Cyclin-dependent Kinase 1 and Aurora Kinase choreograph mitotic storage and redistribution of a growth factor receptor.

Endosomal trafficking of receptors and associated proteins plays a critical role in signal processing. Until recently, it was thought that trafficking was shut down during cell division. Thus, remarkably, the regulation of trafficking during division remains poorly characterized. Here we delineate the role of mitotic kinases in receptor trafficking during asymmetric division. Targeted perturbations reveal that Cyclin-dependent Kinase 1 (CDK1) and Aurora Kinase promote storage of Fibroblast Growth Factor Receptors (FGFRs) by suppressing endosomal degradation and recycling pathways. As cells progress through metaphase, loss of CDK1 activity permits differential degradation and targeted recycling of stored receptors, leading to asymmetric induction. Mitotic receptor storage, as delineated in this study, may facilitate rapid reestablishment of signaling competence in nascent daughter cells. However, mutations that limit or enhance the release of stored signaling components could alter daughter cell fate or behavior thereby promoting oncogenesis.

Transgenic Techniques for Investigating Cell Biology During Development.

Ascidians are increasingly being used as a system for investigating cell biology during development. The extreme genetic and cellular simplicity of ascidian embryos in combination with superior experimental tractability make this an ideal system for in vivo analysis of dynamic cellular processes. Transgenic approaches to cellular and sub-cellular analysis of ascidian development have begun to yield new insights into the mechanisms regulating developmental signaling and morphogenesis. This chapter focuses on the targeted expression of fusion proteins in ascidian embryos and how this technique is being deployed to garner new insights into the cell biology of development.

Mitotic Membrane Turnover Coordinates Differential Induction of the Heart Progenitor Lineage.

In response to microenvironmental cues, embryonic cells form adhesive signaling compartments that influence survival and patterning. Dividing cells detach from the surrounding matrix and initiate extensive membrane remodeling, but the in vivo impact of mitosis on adhesion-dependent signaling remains poorly characterized. We investigate in vivo signaling dynamics using the invertebrate chordate, Ciona intestinalis. In Ciona, matrix adhesion polarizes fibroblast growth factor (FGF)-dependent heart progenitor induction. Here, we show that adhesion inhibits mitotic FGF receptor internalization, leading to receptor enrichment along adherent membranes. Targeted disruption of matrix adhesion promotes uniform FGF receptor internalization and degradation while enhanced adhesion suppresses degradation. Chimeric analysis indicates that integrin ß chain-specific impacts on induction are dictated by distinct internalization motifs. We also found that matrix adhesion impacts receptor enrichment through Caveolin-rich membrane domains. These results redefine the relationship between cell division and adhesive signaling, revealing how mitotic membrane turnover orchestrates adhesion-dependent signal polarization.

Heart genetics in a small package, exploiting the condensed genome of Ciona intestinalis.

Defects in the initial establishment of cardiogenic cell fate are likely to contribute to pervasive human congenital cardiac abnormalities. However, the molecular underpinnings of nascent cardiac fate induction have proven difficult to decipher. In this review we explore the participation of extracellular, cellular and nuclear factors in comprehensive specification networks. At each level, we elaborate on insights gained through the study of cardiogenesis in the invertebrate chordate Ciona intestinalis and propose productive lines of future research. In-depth discussion of pre-cardiac induction is intended to serve as a paradigm, illustrating the potential use of Ciona to elucidate comprehensive networks underlying additional aspects of chordate cardiogenesis, including directed migration and subspecification of cardiac and pharyngeal lineages.

Matrix adhesion polarizes heart progenitor induction in the invertebrate chordate Ciona intestinalis.

Cell-matrix adhesion strongly influences developmental signaling. Resulting impacts on cell migration and tissue morphogenesis are well characterized. However, the in vivo impact of adhesion on fate induction remains ambiguous. Here, we employ the invertebrate chordate Ciona intestinalis to delineate an essential in vivo role for matrix adhesion in heart progenitor induction. In Ciona pre-cardiac founder cells, invasion of the underlying epidermis promotes localized induction of the heart progenitor lineage. We found that these epidermal invasions are associated with matrix adhesion along the pre-cardiac cell/epidermal boundary. Through targeted manipulations of RAP GTPase activity, we were able to manipulate pre-cardiac cell-matrix adhesion. Targeted disruption of pre-cardiac cell-matrix adhesion blocked heart progenitor induction. Conversely, increased matrix adhesion generated expanded induction. We were also able to selectively restore cell-matrix adhesion and heart progenitor induction through targeted expression of Ci-Integrin ß2. These results indicate that matrix adhesion functions as a necessary and sufficient extrinsic cue for regional heart progenitor induction. Furthermore, time-lapse imaging suggests that cytokinesis acts as an intrinsic temporal regulator of heart progenitor adhesion and induction. Our findings highlight a potentially conserved role for matrix adhesion in early steps of vertebrate heart progenitor specification.

The ENU-induced cetus mutation reveals an essential role of the DNA helicase DDX11 for mesoderm development during early mouse embryogenesis.

BACKGROUND: DDX11 is a DNA helicase of the conserved FANCJ/RAD3/XPD family involved in maintaining genome stability. Studies in yeast and humans have shown requirements for DDX11 in sister chromatid cohesion and DNA repair. In mouse, loss of Ddx11 results in embryonic lethality. However, the developmental defects of Ddx11 mutants are poorly understood. RESULTS: We describe the characterization and positional cloning of cetus, a mouse ENU-induced mutation in Ddx11. We demonstrate that cetus causes a nonconservative amino acid change in DDX11 motif V and that this mutation is a null allele of Ddx11. cetus mutant embryos failed to thrive beyond embryonic day 8.5 and displayed placental defects similar to those described in Ddx11 null embryos. Additionally, our characterization of Ddx11(cetus) mutants identified embryonic phenotypes that had not been previously reported in Ddx11(KO) embryos, including loss of somitic mesoderm, an open kinked neural tube and abnormal heart looping. We show that loss of Ddx11 causes widespread apoptosis from early embryonic stages and that loss of Ddx11 disrupts somitic mesoderm more dramatically than other embryonic cells. CONCLUSIONS: Our results identify novel roles of Ddx11 during embryo morphogenesis and demonstrate that the activity of its motif V is essential for DDX11 function.

Abnormal regulation of TSG101 in mice with spongiform neurodegeneration.

Spongiform neurodegeneration is characterized by the appearance of vacuoles throughout the central nervous system. It has many potential causes, but the underlying cellular mechanisms are not well understood. Mice lacking the E3 ubiquitin ligase Mahogunin Ring Finger-1 (MGRN1) develop age-dependent spongiform encephalopathy. We identified an interaction between a "PSAP" motif in MGRN1 and the ubiquitin E2 variant (UEV) domain of TSG101, a component of the endosomal sorting complex required for transport I (ESCRT-I), and demonstrate that MGRN1 multimonoubiquitinates TSG101. We examined the in vivo consequences of loss of MGRN1 on TSG101 expression and function in the mouse brain. The pattern of TSG101 ubiquitination differed in the brains of wild-type mice and Mgrn1 null mutant mice: at 1 month of age, null mutant mice had less ubiquitinated TSG101, while in adults, mutant mice had more ubiquitinated, insoluble TSG101 than wild-type mice. There was an associated increase in epidermal growth factor receptor (EGFR) levels in mutant brains. These results suggest that loss of MGRN1 promotes ubiquitination of TSG101 by other E3s and may prevent its disassociation from endosomal membranes or cause it to form insoluble aggregates. Our data implicate loss of normal TSG101 function in endo-lysosomal trafficking in the pathogenesis of spongiform neurodegeneration in Mgrn1 null mutant mice.

Genetic and phenotypic studies of the dark-like mutant mouse.

The dark-like (dal) mutant mouse has a pleiotropic phenotype that includes dark dorsal hairs and reproductive degeneration. Their pigmentation phenotype is similar to Attractin (Atrn) mutants, which also develop vacuoles throughout the brain. In further characterizing the testicular degeneration of dal mutant males, we found that they had reduced serum testosterone and developed vacuoles in their testes. Genetic crosses placed dal upstream of the melanocortin 1 receptor (Mc1r) and downstream of agouti, although dal suppressed the effect of agouti on pigmentation but not body weight. Atrn(mg-3J) and dal showed additive effects on pigmentation, testicular vacuolation, and spongiform neurodegeneration, but transgenic overexpression of Attractin-like-1 (Atrnl1), which compensates for loss of ATRN, did not rescue dal mutant phenotypes. Our results suggest dal and Atrn function in the same pathway and that identification of the dal gene will provide insight into molecular mechanisms of vacuolation in multiple cell types.

Characterization of Mahogunin Ring Finger-1 expression in mice.

Mutations in mouse Mahogunin Ring Finger-1 (Mgrn1) were first recognized for their effect on agouti-mediated pigment-type switching. Mgrn1 null mutants are completely black and develop spongiform degeneration of the brain. Mgrn1 hypomorphs have dark fur but do not develop neurodegeneration. We characterized a new Mgrn1 hypomorphic allele caused by a gene-trap insertion. Mice homozygous for this mutation are slightly darker than non-mutant animals. They show reduced overall expression of Mgrn1 and two of the four normal Mgrn1 isoforms are replaced by beta-GEO fusion proteins that differ from the normal proteins at their carboxy termini. To investigate the role of different Mgrn1 isoforms in pigment-type switching, we used quantitative relative reverse transcriptase polymerase chain reaction to examine their expression in the skin of Mgrn1 mutant and control mice. Most Mgrn1 mutants produce little or no normal Mgrn1 in the skin. Mgrn1 null mutant mice overexpressing isoform I or III, which are normally absent or weakly expressed in adult skin, had normal agouti-banded hairs. Our results indicate that reduced levels of MGRN1 cause the pigmentation phenotypes of Mgrn1 mutant mice and that there are no significant differences in the function of the four MGRN1 isoforms in pigment-type switching.

Mice with mutations in Mahogunin ring finger-1 (Mgrn1) exhibit abnormal patterning of the left-right axis.

Mahogunin Ring Finger 1 (Mgrn1) encodes a RING-containing protein with ubiquitin ligase activity that has been implicated in pigment-type switching. In addition to having dark fur, mice lacking MGRN1 develop adult-onset spongy degeneration of the central nervous system and have reduced embryonic viability. Observation of complete situs inversus in a small proportion of adult Mgrn1 mutant mice suggested that embryonic lethality resulted from congenital heart defects due to defective establishment and/or maintenance of the left-right (LR) axis. Here we report that Mgrn1 is expressed in a pattern consistent with a role in LR patterning during early development and that many Mgrn1 mutant embryos show abnormal expression of asymmetrically expressed genes involved in LR patterning. A range of complex heart defects was observed in 20-25% of mid-to-late gestation Mgrn1 mutant embryos and another 20% were dead. This finding was consistent with 46-60% mortality of mutants by weaning age. Our results indicate that Mgrn1 acts early in the LR signaling cascade and is likely to provide new insight into this developmental process as Nodal expression was uncoupled from expression of other Nodal-responsive genes in Mgrn1 mutant embryos. Our work identifies a novel role for MGRN1 in embryonic patterning and suggests that the ubiquitination of MGRN1 target genes is essential for the proper establishment and/or maintenance of the LR axis.