Fgf17: A regulator of the mid/hind brain boundary in mammals.

The Fibroblast growth factor (FGFs) family consists of at least 22 members that exert their function by binding and activating fibroblast growth factor receptors (FGFRs). The Fgf8/FgfD subfamily member, Fgf17, is located on human chromosome 8p21.3 and mouse chromosome 14 D2. In humans, FGF17 can be alternatively spliced to produce two isoforms (FGF17a and b) whereas three isoforms are present in mice (Fgf17a, b, and c), however, only Fgf17a and Fgf17b produce functional proteins. Fgf17 is a secreted protein with a cleavable N-terminal signal peptide and contains two binding domains, namely a conserved core region and a heparin binding site. Fgf17 mRNA is expressed in a wide range of different tissues during development, including the rostral patterning centre, midbrain-hindbrain boundary, tailbud mesoderm, olfactory placode, mammary glands, and smooth muscle precursors of major arteries. Given its broad expression pattern during development, it is surprising that adult Fgf17-/- mice displayed a rather mild phenotype; such that mutants only exhibited morphological changes in the frontal cortex and mid/hind brain boundary and changes in certain social behaviours. In humans, FGF17 mutations are implicated in several diseases, including Congenital Hypogonadotropic Hypogonadism and Kallmann Syndrome. FGF17 mutations contribute to CHH/KS in 1.1% of affected individuals, often presenting in conjunction with mutations in other FGF pathway genes like FGFR1 and FLRT3. FGF17 mutations were also identified in patients diagnosed with Dandy-Walker malformation and Pituitary Stalk Interruption Syndrome, however, it remains unclear how FGF17 is implicated in these diseases. Altered FGF17 expression has been observed in several cancers, including prostate cancer, hematopoietic cancers (acute myeloid leukemia and acute lymphoblastic leukemia), glioblastomas, perineural invasion in cervical cancer, and renal cell carcinomas. Furthermore, FGF17 has demonstrated neuroprotective effects, particularly during ischemic stroke, and has been shown to improve cognitive function in ageing mice.

Protocol to establish an oviduct epithelial cell line derived from Gallus gallus using Percoll for in vitro validation of recombinant proteins.

In vitro validation of therapeutic and recombinant proteins expressed from transgenic chickens is limited by the co-culture of fibroblasts. Here, we present a protocol for isolating pure epithelial cells derived from the magnum tubular glands of the chicken oviduct. We describe steps for preparing solutions and buffers, tissue collection, processing, dissociation, and Percoll density centrifugation to separate the epithelial cells from co-isolated fibroblasts. We then detail procedures for expressing a recombinant IgG antibody in the Percoll-derived epithelial cell line.

Update of the GJB2/DFNB1 mutation spectrum in Russia: a founder Ingush mutation del(GJB2-D13S175) is the most frequent among other large deletions.

Although mutations in the GJB2 gene sequence make up the majority of variants causing autosomal-recessive non-syndromic hearing loss, few large deletions have been shown to contribute to DFNB1 deafness. Currently, genetic testing for DFNB1 hearing loss includes GJB2 sequencing and DFNB1 deletion analysis for two common large deletions, del(GJB6-D13S1830) and del(GJB6-D13S1854). Here, we report frequency in Russia, clinical significance and evolutionary origins of a 101 kb deletion, del(GJB2-D13S175), recently identified by us. In multiethnic cohort of 1104 unrelated hearing loss patients with biallelic mutations at the DFNB1 locus, the del(GJB2-D13S175) allele frequency of up to 0.5% (11/2208) was determined and this allele was shown to be predominantly associated with profound sensorineural hearing loss. Additionally, eight previously unpublished GJB2 mutations were described in this study. All patients carrying del(GJB2-D13S175) were of the Ingush ancestry. Among normal hearing individuals, del(GJB2-D13S175) was observed in Russian Republic of Ingushetia with a carrier rate of ~1% (2/241). Analysis of haplotypes associated with the deletion revealed a common founder in the Ingushes, with age of the deletion being ~3000 years old. Since del(GJB2-D13S175) was missed by standard methods of GJB2 analysis, del(GJB2-D13S175) detection has been added to our routine testing strategy for DFNB1 hearing loss.

Evolution of the vertebrate claudin gene family: insights from a basal vertebrate, the sea lamprey.

Claudins are major constituents of tight junctions, contributing both to their intercellular sealing and selective permeability properties. While claudins and claudin-like molecules are present in some invertebrates, the association of claudins with tight junctions has been conclusively documented only in vertebrates. Here we report the sequencing, phylogenetic analysis and comprehensive spatiotemporal expression analysis of the entire claudin gene family in the basal extant vertebrate, the sea lamprey. Our results demonstrate that clear orthologues to about half of all mammalian claudins are present in the lamprey, suggesting that at least one round of whole genome duplication contributed to the diversification of this gene family. Expression analysis revealed that claudins are expressed in discrete and specific domains, many of which represent vertebrate-specific innovations, such as in cranial ectodermal placodes and the neural crest; whereas others represent structures characteristic of chordates, e.g. pronephros, notochord, somites, endostyle and pharyngeal arches. By comparing the embryonic expression of claudins in the lamprey to that of other vertebrates, we found that ancestral expression patterns were often preserved in higher vertebrates. Morpholino mediated loss of Cldn3b demonstrated a functional role for this protein in placode and pharyngeal arch morphogenesis. Taken together, our data provide novel insights into the origins and evolution of the claudin gene family and the significance of claudin proteins in the evolution of vertebrates.

Spatiotemporal expression analysis of Prdm1 and Prdm1 binding partners in early chick embryo.

Prdm1 is a global repressor of transcription that plays multiple important roles during embryonic development, including neural crest specification. Prdm1 acts by repressing large sets of genes via sequence-specific recruitment of co-repressors, many of which are epigenetic modifiers. It is not known whether Prdm1 is expressed during neural crest development in chick embryo. Moreover, the mechanism of Prdm1 action or the nature of possible binding partners that mediate its effects in the neural crest had not yet been addressed. Prdm1 binding partners are known to play important roles during embryonic development, yet in many cases no spatiotemporal expression analysis during early vertebrate development has been performed. In this paper we report the expression patterns of Prdm1 and seven of its known or putative binding partners (Hdac1 and 2, Tle1 and 3, G9a, Prmt5, Lsd1) during early stages of chicken embryogenesis. Prdm1 is expressed in the neural plate border and premigratory neural crest during chick development. Six Prdm1 binding partners (except Tle1) are co-expressed with Prdm1 in the prospective neural plate border at HH4-HH6, and all seven show strong and specific expression in the neural plate border at HH7-HH8, suggesting all of them may cooperate with Prdm1 during neural crest development in chick embryos.

Eye development in the Cape dune mole rat.

Studies on mammalian species with naturally reduced eyes can provide valuable insights into the evolutionary developmental mechanisms underlying the reduction of the eye structures. Because few naturally microphthalmic animals have been studied and eye reduction must have evolved independently in many of the modern groups, novel evolutionary developmental models for eye research have to be sought. Here, we present a first report on embryonic eye development in the Cape dune mole rat, Bathyergus suillus. The eyes of these animals contain all the internal structures characteristic of the normal eye but exhibit abnormalities in the anterior chamber structures. The lens is small but develops normally and exhibits a normal expression of α- and γ-crystallins. One of the interesting features of these animals is an extremely enlarged and highly pigmented ciliary body. In order to understand the molecular basis of this unusual feature, the expression pattern of an early marker of the ciliary zone, Ptmb4, was investigated in this animal. Surprisingly, in situ hybridization results revealed that Ptmb4 expression was absent from the ciliary body zone of the developing Bathyergus eye.

Gene regulatory networks that control the specification of neural-crest cells in the lamprey.

The lamprey is the only basal vertebrate in which large-scale gene perturbation analyses are feasible at present. Studies on this unique animal model promise to contribute both to the understanding of the basic neural-crest gene regulatory network architecture, and evolution of the neural crest. In this review, we summarize the currently known regulatory relationships underlying formation of the vertebrate neural crest, and discuss new ways of addressing the many remaining questions using lamprey as an experimental model.

Postnatal development of the eye in the naked mole rat (Heterocephalus glaber).

The naked mole rat (Heterocephalus glaber) is a subterranean rodent whose eyes are thought to be visually nonfunctional and as such is an ideal animal with which to pursue questions in evolutionary developmental biology. This report is the first in-depth study on the development and morphology of the naked mole rat eye. Using standard histological analysis and scanning and transmission electron microscopy, we describe the structural features of the eye. We further report on the morphological changes that accompany the development of this eye from neonate to adult and compare them with those that occur during mouse eye development. We observed numerous abnormalities in the shape and cellular arrangement of the structures of the anterior chamber, with notable malformations of the lens. Cell proliferation and cell death assays were conducted to investigate the possible causes of lens malformation. We found that neither of these processes appeared abnormal, indicating that they were not responsible for the lens phenotype of the mole rat. In order to investigate the process of lens differentiation, we analyzed the expression of gamma-crystallins using Western blots and immunocytochemistry. At birth, levels of gamma-crystallin appear normal, but soon thereafter, the gamma-crystallin expression is terminated. Absence of detectable gamma-crystallins in adults suggests that there is a gradual degradation and loss of these proteins. The evolutionary factors that could be responsible for the eye morphology of the naked mole rat are discussed. A model for abnormal lens differentiation and the role it plays in the morphogenesis of the rest of the eye in the naked mole rats is proposed.