Annemiek Beverdam

Msx1 and Dlx5 act independently in development of craniofacial skeleton, but converge on the regulation of Bmp signaling in palate formation.

Msx and Dlx homeoproteins control the morphogenesis and organization of craniofacial skeletal structures, specifically those derived from the pharyngeal arches. In vitro Msx and Dlx proteins have opposing transcriptional properties and form heterodimeric complexes via their homeodomain with reciprocal functional repression. In this report we examine the skeletal phenotype of Msx1; Dlx5 double knock-out (DKO) mice in relationship with their expression territories during craniofacial development. Co-expression of Dlx5 and Msx1 is only observed in embryonic tissues in which these genes have independent functions, and thus direct protein interactions are unlikely to control morphogenesis of the cranium. The DKO craniofacial phenotypes indicate a complex interplay between these genes, acting independently (mandible and middle ear), synergistically (deposition of bone tissue) or converging on the same morphogenetic process (palate growth and closure). In the latter case, the absence of Dlx5 rescues in part the Msx1-dependent defects in palate growth and elevation. At the basis of this effect, our data implicate the Bmp (Bmp7, Bmp4)/Bmp antagonist (Follistatin) signal: in the Dlx5(-/-) palate changes in the expression level of Bmp7 and Follistatin counteract the reduced Bmp4 expression. These results highlight the importance of precise spatial and temporal regulation of the Bmp/Bmp antagonist system during palate closure.

Genetics of shoulder girdle formation: roles of Tbx15 and aristaless-like genes

The diverse cellular contributions to the skeletal elements of the vertebrate shoulder and pelvic girdles during embryonic development complicate the study of their patterning. Research in avian embryos has recently clarified part of the embryological basis of shoulder formation. Although dermomyotomal cells provide the progenitors of the scapular blade, local signals appear to have an essential guiding role in this process. These signals differ from those that are known to pattern the more distal appendicular skeleton. We have studied the impact of Tbx15, Gli3, Alx4 and related genes on formation of the skeletal elements of the mouse shoulder and pelvic girdles. We observed severe reduction of the scapula in double and triple mutants of these genes. Analyses of a range of complex genotypes revealed aspects of their genetic relationship, as well as functions that had been previously masked due to functional redundancy. Tbx15 and Gli3 appear to have synergistic functions in formation of the scapular blade. Scapular truncation in triple mutants of Tbx15, Alx4 and Cart1 indicates essential functions for Alx4 and Cart1 in the anterior part of the scapula, as opposed to Gli3 function being linked to the posterior part. Especially in Alx4/Cart1 mutants, the expression of markers such as Pax1, Pax3 and Scleraxis is altered prior to stages when anatomical aberrations are visible in the shoulder region. This suggests a disorganization of the proximal limb bud and adjacent flank mesoderm, and is likely to reflect the disruption of a mechanism providing positional cues to guide progenitor cells to their destination in the pectoral girdle.

Expression patterns of group-I aristaless-related genes during craniofacial and limb development.

Aristaless-related proteins are structurally defined by the presence of a paired-type homeodomain and an additional conserved domain, known as aristaless domain or OAR-domain. These proteins can be further categorized in three groups (Int. J. Dev. Biol., 43 (1999) 651). Group-I aristaless-related genes are linked to functions in the development of the craniofacial and appendicular skeleton and are expressed predominantly in the mesenchyme in stages from gastrulation through at least mid-gestation (Mech. Dev., 48 (1994) 245; Mech. Dev., 52 (1995) 51; Development, 124 (1997) 3999; Dev. Biol., 199 (1998) 11; Development, 126 (1999) 495). In view of the highly redundant character of the functions of these genes in patterning craniofacial and limb structures, we found it important to directly compare their expression patterns at critical stages of craniofacial and limb development.

Molecular characterization of three gonad cell lines

To facilitate the study of the regulation and downstream interactions of genes involved in gonad development it is important to have a suitable cell culture model. We therefore aimed to characterize molecularly three different mouse gonad cell lines. TM3 and TM4 cells were originally isolated from prepubertal mouse gonads and were tentatively identified as being of Leydig cell and Sertoli cell origin, respectively, based upon their morphology and hormonal responses. The third line is a conditionally immortalized cell line, derived from 10.5–11.5 days post-coitum (dpc) male gonads of transgenic embryos carrying a temperature-sensitive SV40 large T-antigen. We studied by reverse transcription-polymerase chain reaction (RT-PCR) the expression profiles of a number of genes known to be important for early gonad development. Moreover, we assessed these cell lines for their capacity to induce Sox9 transcription upon expression of Sry, a key molecular event occurring during sex determination. We found that all three cell lines were unable to upregulate Sox9 expression upon transfection of Sry-expression constructs, even though these cells express many of the studied embryonic gonad genes. These observations point to a requirement for SRY cofactors for direct or indirect upregulation of Sox9 expression during testis determination.

Yap Controls Stem/Progenitor Cell Proliferation in the Mouse Postnatal Epidermis

Tissue renewal is an ongoing process in the epithelium of the skin. We have begun to examine the genetic mechanisms that control stem/progenitor cell activation in the postnatal epidermis. The conserved Hippo pathway regulates stem cell turnover in arthropods through to vertebrates. Here we show that its downstream effector, yes-associated protein (YAP), is active in the stem/progenitor cells of the postnatal epidermis. Overexpression of a C-terminally truncated YAP mutant in the basal epidermis of transgenic mice caused marked expansion of epidermal stem/progenitor cell populations. Our data suggest that the C-terminus of YAP controls the balance between stem/progenitor cell proliferation and differentiation in the postnatal interfollicular epidermis. We conclude that YAP functions as a molecular switch of stem/progenitor cell activation in the epidermis. Moreover, our results highlight YAP as a possible therapeutic target for diseases such as skin cancer, psoriasis, and epidermolysis bullosa.

Sex-specific expression of a novel gene Tmem184a during mouse testis differentiation.

During mouse embryogenesis, the fate of the bipotential gonads is sealed around 10.5 days post coitum (dpc) when the Y-linked gene Sry specifies the differentiation of testes in males, whereas in females, absence of Sry results in ovary formation. Apart from the pivotal action of Sry, many other genes are known to be involved in sex determination and subsequent differentiation. Much is still unknown regarding the regulatory hierarchy governing these events and many more sex differentiation genes are yet to be discovered. In this study, we investigated the expression of Tmem184a, a novel gene encoding a protein of unknown function, but with predicted kinase activity, during mouse embryogenesis. We show that Tmem184a is expressed at high levels in the developing testis from 11.5 dpc, a time of active proliferation and differentiation. Tmem184a expression is further shown to be expressed exclusively within the Sertoli cells of the developing testis cords, suggesting that it may mediate sex-specific signaling events during Sertoli cell differentiation.

The E3 ubiquitin ligase Mycbp2 genetically interacts with Robo2 to modulate axon guidance in the mouse olfactory system

The E3 ubiquitin ligase Mycbp2 and it homologues play an important role in axon guidance and synaptogenesis in Drosophila, Caenorhabditis elegans, zebrafish and mouse. Despite this conserved function, the molecular and cellular basis of Mycbp2-dependent axon guidance remains largely unclear. We have examined here the effect of the loss-of-MYCBP2 function on the topography of the olfactory sensory neuron projection from the nasal cavity to the olfactory bulb in mice. A subpopulation of olfactory sensory axons failed to project to the dorsal surface of the olfactory bulb causing abnormal topography in this neural pathway. These defects were similar to the olfactory bulb phenotype in loss-of-ROBO2 function mice. While mice heterozygous for either Mycbp2 or Robo2 were normal, mice double heterozygous for these two genes produced severe defects in the olfactory system. Therefore, Mycbp2 and Robo2 were found to cooperate within a genetic network that has profound effects on axon guidance. The Mycbp2 phenotype could be partly explained by aberrant patterning of olfactory sensory neurons residing in the dorsal compartment of the nasal cavity. Some of these neurons fail to appropriately express Robo2 which is consistent with their aberrant projection to the ventral olfactory bulb. These results provide the first evidence linking an ubiquitin ligase to an axon guidance receptor during pathfinding in the developing mammalian nervous system.

Positive regulatory interactions between YAP and Hedgehog signalling in skin homeostasis and BCC development in mouse skin in vivo

Skin is a highly plastic tissue that undergoes tissue turnover throughout life, but also in response to injury. YAP and Hedgehog signalling play a central role in the control of epidermal stem/progenitor cells in the skin during embryonic development, in postnatal tissue homeostasis and in skin carcinogenesis. However, the genetic contexts in which they act to control tissue homeostasis remain mostly unresolved. We provide compelling evidence that epidermal YAP and Hedgehog/GLI2 signalling undergo positive regulatory interactions in the control of normal epidermal homeostasis and in basal cell carcinoma (BCC) development, which in the large majority of cases is caused by aberrant Hedgehog signalling activity. We report increased nuclear YAP and GLI2 activity in the epidermis and BCCs of K14-CreER/Rosa-SmoM2 transgenic mouse skin, accompanied with increased ROCK signalling and ECM remodelling. Furthermore, we found that epidermal YAP activity drives GLI2 nuclear accumulation in the skin of YAP2-5SA-ΔC mice, which depends on epidermal β-catenin activation. Lastly, we found prominent nuclear activity of GLI2, YAP and β-catenin, concomitant with increased ROCK signalling and stromal fibrosis in human BCC. Our work provides novel insights into the molecular mechanisms underlying the interplay between cell signalling events and mechanical force in normal tissue homeostasis in vivo, that could potentially be perturbed in BCC development.

The Expression Pattern of EVA1C, a Novel Slit Receptor, Is Consistent with an Axon Guidance Role in the Mouse Nervous System

The Slit/Robo axon guidance families play a vital role in the formation of neural circuitry within select regions of the developing mouse nervous system. Typically Slits signal through the Robo receptors, however they also have Robo-independent functions. The novel Slit receptor Eva-1, recently discovered in C. elegans, and the human orthologue of which is located in the Down syndrome critical region on chromosome 21, could account for some of these Robo independent functions as well as provide selectivity to Robo-mediated axon responses to Slit. Here we investigate the expression of the mammalian orthologue EVA1C in regions of the developing mouse nervous system which have been shown to exhibit Robo-dependent and -independent responses to Slit. We report that EVA1C is expressed by axons contributing to commissures, tracts and nerve pathways of the developing spinal cord and forebrain. Furthermore it is expressed by axons that display both Robo-dependent and -independent functions of Slit, supporting a role for EVA1C in Slit/Robo mediated neural circuit formation in the developing nervous system.

Sox9-dependent expression of Gstm6 in Sertoli cells during testis development in mice

Glutathione S-transferases (GSTs) are an important family of multifunctional enzymes that play a role in the protection of tissues by the detoxification of hazardous and carcinogenic compounds. We found previously that Gstm6 is upregulated in the somatic cells of male mouse fetal gonads relative to female gonads. In this study, we describe the spatial and temporal expression pattern of Gstm6 during mouse development. We show that Gstm6 is predominantly expressed in the reproductive system, at significantly higher levels in XY gonads compared with XX gonads from 11.5 dpc onwards, and remains expressed in the testes in adult mice. Its expression is associated with the Sertoli cell lineage, and is dependent on the expression of the male sex-determining gene Sox9. Our data suggest that Gstm6 plays a male-specific role in gonad development or function, possibly by modulating the exposure of somatic tissue and/or germ cells to endogenous or exogenous toxicants.