T lymphocyte markers including CD3/T-cell receptor (TCR), CD4 and CD8. In contrast, we have been unable to document the presence of stem cells within the embryo itself at these early stages. These results support the hypothesis that the stem cells capable of populating the thymic rudiment originate in the yolk sac, and that their presence as early as at the 2-to 9-somite stage may indicate that prethymic stem cells found elsewhere in the embryo at later times may have been derived by migration from this extra-embryonic site. Our experimental design does not exclude the possibility of multiple origins of prethymic stem cells of which the yolk sac may provide the first wave of stem cells in addition to other later waves of cells.
The branchial Hox code and its implications for gene regulation, patterning of the nervous system and head evolution
Authors: Hunt Paul , Whiting Jenny , Nonchev Stefan , Sham Mai-Har , Marshall Heather , Graham Anthony , Cook Martyn , Allemann Rudolf , Rigby Peter W. J. , Gulisano Massimo , Faiella Antonio , Boncinelli Edoardo , Krumlauf Robb
Published: April 2021

In this study we have examined the expression of murine Hox homeobox containing genes by in situ hybridisation in the branchial region of the head. Genes from the Hox complexes display segmentally restricted domains of expression in the developing hindbrain, which are correlated with similar restricted domains in the neural crest and surface ectoderm of the branchial arches. Comparison of related genes from the different clusters shows that subfamily members are expressed in identical rhombomeres and branchial arches. These patterns suggest a combinatorial system for specifying regional variation in the head, which we refer to as a Hox code. The Hox genes also display dynamic dorso-ventral (D-V) restrictions in the developing neural tube which mirror the timing and spatial distributions of the birth of major classes of neurons in the CNS. Genes in the Hox-2 cluster all have a similar D-V distribution that differs from that of genes from the other Hox clusters, and suggests that members of a subfamily may be used to specify positional values to different subsets of cells at the same axial level. These results are discussed in terms of a system for patterning the branchial regions of the vertebrate head, and evolution of head structures.

We have also examined aspects of the transcriptional regulation of Hox-2 genes in transgenic mice using a lacZ reporter gene. We have been able to reconstruct the major pattern of the Hox-2.6 gene on the basis of identical expression of the transgene and the endogenous gene with respect to timing, spatial restrictions and tissue-specific distributions. Deletion analysis has enabled us to identify three regions involved in generating this pattern. Two of these regions have the properties of enhancers which are capable of imposing spatially-restricted domains of expression on heterologous promoters. We have generated similar Hox-lacZ fusions that reconstruct the highly restricted patterns of the Hox-2.1 and Hox-2.8 genes in the developing nervous system, supporting our in situ analysis and the idea of a Hox code. These transgenic experiments are a useful step in examining regulation in the Hox cascade.
Organogenesis in Drosophila melanogaster. embryonic salivary gland determination is controlled by homeotic and dorsoventral patterning genes
Authors: Panzer Scott , Weigel Detlef , Beckendorf Steven K.
Published: April 2021

We have investigated Drosophila salivary gland determination by examining the effects of mutations in pattern forming genes on the salivary gland primordium. We find that the anterior-posterior extent of the primordium, a placode of columnar epithelial cells derived from parasegment 2, is established by the positive action of the homeotic gene Sex combs reduced (Scr). Embryos mutant for Scr lack a detectable placode, while ectopic Scr expression leads to the formation of ectopic salivary glands. In contrast, the dorsal-ventral extent of the placode is regulated negatively. Functions dependent on the decapentaplegic product place a dorsal limit on the placode, while dorsal-dependent genes act to limit the placode ventrally. We propose a model in which these pattern forming genes act early to determine the salivary gland anlage by regulating the expression of salivary gland determining genes, which in turn control genes that are involved in salivary gland morphogenesis.
The giant gene of Drosophila encodes a b-ZIP DNA-binding protein that regulates the expression of other segmentation gap genes
Authors: Capovilla Maria , Eldon Elizabeth D. , Pirrotta Vincenzo
Published: April 2021

The sequence of a cDNA from the giant gene of Drosophila shows that its product has a basic domain followed by a leucine zipper motif. Both features contain characteristic conserved elements of the b-ZIP family of DNA-binding proteins. Expression of the gene in bacteria or by in vitro translation yields a protein that migrates considerably faster than the protein extracted from Drosophila embryos. Treatment with phosphatase shows that this difference is due to multiple phosphorylation of the giant protein in the embryo. Ectopic expression of the protein in precellular blastoderm embryos produces abnormal phenotypes with a pattern of segment loss closely resembling that of Krüppel mutant embryos. Immunological staining shows that giant, ectopically expressed from the hsp70 promoter, represses the expression of both the Krüppel and knirps segmentation gap genes. The analysis of the interactions between Krüppel, knirps and giant reveals a network of negative regulation. We show that the apparent positive regulation of knirps by Krüppel is in fact mediated by a negative effect of Krüppel on giant and a negative effect of giant on knirps. giant protein made in bacteria or in embryos binds in vitro to the Krüppel regulatory elements CD1 and CD2 and recognizes a sequence resembling the binding sites of other b-ZIP proteins.
Mechanism of anteroposterior axis specification in vertebrates Lessons from the amphibians
Authors: Slack J. M. W. , Tannahill D.
Published: April 2021

Interest in the problem of anteroposterior specification has quickened because of our near understanding of the mechanism in Drosophila and because of the homology of Antennapedia-like homeobox gene expression patterns in Drosophila and vertebrates. But vertebrates differ from Drosophila because of morphogenetic movements and interactions between tissue layers, both intimately associated with anteroposterior specification. The purpose of this article is to review classical findings and to enquire how far these have been confirmed, refuted or extended” by modern work.

The “pre-molecular” work suggests that there are several steps to the process: Formation of anteroposterior pattern in mesoderm during gastrulation with posterior dominance.Regional specific induction of ectoderm to form neural plate.Reciprocal interactions from neural plate to mesoderm.Interactions within neural plate with posterior dominance.

Unfortunately, almost all the observable markers are in the CNS rather than in the mesoderm where the initial specification is thought to occur. This has meant that the specification of the mesoderm has been assayed indirectly by transplantation methods such as the Einsteckung.

New molecular markers now supplement morphological ones but they are still mainly in the CNS and not the mesoderm. A particular interest attaches to the genes of the Antp-like HOX clusters since these may not only be markers but actual coding factors for anteroposterior levels.

We have a new understanding of mesoderm induction based on the discovery of activins and fibroblast growth factors (FGFs) as candidate inducing factors. These factors have later consequences for anteroposterior pattern with activin tending to induce anterior, and FGF posterior structures.

Recent work on neural induction has implicated cAMP and protein kinase C (PKC) as elements of the signal transduction pathway and has provided new evidence for the importance of tangential neural induction. The regional specificity of neural induction has been reinvestigated using molecular markers and provides conclusions rather similar to the classical work.

Defects in the axial pattern may be produced by retinoic acid but it remains unclear whether its effects are truly coordinate ones or are concentrated in certain regions of high sensitivity.

In general the molecular studies have supported and reinforced the “pre-molecular ones”. Important questions still remain: How much pattern is there in the mesoderm (how many states?)How is this pattern generated by the invaginating organizer?Is there one-to-one transmission of codings to the neural plate?What is the nature of the interactions within the neural plate?Are the HOX cluster genes really the anteroposterior codings?
Embryonic stem cell-derived cystic embryoid bodies form vascular channels: an in vitro model of blood vessel development
Authors: Wang Rong , Clark Rebecca , Bautch Victoria L.
Published: April 2021

Murine embryonic stem cells can differentiate in vitro to form cystic embryoid bodies (CEB) that contain different structures and cell types. The blood islands are one such structure that consist of immature hematopoietic cells surrounded by endothelial cells, the first identifiable vascular cells. CEBs differentiated in vitro developed blood islands initially, and subsequently these blood islands matured to form vascular channels containing hematopoietic cells. Phase contrast microscopy demonstrated the presence of channels in mature CEBs grown in suspension culture, and high resolution light and electron microscopy showed that the cells lining these channels were endothelial cells. The channels appeared less organized than the vasculature of the mature yolk sac. The hematopoietic cells were occasionally seen ‘flowing’ through the CEB channels, although their numbers were reduced relative to the yolk sac. Analysis of primary CEB cultures showed the presence of cells with two characteristics of endothelial cells: approximately 30% of the cells labelled with fluorescent acetylated low density lipoprotein and a small number of cells were positive for von Willebrand’s factor by immunostaining. Thus we conclude that a primitive vasculature forms in CEBs differentiated in vitro, and that not only primary differentiation of endothelial cells but also some aspects of vascular maturation are intrinsic to this cell culture system. CEBs are therefore a useful model for the study of developmental blood vessel formation.
Avian neural crest cells express a melanogenic trait during early migration from the neural tube: observations with the new monoclonal antibody, “MEBL-1”
Authors: Kitamura Kunio , Takiguchi-Hayashi Keiko , Sezaki Mariko , Yamamoto Hiroaki , Takeuchi Takuji
Published: April 2021

A new monoclonal antibody, MEBL-1, which specifically recognizes melanocytes and their precursors, was used to examine the differentiation of neural crest cells into melanocytes in situ. MEBL-l-positive precursors of melanocytes in the trunk were first detected in mesenchyme located above the neural tube at the level of a wing bud at stage 19, when active migration of neural crest cells along the ventral route was almost complete. DiO-labelled neural crest cells, which begin to emigrate from the neural tube at stages 17–18 and then migrate along the dorsolateral route, were found to become MEBL-l-positive. This indicates that MEBL-1 antigens are expressed by neural crest cells immediately following their emigration and neural crest cells become melanogenic. Expression of the MEBL-1 antigens was followed by decrease in, and finally the loss of, HNK-1 antigen expression.

At the cephalic level, MEBL-l-positive precursors of melanocytes were first detected in mesenchyme located above the dorsal side of the mesencephalic neural tube at stage 16. At the vagal level, the precursors were noted first in mesenchyme located above the dorsal side of the rhombencephallc neural tube at stage 18.

MEBL-1 reacted with the cell membrane and preme-lanosome-like granules in addition to the nuclear membrane, endoplasmic reticulum and Golgi apparatus of the migratory precursors. The antigens recognized by MEBL-1 were proteins with relative molecular masses of 135 × 103 and 115 × 103. We suggest that MEBL-1 may be the earliest marker for melanogenic neural crest cells.