bluuebunny/crossref_metadata_2025 huggingface dataset:
Origins of the insect enteric nervous system: differentiation of the enteric ganglia from a neurogenic epithelium
Authors: Copenhaver Philip F. , Taghert Paul H.
Published: April 2021

The enteric nervous system (ENS) of the moth Manduca sexta is organized into two distinct cellular domains: an anterior domain that includes several small ganglia on the surface of the foregut, and a more posterior domain consisting of a branching nerve plexus (the enteric plexus) that spans the foregut-midgut boundary. Previously, we showed that the neurons of the posterior domain, the enteric plexus, are generated from a large placode that invaginates from the caudal lip of the foregut; subsequently, the cells become distributed throughout the enteric plexus by a sequence of active migration. We now demonstrate that the neurons of the anterior domain, the cells of the enteric ganglia, arise via a distinct developmental sequence. Shortly after the foregut has begun to form, three neurogenic zones differentiate within the foregut epithelium and give rise to chains of cells that emerge onto the foregut surface. The three zones are not sites of active mitosis, as indicated by the absence of labelling with a thymidine analogue and by clonal analyses using intracellularly injected dyes. Rather, the zones serve as loci through which epithelial cells are recruited into a sequence of delamination and neuronal differentiation. As they emerge from the epithelium, the cells briefly become mitotically active, each cell dividing once or twice. In this manner, they resemble the midline precursor class of neural progenitors in the insect central nervous system more than neuroblast stem cells. The progeny of these zone-derived precursors then gradually coalesce into the ganglia and nerves of the anterior ENS. Although this reorganization results in some variability in the precise configuration of neurons within the ganglia, the overall morphology of the ganglia is highly stereotyped, consisting of cortical layers of cells that surround a ventral neuropil. In addition, a number of the neurons within the frontal and hypocerebral ganglia express identifiable phenotypes in a manner that is similar to many cells of the insect central nervous system. These observations indicate that the differentiation of the enteric ganglia in Manduca involves an unusual combination of features seen during the formation of other regions of the nervous system and, as such, constitutes a distinct program of neurogenesis.
Expression and function of the Drosophila gene runt in early stages of neural development
Authors: Duffy J. B. , Kania M. A. , Gergen J. P.
Published: April 2021

The Drosophila gene runt was initially identified on the basis of its role during segmentation. Recent molecular and genetic studies have demonstrated that the runt gene encodes a novel nuclear protein whose developmental importance is not exclusive to segmentation. This report addresses the functional relevance of runt expression in the developmental pathway of neurogenesis. Antibodies against the runt protein reveal that it is expressed in a subset of neuroblasts, ganglion-mother cells and neurons. A subset of these neurons also co-express the segmentation gene even-skipped (eve). Using eve as a marker, we show that runt is required for the normal development of these neurons. A runt P-transposon that lacks neural eis-regulatory elements is used to show that these neurons require runt activity independent of its activity during segmentation. These results are confirmed using a temperature-sensitive runt allele. Further temperature-shift experiments indicate that the requirement for runt is during an early stage of neurogenesis. Based on its pattern of expression and its temporal requirements, runt is distinguished as one of the earliest acting genes involved in the generation of diverse cell fates in the developing Drosophila nervous system.
hairy gene function in the Drosophila eye: normal expression is dispensable but ectopic expression alters cell fates
Authors: Brown Nadean L. , Sattler Carol A. , Markey Donald R. , Carroll Sean B.
Published: April 2021

The regulatory gene hairy is expressed and required during early embryogenesis to control segmentation gene expression properly and during larval and pupal development to control the pattern of certain adult sensory structures. We have found the hairy protein to be expressed transiently during two stages of eye imaginai disc development, including all cells immediately anterior to the morphogenetic furrow that traverses the developing eye disc, and again in the presumptive R7 photoreceptor cells of the developing ommatidia. This pattern is conserved in a significantly diverged Drosophila species. We show that, surprisingly, ommatidia formed by homozygous hairy- mutant clones are apparently normal, indicating that hairy function in the eye is dispensable. However, we do find that ectopic expression of hairy causes numerous structural abnormalities and the alteration of cell fates. Thus, proper regulation of hairy is still essential for normal eye development. We suggest that the loss of hairy function may be compensated by other regulatory proteins, as has been observed previously for several structurally and functionally related genes involved in sensory organ development. The effects of ectopic hairy expression may result from interactions with proneural genes involved in the development of the eye and other sensory organs.
Pax-6, a murine paired box gene, is expressed in the developing CNS
Authors: Walther Claudia , Gruss Peter
Published: April 2021

A multigene family of paired-box-containing genes (Pax genes) has been identified in the mouse. In this report, we describe the expression pattern of Pax-6 during embryogenesis and the isolation of cDNA clones spanning the entire coding region. The Pax-6 protein consists of 422 amino acids as deduced from the longest open reading frame and contains, in addition to the paired domain, a paired-type homeodomain. Beginning with day 8 of gestation, Pax-6 is expressed in discrete regions of the forebrain and the hindbrain. In the neural tube, expression is mainly confined to mitotic active cells in the ventral ventricular zone along the entire anteroposterior axis starting at day 8.5 of development. Pax-6 is also expressed in the developing eye, the pituitary and the nasal epithelium.
TGFβ1 inhibits proliferation and has a chemotropic effect on mouse primordial germ cells in culture
Authors: Godin Isabelle , Wylie C. C.
Published: April 2021

Primordial germ cells are the stem cells that provide the functional gametes of adult animals. In many animal groups they are set aside at the earliest stages of development, and migrate from their sites of first appearance to the sites where the gonad will form, the genital ridges. During this migration they proliferate. In the mouse embryo their numbers increase from less than one hundred to approximately four thousand during the period of their migration. In a previous paper we showed that both the proliferation and the direction of migration of mouse PGCs in culture were influenced by soluble factors released from their target tissue, the genital ridges. Studies on other stem cell populations have shown that complex combinations of growth factors control their proliferation, migration and differentiation. In this paper, we show that TGFβ1 inhibits proliferation of PGCs taken from 8.5 day old embryos and cultured on embryonic fibroblast feeder layers. We also show that the previously reported chemotropic effect of genital ridges in this culture system is mediated by TGFβ1, or a closely related molecule, released from the genital ridges.
Homeobox genes and models for patterning the hindbrain and branchial arches
Authors: Hunt Paul , Whiting Jenny , Muchamore Ian , Marshall Heather , Krumlauf Robb
Published: April 2021

Antennapedia class homeobox genes, which in insects are involved in regional specification of the segmented central regions of the body, have been implicated in a similar role in the vertebrate hindbrain. The development of the hindbrain involves the establishment of compartments which are subsequently made distinct from each other by Hox gene expression, implying that the lineage of neural cells may be an important factor in their development. The hindbrain produces the neural crest that gives rise to the cartilages of the branchial skeleton. Lineage also seems to be important in the neural crest, as experiments have shown that the crest will form cartilages appropriate to its level of origin when grafted to a heterotopic location. We show how the Hox genes could also be involved in patterning the mesenchymal structures of the branchial skeleton.

Recently it has been proposed that the rhombomere-restricted expression pattern of Hox 2 genes is the result of a tight spatially localised induction from underlying head mesoderm, in which a prepattern of Hox expression is visible. We find no evidence for this model, our data being consistent with the idea that the spatially localised expression pattern is a result of segmentation processes whose final stages are intrinsic to the neural plate.

We suggest the following model for patterning in the branchial region. At first a segment-restricted code of Hox gene expression becomes established in the neuroepithelium and adjacent presumptive neural crest. This expression is then maintained in the neural crest during migration, resulting in a Hox code in the cranial ganglia and branchial mesenchyme that reflects the crest’s rhombomere of origin. The final stage is the establishment of Hox 2 expression in the surface ectoderm which is brought into contact with neural crest-derived branchial mesenchyme. The Hox code of the branchial ectoderm is established later in development than that of the neural plate and crest, and involves the same combination of genes as the underlying crest. Experimental observations suggest the idea of an instructive interaction between branchial crest and its overlying ectoderm, which would be consistent with our observations.

The distribution of clusters of Antennapedia class genes within the animal kingdom suggests that the primitive chordates ancestral to vertebrates had at least one Hox cluster. The origin of the vertebrates is thought to have been intimately linked to the appearance of the neural crest, initially in the branchial region. Our data are consistent with the idea that the branchial region of the head arose in evolution before the more anterior parts, the development of the branchial region employing the Hox genes in a more determinate patterning system. In this scenario, the anterior parts of the head arose subsequently, which may explain the greater importance of interactions in their development, and the fact that Antennapedia class Hox genes are not expressed there.
Scarless wound repair: a human fetal skin model
Authors: Lorenz H. Peter , Longaker Michael T. , Perkocha Luke A. , Jennings Russell W. , Harrison Michael R. , Adzick N. Scott
Published: April 2021

Animal studies demonstrate that the fetus heals cutaneous wounds by reformation of normal tissue architecture without scar formation. We have developed a new model to study human fetal skin wound healing. Grafts of human fetal skin placed onto athymie mice retain the morphologic features of normal development, although they differentiate at an accelerated rate when placed cutaneously compared to subcutaneously. Fullthickness skin grafts from human fetuses at 15 (n=12), 17 (n=ll), 18 (n=25), 19 (n=20) and 22 (n=13) weeks gestational age were placed onto athymie (nu/nu) mice in 2 locations: (1) cutaneously onto a fascial bed and thereby exposed to air or (2) subcutaneously in a pocket under the murine panniculus carnosus. Linear incisions were made in each graft 7 days after transplantation. Grafts were harvested at 7, 14 and 21 days postwounding and analyzed histologically for scar formation.

By hematoxylin & eosin and Mallory’s trichrome stains, complete epidermal and dermal graft wound healing without scar formation was demonstrated in the subcutaneous grafts at each gestational age studied. In contrast, scar was seen at all time points in the cutaneous grafts in both the incisional wound and at the interface of the fetal human skin graft and adult mouse skin, regardless of fetal skin gestational age.

Our results demonstrate that: (1) human fetal skin can heal without a scar in a subcutaneous adult environment; (2) scarless repair does not depend upon either perfusion by fetal serum or the unique amniotic fluid intrauterine environment; (3) in contrast to subcutaneous grafts, cutaneous grafts of 15, 17, 18, 19 and 22 week gestation fetal skin heal with scar.
Transfer and tissue-specific accumulation of cytoplasmic components in embryos of Caenorhabditis elegans and Rhabditis dolichura: in vivo analysis with a low-cost signal enhancement device
Authors: Bossinger Olaf , Schierenberg Einhard
Published: April 2021

The pattern of autofluorescence in the two free-living soil nematodes Rhabditis dolichura and Caenorhabditis elegans has been compared. In C. elegans, during later embryogenesis the prospective gut cells develop a typical bluish autofluorescence as seen under UV illumination, while in Rh. dolichura a strong autofluorescence is already present in the unfertilized egg. Using a new, lowcost signal enhancement device, we have been able to follow in vivo the dramatic change in the pattern of autofluorescence during embryogenesis of Rh. dolichura. Autofluorescent material accumulates progressively in the gut primordium and disappears completely from all other cells. To investigate whether such an accumulation of cytoplasmic components also takes place in the C. elegans embryo, we labeled the cytoplasm of the egg with the fluorescing tracer dyes Lucifer Yellow (LY) or Rhodamine 6G (R6G). While LY appears to bind to yolk and progressively accumulates in the developing gut primordium, R6G does not show any such binding and remains equally distributed over all cells. Measurements in early and late stages indicate a significant increase in the volume of the gut cells during embryogenesis, while the embryo as a whole does not grow. Moreover, in cleavage-blocked 2-cell stages after development overnight, a reversal of cell size relationship to the benefit of the gut precursor cell takes place. In summary, our observations suggest a previously unknown massive transfer of yolk components in the nematode embryo from non-gut cells into lysosomes of the gut primordium, where they are further metabolized during postembryonic development.
Communication compartments in hair follicles and their implication in differentiative control
Authors: Kam Ephraim , Hodgins Malcolm B.
Published: April 2021

Observations on hair follicles presented in this paper show that boundaries to junctional communication are formed between groups of cells following different pathways of differentiation. The patterns of junctional communication in the bulbs of rat vibrissa follicles and human hair follicles were studied by microinjection of the fluorescent tracer dye Lucifer Yellow CH. Dye spread was extensive between undifferentiated cells of the hair bulb matrix but communication boundaries were found between groups of morphologically distinct cells. For example, boundaries to dye spread were observed between undifferentiated matrix cells and cells in the early stage of differentiation into the inner root sheath, between Huxley’s and Henle’s layers in the early inner root sheath and between cells of the cuticle and cortex of the hair. Dye did not spread between epithelial cells of the hair bulb and mesenchymal cells of the connective tissue sheath or dermal papilla. The patterns of dye spread became more complex (increased boundary formation and subcompartmentation) as differentiation progressed in higher regions of the hair bulb. The observed communication can be related to previous ultrastructural studies by others on the distribution of gap junctions in the wool follicle. These results show that junctional communication, with its consequent intercellular spread of small ions and molecules, is associated with uniformity of expression and behaviour within cell populations and that interruption of communication through the formation of boundaries and communication compartments is temporally and spatially related to the production of subpopulations of cells committed to the expression of different phenotypes.
Neural induction and regionalisation in the chick embryo
Authors: Storey Kate G. , Crossley James M. , Robertis Eddy M. De , Norris Wendie E. , Stern Claudio D.
Published: April 2021

Induction and regionalisation of the chick nervous system were investigated by transplanting Hensen’s node into the extra-embryonic region (area opaca margin) of a host embryo. Chick/quail chimaeras were used to determine the contributions of host and donor tissue to the supernumerary axis, and three molecular markers, Engrailed, neurofilaments (antibody 3A10) and XlHboxl/Hox3.3 were used to aid the identification of particular regions of the ectopic axis.

We find that the age of the node determines the regions of the nervous system that form: young nodes (stages 2-4) induce both anterior and posterior nervous system, while older nodes (stages 5-6) have reduced inducing ability and generate only posterior nervous system. By varying the age of the host embryo, we show that the competence of the epiblast to respond to neural induction declines after stage 4.

We conclude that during normal development, the initial steps of neural induction take place before stage 4 and that anteroposterior regionalisation of the nervous system may be a later process, perhaps associated with the differentiating notochord. We also speculate that the mechanisms responsible for induction of head CNS differ from those that generate the spinal cord: the trunk CNS could arise by homeogenetic induction by anterior CNS or by elongation of neural primordia that are induced very early.
Pathfinding by cranial nerve VII (facial) motorneurons in the chick hindbrain
Authors: Chang Susannah , Fan Jinhong , Nayak Jayakar
Published: April 2021

Cranial nerve VII (facial) motorneurons begin extending axons through rhombomeres 4 and 5 (R4 and RS) in the chick hindbrain on the second day of incubation. Without crossing the midline, facial motorneuron axons extend laterally from a ventromedial cell body location. All facial motorneuron axons leave the hindbrain through a discrete exit site in R4. To examine the importance of the exit site in R4 on motorneuron pathfinding, we ablated R4 before motorneuron axonogenesis. We find that mechanisms intrinsic to RS direct the initial lateral orientation of RS motorneuron axons. Upon reaching a particular lateral position, all R5 motorneuron axons must turn. In normal embryos the axons all turn rostrally to reach the nerve exit in R4. In embryos with R4 ablated, sometimes the axons turn rostrally and sometimes they turn caudally. A model combining permissive fields and chemotropic cues is presented to account for our observations.
Cellular and extracellular involvement in the regeneration of the rat lower vibrissa follicle
Authors: Jahoda Colin A. B. , Horne Kenneth A. , Mauger Annick , Bard Sylviane , Sengel Philippe
Published: April 2021

The sequence of events leading to the reconstruction of a fibre-producing hair follicle, after microsurgical amputation of the lower follicle bulb, has been detailed by immunohistology and electron microscopy. The initial response was essentially found to be a wound reaction, in that hyperproliferative follicle epidermis quickly spread to below the level of amputation - associated with downward movement of mesenchymal (or dermal) sheath cells. Fibronectin was prominent in both dermis and epidermis at this stage and, as in wound repair, preceded laminin and type IV collagen in covering the lower dermal-epidermal junction. Once a new basal line of epidermis and a complete basement membrane were established, laminin and type IV collagen were detected below this junction and within the prospective papillaforming mesenchyme. This coincided with ultrastructural observations of profuse sub-basement membrane extracellular material in the region of new papilla formation. The glassy membrane displayed extensive ultrastructural modifications at its lower level, and these corresponded with localized variations in staining intensities for all three antibodies over time. The membrane hung below the level of the epidermis, and was crossed by migrating cells from the mesenchymal dermal sheath of the follicle - it acted to segregate the inner group of follicular dermal cells from wound fibroblasts. Extracellular matrix may be a mediator of the dermal-epidermal interactions associated with this hair follicle regeneration phenomenon.
Homeotic genes have specific functional roles in the establishment of the Drosophila embryonic peripheral nervous system
Authors: Heuer J.G. , Kaufman T.C.
Published: April 2021

The Drosophila embryonic peripheral nervous system (PNS) contains segment-specific spatial patterns of sensory organs which derive from the ectoderm. Many studies have established that the homeotic genes of Drosophila control segment specific characteristics of the epidermis, and more recently these genes have also been shown to control gut morphogenesis through their expression in the visceral mesoderm (Tremml, G. and Bienz, M. (1989), EMBO J. 8, 2677–2685). We report here the roles of homeotic genes in establishing the spatial patterns of sensory organs in the embryonic PNS. The PNS was examined in embryos homozygous for mutations in the homeotic genes Sex combs reduced (Scr), Antennapedia (Antp), Ultrabithorax (Ubx), abdominal-A (abd-A) and Abdominal-B (Abd-B) with antibodies that label specific subsets of sensory organs. Our results suggest that the homeotic genes have specific roles in establishing the correct spatial patterns of sensory organs in their normal domains of expression. In addition, we also report the effects of ectopic expression of the homeotic genes labial (lab), Deformed (Dfd), Scr, Antp or Ubx on the normal development of sensory organs in the embryonic PNS. Interestingly, while previous studies have concluded that ectopic expression of the homeotic genes Dfd, Scr and Antp has no effect on the segmental identity of the abdominal segments, our results demonstrate that this is not true. We show that ectopic expression of these genes does result in the disruption of the developing PNS in the abdomen. Our results are suggestive of a role for the homeotic gene products in regulating genes which are necessary for generating sensory progenitor cells in the developing PNS.
Interactions between retinoids and TGF βs in mouse morphogenesis
Authors: Mahmood Radma , Flanders Kathleen C. , Morriss-Kay Gillian M.
Published: April 2021

Using immunocytochemical methods we describe the distribution of different TGF β isoforms and the effects of excess retinoic acid on their expression during early mouse embryogenesis ( days of development). In normal embryos at 9 days, intracellular TGF β1 is expressed most intensely in neuroepithelium and cardiac myocardium whereas extracellular TGF β1 is expressed in mesenchymal cells and in the endocardium of the heart. At later stages, intracellular TGF β1 becomes very restricted to the myocardium and to a limited number of head mesenchymal cells; extracellular TGF β1 continues to be expressed widely in cells of mesenchymal origin, particularly in head and trunk mesenchyme, and also in endocardium. TGF β1 is widely expressed at all stages investigated while TGF β3 is not expressed strongly in any tissue at the stages examined.

Exposure of early neural plate stage embryos to retinoic acid caused reduced expression of TGF β1 and TGF β2 proteins but had no effect on TGF β1. Intracellular TGF β1 expression was reduced in all tissues except in the myocardium, while extracellular TGF β1 was specifically reduced in neuroepithelium and cranial neural crest cells at early stages. TGF β1 was reduced in all embryonic tissues. The down-regulation of intracellular TGF β1 was observed up to 48 hours after initial exposure to retinoic acid while some downregulation of TGF β1 was still seen up to 60 hours after initial exposure.

TGF βs are known to modulate the expression of various extracellular matrix molecules involved in cell growth, differentiation and morphogenesis. The interaction between retinoic acid and TGF β is discussed in relation to morphogenesis.
Characterization of HNK-1 antigens during the formation of the avian enteric nervous system
Authors: Luider Theo M. , Peters-Van Der Sanden Marjo J. H. , Molenaar Jan C. , Tibboel Dick , Kamp Arthur W. M. Van Der , Melters Carel
Published: April 2021

During vertebrate embryogenesis, interaction between neural crest cells and the enteric mesenchyme gives rise to the development of the enteric nervous system. In birds, monoclonal antibody HNK-1 is a marker for neural crest cells from the entire rostrocaudal axis. In this study, we aimed to characterize the HNK-1 carrying cells and antigen(s) during the formation of the enteric nervous system in the hindgut. Immunohistological findings showed that HNK-l-positive mesenchymal cells are present in the gut prior to neural crest cell colonization. After neural crest cell colonization this cell type cannot be visualized anymore with the HNK-1 antibody. We characterized the HNK-1 antigens that are present before and after neural crest cell colonization of the hindgut. Immunoblot analysis of plasma membranes from embryonic hindgut revealed a wide array of HNK-1-carrying glycoproteins. We found that two HNK-1 antigens are present in E4 hindgut prior to neural crest cell colonization and that the expression of these antigens disappears after neural crest colonization. These two membrane glycoproteins, G-42 and G-44, have relative molecular masses of 42,000 and 44,000, respectively, and they both have isoelectric points of 5.5 under reducing conditions. We suggest that these HNK-1 antigens and the HNK-l-positive mesenchymal cells have some role in the formation of the enteric nervous system.
Expression pattern of Motch, a mouse homolog of Drosophila Notch, suggests an important role in early postimplantation mouse development
Authors: Amo Francisco Franco Del , Smith David E. , Swiatek Pamela J. , Gendron-Maguire Maureen , Greenspan Ralph J. , Mcmahon Andrew P. , Gridley Thomas
Published: April 2021

The Notch gene of Drosophila encodes a large transmembrane protein involved in cell-cell interactions and cell fate decisions in the Drosophila embryo. To determine if a gene homologous to Drosophila Notch plays a role in early mouse development, we screened a mouse embryo cDNA library with probes from the Xenopus Notch homolog, Xotch. A partial cDNA clone encoding the mouse Notch homolog, which we have termed Motch, was used to analyze expression of the Motch gene. Motch transcripts were detected in a wide variety of adult tissues, which included derivatives of all three germ layers. Differentiation of P19 embryonal carcinoma cells into neuronal cell types resulted in increased expression of Motch RNA. In the postimplantation mouse embryo Motch transcripts were first detected in mesoderm at 7.5 days post coitum (dpc). By 8.5 dpc, transcript levels were highest in presomitic mesoderm, mesenchyme and endothelial cells, while much lower levels were detected in neuroepithelium. In contrast, at 9.5 dpc, neuroepithelium was a major site of Motch expression. Transcripts were also abundant in cell types derived from neural crest. These data suggest that the Motch gene plays multiple roles in patterning and differentiation of the early postimplantation mouse embryo.
A developmentally regulated switch in neuronal responsiveness to NCAM and N-cadherin in the rat hippocampus
Authors: Doherty Patrick , Skaper Stephen D. , Moore Stephen E. , Leon Alberta , Walsh Frank S.
Published: April 2021

Monolayers of control 3T3 fibroblasts and 3T3 cells expressing transfected NCAM or N-cadherin have been used as a culture substratum for rat hippocampal neurons. Both NCAM and N-cadherin are expressed in the hippocampus through embryonic day 17 (E17) to postnatal day 4 (PND4); however, whereas E17 neurons responded to transfected NCAM by extending considerably longer neurites, PND4 neurons responded very poorly. The converse was true for responsiveness to N-cadherin. These data demonstrate a switch in neuronal responsiveness to NCAM and N-cadherin in the developing hippocampus. NCAM-dependent neurite outgrowth from E17 neurons was largely dependent on the presence of α2–8-linked polysialic acid (PSA) on neuronal NCAM. NCAM-dependent neurite outgrowth could be fully inhibited by pertussis toxin or a combination of L- and N-type calcium channel antagonists thus providing direct evidence concerning the nature of the second messenger pathway activated in primary neurons by cell adhesion molecules (CAMs).
Embryonic alkaline phosphatase is expressed at M-phase in the spermatogenic lineage of the mouse
Authors: Narisawa Sonoko , Hofmann Marie-Claude , Ziomek Carol A. , Luis Millán José
Published: April 2021

We have recently cloned and characterized a novel embryonic alkaline phosphatase (EAP) expressed at the two-cell to blastocyst stage of preimplantation development in the mouse. The isozyme is re-expressed in trace amounts in the thymus, intestine and testis during adult life. In the present report, we find that EAP transcripts can be detected, by RT-PCR analysis, in very low amounts in the testes of newborn mice, but at 24 days of age EAP mRNA levels reach the highest concentrations, remaining high at 40 and even 117 days of age. We produced a synthetic peptide and a corresponding rabbit anti-peptide antiserum (Rb-1434), which was characterized by enzyme antigen immunoassays and reactivity with chinese hamster ovary cell transfectants, as reacting specifically with EAP. The Rb-1434 antibody enabled us to examine immunohistochemically what cell types in the testis are responsible for the expression of EAP during different developmental stages. No positive cells were recognized in the testis of newborns (day 0) and 8-day-old mice. Positive cells were first observed at day 15 and, at 24 days of age, many positive M-phase cells, morphologically corresponding to spermatocytes in mid to late prophase of meiotic division I, were strongly positive for EAP expression. Positive M-phase cells were also observed at 40 days and 151 days of age. Transgenic mice expressing the human GCAP isozyme in a tissue-specific manner in the testis, showed equivalent stages of M-phase figures when stained immunohistochemically with a specific rabbit polyclonal antiserum. Our results demonstrate that EAP is expressed in M-phase spermatogenic cells during postnatal development, suggesting a role for this AP isozyme during meiosis.
Why we have (only) five fingers per hand: Hox genes and the evolution of paired limbs
Authors: Tabin Clifford J.
Published: April 2021

Limb development has long been a model system for studying vertebrate pattern formation. The advent of molecular biology has allowed the identification of some of the key genes that regulate limb morphogenesis. One important class of such genes are the homeobox-containing, or Hox genes. Understanding of the roles these genes play in development additionally provides insights into the evolution of limb pattern. Hox gene expression patterns divide the embryonic limb bud into five sectors along the anterior/posterior axis. The expression of specific Hox genes in each domain specifies the developmental fate of that region. Because there are only five distinct Hox-encoded domains across the limb bud there is a developmental constraint prohibiting the evolution of more than five different types of digits. The expression patterns of Hox genes in modern embryonic limb buds also gives clues to the shape of the ancestral fin field from which the limb evolved, hence elucidating the evolution of the tetrapod limb.
Vital dye analysis of cranial neural crest cell migration in the mouse embryo
Authors: Serbedzija George N. , Bronner-Fraser Marianne , Fraser Scott E.
Published: April 2021

The spatial and temporal aspects of cranial neural crest cell migration in the mouse are poorly understood because of technical limitations. No reliable cell markers are available and vital staining of embryos in culture has had limited success because they develop normally for only 24 hours. Here, we circumvent these problems by combining vital dye labelling with exo utero embryological techniques. To define better the nature of cranial neural crest cell migration in the mouse embryo, premigratory cranial neural crest cells were labelled by injecting DiI into the amniotic cavity on embryonic day 8. Embryos, allowed to develop an additional 1 to 5 days exo utero in the mother before analysis, showed distinct and characteristic patterns of cranial neural crest cell migration at the different axial levels. Neural crest cells arising at the level of the forebrain migrated ventrally in a contiguous stream through the mesenchyme between the eye and the diencephalon. In the region of the midbrain, the cells migrated ventrolaterally as dispersed cells through the mesenchyme bordered by the lateral surface of the mesencephalon and the ectoderm. At the level of the hindbrain, neural crest cells migrated ventrolaterally in three subectodermal streams that were segmentally distributed. Each stream extended from the dorsal portion of the neural tube into the distal portion of the adjacent branchial arch. The order in which cranial neural crest cells populate their derivatives was determined by labelling embryos at different stages of development. Cranial neural crest cells populated their derivatives in a ventral-to-dorsal order, similar to the pattern observed at trunk levels. In order to confirm and extend the findings obtained with exo utero embryos, DiI (1,1-dioctadecyl-3,3,3′,3′-tetramethylindo-carbocyanine perchlorate) was applied focally to the neural folds of embryos, which were then cultured for 24 hours. Because the culture technique permitted increased control of the timing and location of the DiI injection, it was possible to determine the duration of cranial neural crest cell emigration from the neural tube. Cranial neural crest cell emigration from the neural folds was completed by the 11-somite stage in the region of the rostral hindbrain, the 14-somite stage in the regions of the midbrain and caudal hindbrain and not until the 16-somite stage in the region of the fore-brain. At each level, the time between the earliest and latest neural crest cells to emigrate from the neural tube appeared to be 9 hours. Some aspects of cranial neural crest cell migration were similar to those observed in avian embryos; however, the exact pathways, the timing and the axial levels that contribute to the neural crest cells were distinctly different.
Clox, a mammalian homeobox gene related to Drosophila cut, encodes DNA-binding regulatory proteins differentially expressed during development
Authors: Andres Vicente , Nadal-Ginard Bernardo , Mahdavi Vijak
Published: April 2021

We report the isolation of a cDNA encoding a mammalian homeoprotein related to the Drosophila cut gene product, called Clox, for Cut like homeobox. In addition to the homeodomain, three 73-amino acid repeats, the so-called cut repeats, are also conserved between Cut and the mammalian counterpart described here. This conservation suggests that the cut repeat motif may define a new class of homeoproteins. Both cloned and endogenous Clox proteins are nuclear DNA-binding proteins with very similar sequence specificity. Western blot analysis revealed several distinct Clox protein species in a variety of tissues and cell types. The relative abundance of these proteins is regulated during mouse development and cell differentiation in culture. Interestingly, ∼180-190×103Mr Clox proteins predominate in early embryos and are upregulated in committed myoblasts and chondrocytes, but downregulated upon terminal differentiation. Clox DNA-binding activity is correlated with the abundance of these proteins. In contrast, larger Clox protein species (∼230-250×103Mr) are detected mainly in adult tissues and in terminally differentiated cells. Cotransfection experiments show that Clox proteins can function as repressors of tissue-specific gene transcription. Thus, Clox, like their Drosophila counterparts, are candidate regulators of cell-fate specification in diverse differentiation programs.
Determination of epithelial half-somites in skeletal morphogenesis
Authors: Goldstein Ronald S. , Kalcheim Chaya
Published: April 2021

The segmental body plan of vertebrates arises from the metameric organization of the paraxial mesoderm into somites. Each mesodermal somite is subdivided into at least two distinct domains: rostral and caudal. The segmental pattern of dorsal root ganglia, sympathetic ganglia and nerves is imposed by differential properties of either somitic domain. In the present work, we have extended these studies by investigating the contribution of rostral or caudal-half somites to vertebral development using grafts of multiple somite halves. In both rostral and caudal somitic implants, the grafted mesoderm dissociates normally into sclerotome and dermomyotome, and the sclerotome further develops into vertebrae. However, the morphogenetic capabilities of each somitic half differ. The pedicle of the vertebral arch is almost continuous in caudal half-somite grafts and is virtually absent in rostral half-somite implants. Simi-larly, the intervertebral disk is present in rostral half-somite chimeras, and much reduced or virtually absent in caudal somite chimeras. Thus, only the caudal half cells are committed to give rise to the vertebral pedicle, and only the rostral half cells are committed to give rise to the fibrocartilage of the intervertebral disk.

Each vertebra is therefore composed of a pedicle-containing area, apparently formed by the caudal half-somite, followed by a pedicle-free zone, the intervertebral foramen, derived from the rostral somite. These data directly support the hypothesis of resegmentation, in which vertebrae arise by fusion of the caudal and rostral halves of two consecutive somites.
Induction of labial expression in the Drosophila endoderm: response elements for dpp signalling and for autoregulation
Authors: Tremml Gabi , Bienz Mariann
Published: April 2021

Extracellular signal proteins induce the homeotic gene labial (lab) to high levels of localised expression in the endoderm of Drosophila embryos. We aimed to identify cis-regulatory elements within the lab gene that respond to this induction by analysing the activity of stably integrated reporter gene constructs. Dissection of lab 5 flanking sequences reveals two types of response elements. One of these mediates lab dependent activity, providing evidence that lab induction in the endoderm is autoregulatory. The other element, to a large extent independent of lab function, responds to decapentaplegic (dpp), a signal molecule related to mammalian TGF-. Our evidence suggests that lab induction in the endoderm reflects coordinate action of two distinct factors one of which may be lab protein itself, and another whose localised activity or expression in the midgut depends on the dpp signal.
Molecular gradients along the proximal-distal axis of embryonic insect legs: possible guidance cues of pioneer axon growth
Authors: Norbeck Betty A. , Feng Yun , Denburg Jeffrey L.
Published: April 2021

It has been proposed that gradients of environmental cues direct the proximal growth of pioneer axons in embryonic insect legs. Hybridoma techniques have been used to produce 3 monoclonal antibodies (mAbs) that bind to components associated with the basal lamina/extracellular matrix that are non-uniformly distributed along the proximal-distal axis of cockroach legs at the time of pioneer axon growth. Two of these mAbs, PROD-1 and PROD-2, label the proximal parts of the leg more intensely than the distal ends. The other mAb, DIP-1, has the reverse pattern of binding with the distal parts of the leg labeled more intensely. The graded distribution of these antigens only occurs just prior to and during the growth period of the Ti1 pioneer axons. Western blot analyses and immunoprecipitations have identified the protein antigens recognized by these mAbs. The spatial and temporal distributions of these molecules in the legs and the CNS make them good candidates for environmental guidance cues of pioneer axon growth.
Ontogeny of calbindin-D28K and calbindin-D9K in the mouse kidney, duodenum, cerebellum and placenta
Authors: Shamley D. R. , Opperman L. A. , Buffenstein R. , Ross F. P.
Published: April 2021

The appearance of the calcium-binding proteins (CaBP-D28K and CaBP-D9K) in embryonic mice tissues was determined using a sensitive immunohistochemical assay. CaBP-D28K first appears in myenteric nerve plexuses of the duodenum on day E15, in duodenal villus cells on day E16, in Purkinje cells of the cerebellum on day E19, in cells of the mesonephric duct on day E11 and in the metanephric duct on day E12. CaBP-D9K first appears in enterocytes of the duodenum on day E18, in trophoblastic giant cells (TGC) of the placenta on day E10, and in the metanephric duct on day E15. A differential time of appearance and colocalization of the two CaBPs is demonstrated in the embryonic mouse kidney, suggesting either that vitamin D does not control both CaBPs in the foetus or that the vitamin D control is unequal. The early appearance and location of CaBP-D9K in TGCs may suggest that these cells play an important role in transplacental transfer of calcium.
Differential screening of a PCR-generated mouse embryo cDNA library: glucose transporters are differentially expressed in early postimplantation mouse embryos
Authors: Smith David E. , Gridley Thomas
Published: April 2021

Differential screening of a cDNA library constructed using PCR amplification techniques from RNA isolated from the distal portion (embryonic ectoderm, mesoderm and visceral endoderm) of 7.5 days post coitum (dpc) mouse embryos led to the isolation of two cDNA clones expressed at higher levels in 7.5 dpc embryos than 12.5 dpc embryos. Nucleotide sequence analysis revealed that each of these clones was a different member of the family of facilitative glucose transporters (Glut genes). The differentially expressed cDNA clones represent mouse Glut-1 and Glut-3. Levels of the Glut-3 mRNA declined 14-fold between days 7.5 and 12.5 of gestation, and were under our limits of detetction by 14.5 dpc. The levels of the Glut-1 mRNA declined about 3-fold between days 7.5 and 12.5 of gestation.

Analysis of the expression of these genes by in situ hybridization revealed striking differences in transcript localization in early postimplantation mouse embryos. At 7.5 dpc, both transporters were expressed more strongly in extraembryonic tissues than in the embryo proper. While both transporters were expressed in the amnion and chorion, only Glut-1 was expressed in the ectoplacental cone. In the yolk sac, Glut-3 appeared to be expressed only in the endoderm while Glut-1, although expressed in both layers, was expressed more strongly in the mesoderm layer. Thus, the two transporters have relatively reciprocal sites of expression in the developing extraembryonic membranes. Expression of Glut-1 was fairly widespread in the embryo at 8.5 dpc, but by 10.5 dpc expression was down-regulated and was observed in the eye and the spinal cord. Expression of Glut-3 was largely confined to non-neural surface ectoderm and was also substantially down-regulated by 10.5 dpc.

These results prompted an examination of the RNA expression pattern of two other glucose transporter isoforms, Glut-2 and Glut-4. We did not detect Glut-4 expression, while Glut-2 expression was largely confined to extraembryonic visceral yolk sac endoderm. These data suggest differential roles for these glucose transporter family members during early postimplantation development of mice.
Expression of Pax-3- and neuroectoderm-inducing activities during differentiation of P19 embryonal carcinoma cells
Authors: Pruitt Steven C.
Published: April 2021

A P19 embryonal carcinoma stem cell line carrying an insertion of the E. coli LacZ gene in an endogenous copy of the Pax-3 gene was identified. Expression of the Pax-3/LacZ fusion gene in neuroectodermal and mesodermal lineages following induction of differentiation by chemical treatments (retinoic acid and dimethylsulfoxide) was characterized using this line and is consistent with the previous localization of Pax-3 expression in the embryo to mitotically active cells of the dorsal neuroectoderm and the adjacent segmented dermomyotome. Pax-3/LacZ marked stem cells were also utilized as target cells in mixing experiments with unmarked P19 cells that had been differentiated by pretreatment with chemical inducers. Induction of -galactosidase and neuroectodermal markers in the target cells demonstrates that: (1) some differentiated P19 cell derivatives transiently express endogenous Pax-3- and neuroectoderm-inducing activities, (2) undifferentiated target stem cells respond to these activities even in the presence of leukemia inhibitory factor and (3) the endogenous activities can be distinguished from, and are more potent than, retinoic acid treatment in inducing neuroectoderm. These observations demonstrate that P19 embryonal carcinoma cells provide a useful in vitro system for analysis of the cellular interactions responsible for neuroectoderm induction in mammals.
Expression of α1 integrin, a laminin-collagen receptor, during myogenesis and neurogenesis in the avian embryo
Authors: Duband Jean-Loup , Belkin Alexey M. , Syfrig Josef , Thiery Jean Paul , Koteliansky Victor E.
Published: April 2021

In this study, we have examined the spatiotemporal distribution of the 1 integrin subunit, a putative laminin and collagen receptor, in avian embryos, using immunofluorescence microscopy and immunoblotting techniques. We used an antibody raised against a gizzard 175 × 103Mr membrane protein which was described previously and which we found to be immunologically identical to the chicken α1 integrin subunit. In adult avian tissues, α1 integrin exhibited a very restricted pattern of expression; it was detected only in smooth muscle and in capillary endothelial cells. In the developing embryo, α1 integrin subunit expression was discovered in addition to smooth muscle and capillary endothelial cells, transiently, in both central and peripheral nervous systems and in striated muscles, in association with laminin and collagen IV. α1 integrin was practically absent from most epithelial tissues, including the liver, pancreas and kidney tubules, and was weakly expressed by tissues that were not associated with laminin and collagen IV. In the nervous system, 1 integrin subunit expression occurred predominantly at the time of early neuronal differentiation. During skeletal muscle development, α1 integrin was expressed on myogenic precursors, during myoblast migration, and in differentiating myotubes. α1 integrin disappeared from skeletal muscle cells as they became contractile. In visceral and vascular smooth muscles, α1 integrin appeared specifically during early smooth muscle cell differentiation and, later, was permanently expressed after cell maturation. These results indicate that (i) the expression pattern of α1 integrin is consistent with a function as a laminin/collagen IV receptor; (ii) during avian development, expression of the α1 integrin subunit is spatially and temporally regulated; (iii) during myogenesis and neurogenesis, expression of α1 integrin is transient and correlates with cell migration and differentiation.