TCAV-1, a monoclonal antibody specific to epithelial pharyngeal cells in the planarian Dugesia (Girardia) tigrina. Application to pattern formation of the pharynx during regeneration

During regeneration in planarians, anterior (head and prepharyngeal) and posterior (postpharyngeal and tail) fragments rebuild one of the most peculiar structures of planarians: the pharynx and the pharynx cavity. Previous studies (see Brønsted, 1969, for a general review, and Asai, 1990, 1991, for anterior regeneration) have shown that within postpharyngeal pieces both structures appear in the old stump from clusters of undifferentiated cells. However, the lineage and differentiation of their elements (inner and outer epithelial cells, muscle layers, gland cells, nerve rings) and the overall pattern of growth and differentiation is not clear.     

Circular form of regeneration in an unidentified species of land planarians, Bipalium sp

The communication describes an interesting and possibly novel finding regarding a species of land planarians (Bipalium sp.) from a high altitude of Himalayan range. The regeneration in Bipalium sp. is highly interesting and the process takes about a fortnight for the completion. Accumulation of numerous basophilic cells in parenchyma at the blastema region occurs within 5 days after excision and in 7 to 9 days the head and tail regions become reorganized with the formation of functional pharynx in the tail piece. The neoblast cells in the mesenchyme and the gastrodermis cells proliferate continuously replenishing the population of stem cell for growth, reproduction, and regeneration. Many undifferentiated cells are present even in completely regenerated specimens. Sometimes middle piece regenerates apparently fuse at the blastema surface forming a peculiar circular form of Bipalium sp. where both the head and tail cut ends join completely. These regenerated individuals excepting the middle piece regenerate behave in a normal fashion within 12-14 days.     

Proportion regulation in the planarian,Dugesia tigrina following regeneration of structures

Planarian regenerates with abnormal body proportioning were followed after structure formation in order to determine if body proportion will ‘normalize’ over time. Results show proportioning will occur following structure formation since the pharynx in regenerates with proportionally larger heads and prepharyngeal area moved anteriorly over time. This occurred regardless of whether regenerates were provided with a normal feeding regime which allowed for an increase in body area or if they were on a maintenance diet which did not permit growth. An examination of mitotic indices did not demonstrate significant differences in the level of mitotic activity between pre- and postpharyngeal regions. It is concluded that the ‘normalizing’ of proportion in these abnormal regenerates does occur, but the process by which it occurs is not solely explained through normal growth. This revised version was published online in July 2006 with corrections to the Cover Date.     

Multiple phenotypes resulting from a mutagenesis screen for pharynx muscle mutations in Caenorhabditis elegans

We describe a novel screen to isolate pharyngeal cell morphology mutants in Caenorhabditis elegans using myo-2::GFP to rapidly identify abnormally shaped pharynxes in EMS (Ethyl Methanesulfonate) mutagenized worms. We observed over 83 C. elegans lines with distinctive pharyngeal phenotypes in worms surviving to the L1 larval stage, with phenotypes ranging from short pharynx, unattached pharynx, missing cells, asymmetric morphology, and non-adherent pharynx cells. Thirteen of these mutations have been chromosomally mapped using Single Nucleotide Polymorphisms (SNPs) and deficiency strain complementation. Our studies have focused on genetically mapping and functionally testing two phenotypes, the short pharynx and the loss of muscle cohesion phenotypes. We have also identified new alleles of sma-1, and our screen suggests many genes directing pharynx assembly and structure may be either pharynx specific or less critical in other tissues.     

Earthworm leukocyte interactions during early stages of graft rejection

The earliest phase of graft rejection in earthworms, the recognition of foreign tissue antigens, has been subjected to analysis by confronting host leukocytes with foreign erythrocytes. Only rabbit and rat erythrocytes significantly prevented healing of allografts when grafts were transplanted and erythrocytes injected simultaneously. In contrast, autografts and allografts transplanted on worms injected 1 or 2 days before grafting were never affected. Since earthworms readily produce higher titers of erythrocyte agglutinins at 24 h postinjection than at later times, we propose a hypothetical scheme of earthworm leukocyte interactions that may occur during the early phases of graft healing and of agglutinin synthesis.     

Dorsal Blastomeres in the Equatorial Region of the 32-Cell Xenopus Embryo Autonomously Produce Progeny Committed to the Organizer

For testing the autonomic differentiation abilities of dorsal equatorial blastomeres of 32-cell Xenopus embryos, their roles in head formation in normal development and the organizer-inducing capabilities of the dorsal-most vegetal cells, interspecific transplantations were made using Xenopus borealis and X. laevis . When transplanted into the ventral region, the dorsal blastomeres produced descendants that differentiated into prechordal mesoderm, notochord and somites in the recipient according to their fates. They induced formation of the secondary embryo with the head and tail. The prechordal mesoderm and notochord in the secondary structure consisted of progeny of the graft, whereas somites and the CNS were chimeric and the pronephros was composed of host cells. Replacement of the dorsal blastomeres by ventral equatorial cells caused complete arrest of head formation in the recipient. Without exception, the notochord was completely absent or very thin. These results confirm the assumption that the presumptive head organizer in the Xenopus embryo is localized in the dorsal equatorial region at the 32-cell stage and comes into existence not under the inductive influence of the dorsal-most vegetal cells, but owing to allocation of morphogenetic determinants residing in the fertilized egg to the dorsal equatorial blastomeres of the 32-cell embryo.     

Transplantation phenomena in hydra: Cooperation of position-dependent and structure-dependent factors determines the transplantation result

Hydra transplantation phenomena were examined using lateral grafting (Browne, 1909; Wolpert et al., 1974) and axial grafting (Shostak, 1972, Shostak, 1973, Shostak, 1974; Hicklin et al., 1973) procedures. When a small piece of tissue excised from one hydra is transplanted to another by the former procedure, the transplanted tissue forms a small protrusion on the body column of the host animal, whereas such a protrusion is not produced by the latter procedure. It was found that transplantation experiments carried out by the two procedures gave significantly different results. The frequency of head formation by the transplanted tissue on the host was significantly higher by the lateral than by the axial grafting procedure. This suggests that the small protrusion introduced onto the smooth cylindrical structure of the host body column by the former, but not by the latter, procedure plays an important role in determining the fate of the transplant. It is suggested that the cooperation of two factors determines the transplantation result. One is the positional factor described and discussed by previous workers (e.g., Wolpert et al., 1974; Sugiyama, 1982; MacWilliams, 1983, MacWilliams, 1983; Takano and Sugiyama, 1983) and the other is the structural factor revealed in this study. The underlying mechanisms responsible for the latter factor are discussed.     

The process of pharynx regeneration in planarians.

To understand the cellular events during planarian regeneration, we analyzed the process of pharynx regeneration in both head and tail pieces using cell-type-specific markers. Interestingly, cells expressing the pharynx-muscle-specific myosin heavy chain gene (DjMHC-A) appeared within 24 h after amputation (prior to the formation of a pharynx rudiment) in the mesenchymal space of the stump, not in the blastema region. These DjMHC-A-positive cells migrated to the midline and formed the pharynx rudiment. Even after formation of the pharynx rudiment, DjMHC-A-positive cells constantly appeared in the mesenchymal space in the region surrounding the pharynx rudiment and participated in the growth of the pharynx rudiment. These observations clearly indicated that the cells involved in pharynx-muscle formation are committed in the mesenchymal space of the stump, rather than in the blastema region or the pharynx rudiment during planarian regeneration. We also analyzed the process of regeneration of the pharynx epithelia using a monoclonal antibody and investigated the origin of the pharynx epithelia.     

A developmental pathway controlling outgrowth of the Xenopus tail bud

We have developed a new assay to identify factors promoting formation and outgrowth of the tail bud. A piece of animal cap filled with the test mRNAs is grafted into the posterior region of the neural plate of a host embryo. With this assay we show that expression of a constitutively active Notch (Notch ICD) in the posterior neural plate is sufficient to produce an ectopic tail consisting of neural tube and fin. The ectopic tails express the evenskipped homologue Xhox3, a marker for the distal tail tip. Xhox3 will also induce formation of an ectopic tail in our assay. We show that an antimorphic version of Xhox3, Xhox3VP16, will prevent tail formation by Notch ICD, showing that Xhox3 is downstream of Notch signalling. An inducible version of this reagent, Xhox3VP16GR, specifically blocks tail formation when induced in tailbud stage embryos, comfirming the importance of Xhox3 for tail bud outgrowth in normal development. Grafts containing Notch ICD will only form tails if placed in the posterior part of the neural plate. However, if Xwnt3a is also present in the grafts they can form tails at any anteroposterior level. Since Xwnt3a expression is localised appropriately in the posterior at the time of tail bud formation it is likely to be responsible for restricting tail forming competence to the posterior neural plate in our assay. Combined expression of Xwnt3a and active Notch in animal cap explants is sufficient to induce Xhox3, provoke elongation and form neural tubes. Conservation of gene expression in the tail bud of other vertebrates suggests that this pathway may describe a general mechanism controlling tail outgrowth and secondary neurulation.     

Xenopus Wnt-5a induces an ectopic larval tail at injured site, suggesting a crucial role for noncanonical Wnt signal in tail regeneration

Amputation of the larval tail of Xenopus injures the notochord, spinal cord, muscle masses, mesenchyme, and epidermis, induces the growth and differentiation of cells in those tissues, and results in tail regeneration. A dorsal incision in the larval tail injures the same tissues and induces cell growth and differentiation, but never results in the formation of any extra appendages. The first sign of tail regeneration is the multilayered wound epidermis and Xwnt-5a expression in the distal region, neither of which is observed in the recovering region after a dorsal incision. To evaluate the role of Xwnt-5a in tail regeneration, Xwnt-5a was overexpressed in the recovering region. When an animal cap injected with Xwnt-5a mRNA was grafted into the dorsal incision, an ectopic protrusion was formed. Morphological and molecular analyses revealed that the protrusion was an ectopic larval tail, which was equivalent to the regenerating tail but different from the tail that develops from the embryonic tail bud. Lineage labeling revealed that the major differentiated structures of the ectopic tail were formed from host cells, suggesting that Xwnt-5a induced host cells to make a complete tail. The ectopic tail was not induced by Xwnt-8 or Xwnt-11, demonstrating the specificity of Xwnt-5a in this process. A pharmacological study showed that JNK signaling is required in tail regeneration. These results support the proposition that Xwnt-5a plays an instructive role in larval tail regeneration via Wnt/JNK signaling.     

Feather buds exert a polarizing activity when transplanted to chick limb buds

Homeoproteins have been shown to be expressed in a position-specific manner along the anterior-posterior axis in the developing chick feather bud, as seen also in the developing limb bud. These facts raise the possibility that there may be common mechanistic features in the establishment of the anterior-posterior polarity between both organs. In order to investigate this possibility, feather bud tissues were transplanted into the anterior region of limb buds to determine whether feather bud tissues possess properties such as the zone of polarizing activity of the limb bud. The manipulated limb bud formed a mirror image duplication of the skeletal elements, mainly (2)2234 digit pattern or sometimes 3(2)234. Both the anterior and posterior halves of feather bud tissue exhibited almost equal activity in inducing ectopic skeletal elements. Hox d-12 and Hox a-13 were expressed coordinately around the transplanted site of the operated limb bud. This secondary axis-inducing activity of the feather bud was enhanced when grafts were pretreated with trypsin. In contrast, the presumptive feather bud tissue and inter-feather bud tissue did not induce a secondary axis of the limb bud. These results suggest that the feather bud contains a region that exerts polarizing activity and that this region may play key roles in the formation of the anterior-posterior and, if it exists, proximal-distal axis of the feather bud, possibly via the regulation of region specific expression of Hox genes.     

Behavioral plasticity and central regeneration of locomotor reflexes in the freshwater oligochaete, Lumbriculus variegatus. II: Ablation studies

Abstract. After 8–10 segments of posterior ventral nerve cord were ablated in Lumbriculus variegatus , touch-evoked locomotor responses were evident both in segments anterior and posterior to the ablation site. However, responses in these two regions were independent and uncoupled. During recovery, four outcomes were observed at the ablation site: (Group 1) recovery of normal functions with no growth of new segments; (Group 2) formation of a laterally protruding, multi-segmented, ectopic head; (Group 3) formation of a laterally protruding, amorphous, and multi-segmented outgrowth; and (Group 4) segmental autotomy. In Groups 1 and 2, touch-evoked swimming and body reversal were studied. In addition, sensory fields and conduction properties of giant nerve fibers were examined near the ablation site. In some Group 1 worms, clear-cut behavioral and electrical signs of recovery and reconnection were seen by 3 d after ablation. By 8 d, all worms had recovered and exhibited response patterns comparable to those of normal worms. In Group 2 worms, with an ectopic head, segments posterior to the ablation (together with those in the ectopic head), exhibited touch-evoked swimming and body reversal responses resembling those of a complete worm. Segments anterior to the ectopic head were independently capable of locomotor responses. Medial and lateral giant fiber sensory fields in worms with ectopic heads reflected a pattern expected for two worms. Thus, through apparent morphallactic reorganization, a medial giant fiber sensory field emerged which included the ectopic head and 10–15 adjacent posterior segments. In contrast, electrical recordings showed longitudinal through-conduction of giant fiber spikes, across the ablation site. Histological examination revealed that the giant nerve fibers in the ectopic head were complexly interconnected with those in the main body axis.     

Secondary embryonic axis formation by transplantation of D quadrant micromeres in an oligochaete annelid

Among spiral cleaving embryos (e.g. mollusks and annelids), it has long been known that one blastomere at the four-cell stage, the D cell, and its direct descendants play an important role in axial pattern formation. Various studies have suggested that the D quadrant acts as the organizer of the embryonic axes in annelids, although this has never been demonstrated directly. Here we show that D quadrant micromeres (2d and 4d) of the oligochaete annelid Tubifex tubifex are essential for embryonic axis formation. When 2d and 4d were ablated the embryo developed into a rounded cell mass covered with an epithelial cell sheet. To examine whether 2d and 4d are sufficient for axis formation they were transplanted to an ectopic position in an otherwise intact embryo. The reconstituted embryo formed a secondary embryonic axis with a duplicated head and/or tail. Cell lineage analyses showed that neuroectoderm and mesoderm along the secondary axis were derived from the transplanted D quadrant micromeres and not from the host embryo. However, endodermal tissue along the secondary axis originated from the host embryo. Interestingly, when either 2d or 4d was transplanted separately to host embryos, the reconstituted embryos failed to form a secondary axis, suggesting that both 2d and 4d are required for secondary axis formation. Thus, the Tubifex D quadrant micromeres have the ability to organize axis formation, but they lack the ability to induce neuroectodermal tissues, a characteristic common to chordate primary embryonic organizers.     

Embryonic origin and Hox status determine progenitor cell fate during adult bone regeneration

The fetal skeleton arises from neural crest and from mesoderm. Here, we provide evidence that each lineage contributes a unique stem cell population to the regeneration of injured adult bones. Using Wnt1Cre::Z/EG mice we found that the neural crest-derived mandible heals with neural crest-derived skeletal stem cells, whereas the mesoderm-derived tibia heals with mesoderm-derived stem cells. We tested whether skeletal stem cells from each lineage were functionally interchangeable by grafting mesoderm-derived cells into mandibular defects, and vice versa. All of the grafting scenarios, except one, healed through the direct differentiation of skeletal stem cells into osteoblasts; when mesoderm-derived cells were transplanted into tibial defects they differentiated into osteoblasts but when transplanted into mandibular defects they differentiated into chondrocytes. A mismatch between the Hox gene expression status of the host and donor cells might be responsible for this aberration in bone repair. We found that initially, mandibular skeletal progenitor cells are Hox-negative but that they adopt a Hoxa11-positive profile when transplanted into a tibial defect. Conversely, tibial skeletal progenitor cells are Hox-positive and maintain this Hox status even when transplanted into a Hox-negative mandibular defect. Skeletal progenitor cells from the two lineages also show differences in osteogenic potential and proliferation, which translate into more robust in vivo bone regeneration by neural crest-derived cells. Thus, embryonic origin and Hox gene expression status distinguish neural crest-derived from mesoderm-derived skeletal progenitor cells, and both characteristics influence the process of adult bone regeneration.     

On the Role of the Reticular-epithelial Complex in Transplantation of the Thymus

Transplantation of the neonatal thymus, into young, adult hosts, resulted in massive cell death of graft cortical lymphoid tissue with apparent selective survival of the reticular-epithelial cells. The central area of the graft was progressively cleared of cell debris and the characteristic thymic architecture restored within fourteen days of grafting. Evidence obtained from the regeneration of different-sized transplants suggested that the size and shape attained by the regenerated graft was closely related to the size and shape of the donor tissue.
When donor rat thymuses were transplanted in Millipore chambers, the lymphocyte population did not reappear and after seven days only reticular-epithelial cells remained, retaining their normal appearance. However, when these thymic remnants were removed from the chambers and transplanted into secondary hosts, the thymus regenerated normally, suggesting that the lymphocytes in the regenerated gland were derived from the host. Thymic remnants after cortisol treatment of donors also formed distinct organs after grafting despite the fact that they contained few donor lymphocytes. From the differential effects of cortisol on host and transplanted thymus and the different growth characteristics of transplants it appears that transplants differ in their growth/involution control system from the host thymus.     

A Hox 3.3-lacZ transgene expressed in developing limbs.

We describe transgenic mouse lines that express lacZ under the control of the Hox 3.3 Promoter II. The correct anterior boundary can be fixed by 3.6 kb of promoter DNA (plus 1.6 kb of 5' transcribed sequences), both in tissues of ectodermal and mesodermal origin. The posterior border, however, is not respected, and lacZ expression continues into the tail region. One line has particularly strong graded expression in the anterior proximal limb bud. Other lines, containing a shorter promoter fragment (0.6 kb), have ectopic expression in the head region, including one line that has expression in the anterior half of the retina. Such mouse lines make it possible to molecularly distinguish cells in regions of the embryo that look otherwise identical and may be useful in studying the establishment of molecular differences in the mouse embryo.     

Time Space Translation: A Hox Mechanism for Vertebrate A-P Patterning

The vertebrate A-P axis is a time axis. The head is made first and more and more posterior levels are made at later and later stages. This is different to the situation in most other animals, for example, in Drosophila. Central to this timing is Hox temporal collinearity (see below). This occurs rarely in the animal kingdom but is characteristic of vertebrates and is used to generate the primary axial Hox pattern using time space translation and to integrate successive derived patterns (see below). This is thus a different situation than in Drosophila, where the primary pattern guiding Hox spatial collinearity is generated externally, by the gap and segmentation genes.     

Ultrastructural localization of epididymal secretory proteins associated with the surface of spermatozoa from rabbit cauda epididymis

Summary Antibodies against whole rabbit epididymal fluid as well as against three purified proteins from this fluid (namely EP21, EP35 and uteroglobin) were prepared and characterized by Western immunoblot. These antibodies were used to study the association of those proteins to the spermatozoon by means of immunoelectron microscopy using a colloidal gold-labelling technique. Antibodies against whole fluid intensely stained the spermatozoon surface at the acrosomal and postacrosomal regions of the head and at the middle piece of the tail. The equatorial region and the principal piece were much less labelled. The EP21 antigen associated with the whole surface of the head and the middle piece but not with the principal piece of the tail. EP35 was distributed over the acrosomal but not the postacrosomal region. The principal piece also contained this antigen in considerable amounts. The antibody against uteroglobin did not stain the head surface but intensely labelled both the middle and principal pieces.     

Males of a solitary wasp possess a postpharyngeal gland

The postpharyngeal gland has long been thought to occur only in ants. Here we characterize, by use of light and electron microscopy as well as 3D reconstruction based on nuclear magnetic resonance (NMR) imaging data, a large cephalic gland reservoir of males of a solitary digger wasp, the European beewolf, Philanthus triangulum. Several lines of evidence suggest that this reservoir is a postpharyngeal gland. The gland reservoir originates from the posterior part of the pharynx and consists of two pairs of unbranched tubular structures that occupy a large portion of the head capsule. Its wall is composed of a unicellular epithelium that is lined by a cuticle. The gland contains a blend of hydrocarbons and compounds with functional groups, and we show that the hydrocarbon fraction of the pheromone is congruent with the hydrocarbons on the cuticle. We discuss the implications of our findings for the evolution of the postpharyngeal gland in ants.