Brigitte Galliot

Apoptotic Cells Provide an Unexpected Source of Wnt3 Signaling to Drive Hydra Head Regeneration

Decapitated Hydra regenerate their heads via morphallaxis, i.e., without significant contributions made by cell proliferation or interstitial stem cells. Indeed, Hydra depleted of interstitial stem cells regenerate robustly, and Wnt3 from epithelial cells triggers head regeneration. However, we find a different mechanism controlling regeneration after midgastric bisection in animals equipped with both epithelial and interstitial cell lineages. In this context, we see rapid induction of apoptosis and Wnt3 secretion among interstitial cells at the head- (but not foot-) regenerating site. Apoptosis is both necessary and sufficient to induce Wnt3 production and head regeneration, even at ectopic sites. Further, we identify a zone of proliferation beneath the apoptotic zone, reminiscent of proliferative blastemas in regenerating limbs and of compensatory proliferation induced by dying cells in Drosophila imaginal discs. We propose that different types of injuries induce distinct cellular modes of Hydra head regeneration, which nonetheless converge on a central effector, Wnt3.

Evolution of homeobox genes: Q50 Paired-like genes founded the Paired class

Abstract The genes belonging to the Paired class exert primary developmental functions. They are characterized by six invariant amino acid residues in the homeodomain, while the residue at position 50 can be a serine, glutamine or lysine as in the Pax-type, Q50 Paired-like or the K50 Paired-like homeodomains respectively. Genes in this class emerged early in animal evolution: three distinct Pax genes and two Q50 Paired-like genes have recently been characterised from cnidarians. Phylogenetic molecular reconstructions taking into account homeodomain and paired-domain sequences provide some new perspectives on the evolution of the Paired-class genes. Analysis of 146 Paired-class homeodomains from a wide range of metazoan taxa allowed us to identify 18 families among the three sub-classes from which the aristaless family displays the least diverged position. Both Pax-type and K50 families branch within the Q50 Paired-like sequences implying that these are the most ancestral. Consequently, most Pax genes arose from a Paired-like ancestor, via fusion of a Paired-like homebox gene with a gene encoding only a paired domain; the Cnidaria appear to contain genes representing the ’before’ and ’after’ fusion events.

HOM/HOX homeobox genes are present in hydra (Chlorohydra viridissima) and are differentially expressed during regeneration.

Hydra, a diblastic animal consisting of two cell layers, ectoderm and endoderm, is one of the most ancient animals displaying an anteroposterior axis with a head and a foot developing from an uncommitted gastric region. As such, hydra is an interesting model for studying the presence and function of homeobox genes in a phylogenetically old organism. By screening a Chlorohydra viridissima cDNA library with a ‘guessmer’ oligonucleotide, we have cloned several such cnidarian homeobox‐containing genes (cnox genes). Two of these, cnox1 and cnox2, display labial and Deformed type homeodomains respectively and could represent two ancestral genes of the HOM/HOX complexes; cnox3 exhibits some similarity to the BarH1 and the distal‐less type homeodomains and a fourth gene is highly related to the msh/Hox7 type of homeodomain. We used quantitative PCR to study levels of expression of these genes along the body axis and during head regeneration. In all cases, the expression in heads was stronger than that in the gastric region. cnox1 transcripts dramatically peaked within the first hours of head regeneration, whereas cnox2 and cnox3 reached their maximal levels 1 and 2 days after cutting respectively. This differential expression of homeobox genes at various stages of regeneration suggests that they play specific roles in regenerative processes.

The segment-specific gene Krox-20 encodes a transcription factor with binding sites in the promoter region of the Hox-1.4 gene

Krox‐20 is a mouse zinc finger gene expressed in a segment‐specific manner in the early central nervous system, which makes it a potential developmental control gene. In this report, we show that the Krox‐20 protein binds in vitro to two specific DNA sites located upstream from the homeobox containing gene Hox‐1.4. The nucleotide sequence recognized by Krox‐20 is closely related to the Sp1 target sequence, which is consistent with the similarity existing between the zinc fingers of the two proteins. In co‐transfection experiments in cultured cells, Krox‐20 dramatically activates transcription from the herpes simplex virus thymidine kinase promoter when an oligomer of its binding site is present in cis close to the promoter. Analysis of mutated binding sites demonstrates that the level of activation by Krox‐20 correlates with the affinity of the protein for the mutant sequence. These data indicate that Krox‐20 constitutes a sequence‐specific DNA‐binding transcription factor. Parallel analysis of the expression of Krox‐20 and Hox‐1.4 in the neural tube by in situ hybridization revealed no overlap, arguing against direct interactions between these two genes. The possible involvement of Krox‐20 in the regulation of the transcription of other homeobox genes is discussed in view of their respective patterns of expression.

A new homeo-box is present in overlapping cosmid clones which define the mouse Hox-1 locus

A new murine homeo‐box (Hox1‐3) has been isolated and studied with respect to its structure and transcriptional pattern. This homeo‐box is part of a gene which is specifically regulated during mouse prenatal development and expressed in a restricted number of teratocarcinoma tumours as well as in adult testis. Hox1‐3 is shown to be a member of a cluster of at least four homeo‐boxes lying within a 75‐kb segment of DNA on mouse chromosome 6. The structure of the whole cluster, the Hox‐1 locus is presented.

Silencing of the hydra serine protease inhibitor Kazal1 gene mimics the human SPINK1 pancreatic phenotype.

In hydra, the endodermal epithelial cells carry out the digestive function together with the gland cells that produce zymogens and express the evolutionarily conserved gene Kazal1. To assess the hydra Kazal1 function, we silenced gene expression through double-stranded RNA feeding. A progressive Kazal1 silencing affected homeostatic conditions as evidenced by the low budding rate and the induced animal death. Concomitantly, a dramatic disorganization followed by a massive death of gland cells was observed, whereas the cytoplasm of digestive cells became highly vacuolated. The presence of mitochondria and late endosomes within those vacuoles assigned them as autophagosomes. The enhanced Kazal1 expression in regenerating tips was strongly diminished in Kazal1(–) hydra, and the amputation stress led to an immediate disorganization of the gland cells, vacuolization of the digestive cells and death after prolonged silencing. This first cellular phenotype resulting from a gene knock-down in cnidarians suggests that the Kazal1 serine-protease-inhibitor activity is required to prevent excessive autophagy in intact hydra and to exert a cytoprotective function to survive the amputation stress. Interestingly, these functions parallel the pancreatic autophagy phenotype observed upon mutation within the Kazal domain of the SPINK1 and SPINK3 genes in human and mice, respectively.

Hox-1.6: a mouse homeo-box-containing gene member of the Hox-1 complex.

Hox‐1.6, a mouse homeo‐box‐containing gene member of the Hox‐1 complex, is described. The Hox‐1.6 homeo‐box shows more divergence than the other members of the complex with the Drosophila Antennapedia‐like homeo‐box class. This previously undescribed gene was studied with respect to its transcription pattern and was found to be expressed during mouse fetal development in an intestine‐specific manner in adults, and in tumours or cell types exhibiting early endodermal‐like differentiation. The study of embryonic partial Hox‐1.6 cDNA clones revealed structural features common to other Drosophila and vertebrate homeo‐box‐containing genes, but also indicated that Hox‐1.6 transcripts might display splicing patterns more complex than those known for other vertebrate homeo‐genes. One of these cDNA clones contains a rather short open reading frame which would encode a protein of approximately 14.5 kd. The use of this clone as a probe for S1 nuclease mapping confirmed that different Hox‐1.6 transcripts were present both in embryonic total RNA and in embryonal carcinoma cell cytoplasmic RNA. These various transcripts are probably generated by an alternative splicing mechanism and may thus encode a set of related proteins.

Origin of anterior patterning: how old is our head?

Most animals that display a bilateral symmetry (bilaterians) share homologous regulatory genes involved in head development. Recently, homologues of several of these genes have been cloned from animals that are radially organized, such as coral, sea anemones, jellyfish or hydra (cnidarians). Surprisingly, some of these are expressed apically and/or during apical patterning in hydrozoans, suggesting that head patterning is much older than previously thought.

The Hydra model: disclosing an apoptosis-driven generator of Wnt-based regeneration.

The Hydra model system is well suited for the eludication of the mechanisms underlying regeneration in the adult, and an understanding of the core mechanisms is likely to cast light on pathways conserved in other species. Recent detailed analyses of the activation of the Wnt-beta-catenin pathway in bisected Hydra shows that the route taken to regenerate a structure as complex as the head varies dramatically according to the level of the amputation. When decapitation induces direct re-development due to Wnt3 signaling from epithelial cells, head regeneration after mid-gastric section relies first on Wnt3 signaling from interstitial cells, that undergo apoptosis-induced compensatory proliferation, and subsequently on activation of Wnt3 signaling in the epithelial cells. The relative distribution between stem cells and head progenitor cells is strikingly different in these two contexts, indicating that the pre-amputation homeostatic conditions define and constrain the route that bridges wound-healing to the re-development program of the missing structure.

Conserved and divergent genes in apex and axis development of cnidarians

Despite their radial organization and their sister group position in the life tree, cnidarian species express during morphogenesis a large number of genes that are related to bilaterian developmental genes. Among those, homologs to forkhead, emx, aristaless, goosecoid, brachyury, wnt and nanos genes are regulated during apical patterning in cnidarians, suggesting that key components of early organizer activity were conserved across evolution and recruited for either anterior, axial, or dorso-ventral patterning in bilaterians. In contrast, the expression patterns of the cnidarian Hox-related genes suggest that the apical-basal axis of the cnidarian polyp and the anterior-posterior axis of bilaterians do not differentiate following homologous processes.