Katie L. Ayers

Evaluating Smoothened as a G-protein-coupled receptor for Hedgehog signalling

The Hedgehog signalling pathway controls numerous developmental processes. In response to Hedgehog, Smoothened (Smo), a seven-pass transmembrane protein, orchestrates pathway signalling and controls transcription factor activation. In the absence of Hedgehog, the receptor Patched indirectly inhibits Smo in a catalytic manner. Many questions surrounding Smo activation and signalling remain. Recent findings in Drosophila and vertebrate systems have provided strong evidence that Smo acts as a G-protein-coupled receptor. We discuss the role and regulation of Smo and reassess similarities between Smo and G-protein-coupled receptors. We also examine recently identified members of the invertebrate and vertebrate Smo signalling cascades that are typical components of G-protein-coupled receptor pathways. Greater understanding of the mechanisms of Smo activation and its signalling pathways will allow implementation of novel strategies to target disorders related to disruption of Hh signalling.

The long-range activity of Hedgehog is regulated in the apical extracellular space by the glypican Dally and the hydrolase Notum.

Cell fate determination during developmental patterning is often controlled by concentration gradients of morphogens. In the epithelial field, morphogens like the Hedgehog (Hh) peptides diffuse both apically and basolaterally; however, whether both pools of Hh are sensed at the cellular level is unclear. Here, we show that interfering with the amount of apical Hh causes a dramatic change in the long-range activation of low-threshold Hh target genes, without similar effect on short-range, high-threshold targets. We provide genetic evidence that the glypican Dally upregulates apical Hh levels, and that the release of Dally by the hydrolase Notum promotes apical Hh long-range activity. Our data suggest that several pools of Hh are perceived in epithelial tissues. Thus, we propose that the overall gradient of Hh is a composite of pools secreted by different routes (apical and basolateral), and that a cellular summation of these components is required for appropriate developmental patterning.

RNA sequencing reveals sexually dimorphic gene expression before gonadal differentiation in chicken and allows comprehensive annotation of the W-chromosome

BackgroundBirds have a ZZ male: ZW female sex chromosome system and while the Z-linked DMRT1 gene is necessary for testis development, the exact mechanism of sex determination in birds remains unsolved. This is partly due to the poor annotation of the W chromosome, which is speculated to carry a female determinant. Few genes have been mapped to the W and little is known of their expression.ResultsWe used RNA-seq to produce a comprehensive profile of gene expression in chicken blastoderms and embryonic gonads prior to sexual differentiation. We found robust sexually dimorphic gene expression in both tissues pre-dating gonadogenesis, including sex-linked and autosomal genes. This supports the hypothesis that sexual differentiation at the molecular level is at least partly cell autonomous in birds. Different sets of genes were sexually dimorphic in the two tissues, indicating that molecular sexual differentiation is tissue specific. Further analyses allowed the assembly of full-length transcripts for 26 W chromosome genes, providing a view of the W transcriptome in embryonic tissues. This is the first extensive analysis of W-linked genes and their expression profiles in early avian embryos.ConclusionSexual differentiation at the molecular level is established in chicken early in embryogenesis, before gonadal sex differentiation. We find that the W chromosome is more transcriptionally active than previously thought, expand the number of known genes to 26 and present complete coding sequences for these W genes. This includes two novel W-linked sequences and three small RNAs reassigned to the W from the Un_Random chromosome.

The role of kinases in the Hedgehog signalling pathway.

The Hedgehog (Hh) signalling pathway has a crucial role in several developmental processes and is aberrantly activated in a variety of cancers. In Drosophila, many of the canonical Hh pathway components are phosphorylated, yet the precise role of these phosphorylation events in the regulation of Hh signal transduction is unclear. Furthermore, the Hh pathway receives input from several kinases that have well‐described roles in other cellular functions, some of which have both positive and negative effects on Hh signalling. Several recent studies have characterized the role of specific phosphorylation events in the Hh pathway, and have begun to shed light on how phosphorylation of Hh signalling components affects their subcellular location, stability and activity to mediate the transcriptional response to the Hh gradient.

Disorders of sex development: Insights from targeted gene sequencing of a large international patient cohort

BackgroundDisorders of sex development (DSD) are congenital conditions in which chromosomal, gonadal, or phenotypic sex is atypical. Clinical management of DSD is often difficult and currently only 13% of patients receive an accurate clinical genetic diagnosis. To address this we have developed a massively parallel sequencing targeted DSD gene panel which allows us to sequence all 64 known diagnostic DSD genes and candidate genes simultaneously.ResultsWe analyzed DNA from the largest reported international cohort of patients with DSD (278 patients with 46,XY DSD and 48 with 46,XX DSD). Our targeted gene panel compares favorably with other sequencing platforms. We found a total of 28 diagnostic genes that are implicated in DSD, highlighting the genetic spectrum of this disorder. Sequencing revealed 93 previously unreported DSD gene variants. Overall, we identified a likely genetic diagnosis in 43% of patients with 46,XY DSD. In patients with 46,XY disorders of androgen synthesis and action the genetic diagnosis rate reached 60%. Surprisingly, little difference in diagnostic rate was observed between singletons and trios. In many cases our findings are informative as to the likely cause of the DSD, which will facilitate clinical management.ConclusionsOur massively parallel sequencing targeted DSD gene panel represents an economical means of improving the genetic diagnostic capability for patients affected by DSD. Implementation of this panel in a large cohort of patients has expanded our understanding of the underlying genetic etiology of DSD. The inclusion of research candidate genes also provides an invaluable resource for future identification of novel genes.

The Molecular Genetics of Ovarian Differentiation in the Avian Model

In birds as in mammals, sex is determined at fertilization by the inheritance of sex chromosomes. However, sexual differentiation – development of a male or female phenotype – occurs during embryonic development. Sex differentiation requires the induction of sex-specific developmental pathways in the gonads, resulting in the formation of ovaries or testes. Birds utilize a different sex chromosome system to that of mammals, where females are the heterogametic sex (carrying Z and W chromosomes), while males are homogametic (carrying 2 Z chromosomes). Therefore, while some genes essential for testis and ovarian development are conserved, important differences also exist. Namely, the key mammalian male-determining factor SRY does not exist in birds, and another transcription factor, DMRT1, plays a central role in testis development. In contrast to our understanding of testis development, ovarian differentiation is less well-characterized. Given the presence of a female-specific chromosome, studies in chicken will provide insight into the induction and function of female-specific gonadal pathways. In this review, we discuss sexual differentiation in chicken embryos, with emphasis on ovarian development. We highlight genes that may play a conserved role in this process, and discuss how interaction between ovarian pathways may be regulated.

The Genetic and Environmental Factors Underlying Hypospadias

Hypospadias results from a failure of urethral closure in the male phallus and affects 1 in 200-300 boys. It is thought to be due to a combination of genetic and environmental factors. The development of the penis progresses in 2 stages: an initial hormone-independent phase and a secondary hormone-dependent phase. Here, we review the molecular pathways that contribute to each of these stages, drawing on studies from both human and mouse models. Hypospadias can occur when normal development of the phallus is disrupted, and we provide evidence that mutations in genes underlying this developmental process are causative. Finally, we discuss the environmental factors that may contribute to hypospadias and their potential immediate and transgenerational epigenetic impacts.

Dally and Notum regulate the switch between low and high level Hedgehog pathway signalling.

During development, secreted morphogens, such as Hedgehog (Hh), control cell fate and proliferation. Precise sensing of morphogen levels and dynamic cellular responses are required for morphogen-directed morphogenesis, yet the molecular mechanisms responsible are poorly understood. Several recent studies have suggested the involvement of a multi-protein Hh reception complex, and have hinted at an understated complexity in Hh sensing at the cell surface. We show here that the expression of the proteoglycan Dally in Hh-receiving cells in Drosophila is necessary for high but not low level pathway activity, independent of its requirement in Hh-producing cells. We demonstrate that Dally is necessary to sequester Hh at the cell surface and to promote Hh internalisation with its receptor. This internalisation depends on both the activity of the hydrolase Notum and the glycosyl-phosphatidyl-inositol (GPI) moiety of Dally, and indicates a departure from the role of the second glypican Dally-like in Hh signalling. Our data suggest that hydrolysis of the Dally-GPI by Notum provides a switch from low to high level signalling by promoting internalisation of the Hh-Patched ligand-receptor complex.

The molecular genetics of avian sex determination and its manipulation

The chicken (Gallus gallus domesticus) has long been a useful model for developmental biologists. The developing avian embryo is easily accessible and fertile eggs are widely available. In addition, the embryo is also amenable to genetic manipulation allowing studies on many important morphological and cellular processes. More recently, the ability to directly manipulate gene expression through the production of transgenic or mutant chicken embryos by viral delivery methods has been useful to analyse gene function in a wide range of tissues, including the developing gonads. Chickens are amniotes and their development closely resembles that of mammals, implying underlying genetic conservation of key pathways, including sex development. Studies of sex determination and gonadal development in this model are providing insight into avian ovarian and testis developmental pathways and their evolution. Indeed, the chicken embryo is a suitable model for the functional analysis of genes implicated in human disorders of sex development, and studies in this model will complement those carried out in mammalian models such as the mouse. In this review we discuss the current knowledge of sex determination and sexual differentiation in avians, using chicken as model. We review how sex chromosomes contribute to this process and provide current information on our understanding of gonadal sexual differentiation at both the cellular and molecular level in the chicken embryo. Finally, we review the methods currently used to investigate the role of genes and signaling pathways during sexual differentiation, and discuss how these methods may contribute to further understanding of vertebrate gonadogenesis. genesis 51:325–336.

Identification of candidate gonadal sex differentiation genes in the chicken embryo using RNA-seq.

BackgroundDespite some advances in recent years, the genetic control of gonadal sex differentiation during embryogenesis is still not completely understood. To identify new candidate genes involved in ovary and testis development, RNA-seq was used to define the transcriptome of embryonic chicken gonads at the onset of sexual differentiation (day 6.0/stage 29).ResultsRNA-seq revealed more than 1000 genes that were transcribed in a sex-biased manner at this early stage of gonadal differentiation. Comparison with undifferentiated gonads revealed that sex biased expression was derived primarily from autosomal rather than sex-linked genes. Gene ontology and pathway analysis indicated that many of these genes encoded proteins involved in extracellular matrix function and cytoskeletal remodelling, as well as tubulogenesis. Several of these genes are novel candidate regulators of gonadal sex differentiation, based on sex-biased expression profiles that are altered following experimental sex reversal. We further characterised three female-biased (ovarian) genes; calpain-5 (CAPN5), G-protein coupled receptor 56 (GPR56), and FGFR3 (fibroblast growth factor receptor 3). Protein expression of these candidates in the developing ovaries suggests that they play an important role in this tissue.ConclusionsThis study provides insight into the earliest steps of vertebrate gonad sex differentiation, and identifies novel candidate genes for ovarian and testicular development.Despite some advances in recent years, the genetic control of gonadal sex differentiation during embryogenesis is still not completely understood. To identify new candidate genes involved in ovary and testis development, RNA-seq was used to define the transcriptome of embryonic chicken gonads at the onset of sexual differentiation (day 6.0/stage 29). RNA-seq revealed more than 1000 genes that were transcribed in a sex-biased manner at this early stage of gonadal differentiation. Comparison with undifferentiated gonads revealed that sex biased expression was derived primarily from autosomal rather than sex-linked genes. Gene ontology and pathway analysis indicated that many of these genes encoded proteins involved in extracellular matrix function and cytoskeletal remodelling, as well as tubulogenesis. Several of these genes are novel candidate regulators of gonadal sex differentiation, based on sex-biased expression profiles that are altered following experimental sex reversal. We further characterised three female-biased (ovarian) genes; calpain-5 (CAPN5), G-protein coupled receptor 56 (GPR56), and FGFR3 (fibroblast growth factor receptor 3). Protein expression of these candidates in the developing ovaries suggests that they play an important role in this tissue. This study provides insight into the earliest steps of vertebrate gonad sex differentiation, and identifies novel candidate genes for ovarian and testicular development.