Genetic analysis of developmental mechanisms in hydra: XIV. Identification of the cell lineages responsible for the altered developmental gradients in a mutant strain,reg-16

The developmental gradients of six chimeric strains of hydra produced from a normal strain (105) and a regeneration-deficient strain (reg-16) were analyzed. The reg-16 mutant has been shown to have a lower gradient of head-activation potential and a higher gradient of head-inhibition potential than the normal 105 strain. The chimeric animals consisted of different combinations of the three self-renewing cell lineages found in hydra (the ectodermal and endodermal epithelial cell lineages and the interstitial cell lineage) from each of the parental origins. To identify the cell lineages responsible for the abnormal gradients in reg-16, the head-activation and head-inhibition potentials of these cell lineage chimeras were assayed by lateral transplantation of tissue. The results obtained have provided evidence which indicates that the defect responsible for the low head-activation potential in reg-16 resides in its ectodermal and endodermal epithelial cell lineages, whereas the defect responsible for its high head-inhibition potential resides in its endodermal epithelial and interstitial cell lineages. The cellular localization of these defects is not identical but very similar to the cellular localization of the regenerative defects in reg-16. This finding is consistent with and supports the view that the abnormalities of the developmental gradients are correlated to the reduced head regenerative capacity in reg-16.     

Role of interstitial cell migration in generating position-dependent patterns of nerve cell differentiation in Hydra

The role of interstitial cell migration in the formation of newly differentiated nerve cells was examined during head regeneration in Hydra magnipapillata. When distal tissue was removed from the body of a wild-type strain (105), nerve cell differentiation occurred at a rapid rate during the first 48 hr of regeneration, slowing after this point. Rapid nerve cell differentiation was due primarily to migration of interstitial cells, some of which appeared to be nerve cell precursors, into the regenerating head. The migration decreased considerably after the first 48 hr of regeneration. In reg-16, a mutant strain deficient in head regeneration, no migration of interstitial cells and hence no new nerve cell differentiation were observed in the regenerating tip. However, the interstitial cells of reg-16 were observed to migrate into regenerating tissue of strain 105. These observations suggest that the migration of nerve cell precursors plays an important role when the new nerve net is being established during head regeneration.     

Analysis of morphogenetic mutants of hydra

The mutantreg-16 is deficient in head regeneration and abnormal in size regulation. The gastric region becomes twice as long as that of normal animals before the first bud is produced. Both mutant characteristics are due to changes in head-specific morphogen concentrations.Reg-16 contains twice as much head inhibitor and only half as much head activator in its head as normal animals. This leads to a higher level of free head inhibitor in the whole animal resulting on one hand in a greater distance of buds from the head, and on the other hand in a total blockage of release of head activator and head inhibitor which would be necessary to initiate head regeneration.     

Genetic Analysis of Developmental Mechanisms in Hydra. XV. Nematocyte Differentiation in Strains with Altered Developmental Gradients*

Nematocyte differentiation from the interstitial stem cells in hydra occurs non-uniformly along the body column. The relative ratios of the 4 nematocyte types produced vary gradually from head to foot along the body axis (Bode and Smith, 1977). To find out whether this regional variation in nematocyte differentiation along the body column is related to the gradients of the head-activation and head-inhibition potentials, nematocyte differentiation patterns were examined in strains which have significantly different developmental gradients along their body columns. Five strains of hydra, including a wild-type, two mutant strains and two chimeric (mutnt/wild-type) strains, were investigated. It was found that the regional variations in the nematocyte differentiation were similar in all the strains examined, and that no significant differences of the variation existed that could be attributed to the differences of the developmental gradients in these strains. This suggests that nematocyte differentiation is strongly affected by the axial position along the body column, but that the gradients of the morphogenetic potentials involved in head formation are not involved in this effect. Instead, some other parameter(s) of axial position not directly associated with these gradients must be responsible for the positional effect on nematocyte differentiation.     

Inhibitory and stimulatory effects of limb ectoderm on in vitro chondrogenesis

Previous studies have indicated possible dual effects of the limb ectoderm in cartilage differentiation. On one hand, explants from early (stage 15) wing buds are dependent on contact with the limb ectoderm for cartilage differentiation (Gumpel-Pinot, J. Embryol. Exp. Morph. 59:157-173, 1980). On the other hand, limb ectoderm from stage 23/24 wing buds inhibits cartilage differentiation by cultured limb mesenchyme cells even without direct contact (Solursh et al., Dev. Biol. 86:471-482, 1981). In the present study, ectoderms from both stage 15/16 and stage 23/24 wings are cultured under the same conditions, and ectoderms from each source are shown to have two effects. Each stimulates chondrogenesis in stage 15 wing bud mesenchyme, and each inhibits chondrogenesis in older wing mesenchyme. The results suggest that the limb ectoderm has at least dual effects on cartilage differentiation, depending on the stage of the mesenchyme. One effect involves an early mesenchymal dependence on the ectoderm. This effect requires contact between the ectoderm and mesoderm (Gumpel-Pinot, J. Embryol. Exp. Morphol. 59:157-173, 1980) but also can be observed at a distance from the ectoderm. Later, the ectoderm can act without direct contact between the ectoderm and mesoderm to inhibit chondrogenesis over some distance.     

Roles of Head-Activation and Head-Inhibition Potentials in Pattern Formation of Hydra: Analysis of a Multi-Headed Mutant Strain

SYNOPSIS. Lateral grafting of tissue was used to compare therelative head-activation and head-inhibition potentials of differentHydra strains. A small piece of tissue taken from one polyp,when grafted to another polyp, induces formation of a head structurewhen the relative head-activation potential of the donor tissueis sufficiently (i.e., more than some threshold value) higherthan the relative head-inhibition potential of the recipienttissue. It was found that a multi-headed mutant strain (mh-1),which produces many extra heads along its body column, has significantlyhigher head-activation and significantly lower head-inhibitionpotentials than the standard wild-type strain. This suggeststhat these potentials play important roles in hydra morphogenesis,and that an imbalance between the two potentials is responsiblefor the developmental abnormality of mh-1. The significanceof this finding is discussed in light of the "positional information"model proposed by Wolpert and his associates and the "lateralinhibition" model proposed by Gierer and Meinhardt.     

Cytoplasmic factors determining anteroposterior polarity in Drosophila embryos

Summary Cytoplasm removal/transplant techniques applied to Drosophila cleavage-stage embryos induced changes in anteroposterior polarity. Removal of anterior cytoplasm or anterior transplantation of posterior cytoplasm caused the anterior formation of posterior (telson) structures, and the replacement of anterior cytoplasm with posterior cytoplasm induced double-abdomen embryos, as reported by Frohnhöfer et al. [J Embryol Exp Morphol 97 (suppl):169–179 (1986)]. Changing the conditions of anterior cytoplasm removal we showed that greater volumes, earlier stages, and removal from the periphery were efficient. In addition we found that double-cephalon embryos are induced by replacing posterior cytoplasm with anterior cytoplasm, while removal of posterior cytoplasm or the posterior transplantation of anterior cytoplasm was without effect. However, introduction of anterior cytoplasm into the posterior of nanos embryos, which are mutants not developing abdominal segments, caused the formation of double-cephalon embryos. Similarly, double-abdomen embryos are produced by introducing posterior cytoplasm into the anterior of bicoid embryos, which are mutants not forming cephalic and thoracic structures. These results are compatible with the initial involvement of separate anterior, posterior and terminal cytoplasmic factors deduced from mutant analysis (Nüsslein-Volhard and Roth 1989).     

Pattern regulation properties of a Hydra strain which produces additional heads along the body axis

The multiheaded one (mh-1) strain, isolated from inbred crossings of wild type Hydra magnipapillata, develops additional heads along the body axis. This strain reproduces asexually by budding like the wild type (wt) does. We found that young polyps have a wt-like shape and display wt-like properties. When they grow in size and before they produce extra heads along the body axis, the tissue between the head and the budding zone changes its property: in this region, where later on the extra heads preferentially form, foot regeneration is significantly delayed while head regeneration remains unaffected. Further, following various transplantations additional heads form under conditions under which the wild type did not. The observed changes in pattern control and regulation indicate a two-step process of pattern formation. Morphogenetic signalling is suggested to cause the positional value to increase slowly in the form of patches and preferentially in the region between the head and the budding zone. This increase causes an altered morphogenetic signalling, which is eventually responsible for additional head formation.     

A mutant strain of Polysphondylium pallidum deficient in production of cyclic AMP.

A mutant strain (PN507) of the cellular slime mold Polysphondylium pallidum is described which: (a) is morphogenetically abnormal in stalk formation; (b) secretes unusually low quantities of cyclic AMP; (c) responds to exogenous cyclic AMP in the same manner as wild type, by differentiating stalk cells and synthesizing several specific proteins; (d) complements with other morphogenetic mutants secreting normal amounts of cyclic AMP to produce fruiting structures resembling wild type. The tentative conclusion is that the critical defect of PN507 is low production of cyclic AMP.     

The role of tissue interactions in the skeletogenic differentiation of avian neural crest cells

In the avian embryo, cranial neural crest (NC) cells migrate extensively throughout the head region and give rise to most of the cranial skeleton (Le Lievre, C. S. (1978). J. Embryol. Exp. Morphol.47, 17–37). To investigate the skeletogenic differentiation of these cells, NC explants from the mesencephalic level of st. 9+ embryos were grown in standard organ culture on Millipore filter substrates either in isolation or in combination with those tissues with which the cells normally associate during their in vivo migration and at their final tissue sites. The results demonstrate that interaction between premigratory NC and cranial ectoderm leads to chondrogenic differentiation of NC cells. Combination of premigratory NC with presumptive site tissues led to a pattern of NC cell differentiation normally expressed after in vivo migration: Combinations of NC with retinal pigmented epithelia gave cartilage, whereas NC with maxillary ectoderm formed cartilage and membrane bone. Both resulting skeletal tissues possessed their characteristic collagen types (II in cartilage and I in bone) as shown by indirect immunofluorescence using antibodies raised against specific types of collagen. It is concluded that avian cephalic NC cells require tissue interactions if chondrogenic and osteogenic differentiation is to ensue, but that migration per se is not an absolute prerequisite for these types of differentiation. The degree of specificity underlying such interactions is discussed.     

Separation and specificity of action of four morphogens from hydra

Summary A procedure is presented by which four previously described morphogenetic substances can be purified from hydra: an activator and an inhibitor of head formation and an activator and an inhibitor of foot formation. We show that all four substances act specifically. At low concentrations, the head factors only influence head and not foot formation, and the foot factors only influence foot and not head formation.     

An ultrastructural study of differentiation of pyriform cells and their contribution to oocyte growth in representative squamata.

An electron microscopic study of the differentiation of pyriform cells and their contribution to oocyte growth in three lizards (Tarentola mauritanica, Cordylus wittifer, Platysaurus intermedius) and one colubrid snake (Coluber viridiflavus) revealed that pyriform cells differentiate from small follicle cells via intermediate cells after establishing an intercellular bridge with the oocyte (see also Hubert: Bull Soc Zool Fr 102:151-158, 1977; Filosa et al: J Embryol Exp Morphol 54:5-15 1979; Klosterman: J Morphol 192:125-144, 1987). Once differentiated, pyriform cells display ultrastructural features indicative of synthetic activity, including abundant ribosomes, Golgi membranes, vacuoles, mitochondria, and lipid droplets. These cellular components extend to the apex of the cell at the level of the intercellular bridge, suggesting that constituents of pyriform cells may be transferred to the oocyte. Furthermore, we demonstrate for the first time that pyriform cells incorporate exogenous yolk. The yolk is segregated inside maturing yolk granules that form in the pyriform cell in the same manner as described for vitellogenic oocytes in non-mammalian vertebrates (see Wallace: Developmental Biology, A Comprehensive Synthesis 127-177, 1985). It is the first clear evidence that pyriform cells and the oocyte may fulfill similar vitellogenic functions. The establishment of an intercellular bridge may represent a crucial event in the development of an integrated system in which pyriform cells and oocyte cooperate.     

Formation of the epicardium studied with the scanning electron microscope

The topographical characteristics of epicardial and myocardial cells of the embryonic chick heart are sufficiently distinct to enable reproducible identification by scanning electron microscopy. This made it possible to observe the development of the epicardial investment on the myocardial wall. The epicardial cells migrate from the mesothelium of the sinus venosus, cover first circularly the ventricular wall, and then extend cranially and caudally to ensheath the entire heart surface. Our observation argues against the generally accepted concept that the epicardium is a derivative of the outer myocardial layer. We strongly support the suggestion that the term “epimyocardium” is a misnomer [Manasek, F. J. (1969). J. Embryol. Exp. Morphol.22, 333–348].     

The extracellular matrix between the optic vesicle and presumptive lens during lens morphogenesis in an anophthalmic strain of mice

Extracellular matrix material present during early lens morphogenesis in anophthalmic strain ZRDCT-Ch mice was studied histochemically by the Alcian blue 8GX pH 2.5, Alcian blue 8GX pH 2.5/periodic acid-Schiff combined, high iron diamine, and Van Gieson methods. Observed staining patterns were compared with results from an analysis of a normal strain of mice (E.H. Webster, Jr., A.F. Silver, and N.I. Gonsalves, 1983, Develop. Biol. 100, 147-157). No differences in constituents were found between the strains in staining patterns of the ectodermal basal lamina. However, the optic vesicle basal lamina in the anophthalmic strain was found to have a relatively lower staining intensity for sulfated glycosaminoglycan associated with it than was observed in the normal strain, although these mutant optic vesicles were morphologically normal. Results from this and the earlier study on normal mice indicate that one function of sulfated glycosaminoglycan in early lens morphogenesis may be to serve as a cementing medium between the optic and lens rudiments. This sulfated glycosaminoglycan deficiency on the anophthalmic optic vesicle basal lamina is temporally correlated with and may be causally related to precocious lens cup formation and frequently observed separation of the normally adherent eye rudiments. Conclusions drawn from this study are consistent with the speculation of H.B. Chase and E.B. Chase (1941, J. Morphol. 68, 279-301) that there may be abnormal contact between the optic vesicle and presumptive lens ectoderm in the mutant strain, although there is a differing view on the cause of the abnormal contact.     

Skin sensory innervation patterns in embryonic chick hindlimbs deprived of motoneurons

During normal development and following a variety of experimental manipulations (e.g., neural tube rotations, limb shifts), sensory neurons in the chick grow to their correct targets. L. Landmesser and M. G. Honig (1986, Dev. Biol. 118, 511-531) have suggested that sensory innervation may be precise, not because sensory neurons respond to limb-derived guidance cues, but because sensory neurons interact with motoneurons, which do respond to such cues. To test this hypothesis for skin sensory neurons, the ventral neural tube, including the motoneuron precursors, was removed from chick embryos prior to sensory axon outgrowth and the resulting patterns of dermatomes and axonal projections were mapped physiologically and anatomically. As reported previously, dorsal root ganglia (DRGs) and cutaneous nerves formed in their usual locations following the early removal of motoneurons, while most muscle nerves and the plexus region were reduced substantially (A. C. Taylor, 1944, J. Exp. Zool. 96, 159-185; L. Landmesser and M. G. Honig, 1986, Dev. Biol. 118, 511-531; G. J. Swanson and J. Lewis, 1986, J. Embryol. Exp. Morphol. 95, 37-52). The patterns of axonal projections and dermatomes were surprisingly, although not entirely, normal. In particular, cutaneous nerves in motoneuron-depleted embryos were derived from the same DRGs in approximately the same proportions as normal. Thus, while motoneurons may play a facilitative role in the development of the segmental pattern of skin sensory innervation, they do not appear to be essential.     

Mechanics of head fold formation: investigating tissue-level forces during early development

During its earliest stages, the avian embryo is approximately planar. Through a complex series of folds, this flat geometry is transformed into the intricate three-dimensional structure of the developing organism. Formation of the head fold (HF) is the first step in this cascading sequence of out-of-plane tissue folds. The HF establishes the anterior extent of the embryo and initiates heart, foregut and brain development. Here, we use a combination of computational modeling and experiments to determine the physical forces that drive HF formation. Using chick embryos cultured ex ovo, we measured: (1) changes in tissue morphology in living embryos using optical coherence tomography (OCT); (2) morphogenetic strains (deformations) through the tracking of tissue labels; and (3) regional tissue stresses using changes in the geometry of circular wounds punched through the blastoderm. To determine the physical mechanisms that generate the HF, we created a three-dimensional computational model of the early embryo, consisting of pseudoelastic plates representing the blastoderm and vitelline membrane. Based on previous experimental findings, we simulated the following morphogenetic mechanisms: (1) convergent extension in the neural plate (NP); (2) cell wedging along the anterior NP border; and (3) autonomous in-plane deformations outside the NP. Our numerical predictions agree relatively well with the observed morphology, as well as with our measured stress and strain distributions. The model also predicts the abnormal tissue geometries produced when development is mechanically perturbed. Taken together, the results suggest that the proposed morphogenetic mechanisms provide the main tissue-level forces that drive HF formation.     

Analysis of head and foot formation inHydra by means of an endogenous inhibitor

Summary In tissue regenerating the head, the ability to initiate head formation in a host increases with the time allowed for regeneration before grafting, while the foot-initiating ability decreases concomitantly. The reverse was found for tissue about to regenerate a foot. The early divergent changes thus indicated are counteracted in both head and foot regeneration by treatment with an inhibitor (Berking, 1977) in low concentrations.The inhibitor also interferes with processes which determine wether or not hypostome and tentacles are formed, and how many tentacles (if any) appear. The circumferential spacing of the tentacles was regular whether their number was normal or below normal.Secondary axes caused by implanted tissue either detach after having formed a head and a foot (i.e. behave like buds) or do not detach, having only formed a head. This alternative depends on the origin and amount of the implanted tissue and on the position of the implant within the host.The following model based on these findings is proposed: Head and foot formation start with pre-patterns which cause a continuously increasing change of the tissue's ability to initiate a head or a foot. Along the body axis this ability is determined by a graded distribution of sources. As development progresses, the high source density which accumulates in the head region causes the formation of a hypostome and tentacles; the angular spacing of tentacles is also dependent on source density. At a certain low source density foot-formation is initiated. The inhibitor counteracts the increase of source density in head-forming tissue as well as the decrease of source density in foot-forming tissue. It thus appears to be part of the mechanism which controls morphogenesis in hydra.     

Isolation of a substance activating foot formation in hydra

We have developed an assay for a substance from hydra that accelerates foot regeneration in the animal. This substance is specific for the foot as evidenced by the following findings: (1) It is present in the animal as a steep gradient descending from foot to head, paralleling the foot-forming potential of the tissue (2) It does not accelerate head regeneration, nor do the head factors of hydra discovered by Schaller (1973) and Berking (1977) accelerate foot regeneration. We propose that the foot-activating substance is a morphogen responsible for foot formation in hydra. The foot activator can be extracted from hydra tissue with methanol and separated from other known morphogens of hydra by gel filtration and ion-exchange chromatography. A substance with similar biological and physicochemical properties can be isolated from sea anemones.     

Interactions between the foot and bud patterning systems in Hydra vulgaris.

In the freshwater coelenterate, hydra, asexual reproduction via budding occurs at the base of the gastric region about two-thirds of the distance from the head to the foot. Developmental gradients of head and foot activation and inhibition originating from these organizing centers have long been assumed to control budding in hydra. Much has been learned over the years about these developmental gradients and axial pattern formation, and in particular, the inhibitory influence of the head on budding is well documented. However, understanding of the role of the foot and potential interactions between the foot, bud, and head patterning systems is lacking. The purpose of this study was to investigate the role of the foot in the initiation of new axis formation during budding by manipulating the foot and monitoring effects on the onset of first bud evagination and the time necessary to reach the 50% budding point. Several experimental situations were examined: the lower peduncle and foot (PF) were injured or removed, a second PF was laterally grafted onto animals either basally (below the budding zone) or apically (above the budding zone), or both the head and PF were removed simultaneously. When the PF was injured or removed, the onset of first bud evagination was delayed and/or the time until the 50% budding point was reached was longer. The effects were more pronounced when the manipulation was performed closer to the anticipated onset of budding. When PF tissue was doubled, precocious bud evagination was induced, regardless of graft location. Removal of the PF at the same time as decapitation reduced the inductive effect of decapitation on bud evagination. These results are discussed in light of potential signals from the foot or interactions between the foot and head patterning systems that might influence bud axis initiation.