Mechanisms regulating the p120-catenin/Kaiso pathway

The Wnt pathways contribute to many processes in cancer and developmental biology, with β-catenin being a key canonical component. P120-catenin, which is structurally similar to β-catenin, regulates the expression of certain Wnt target genes, relieving repression conferred by the POZ/ zinc-finger transcription factor Kaiso. In my first project, employing Xenopus embryos and mammalian cell lines, I found that the degradation machinery of the canonical Wnt pathway modulates p120-catenin protein stability, especially p120 isoform-1, through mechanisms shared with b-catenin. Exogenous expression of destruction-complex components such as GSK3b or Axin promotes p120-catenin degradation, and consequently, is able to rescue developmental phenotypes resulting from p120 over-expression during early Xenopus embryonic development. Conversely, as predicted, the in vivo depletion of either Axin or GSK3b coordinately increased p120 and b-catenin levels, while p120 levels decreased upon LRP5/6 depletion, which are positive modulators in the canonical Wnt pathway. At the primary sequence level, I resolved conserved GSK3b phosphorylation sites in p120’s (isoform 1) amino-terminal region. Point-mutagenesis of these residues inhibited the association of destruction complex proteins including those involved in ubiquitination, resulting in p120-catenin stabilization. Importantly, we found that two additional p120-catenin family members, ARVCF-catenin and d-catenin, in common with b-catenin and p120, associate with Axin, and are degraded in Axin’s presence. Thus, by similar means, it appears that canonical Wnt signals coordinately modulate multiple catenin proteins having roles in development and conceivably disease states. In my second project, I found that the Dyrk1A kinase exhibits a positive effect upon p120-catenin levels. That is, unlike the negative regulator GSK3b kinase, a candidate screen revealed that Dyrk1A kinase enhances p120-catenin protein levels via increased half-life. Dyrk1A is encoded by a gene located within the trisomy of chromosome 21, which contributes to mental retardation in Down Syndrome patients. I found that Dyrk1A expression results in increased p120 protein levels, and that Dyrk1A specifically associates with p120 as opposed to other p120-catenin family members or b-catenin. Consistently, Dyrk1A depletion in mammalian cell lines and Xenopus embryos decreased p120-catenin levels. I further confirmed that Dyrk overexpression and knock-down modulates both Siamois and Wnt11 gene expression in the expected manner based upon the resulting latered levels of p120-catenin. I determined that Dyrk expression rescues Kaiso depletion effects (gastrulation failure; increased endogenous Wnt11 expression), and vice versa. I then identified a putative Dyrk phosphorylation region within the N-terminus of p120-catenin, which may also be responsible for Dyrk1A association. I went on to make a phosphomimic mutant, which when over-expressed, had the predicted enhanced capacity to positively modulate endogenous Wnt11 and Siamois expression, and thereby generate gastrulation defects. Given that Dyrk1A modulates Siamois expression through stabilization of p120-catenin, I further observed that ectopic expression of Dyrk can positively influence b-catenin’s capacity to generate ectopic dorsal axes when ventrally expressed in early Xenopus embryos. Future work will investigate how Dyrk1A modulates the Wnt signaling pathway through p120-catenin, and possibly begin to address how dysfunction of Dyrk1A with respect to p120-catenin might relate to aspects of Down syndrome. In summary, the second phase of my graduate work appears to have revealed a novel aspect of Dyrk1A/p120-catenin action in embryonic development, with a functional linkage to canonical Wnt signaling. What I have identified as a “Dyrk1A/p120-catenin/Kaiso pathway” may conceivably assist in our larger understanding of the impact of Dyrk1A dosage imbalance in Down syndrome.

A family of fibrinogen-binding MSCRAMMs from Enterococcus faecalis.

We report that three (EF0089, EF2505 and EF1896, renamed here Fss1, Fss2 and Fss3, respectively, for Enterococcus faecalis surface protein) of the recently predicted MSCRAMMs (microbial surface components recognizing adhesive matrix molecules) in E. faecalis strain V583 bind fibrinogen (Fg). Despite an absence of extensive primary sequence homology, the three proteins appear to be related structurally. Within the N-terminal regions of the three enterococcal proteins, we identified pairs of putative IgG-like modules with a high degree of predicted structural similarity to the Fg-binding N2 and N3 domains of the staphylococcal MSCRAMMs ClfA and SdrG. A second N2N3-like segment was predicted in Fss1. Far-UV circular dichroism spectroscopy revealed that all four predicted N2N3-like regions are composed mainly of beta-sheets with only a minor proportion of alpha-helices, which is characteristic of Ig-like folded domains. Three of the four identified enterococcal N2N3-like regions showed potent dose-dependent binding to Fg. However, the specificity of the Fg-binding MSCRAMMs differs, as indicated by far-Western blots, which showed that recombinant segments of the MSCRAMMs bound different Fg polypeptide chains. Enterococci grown in serum-supplemented broth adhere to Fg-coated surfaces, and inactivation in strain OG1RF of the gene encoding Fss2 resulted in reduced adherence, whilst complementation of the mutant restored full Fg adherence. Thus, E. faecalis contains a family of MSCRAMMs that structurally and functionally resemble the Fg-binding MSCRAMMs of staphylococci.

Mutations affecting beta-tubulin folding and degradation.

Revertants of a colcemid-resistant Chinese hamster ovary cell line with an altered (D45Y) beta-tubulin have allowed the identification of four cis-acting mutations (L187R, Y398C, a 12-amino acid in-frame deletion, and a C-terminal truncation) that act by destabilizing the mutant tubulin and preventing it from incorporating into microtubules. These unstable beta-tubulins fail to form heterodimers and are predominantly found in association with the chaperonin CCT, suggesting that they cannot undergo productive folding. In agreement with these in vivo observations, we show that the defective beta-tubulins do not stably interact with cofactors involved in the tubulin folding pathway and, hence, fail to exchange with beta-tubulin in purified alphabeta heterodimers. Treatment of cells with MG132 causes an accumulation of the aberrant tubulins, indicating that improperly folded beta-tubulin is degraded by the proteasome. Rapid degradation of the mutant tubulin does not elicit compensatory changes in wild-type tubulin synthesis or assembly. Instead, loss of beta-tubulin from the mutant allele causes a 30-40% decrease in cellular tubulin content with no obvious effect on cell growth or survival.

Cross currents in protein misfolding disorders: interactions and therapy.

Protein Misfolding Disorders (PMDs) are a group of diseases characterized by the accumulation of abnormally folded proteins. Despite the wide range of proteins and tissues involved, PMDs share similar molecular and pathogenic mechanisms. Several epidemiological, clinical and experimental reports have described the co-existence of PMDs, suggesting a possible cross-talk between them. A better knowledge of the molecular basis of PMDs could have important implications for understanding the mechanism by which these diseases appear and progress and ultimately to develop novel strategies for treatment. Due to their similar molecular mechanisms, common therapeutic strategies could be applied for the diseases in this group.

Regulation and function of {\it Xparaxis\/} during the development of paraxial mesoderm in {\it Xenopus laevis\/}

Genes of the basic helix-loop-helix transcription factor family have been implicated in many different developmental processes from neurogenesis to myogenesis. The recently cloned bHLH transcription factor, paraxis, has been found to be expressed in the paraxial mesoderm of the mouse suggesting a role for paraxis in the development of this mesodermal subtype which gives rise to the axial muscle, skeleton, and dermis of the embryo. In order to perform in vivo gain of function assays and obtain a better understanding of the possible roles of paraxis in mesodermal and somitic development, we have successfully identified homologues of paraxis in the frog, Xenopus laevis, where the process of mesodermal induction and development is best understood. The two homologues, Xparaxis-a and Xparaxis-b, are conserved with respect to their murine homologue in structure and expression within the embryo. Xparaxis genes are expressed immediately after gastrulation in the paraxial mesoderm of Xenopus embryos and are down regulated in the myotome of the mature somite with continued expression in the undifferentiated dermatome. Overexpression of Xparaxis-b in Xenopus embryos caused defects in the organization and morphology of the somites. This effect was not dependent on DNA binding of Xparaxis but is likely due to its dimerization with other bHLH factors. Co-injections with XE12 did not diminish the effects indicating that the defects were not the result of limiting amounts of XE12. We also demonstrated that Xparaxis does not cause obvious defects in the cell adhesions and movements required for proper mesoderm patterning during gastrulation. The paraxis proteins also lacked the ability to activate transcription as GAL4 fusion proteins in a GAL4 reporter assay, indicating that the genes may function more as modulators of the activity of dimerization partners than as positively acting cell determination factors. In agreement with this, Xparaxis is regulated in response to other pathways of bHLH gene action, in that XE12 can activate Xparaxis-b, in vivo. In addition we show regulation of Xparaxis in response to mMyoD induced myogenesis pathways, again suggesting Xparaxis plays an important role in the patterning and organization of the paraxial mesoderm. ^

Functional studies of the homeobox gene {\it Goosecoid\/} during mouse embryogenesis

A fundamental question in developmental biology is to understand the mechanisms that govern the development of an adult individual from a single cell. Goosecoid (Gsc) is an evolutionarily conserved homeobox gene that has been cloned in vertebrates and in Drosophila. In mice, Gsc is first expressed during gastrulation stages where it marks anterior structures of the embryo, this pattern of expression is conserved among vertebrates. Later, expression is observed during organogenesis of the head, limbs and the trunk. The conserved pattern of expression of Gsc during gastrulation and gain of function experiments in Xenopus suggested a function for Gsc in the development of anterior structures in vertebrates. Also, its expression pattern in mouse suggested a role in morphogenesis of the head, limbs and trunk. To determine the functional requirement of Gsc in mice a loss of function mutation was generated by homologous recombination in embryonic stem cells and mice mutant for Gsc were generated.^ Gsc-null mice survived to birth but died hours after delivery. Phenotypic analysis revealed craniofacial and rib cage abnormalities that correlated with the second phase of Gsc expression in the head and trunk but no anomalies were found that correlated with its pattern of expression during gastrulation or limb development.^ To determine the mode of action of Gsc during craniofacial development aggregation chimeras were generated between Gsc-null and wild-type embryos. Chimeras were generated by the aggregation of cleavage stage embryos, taking advantage of two different Gsc-null alleles generated during gene targeting. Chimeras demonstrated a cell-autonomous function for Gsc during craniofacial development and a requirement for Gsc function in cartilage and mesenchymal tissues.^ Thus, during embryogenesis in mice, Gsc is not an essential component of gastrulation as had been suggested in previous experiments. Gsc is required for craniofacial development where it acts cell autonomously in cartilage and mesenchymal tissues. Gsc is also required for proper development of the rib cage but it is dispensable for limb development in mice. ^

STABILITY AND FOLDING OF MUTANT FORMS OF ISO-1 CYTOCHROME-C FROM SACCHAROMYCES CEREVISIAE

Partially functional forms of iso-1-cytochrome c from Saccharomyces cerevisiae were obtained by replacements of the evolutionarily conserved proline 71 with valine, isoleucine and threonine (Ernst et.al.,1985). Pro-71 lies at the juncture of two short helical regions and is believed to be important for proper local polypeptide chain folding within the iso-1-cytochrome c structure.^ To study folding in the absence of intermolecular disulfide dimer formation the free sulfhydryl group of Cys-102 was modified in both wild type and mutant proteins with an alkylating reagent, methyl methanethiosulfonate. Spectral analysis of the wild type and mutant proteins shows that the native-like functional (or partially functional) folded structure of cytochrome c is retained in the chemically modified derivatives. The replacement of Pro-71 with valine, isoleucine or threonine reduces the intensity of the 696 nm absorbance band which is an indicator of the Met-80 ligation to the heme. Thermal stability and guanidine hydrochloride unfolding studies of the mutant proteins shows a destabilization of the protein as a result of mutation. The degree of destabilization depends on the chemical nature of the substituent amino acid in the mutant protiens.^ Kinetics of folding/unfolding reactions of the proteins were monitored by fluorescence changes using stopped flow mixing to obtain guanidine hydrochloride concentration jumps ending below, within, and above the transition zone. The replacement of Pro-71 alters the rate on one of the fastest phases, $\tau\sb3$, while the two other phases, $\tau\sb1$ & $\tau\sb2$, remain the same.^ Slow refolding kinetic studies indicate that replacement of Pro-71 does not completely eliminate the absorbance or fluorescence detected slow phases leading to the conclusion that Pro-71 is not involved in the generation of the slow phases in the folding kinetics of iso-1-cytochrome c.^ The alkaline conformational change involving the disappearance of the 696 nm absorbance band occurs with increasing pH in the alkaline pH region (Davis et al., 1974). The apparent pK of this conformational change in mutant proteins is shifted as much as two pH units compared to wild type. The equilibrium and kinetic data of alkaline transition for the wild type follows a simple mechanism proposed by Davis et al., (1974) for horse heart cytochrome c. A more complex mechanism is proposed for the behavior of the mutant proteins. ^

The pre- and post-somatic segments of the human type I spiral ganglion neurons--structural and functional considerations related to cochlear implantation.

Human auditory nerve afferents consist of two separate systems; one is represented by the large type I cells innervating the inner hair cells and the other one by the small type II cells innervating the outer hair cells. Type I spiral ganglion neurons (SGNs) constitute 96% of the afferent nerve population and, in contrast to other mammals, their soma and pre- and post-somatic segments are unmyelinated. Type II nerve soma and fibers are unmyelinated. Histopathology and clinical experience imply that human SGNs can persist electrically excitable without dendrites, thus lacking connection to the organ of Corti. The biological background to this phenomenon remains elusive. We analyzed the pre- and post-somatic segments of the type I human SGNs using immunohistochemistry and transmission electron microscopy (TEM) in normal and pathological conditions. These segments were found surrounded by non-myelinated Schwann cells (NMSCs) showing strong intracellular expression of laminin-β2/collagen IV. These cells also bordered the perikaryal entry zone and disclosed surface rugosities outlined by a folded basement membrane (BM) expressing laminin-β2 and collagen IV. It is presumed that human large SGNs are demarcated by three cell categories: (a) myelinated Schwann cells, (b) NMSCs and (c) satellite glial cells (SGCs). Their BMs express laminin-β2/collagen IV and reaches the BM of the sensory epithelium at the habenula perforata. We speculate that the NMSCs protect SGNs from further degeneration following dendrite loss. It may give further explanation why SGNs can persist as electrically excitable monopolar cells even after long-time deafness, a blessing for the deaf treated with cochlear implantation.

Inhibition of DNA polymerase reaction by pyrimidine nucleotide analogues lacking the 2-keto group

To investigate the influence of the pyrimidine 2-keto group on selection of nucleotides for incorporation into DNA by polymerases, we have prepared two C nucleoside triphosphates that are analogues of dCTP and dTTP, namely 2-amino-5-(2'-deoxy-beta-d-ribofuranosyl)pyridine-5'-triphosphate (d*CTP) and 5-(2'-deoxy- beta-d-ribofuranosyl)-3-methyl-2-pyridone-5'-triphosphate (d*TTP) respectively. Both proved strongly inhibitory to PCR catalysed by Taq polymerase; d*TTP rather more so than d*CTP. In primer extension experiments conducted with either Taq polymerase or the Klenow fragment of Escherichia coli DNA polymerase I, both nucleotides failed to substitute for their natural pyrimidine counterparts. Neither derivative was incorporated as a chain terminator. Their capacity to inhibit DNA polymerase activity may well result from incompatibility with the correctly folded form of the polymerase enzyme needed to stabilize the transition state and catalyse phosphodiester bond formation.

Valle d’Aosta section of the Sesia Zone: multi-stage HP metamorphism and assembly of a rifted continental margin

The spectrum characteristic of the EMC ranges from eclogites (containing omphacite and/or jadeite, garnet, phengite, glaucophane, zoisite, chloritoid, rutile) to phengite schists, calcschists, and marbles, as well as a variety of orthogneisses. Despite the intense polyphase deformation and HP-metamorphic recrystallization, it is possible in some locations to recognize pre-Alpine characteristics in some of the protoliths. For instance, two types of felsic orthogneiss can be distinguished in the Aosta Valley, one derived from Permian granitoids (with local preservation of intrusive contacts, magmatic inclusions, leucocratic veins and other magmatic structures; Stop 3), the other derived from pre-Variscan leuco-monzogranite, such as the building stone mined at the “Argentera” quarry near Settimo Vittone / Montestrutto (Stop 2; so-called “Verde Argento” contains jadeite, phengite, K-feldspar, quartz). Polycyclic and more rarely monocyclic metasediments contain evidence of a complex Alpine PTDt-evolution, locally including relics of their prograde history from blueschist, one or more stages at eclogite facies. Recent petrochronological studies have dated this HP-evolution of the Sesia Zone in some detail. In the area visited, clear evidence of HP-cycling has been identified in one km-size tectonic slice (Stop 1), but not in adjacent parts of the EMC, indicating “yo-yo tectonics”. Partial retrogression and attendant ductile to brittle deformation of the HP-rocks is evident in one of the outcrops (Stop 4). Apart from the four localities in the Sesia Zone, a final outcrop introduces HP-rocks of the adjacent Piemonte oceanic unit, specifically calc-schists and ophiolite members of the “Zermatt-Saas” zone. The hilltop outcrop (Stop 5) displays foliated antigorite schist with peridotite relics (clinopyroxene, spinel) containing lenses derived from doleritic dykes. These fine-grained metarodingites and the folded veins containing Mg-chlorite and titanoclinohumite within serpentinite once again indicate equilibration under low-temperature eclogite facies conditions. However, these units reached that HP stage more than 20 Ma after the youngest eclogite facies imprint recognized in the Sesia Zone. Despite nearly half a century of intense study in the Sesia Zone, the complex assembly of its HP-terranes and their relation to more external parts of the Western Alps remains incompletely understood. This field guide merely introduces a few of the classic outcrops and discusses some of the critical evidence they contain, but it could not incorporate details on each stage of the evolution recognized so far.

Stereoselective virtual screening of the ZINC database using atom pair 3D-fingerprints

Background Tools to explore large compound databases in search for analogs of query molecules provide a strategically important support in drug discovery to help identify available analogs of any given reference or hit compound by ligand based virtual screening (LBVS). We recently showed that large databases can be formatted for very fast searching with various 2D-fingerprints using the city-block distance as similarity measure, in particular a 2D-atom pair fingerprint (APfp) and the related category extended atom pair fingerprint (Xfp) which efficiently encode molecular shape and pharmacophores, but do not perceive stereochemistry. Here we investigated related 3D-atom pair fingerprints to enable rapid stereoselective searches in the ZINC database (23.2 million 3D structures). Results Molecular fingerprints counting atom pairs at increasing through-space distance intervals were designed using either all atoms (16-bit 3DAPfp) or different atom categories (80-bit 3DXfp). These 3D-fingerprints retrieved molecular shape and pharmacophore analogs (defined by OpenEye ROCS scoring functions) of 110,000 compounds from the Cambridge Structural Database with equal or better accuracy than the 2D-fingerprints APfp and Xfp, and showed comparable performance in recovering actives from decoys in the DUD database. LBVS by 3DXfp or 3DAPfp similarity was stereoselective and gave very different analogs when starting from different diastereomers of the same chiral drug. Results were also different from LBVS with the parent 2D-fingerprints Xfp or APfp. 3D- and 2D-fingerprints also gave very different results in LBVS of folded molecules where through-space distances between atom pairs are much shorter than topological distances. Conclusions 3DAPfp and 3DXfp are suitable for stereoselective searches for shape and pharmacophore analogs of query molecules in large databases. Web-browsers for searching ZINC by 3DAPfp and 3DXfp similarity are accessible at www.gdb.unibe.ch webcite and should provide useful assistance to drug discovery projects.

Polydnavirus DNA of the braconid wasp Chelonus inanitus is integrated in the wasp's genome and excised only in later pupal and adult stages of the female

Many endoparasitic wasps inject, along with the egg, polydnavirus into their insect hosts, the virus being a prerequisite for successful parasitoid development. The genome of polydnaviruses consists of multiple circular dsDNA molecules of variable size. We show for a 12 kbp segment of the braconid Chelonus inanitus (CiV12) that it is integrated into the wasp genome. This is the first direct demonstration of integration for a bracovirus. PCR data indicated that the integrated form of CiV12 was present in all male and female stages investigated while the excised circular virus DNA only appeared in females after a specific stage in pupal-adult development. The data also indicated that after excision of virus DNA the genomic DNA was rejoined. This has not yet been reported for any polydnavirus. Sequence analyses in the junction regions revealed the presence of an imperfect consensus sequence of 15 nucleotides in CiV12, in each terminus of the integrated virus DNA and in the rejoined genomic DNA. Within these repeats two sequence types (ATA, TAC) were observed in the various virus clones and in the clones encompassing the rejoined genomic DNA; they corresponded to the sequence type in the right and left junction, respectively. To explain this, we propose a model of virus DNA replication in which the genomic DNA is folded to juxtapose the direct repeat of the left with that of the right junction; recombination at specific sites would then yield the two types of virus and rejoined genomic DNA.

Developmental functions of XKaiso, a transcriptional repressor associating with the p120 catenin in Xenopus laevis

The Armadillo family catenin proteins function in multiple capacities including cadherin-mediated cell-cell adhesion and nuclear signaling. The newest catenin, p120 catenin, differs from the classical catenins and binds to the membrane-proximal domain of cadherins. Recently, a novel transcription factor Kaiso was found to interact with p120 catenin, suggesting that p120 catenin also possesses a nuclear function. We isolated the Xenopus homolog of Kaiso, XKaiso, from a Xenopus stage 17 cDNA library. XKaiso contains an amino-terminal BTB/POZ domain and three carboxyl-terminal zinc fingers. The XKaiso transcript was present maternally and expressed throughout early embryonic development. XKaiso's spatial expression was defined via in situ hybridization and was found localized to the brain, eye, ear, branchial arches, and spinal cord. Co-immunoprecipitation of Xenopus p120 catenin and XKaiso demonstrated their mutual association, while related experiments employing differentially epitope-tagged XKaiso constructs suggest that XKaiso also self-associates. On the functional level, reporter assays employing a chimera of XKaiso fused to the GAL4 DNA binding domain indicated that XKaiso is a transcriptional repressor. To better understand the significance of the Kaiso-p120 catenin complex in vertebrate development, Kaiso knock-down experiments were undertaken, and the modulatory role of p120 catenin in Kaiso function examined during Xenopus development. Using morpholino antisense oligonucleotides to block translation of XKaiso, XKaiso was found to be essential for Xenopus gastrulation, being required for correct morphogenetic movements in early embryogenesis. Molecular marker analyses indicated that one target gene of the Wnt/β-catenin pathway, Siamois, is significantly increased in embryos depleted for XKaiso, while other dorsal, ventral, and mesodermal cell fate markers were unaltered. In addition, the non-canonical Wnt-11, known to participate in planar cell polarity/convergent extension processes, was significantly upregulated following depletion of XKaiso. Such increased Wnt-11 expression likely contributed to the XKaiso depletion phenotype because a dominant negative form of Wnt-11 or of the downstream effector Dishevelled partially rescued the observed gastrulation defects. These results show that XKaiso is essential for proper gastrulation movements, resulting at least in part from its modulation of non-canonical Wnt signaling. The significance of the XKaiso-p120 catenin interaction has yet to be determined, but appears to include a role in modulating genes promoting canonical and non-canonical Wnt signals. ^

La homogeneidad varietal en viveros de olivo estudiada con marcadores moleculares

La difusión de variedades tradicionales de olivo (Olea europea L.) y la obtención de nuevas variantes fenotípicas han generado confusión en la correcta identificación y denominación de algunas de las aprox. 2 000 variedades conocidas a nivel mundial. En la Argentina, en los últimos años se ha triplicado la superficie implantada con olivo principalmente a partir de plantas de viveros locales e importadas. Con el fin de evaluar la homogeneidad genotípica del material comercializado en Mendoza, se probaron 7 marcadores RAPD altamente reproducibles en muestras de 5 viveros, correspondientes a 5 variedades de olivo. Los marcadores RAPD fueron previamente desarrollados para caracterizar las variedades del INTA Junín. Arbequina y Arauco fueron los materiales más homogéneos. En ambas variedades, todos los individuos compartieron el 100 % de los marcadores utilizados. Los lotes de muestras de Empeltre, Farga y Aloreña no fueron genotípicamente homogéneos, observándose 2-3 patrones diferentes por lote. Todas las variedades ensayadas -en alguna de sus muestras- tuvieron diferencias con su respectiva variedad del INTA Junín. Arbequina y Arauco también compartieron el 100 % de marcadores entre sí, no pudiéndose separar ambos grupos.

Contemporary genetic, historical genetic and morphological data sets for the Yellow-tufted Honeyeater Lichenostomus melanops

Understanding the evolutionary history of threatened populations can improve their conservation management. Re-establishment of past but recent gene flow could re-invigorate threatened populations and replenish genetic diversity, necessary for population persistence. One of the four nominal subspecies of the common yellow-tufted honeyeater, Lichenostomus melanops cassidix, is critically endangered despite substantial conservation efforts over 55 years. Using a combination of morphometric, genetic and modelling approaches we tested for its evolutionary distinctiveness and conservation merit. We confirmed that cassidix has at least one morphometric distinction. It also differs genetically from the other subspecies in allele frequencies but not phylogenetically, implying that its evolution was recent. Modelling historical distribution supported the lack of vicariance and suggested a possibility of gene flow among subspecies at least since the late Pleistocene. Multi-locus coalescent analyses indicated that cassidix diverged from its common ancestor with neighbouring subspecies gippslandicus sometime from the mid-Pleistocene to the Holocene, and that it has the smallest historical effective population size of all subspecies. It appears that cassidix diverged from its ancestor with gippslandicus through a combination of drift and local selection. From patterns of genetic subdivision on two spatial scales and morphological variation we concluded that cassidix, gippslandicus and (melanops + meltoni) are diagnosable as subspecies. Low genetic diversity and effective population size of cassidix may translate to low genetic fitness and evolutionary potential, thus managed gene flow from gippslandicus is recommended for its recovery.