Hox-4 gene expression in mouse/chicken heterospecific grafts of signalling regions to limb buds reveals similarities in patterning mechanisms.

The products of Hox-4 genes appear to encode position in developing vertebrate limbs. In chick embryos, a number of different signalling regions when grafted to wing buds lead to duplicated digit patterns. We grafted tissue from the equivalent regions in mouse embryos to chick wing buds and assayed expression of Hox-4 genes in both the mouse cells in the grafts and in the chick cells in the responding limb bud using species specific probes. Tissue from the mouse limb polarizing region and anterior primitive streak respecify anterior chick limb bud cells to give posterior structures and lead to activation of all the genes in the complex. Mouse neural tube and genital tubercle grafts, which give much less extensive changes in pattern, do not activate 5'-located Hox-4 genes. Analysis of expression of Hox-4 genes in mouse cells in the grafted signalling regions reveals no relationship between expression of these genes and strength of their signalling activity. Endogenous signals in the chick limb bud activate Hox-4 genes in grafts of mouse anterior limb cells when placed posteriorly and in grafts of mouse anterior primitive streak tissue. The activation of the same gene network by different signalling regions points to a similarity in patterning mechanisms along the axes of the vertebrate body.     

The murine Hox-2 genes display dynamic dorsoventral patterns of expression during central nervous system development.

This report demonstrates that the genes in the murine Hox-2 cluster display spatially and temporally dynamic patterns of expression in the transverse plane of the developing CNS. All of the Hox-2 genes exhibit changing patterns of expression that reflect events during the ontogeny of the CNS. The observed expression correlates with the timing and location of the birth of major classes of neurons in the spinal cord. Therefore, it is suggested that the Hox-2 genes act to confer rostrocaudal positional information on each successive class of newly born neurons. This analysis has also revealed a striking dorsal restriction in the patterns of Hox-2 expression in the spinal cord between 12.5 and 14.5 days of gestation, which does not appear to correlate with any morphological structure. The cellular retinol binding protein (CRBP) shows a complementary ventral staining pattern, suggesting that a number of genes are dorsoventrally restricted during the development of the CNS. The expression of Hox-2 genes has also been compared with the Hox-3.1 gene, which exhibits a markedly different dorsoventral pattern of expression. This suggests that, while genes in the different murine Hox clusters may have similar A-P domains of expression, they are responding to different dorsoventral patterning signals in the developing spinal cord.     

Alcaligenes eutrophus hydrogenase genes (Hox)

Mutants of Alcaligenes eutrophus H16 lacking catalytically active soluble hydrogenase (Hos-) grew very slowly lithoautotrophically with hydrogen. Mutants devoid of particulate hydrogenase activity (Hop-) were not affected in growth with hydrogen. The use of Hos- and Hop- mutants as donors of hydrogen-oxidizing ability in crosses with plasmid-free recipients impaired in both hydrogenases (Hox-) resulted in transconjugants which had inherited the plasmid and the phenotype of the donor. This indicates that the structural genes which code for the hydrogenases reside on plasmid pHG1. The Hox function of one class of Hox- mutants could not be restored by conjugation. These mutants exhibited a pleiotropic phenotype since they were unable to grow with hydrogen and also failed to grow heterotrophically with nitrate (Hox- Nit-). Nitrate was scarcely utilized as electron acceptor or as nitrogen source. Hox- Nit- mutants did not act as recipients but could act as donors of the Hox character. Transconjugants derived from those crosses were Hox+ Nit+, indicating that the mutation which leads to the Hox- Nit- phenotype maps on the chromosome. Apparently, the product of a chromosomal gene is involved in the expression of plasmid-encoded Hox genes. We observed that the elimination of plasmid pHG1 coincided with the occurrence of multiple resistances to various antibiotics. Since Hox+ transconjugate retained the antibiotic-resistant phenotype, we conclude that this property is not directly plasmid associated.     

Sequence and embryonic expression of the murine Hox-3.5 gene.

The murine Hox-3.5 gene has been mapped and linked genomically to a position 18 kb 3' of its most 5' locus neighbour, Hox-3.4, on chromosome 15. The sequence of the Hox-3.5 cDNA, together with the position of the gene within the locus, show it to be a paralogue of Hox-2.6, Hox-1.4 and Hox-4.2. The patterns of embryonic expression for the Hox-3.5 gene are examined in terms of three rules, proposed to relate a Hox gene's expression pattern to its position within the locus. The anterior boundaries of Hox-3.5 expression in the hindbrain and prevertebral column lie anterior to those of Hox-3.4 and all other, more 5'-located Hox-3 genes. Within the hindbrain, the Hox-3.5 boundary is seen to lie posterior to that of its paralogue, Hox-2.6, by a distance equal to about the length of one rhombomere. Patterns of Hox-3.5 expression within the oesophagus and spinal cord, but not the testis, are similar to those of other Hox-3 genes, Hox-3.3 and Hox-3.4.     

Treatment of chytridiomycosis with reduced-dose itraconazole

Effective treatment methods to eliminate infection with Batrachochytrium dendrobatidis (Bd) are required for development of sustainable captive survival assurance populations of amphibians and to reduce the risk of introducing Bd to new locations as part of amphibian trade or reintroduction programs. Treatment with itraconazole baths at 100 mg l-1 is commonly used in captive amphibians, but side effects are observed in some amphibian species and life stages. Naturally occurring outbreaks of chytridiomycosis in Wyoming toads Anaxyrus baxteri and White's tree frogs Litoria caerulea were treated with lower-dose itraconazole baths (e.g. 50 mg l-1 for White's tree frogs) and followed post-treatment with serial Taqman PCR testing to confirm elimination of Bd infection. Post-treatment PCR tests were consistently negative for the presence of Bd and treatment was deemed successful. Although this was not a controlled clinical trial, results suggest that lower doses of itraconazole may be effective for treatment of chytridiomycosis with resulting cost savings to amphibian conservation programs and a potential for a reduction in dose-related side effects from itraconazole treatment. Prospective clinical trials of alternative itraconazole treatment protocols are encouraged.     

Separate elements cause lineage restriction and specify boundaries of Hox-1.1 expression.

The Hox genes are a class of putative developmental control genes that are thought to be involved in the specification of positional identity along the anteroposterior axis of the vertebrate embryo. It is apparent from their expression pattern that their regulation is dependent upon positional information. In a previous analysis of the Hox-1.1 promoter in transgenic mice, we identified sequences that were sufficient to establish transgene expression in a specific region of the embryo. The construct used, however, did not contain enough regulatory sequences to reproduce all aspects of Hox-1.1 expression. In particular, neither a posterior boundary nor a restriction of expression to prevertebrae was achieved. Here we show correct regulation by Hox-1.1 sequences in transgenic mice and identify the elements responsible for different levels of control. Concomitant with the subdivision of mesodermal cells into different lineages during gastrulation and organogenesis, Hox-1.1 expression is restricted to successively smaller sets of cells. Distinct elements are required at different stages of development to execute this developmental programme. One position-responsive element (130 bp nontranslated leader) was shown to be crucial for the restriction of expression not only along the anteroposterior axis of the embryo, setting the posterior border, but also along the dorsoventral axis of the neural tube and to the lineage giving rise to the prevertebrae. Thus, Hox-1.1 expression is established in a specific region of the embryo and in a specific lineage of the mesoderm by restricting the activity of the promoter by the combined effect of several regulatory elements.     

Hox C6 expression during development and regeneration of forelimbs in larval Notophthalmus viridescens

 A central theme concerning the epimorphic regenerative potential of urodele amphibian appendages is that limb regeneration in the adult parallels larval limb development. Results of previous research have led to the suggestion that homeobox containing genes are ”re-expressed” during the epimorphic regeneration of forelimbs of adult Notophthalmus viridescens in patterns which retrace larval limb development. However, to date no literature exists concerning expression patterns of any homeobox containing genes during larval development of this species. The lack of such information has been a hindrance in exploring the similarities as well as differences which exist between limb regeneration in adults and limb development in larvae. Here we report the first such results of the localization of Hox C6 (formerly, NvHBox-1) in developing and regenerating forelimbs of N. viridescens larvae as demonstrated by whole-mount in situ hybridization. Inasmuch as the pattern of Hox C6 expression is similar in developing forelimb buds of larvae and epimorphically regenerating forelimb blastemata of both adults and larvae, our results support the paradigm that epimorphic regeneration in adult newts parallels larval forelimb development. However, in contrast with observations which document the presence of Hox C6 in both intact, as well as regenerating hindlimbs and tails of adult newts, our results reveal no such Hox C6 expression during larval development of hindlimbs or the tail. As such, our findings indicate that critical differences in larval hindlimb and tail development versus adult expression patterns of this gene in these two appendages may be due primarily to differences in gene regulation as opposed to gene function. Thus, the apparent ability of urodeles to regulate genes in such a highly co-ordinated fashion so as to replace lost, differentiated, appendicular structures in adult animals may assist, at least in part, in better elucidating the phenomenon of epimorphic regeneration. Received: 6 November 1998 / Accepted: 12 December 1998     

Hox-3.6: isolation and characterization of a new murine homeobox gene located in the 5' region of the Hox-3 cluster.

Most members of the murine Hox gene system can be grouped into two subclasses based on their structural similarity to either one of the Drosophila homeotic genes Antennapedia (Antp) or Abdominal B (AbdB). All the AbdB-like genes reported thus far are located in the 5' region of their respective cluster. We describe here the isolation, structural characterization and spatio-temporal expression pattern of a new AbdB-like homeobox gene designated Hox-3.6 that is located in the 5' region of the Hox-3 cluster. Hox-3.6 has an extreme posterior expression domain in embryos of 12.5 days of gestation, a feature that has thus far only been observed for the 5' most genes of the Hox-4 cluster. Like the other members of the AbdB subfamily, Hox-3.6 exhibits spatially restricted expression in the hindlimb bud, but the expression domain is antero-proximal in contrast to the postero-distal domain reported for its cognate gene Hox-4.5. Structural analysis of the 5' region revealed the presence of a 35 bp sequence which shares homology and relative 5' position with an upstream sequence present in its two nearest downstream neighbors, Hox-3.2 and -3.1.     

Differential expression of two msh-related homeobox genes Chox-7 and Chox-8 during chick limb development.

We have isolated two closely related cDNAs, Chox-7 and Chox-8, encoding homeodomain-containing proteins homologous to Drosophila msh. The Chox-7 and Chox-8 genes are chicken cognates of mouse Hox-7.1 and Hox-8.1, respectively. In situ hybridization using 3' regions of the cDNAs as probes revealed that the Chox-7 gene is highly expressed in the mesenchyme subjacent to the apical ectodermal ridge whereas Chox-8 expression is localized in the anterodistal mesenchymal region at early stages of limb formation, suggesting different roles during limb development. At later stages, both genes are expressed in the anterior and posterior mesenchymes and in the interdigital mesenchyme where programmed cell death occurs.     

Early limb patterning in the direct‐developing salamander Plethodon cinereus revealed by sox9 and col2a1

Direct‐developing amphibians form limbs during early embryonic stages, as opposed to the later, often postembryonic limb formation of metamorphosing species. Limb patterning is dramatically altered in direct‐developing frogs, but little attention has been given to direct‐developing salamanders. We use expression patterns of two genes, sox9 and col2a1, to assess skeletal patterning during embryonic limb development in the direct‐developing salamander Plethodon cinereus. Limb patterning in P. cinereus partially resembles that described in other urodele species, with early formation of digit II and a generally anterior‐to‐posterior formation of preaxial digits. Unlike other salamanders described to date, differentiation of preaxial zeugopodial cartilages (radius/tibia) is not accelerated in relation to the postaxial cartilages, and there is no early differentiation of autopodial elements in relation to more proximal cartilages. Instead, digit II forms in continuity with the ulnar/fibular arch. This amniote‐like connectivity to the first digit that forms may be a consequence of the embryonic formation of limbs in this direct‐developing species. Additionally, and contrary to recent models of amphibian digit identity, there is no evidence of vestigial digits. This is the first account of gene expression in a plethodontid salamander and only the second published account of embryonic limb patterning in a direct‐developing salamander species.     

Pbx1/Pbx2 requirement for distal limb patterning is mediated by the hierarchical control of Hox gene spatial distribution and Shh expression

Vertebrate limb development occurs along three cardinal axes-proximodistal, anteroposterior and dorsoventral-that are established via the organization of signaling centers, such as the zone of polarizing activity (ZPA). Distal limb development, in turn, requires a molecular feedback loop between the ZPA expression of sonic hedgehog (Shh) and the apical ectodermal ridge. The TALE homeoprotein Pbx1 has been shown to be essential for proximal limb development. In this study, we first uncover that Pbx1 and Pbx2 are co-expressed in the lateral plate and early limb field mesoderm. Later, Pbx2 is expressed throughout the limb, unlike Pbx1, which is expressed only in the proximal bud. By exploiting a Pbx1/Pbx2 loss-of-function mouse model, we demonstrate that, despite the lack of limb abnormalities in Pbx2-deficient (Pbx2(-/-)) embryos, compound Pbx1(-/-); Pbx2(+/-) mutants, in addition to their exacerbated proximal limb defects, exhibit novel and severe distal abnormalities. Additionally, we reveal that Pbx1(-/-); Pbx2(-/-) embryos lack limbs altogether. Furthermore, we establish that, unlike in flies, where the leg develops independently of Hox and where the Pbx ortholog Exd is required for specification of proximal (but not distal) limbs, in vertebrates, distal limb patterning is Pbx1/Pbx2 dependent. Indeed, we demonstrate that Pbx genetic requirement is mediated, at least in part, through their hierarchical control of Hox spatial distribution and Shh expression. Overall, we establish that, by controlling the spatial expression of Hox genes in the posterior limb and regulating ZPA function, Pbx1/Pbx2 exert a primary hierarchical function on Hox genes, rather than behaving merely as Hox ancillary factors.     

Why we have (only) five fingers per hand: hox genes and the evolution of paired limbs.

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.     

How common is common? Rapidly assessing population size and structure of the frog Mantidactylus betsileanus at a site in east‐central Madagascar

Population‐level data are urgently needed for amphibians in light of the ongoing amphibian extinction crisis. Studies focused on population dynamics are not only important for rare species but also for common species which shape ecosystems to a greater degree than those that are rare. Some of the greatest global amphibian species diversity is found in Madagascar, yet there are few studies on the ecology of frog species on the island. We carried out a mark‐recapture study on the widespread frog Mantidactylus betsileanus (Mantellidae: Mantellinae: Mantidactylus) at two adjacent rainforest sites in east‐central Madagascar to assess its population size and structure. To do so, we validated and implemented an individual identification protocol using photographs of the ventral patterns of frogs and identified individuals with photographic‐matching software. Using this rapid, non‐invasive survey method, we were able to estimate a density of 26 and 28 frogs per 100 m² at each of the two sites sampled. Our results show the rainforests near the village of Andasibe, Madagascar support remarkably high amphibian abundance, helping illustrate the significant ecological role of frogs in this ecosystem. Further, individual frog markings allowed us to develop more precise estimates than traditional survey methods. This study provides a blueprint to augment existing population studies or develop new monitoring programs in Madagascar and beyond.     

Density dependence in the terrestrial life history stage of two anurans

Populations of species with complex life cycles have the potential to be regulated at multiple life history stages. However, research tends to focus on single stage density-dependence, which can lead to inaccurate conclusions about population regulation and subsequently hinder conservation efforts. In amphibians, many studies have demonstrated strong effects of larval density and have often assumed that populations are regulated at this life history stage. However, studies examining density regulation in the terrestrial stages are rare, and the functional relationships between terrestrial density and vital rates in amphibians are unknown. We determined the effects of population density on survival, growth and reproductive development in the terrestrial stage of two amphibians by raising juvenile wood frogs (Rana sylvatica) and American toads (Bufo americanus) at six densities in terrestrial enclosures. Density had strong negative effects on survival, growth and reproductive development in both species. We fitted a priori recruitment functions to describe the relationship between initial density and the density of survivors after one year, and determined the functional relationship between initial density and mass after one year. Animals raised at the lowest densities experienced growth and survival rates that were over twice as great as those raised at the highest density. All female wood frogs in the lowest density treatment showed signs of reproductive development, compared to only 6% in the highest density treatment. Female American toads reached minimum reproductive size only at low densities, and male wood frogs and American toads reached maturity only in the three lowest density treatments. Our results demonstrate that in the complex life cycle of amphibians, density in the terrestrial stage can reduce growth, survival and reproductive development and may play an important role in amphibian population regulation. We discuss the implications of these results for population regulation in complex life cycles and for amphibian conservation.