The effect of retinoic acid on positional memory in the dorsoventral axis of regenerating axolotl limbs

We investigated the effect of retinoic acid (RA) on pattern regulation in the dorsoventral (DV) axis of regenerating axolotl limbs. Half and double half dorsal and ventral zeugopodia (lower arms or legs) were amputated through their distal ends, and 4 days later the animals were injected intraperitoneally with 50 (large animals) or 100 (small animals) micrograms RA/g body wt. Half and double half dorsal and ventral zeugopodia of uninjected axolotls, and sham-operated zeugopodia of untreated and RA-treated limbs served as controls. Skeletal patterns and the DV muscle patterns of control and experimental regenerates were then analyzed. Sham-operated zeugopodia of uninjected animals regenerated normally. Sham-operated, RA-treated zeugopodia regenerated normally with proximodistal duplications. Sixty percent of uninjected control dorsal half zeugopodia, 80% of control ventral half zeugopodia, and 100% of control double dorsal and double ventral zeugopodia regenerated distally, but the regenerates did not reconstitute the muscle pattern of the missing half. Thirty-eight percent of RA-treated ventral half zeugopodia and 78% of RA-treated double ventral zeugopodia failed to regenerate distally. Of those cases that did regenerate distally, none regenerated the muscle pattern of the missing half. By contrast, 100% of RA-treated dorsal half zeugopodia regenerated distally and all completed the normal DV muscle pattern. Forty-one percent of RA-treated double dorsal zeugopodia failed to regenerate, but of the remainder that did regenerate, 50% completed the normal DV muscle pattern. These represented eight cases, six of which regenerated single limbs, and two of which regenerated twin limbs, each with a normal DV muscle pattern. We interpret these data to mean that RA ventralizes the positional memory of blastema cells in the DV axis.     

Retinoic acid in the formation of the dorsoventral retina and its central projections

Dynamic expression patterns of four retinoid-metabolizing enzymes create rapidly changing retinoic acid (RA) patterns in the emerging eye anlage of the mouse. First, a RA-rich ventral zone is set up, then a RA-poor dorsal zone, and finally a tripartite organization consisting of dorsal and ventral RA-rich zones separated by a horizontal RA-poor stripe. This subdivision of the retina into three RA concentration zones is directly visible as beta-galactosidase labeling patterns in retinas of RA-reporter mice. Because the axons of retinal ganglion cells transport the reporter product anterogradely, the central projections from dorsal and ventral retina can be visualized as two heavily labeled axon bundles. Comparisons of the axonal labeling with physiologic recordings of visual topography in the adult mouse show that the labeled axons represent the upper and the lower visual fields. The RA-poor stripe develops into a broad horizontal zone of higher visual acuity. Comparisons of the retina labeling with eye-muscle insertions show that the axis of the RA pattern lines up with the dorsoventral axis of the oculomotor system. These observations indicate that the dorsoventral axis of the embryonic eye anlage determines the functional coordinates of both vision and eye movements in the adult.     

Separable binding proteins for retinoic acid and retinol in bovine retina.

Binding proteins for retinoic acid and retinol were separated from a supernatant prepared from bovine retina. Fraction IV from DEAE-cellulose chromatography bound exogenous [3H] retinoic acid which could not be effectively displaced by retinol, retinal, retinyl acetate or palmitate, but which was readily displaced with excess retinoic acid. [3H] Retinol was bound by fraction V from DEAE-cellulose chromatography and was not displaced by retinal, retinoic acid, retinyl acetate or retinyl palmitate, but was readily displaced by excess retinol. Unlike bovine serum retinol-binding protein, neither intracellular binding protein formed a complex with purified human serum prealbumin. The supernatant from bovine retinas was estimated to contain five times more retinoic acid binding than retinol binder.     

Extracellular modulation of BMP activity in patterning the dorsoventral axis

Signaling via bone morphogenetic proteins (BMPs) regulates a vast array of diverse biological processes in the developing embryo and in postembryonic life. Many insights into BMP signaling derive from studies of the BMP signaling gradients that pattern cell fates along the embryonic dorsal-ventral (DV) axis of both vertebrates and invertebrates. This review examines recent developments in the field of DV patterning by BMP signaling, focusing on extracellular modulation as a key mechanism in the formation of BMP signaling gradients in Drosophila, Xenopus, and zebrafish.     

Microtubules and specification of the dorsoventral axis in frog embryos

The body plan of the frog is set-up by a rearrangement of the egg cytoplasm shortly after fertilization. Microtubules play several roles in this critical developmental event.     

Conserved mechanism of dorsoventral axis determination in equal-cleaving spiralians

Many members of the spiralian phyla (i.e., annelids, echiurans, vestimentiferans, molluscs, sipunculids, nemerteans, polyclad turbellarians, gnathostomulids, mesozoans) exhibit early, equal cleavage divisions. In the case of the equal-cleaving molluscs, animal-vegetal inductive interactions between the derivatives of the first quartet micromeres and the vegetal macromeres specify which macromere becomes the 3D cell during the interval between fifth and sixth cleavage. The 3D macromere serves as a dorsal organizer and gives rise to the 4d mesentoblast. Even though it has been argued that this situation represents the ancestral condition among the Spiralia, these inductive events have only been documented in equal-cleaving molluscs. Embryos of the nemertean Cerebratulus lacteus also undergo equal, spiral cleavage, and the fate map of these embryos is similar to that of other spiralians. The role of animal first quartet micromeres in the establishment of the dorsal (D) cell quadrant was examined in C. lacteus by removing specific combinations of micromeres at the eight-cell stage. To follow the development of various cell quadrants, one quadrant was labeled with DiI at the four-cell stage, and specific first quartet micromeres were removed from discrete positions relative to the location of the labeled quadrant. The results indicate that the first quartet is required for normal development, as removal of all four micromeres prevented dorsoventral axis formation. In most cases, when either one or two adjacent first quartet micromeres were removed from one side of the embryo, the cell quadrant on the opposite side, with its macromere centered under the greatest number of the remaining animal micromeres, ultimately became the D quadrant. Twins containing duplicated dorsoventral axes were generated by removal of two opposing first quartet micromeres. Thus, any cell quadrant can become the D quadrant, and the dorsoventral axis is established after the eight-cell stage. While it is not yet clear exactly when key inductive interactions take place that establish the D quadrant in C. lacteus, contacts between the progeny of animal micromeres and vegetal macromeres are established during the interval between the fifth and sixth round of cleavage divisions (i.e., 32- to 64-cell stages). These findings argue that this mechanism of cell and axis determination has been conserved among equal-cleaving spiralians.     

Separable binding proteins for retinoic acid and retinol in bovine retina

Binding proteins for retinoic acid and retinol were separated from a supernatant prepared from bovine retina. Fraction IV from DEAE-cellulose chromatography bound exogenous [³H] retinoic acid which could not be effectively displayed by retinol, retinal, retinyl acetate or palmitate, but which was readily displaced with excess retinoic acid. [³H] Retinol was bound by fraction V from DEAE-cellulose chromatography and was not displaced by retinal, retinoic acid, retinyl acetate or retinyl palmitate, but was readily displaced by excess retinol. Unlike bovine serum retinol-binding protein, neither intracellular binding protein formed a complex with purified human serum prealbumin. The supernatant from bovine retinas was estimated to contain five times more retinoic acid binding than retinol binder.     

A retinoic acid synthesizing enzyme in ventral retina and telencephalon of the embryonic mouse

Most retinoic acid (RA) in the embryonic mouse is generated by three retinaldehyde dehydrogenases (RALDHs). RALDH1 (also called E1, AHD2 or ALDH1) is expressed in the dorsal retina, and RALDH2 (V2, ALDH11) generates most RA in the embryonic trunk. The third one, RALDH3 (V1), synthesizes the bulk of RA in the head of the early embryo. We show here that RALDH3 is a mouse homologue to ALDH6, an aldehyde dehydrogenase cloned from adult human salivary gland (Hsu, L.C., Chang, W.-C., Hiraoka, L., Hsien, C.-L., 1994. Molecular cloning, genomic organization, and chromosomal localization of an additional human aldehyde dehydrogenase gene, ALDH6. Genomics 24, 333-341), which was recently reported to act as a RALDH (Yoshida, A., Rzhetsky, A., Hsu, L.C., Chang, C., 1998. Human aldehyde dehydrogenase gene family. Eur. J. Biochem. 251, 549-557). RALDH3 expression begins in the surface ectoderm over the optic recess. In rapidly changing expression patterns it labels the appearance of several ectodermal structures: it marks the formation of the lens and the olfactory organ from ectodermal placodes, and it delineates the beginning eyelid field. Within the optic vesicle, RALDH3 is expressed in the ventral retina and the dorsal pigment epithelium. In the telencephalon, RALDH3 is expressed at high levels in the lateral part of the ganglionic eminence. From here it extends via the piriform cortex into the lower part of the septum. Of the three RALDHs, RALDH3 shows the strongest predilection for epithelia.     

Development of a chordate anterior-posterior axis without classical retinoic acid signaling

Developmental signaling by retinoic acid (RA) is thought to be an innovation essential for the origin of the chordate body plan. The larvacean urochordate Oikopleura dioica maintains a chordate body plan throughout life, and yet its genome appears to lack genes for RA synthesis, degradation, and reception. This suggests the hypothesis that the RA-machinery was lost during larvacean evolution, and predicts that Oikopleura development has become independent of RA-signaling. This prediction raises the problem that the anterior-posterior organization of a chordate body plan can be developed without the classical morphogenetic role of RA. To address this problem, we performed pharmacological treatments and analyses of developmental molecular markers to investigate whether RA acts in anterior-posterior axial patterning in Oikopleura embryos. Results revealed that RA does not cause homeotic posteriorization in Oikopleura as it does in vertebrates and cephalochordates, and showed that a chordate can develop the phylotypic body plan in the absence of the classical morphogenetic role of RA. A comparison of Oikopleura and ascidian evidence suggests that the lack of RA-induced homeotic posteriorization is a shared derived feature of urochordates. We discuss possible relationships of altered roles of RA in urochordate development to genomic events, such as rupture of the Hox-cluster, in the context of a new understanding of chordate phylogeny.     

Noggin and noggin-like genes control dorsoventral axis regeneration in planarians

Planarians regenerate a whole animal from a small body piece within a few days. Recent studies have shown that the bone morphogenetic protein (BMP) pathway is required to reestablish the dorsoventral (DV) axis. In vertebrates, the specification of the DV axis depends on the coordinated action of a dual organizer defined by BMP and antidorsalizing morphogenetic protein (ADMP) under the control of several factors, including the inhibitors chordin and noggin. Planarians have an expanded noggin family (up to ten members), which have been classified as canonical noggin (nog) and noggin-like (nlg) genes, the latter carrying an insertion within the noggin domain. Here we show that a BMP/ADMP organizer governs DV axis reestablishment during planarian regeneration, highlighting a greater-than-thought conservation of the mechanisms that establish this axis in protostomes and deuterostomes. Also, we report that whereas noggin genes function as canonical BMP inhibitors, the silencing of planarian nlg8 induces ectopic neurogenesis and enhances ventralizing bmp(RNAi) phenotypes. Finally, we show that noggin-like genes are conserved from cnidarian to vertebrates and that both planarian nlg8 and Xenopus nlg ventralize Xenopus embryos when overexpressed. Remarkably, this ventralization is not associated with an increase in SMAD1/5/8 phosphorylation.     

Hippocampal-medial entorhinal circuit is differently organized along the dorsoventral axis in rodents

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The complete amino acid sequence of the cellular retinoic acid-binding protein from bovine retina

The complete amino acid sequence of cellular retinoic acid-binding protein from bovine retina was obtained by Edman degradation of peptides obtained from enzymatic and CNBr digests. The 136 residue sequence is identical to that reported recently for the protein from bovine adrenal tissue indicating that the same gene is expressed in both tissues.     

Establishment of the dorsoventral axis in nemertean embryos: Evolutionary considerations of spiralian development

The Nemertea represent one of a number of invertebrate phyla that display a highly conserved pattern of cell division known as spiral cleavage. The fates of the early blastomeres are known for representatives of some spiralian phyla (i.e., molluscs and annelids) and in these species there appears to be a high degree of conservation in the ultimate fates of particular embryonic cells. The first two cleavage planes bear an invariant relationship to the symmetry properties of the future larval and adult body plan. To investigate whether these properties of spiralian embryo-genesis are shared (conserved) amongst members of other spiralian phyla, individual blastomeres in two- and four-cell embryos of the nemertean, Nemertopsis bivittata, were microinjected with bi-otinylated dextran lineage tracers. N. bivittata is a direct-developing hoplonemertean that forms a nonfeeding larva. When individual blastomeres are injected at the two-cell stage, two sets of complementary labeling patterns (a total of four different patterns) were observed in the ectoderm of the larvae. When cells were injected at the four-cell stage, four different patterns were observed that represented subsets of the four patterns observed in the previous experiment. Unlike the case in the annelids and molluscs, in which the first cleavage plane bears a strict 45° angular relationship to the future dorsoventral axis, the first cleavage plane in N. bivittata can bear one of two different relationships relative to the larval/adult dorsoventral axis. In half the cases examined, the first cleavage plane corresponded roughly to the plane of bilateral symmetry, and in the rest, it lay along a frontal plane. A similar result was observed for the embryos of the indirect-developing heteronemertean, Cerebratutus lacteus. These results indicate that the fates of the four cell quadrants in nemerteans are not directly homologous to those in other spira-lians, such as the annelids and molluscs. For instance, no single cell quadrant appears to contribute a greater share to the formation of ectoderm, as is the case in the formation of the post-trochal region by the D-cell quadrant in annelids and mol-luscs. Rather, two adjacent cell quadrants contribute nearly equally to the formation of dorsal or ventral ectoderm in the larvae. Possible explanations for the determination of dorsoventrality in nemerte-ans, as well as implications of these findings regarding the evolution of spiralian development, are discussed. © 1994 Wiley-Liss, Inc.     

Zebrafish chordin-like and chordin are functionally redundant in regulating patterning of the dorsoventral axis

Chordin is the prototype of a group of cysteine-rich domain-containing proteins that bind and modulate signaling of various TGFβ-like ligands. Chordin-like 1 and 2 (CHL1 and 2) are two members of this group that have been described in human, mouse, and chick. However, in vivo roles for CHL1 and 2 in early development are unknown due to lack of loss-of-function analysis. Here we identify and characterize zebrafish, Danio rerio, CHL (Chl). The chl gene is on a region of chromosome 21 syntenic with the area of murine chromosome 7 bearing the CHL2 gene. Inability to identify a separate zebrafish gene corresponding to the mammalian CHL1 gene suggests that Chl may serve roles in zebrafish distributed between CHL1 and CHL2 in other species. Chl is a maternal factor that is also zygotically expressed later in development and has spatiotemporal expression patterns that differ from but overlap those of zebrafish chordin (Chd), suggesting differences but also possible overlap in developmental roles of the two proteins. Chl, like Chd, dorsalizes embryos upon overexpression and is cleaved by BMP1, which antagonizes this activity. Loss-of-function experiments demonstrate that Chl serves as a BMP antagonist with functions that overlap and are redundant with those of Chd in forming the dorsoventral axis.