Formation of extra digits in the interdigital spaces of the chick leg bud is not preceded by changes in the expression of the Msx and Hoxd genes

By in situ hybridization we studied the expression patterns of Msx and Hoxd genes during the late development of the chick leg autopodium (foot) and compared them to patterns during the experimental development of interdigital extra digits. Extra digits are induced in the third interdigital space after various experimental manipulations, such as transient isolation of the interdigit, or removal of the interdigital marginal ectoderm and mesoderm. Msx1 and Msx2 are normally expressed in the interdigital tissue programmed to die. Our experiment changes the fate of the interdigital tissue from cell death to chondrogenesis and provides a good model for studying Msx involvement in defining areas of programmed cell death. Among the proposed roles for Hoxd genes is their involvement in the specification of digit identity early in development. The induction of extra digits allows us to examine whether this new morphogenetic commitment of the interdigital tissue involves changes in the domains of expression of Hoxd genes. Our results show that extra digits develop without a previous modification of the normal pattern of expression of Msx or Hoxd genes. This observation does not support the correlation between the expression of Msx genes and programmed cell death and suggests a role for these genes in maintaining the interdigital tissue in an undifferentiated state. Our results show that an increased number of digits can be formed without modifications in the pattern of expression of the 5′-located Hoxd genes and suggest the existence of latent or residual digit organization mechanisms past the time when digits are normally determined, independent of Hoxd gene expression.     

Formation of extra-digits induced by surgical removal of the apical ectodermal ridge of the chick embryo leg bud in the stages previous to the onset of interdigital cell death

Summary In the present work we have studied the mechanism of formation and the possible morphogenetic significance of the process of ectopic chondrogenesis induced by surgical removal of AER of the interdigital spaces of the chick leg bud at stage 28–30 (Hurle and Gañan 1986). Our results show that ridge removal causes condensation and rounding of the underlaying mesenchymal cells followed by chondrogensis. The long-term study of the fate of these ectopic cartilages shows that in a high percentage of the cases the cartilages undergo morphogenesis taking by day 10 of incubation the appearance of the two distal phalanges of an extra-digit. These extra-digits lack tendons and are joined by thin interdigital membranes to the neighboring digits.     

Interdigital chondrogenesis and extra digit formation in the duck leg bud subjected to local ectoderm removal

Summary In the chick embryo the interdigital tissue in the stages previous to cell death exhibits in vitro a high chondrogenic potential, and forms extra digits when subjected in vivo to local ectodermal removal. In the present work we have analyzed the chondrogenic potential both in vivo and in vitro of the interdigital mesenchyme of the duck leg bud. As distinct from the chick, the interdigital mesenchyme of the duck leg bud exhibits a low degree of degeneration, resulting in the formation of webbed digits. Our results show that duck interdigital mesenchyme exhibits also a high chondrogenic potential in vitro until the stages in which cell death starts. Once cell death is finished chondrogenesis becomes negative and the interdigital mesenchyme forms a fibroblastic tissue. In vivo the interdigital mesenchyme of the duck leg bud subjected to ectoderm removal forms ectopic foci of chondrogenesis with a range of incidence similar to that in the chick. Unlike those of the chick the ectopic cartilages of the duck are rounded and smaller, and appear to be located at the distal margin of the interdigital mesenchyme. Formation of extra digits in the duck occurs with a lower incidence than in the chick. It is concluded that ectopic chondrogenesis and formation of extra digits is related to the intensity of interdigital cell death. The non-degenerating interdigital mesenchymal cells destined to form the interdigital webs of the duck appear to contribute very little to the formation of interdigital cartilages.     

Extracellular matrix modifications in the interdigital spaces of the chick embryo leg bud during the formation of ectopic digits

In previous studies we have observed that the interdigital mesenchyme of the chick leg bud, in the stages preceding the onset of cell death, retains a significant regulatory potential, forming ectopic extra digits under a variety of surgical manipulations. Most evidence suggests that interdigital extra digits are caused by the abolition of local antichondrogenic effects operating in the interdigital spaces under normal conditions rather than by modifications of the signalling mechanisms accounting for the normal patterning of the digits in early stages of development. The interdigital spaces exhibit a complex scaffold of extracellular matrix with well-defined domains of spatial distribution of type I and type VI collagens, tenascin, fibronectin, laminin and elastic matrix components that have been proposed to play a role in the establishment of the non-chondrogenic fate of the interdigital tissue in situ. In an attempt to analyze this possible role of the interdigital extracellular matrix (ECM), in the present work we have studied changes in the pattern of ECM distribution associated with the formation of extra digits. Extra digits were induced by making a T-cut in the third interdigital space of the leg but of stage 29 HH chick embryos. Subsequent modifications of the ECM were detected immunohistochemically in whole-mount specimens using laser confocal microscopy. Our results reveal that in the first hours after the operation, changes in the ECM apparently related to the healing of the wound cause a significant reorganization of the normal ECM scaffold of the interdigit. In addition, chondrogenesis of the interdigital tissue is preceded by disappearance of elastin fibers in the interdigital mesenchyme subjacent to the wound and by an intense deposition of tenascin. Tenascin deposition and loss of the elastin fibrillar scaffold were also observed preceding chondrogenesis in fragments of interdigital tissue explanted to culture conditions. The significance of these observations in relation to the establishment of the skeletal elements of the autopodium is discussed.     

Ultrastructural localization of cholinesterase uring chondrogenesis and myogenesis in the chick limb bud

Summary Cholinesterase (ChE) is transiently expressed in undifferentiated embryonic cells. In the chick limb bud ChE-activity was found in the apical ectodermal ridge and in the subridge mesenchyme. The reaction was localized in the perinuclear cisterna, in an extensive network of narrow profiles of endoplasmic reticulum (ER), and in the Golgi complexThe chondroblasts emerging from the subridge mesenenyme, also showed strong ChE-activity. During differentiation the enzyme first disappeared from the Golgi zone. Then, the narrow ChE-positive ER was successively replaced by ChE-negative extended rough ER characteristic for the differentiated chondrocyte.The myoblasts showed weak ChE-activity with the same ultrastructural localization as in other mesenchymal cells. After fusion the myotubes exhibited strong ChE-activity in the perinuclear cisterna and the developing sarcoplasmic reticulum. In later stages of myogenesis the myoblasts were closely attached to the myotubes and had lost their ChE-activity.During mitosis of ChE-positive cells, ChE-activity was retained in fragments of perinuclear cisterna and ER. In ChE-active mesenchymal cells and chondroblasts we observed specialized contact zones between ER and plasma membrane. ChE-active cisternae of ER run parallel to the plasma membrane with a gap of approximately 10–15 nm. We discuss a possible function of a cholinergic system during morphogenesis.     

Ca2+ channel activities in the limb bud of early embryonic chick

Ca²⁺ channel activities were recorded in the limb bud of embryonic day 4 chick with Ca²⁺ sensitive fluorescence (Fura-2) measurements and patch clamp techniques. Rises in intracellular Ca²⁺ concentrations were evoked by depolarization with the application of 100 mM K⁺ and this Ca²⁺ response was abolished by removing extracellular Ca²⁺. The Ca²⁺ response was blocked by 10 M nifedipine and enhanced by 5 M Bay K 8644. Long-lasting inward currents were revealed by whole-cell patch clamp recordings from dissociated cells of the limb bud. The inward current was also blocked by 10 M nifedipine. Our study suggested the presence of L-type Ca²⁺ channels in the limb bud cells.     

In vivo inhibition of programmed cell death by local administration of FGF-2 and FGF-4 in the interdigital areas of the embryonic chick leg bud

The formation of the digits in amniote vertebrates is accompanied by a massive degeneration process that accounts for the disappearance of the interdigital mesenchyme. The establishment of these areas of interdigital cell death (INZs) is concomitant with the flattening of the apical ectodermal ridge (AER), but a possible causal relationship between these processes has not been demonstrated. Recent studies have shown that the function of the AER can be substituted for by implantation of beads bearing either FGF-2 or FGF-4 into the apical mesoderm of the early limb bud. According to these observations, if the onset of INZs is triggered by the cessation of the AER function, local administration of FGFs to the interdigital tissue prior to cell death should delay or inhibit interdigit degeneration. In the present study we have confirmed this prediction. Implanting Affi-gel blue or heparin beads pre-absorbed with either FGF-2 or FGF-4 into the interdigital tissue of the chick leg bud in the stages prior to cell death stimulates cell proliferation and causes the formation of webbed digits. Vital staining with neutral red confirmed an intense temporal inhibition of interdigital cell death after FGF treatment. This inhibition of interdigital cell death was not accompanied by modifications in the pattern of expression of Msx-1 or Msx-2 genes, which in normal development display a domain of expression in the interdigital tissue preceding the onset of degeneration.     

Scanning and light microscope studies of the development of the chick embryo semilunar heart valves

Summary The development of the semilunar valves of the great arteries was studied by light and scanning electron microscopy in the chick embryo. The results show that three distinct developmental periods can be distinguished. The formation of the anlage of the valves takes place in the first period (stages 26–29). These early anlage consist of three pyramidal shaped cusps formed by a core of loosely packed mesenchymal cells covered by a flattened endothelium. In the second period (stages 30–35) the cusps undergo excavation on their distal face. Morphological evidence is reported suggesting that this excavation process is produced by an initial solid ingrowth of the endothelium of the arterial face of the cusps which is immediately luminated by detachment of cells towards the bloodstream and by cell death. The histogenesis of the valves takes place in the third period (from stage 36 until hatching). It was observed that during this period some myocardial cells of the outflow tracts of the ventricles invade the valvular tissue and that in the upper part of the cusps a prominent fibrous layer is formed.     

Regeneration in theXenopus tadpole optic nerve is preceded by a massive macrophage/microglial response

Summary Changes in the optic nerve following a crush lesion and during axonal regeneration have been studied inXenopus tadpoles, using ultrastructural and immunohistological methods. Degeneration of both unmyelinated and myelinated axons is very rapid and leads to the formation, within 5 days, of a nerve which consists largely of degeneration debris and cells. Immunohistological analysis with monoclonal antibody 5F4 shows that there is a rapid and extensive microglial/macrophage response to crush of the nerve. Regenerating axons have begun to enter the distal stump by 5 days and grow along the outer part of the nerve in close approximation to the astrocytic glia limitans. Between 5 and 10 days after nerve crush, regenerating axons reach and pass the chiasma. Macrophages are seen in the nerve at the site of the lesion within 1 h, and the response peaks between 3–5 days, just before axonal regeneration gets under way.     

The ventralizing effect of the notochord on somite differentiation in chick embryos

The dorso-ventral pattern formation of the somites becomes manifest by the formation of the epithelially organized dorsal dermomyotome and the mesenchymal ventrally situated sclerotome. While the dermomyotome gives rise to dermis and muscle, the sclerotome differentiates into cartilage and bone of the axial skeleton. The onset of muscle differentiation can be visualized by immunohistochemistry for proteins associated with muscle contractility, e.g. desmin. The sclerotome cells and the epithelial ventral half of the somite express Pax-1, a member of a gene family with a sequence similarity to Drosophila paired-box-containing genes. In the present study, changes of Pax-1 expression were studied after grafting an additional notochord into the paraxial mesoderm region. The influence of the notochord and the floor-plate on dermomyotome formation and myotome differentiation has also been investigated. The notochord is found to exert a ventralizing effect on the establishment of the dorso-ventral pattern in the somites. Notochord grafts lead to a suppression of the formation and differentiation of the dorsal somitic derivatives. Simultaneously, a widening of the Pax-1-expressing domain in the sclerotome can be observed. In contrast, grafted roof-plate and aorta do not interfere with dorso-ventral patterning of the somitic derivatives.     

N-Methyl-d-aspartate stimulation of the survival of rat cerebellar granule cells in culture is not dependent upon increased c-fos expression and is not mimicked by protein kinase C activation

The role of c-fos expression and protein kinase C in the survival of primary cultures of rat cerebellar granule cells was investigated. Results from immunocytochemistry and immunoblotting suggest that increased c-fos expression is not essential for the survival of cells grown in low K⁺ media in the presence of N-methyl-d-aspartate (NMDA) at the critical time point when sensitivity to survival requirements develops. In addition the phorbol ester, phorbol 12-myristate 13-acetate failed to bring about survival of cells cultured in low K⁺ media in the absence of NMDA when given chronically, suggesting that protein kinase C activation alone is not sufficient to maintain granule cell survival in culture.     

The histogenesis of the isthmic nuclei in chick embryos (Gallus domesticus)

Summary The neuromeric mes-rhombencephalic boundary runs between the oculomotor and trochlear nuclei. During morphogenesis the m ₂-segment is reduced to a cellfree mantle demarcating a morphological mes-rhombencephalic border. The floor plate and glycogen-containing raphe extend rostralwards to terminate at the level of the m ₂-segment ventromedially.The isthmic migration commences within the dorsal and dorsolateral rhombencephalic cell columns caudal to the emergence of the trochlear nerve. The neuroblasts migrate radially out from the synthetic zone of the neural epithelium into the mantle constituting the isthmic migration. The latter migrate longitudinally en masse rostralwards into the mantle below the optic lobe. The m ₂-segment can, however, be identified as a morphological border between the mesencephalic and isthmic (rhombencephalic) mantles throughout the early embryogenesis.The isthmic migration subdivides into a tectal and a tegmental nuclear group. Both groups contribute to the formation of the isthmic nuclei. The caudal portion of the mesencephalic tectal mantle contributes to a mesencephalic isthmic nucleus: Nucleus isthmi principalis mesencephali (magnocellularis).     

Formation of alternating tiers in the optic chiasm of the chick embryo

Background: When the fibers of the two optic nerves of the chick cross to the contralateral side at the prospective chiasmatic region, they segregate into clearly defined bundles. These bundles form horizontally oriented tiers which alternate between the right and the left optic nerve. Methods: We have analyzed the development of these tiers qualitatively and quantitatively using light and electron microscopy between embryonic days (E) 4 and E19. Results: The formation of the chiasm begins on E4. In the course of E4, tiers become visible for the first time. Their number increases rapidly until E7. Then the increase is slowed down and the final value (32 ± 1) is approximated by E18/19. Growing axons allow one to distinguish three different segments: the growth cone, the distal, and the proximal segment. The latter originates in the perikaryon. Growth cones and distal segments are found predominantly in the ventralmost tiers. Their frequency decreases from ventral to dorsal. Proximal segments which indicate the presence of older axons appear first in the dorsal tiers and later also in more ventrally located tiers. Conclusion: Based on these criteria it is concluded that newly formed axons contribute primarily but not exclusively to the ventral tiers. There is a gradient of maturity of axons from ventral to dorsal whose slope becomes steeper with age until the last growth cones have arrived by E18. Thus, the formation of the chiasm corresponds to the spatiotemporal pattern of ganglion cell formation in the retina. The process of cell death of retinal ganglion cells is also seen in the chiasm but probably does not lead to a transitory diminution in the number of tiers. © 1994 Wiley-Liss, Inc.     

Role for differential cell proliferation in perforation and rupture of chick pharyngeal closing plates

Patterns of cell proliferation show two distinct domains around avian pharyngeal closing plates as they form, perforate, and rupture. Pouch endoderm and groove ectoderm are in one domain showing 81–86% proliferation, which suggests relatively rapid growth. Cells in epithelia that comprise the pharyngeal closing plates contiguous with pouch and groove epithelia are vital but are dividing at a significantly lower percentage (62–64%). This phenomenon is similar to the lag in growth reported for cells in the oral membrane around the time of rupture (Miller and Olcott, 1989: Anat. Rec., 223:204–208), is consistent with Waterman's suggestion (1985: Anat. Rec., 211:450–457) that cellular reorganization, rather than massive degeneration, is a major mechanism of initial perforation, and suggests that differential growth is a contributor to perforation and rupture of chick pharyngeal closing plates. © 1993 Wiley-Liss, Inc.     

Formation of gap junctions by stem cells in the developing retina of the clawed frog (Xenopus laevis)

Summary To ascertain whether gap junctions are preferentially formed by proliferative retinal stem cells, an analysis by electron microscopic autoradiography was made on the retina of the Xenopus embryo at stages 26, 29 and 31, after pulse labelling with ³H-thymidine. When the administration of the isotope was carried out for 0.5 or 3 h, retinal cells in S phase or S to M phase in the cell cycle were marked with the isotope, respectively. In these specimens, most gap junctions were found on the isotope-labelled cells, and few on the unlabelled cells. Several cells in mitosis also formed gap junctions. These results would suggest that gap junctions are formed by retinal stem cells in S to M phases of the cell cycle, but not by cells in G₁ phase and post-mitotic cells.     

Phylogeny and evolution of the NS1 and VP1/VP2 gene sequences from porcine parvovirus

The objective of the present study was to gain new insights on the evolution and phylogeny of the PPV genome, specifically on the NS1 and VP1/VP2 genes. Moreover, two new complete sequences from PPV isolates from China (BQ and ZJ strains) were generated and included in the study. The data set studied contained available NS1 and VP1/VP2 sequences at the GenBank database, plus those corresponding to the mentioned Chinese isolates. PPV sequences were divided into two major groups, with one group separated into two branches. Both phylogenetic groups were homogeneous and several marker aminoacidic changes and synapomorphic positions were identified along both genes. Despite the two genes were satisfactory molecular markers, the absence of selection pressure on the VP1/VP2 fragment makes it a preferential option compared to the NS1 one. Furthermore, NS1 gene showed a biased mutation pattern compared with VP1/VP2 genes, which is compatible with the existence of selection in the first but not in the second gene (as indicated by the negative difference between non-synonymous and synonymous values). No correlation between NS1 and VP1/VP2 phylogenetic groups and/or branches and health status was observed. However, a relationship among virulence and the absence of the 127-bp repeat located downstream the part of ORF2 encoding the structural proteins VP1 and VP2 cannot be excluded.