Michael R. Kuehn

A molecular pathway determining left-right asymmetry in chick embryogenesis

While significant progress has been made in understanding the molecular events underlying the early specification of the antero-posterior and dorso-ventral axes, little information is available regarding the cellular or molecular basis for left-right (LR) differences in animal morphogenesis. We describe the expression patterns of three genes involved in LR determination in chick embryos: activin receptor IIa, Sonic hedgehog (Shh), and cNR-1 (related to the mouse gene nodal). These genes are expressed asymmetrically during and after gastrulation and regulate the expression of one another in a sequential pathway. Moreover, manipulation of the sidedness of either activin protein or Shh expression alters heart situs. Together, these observations identify a cascade of molecular asymmetry in that determines morphological LR asymmetry in the chick embryo.

The SIL gene is required for mouse embryonic axial development and left-right specification

The establishment of the main body axis and the determination of left–right asymmetry are fundamental aspects of vertebrate embryonic development. A link between these processes has been revealed by the frequent finding of midline defects in humans with left–right anomalies. This association is also seen in a number of mutations in mouse and zebrafish,, and in experimentally manipulated Xenopus embryos. However, the severity of laterality defects accompanying abnormal midline development varies, and the molecular basis for this variation is unknown. Here we show that mouse embryos lacking the early-response gene SIL have axial midline defects, a block in midline Sonic hedgehog (Shh) signalling and randomized cardiac looping. Comparison with Shh mutant embryos, which have axial defects but normal cardiac looping, indicates that the consequences of abnormal midline development for left–right patterning depend on the time of onset, duration and severity of disruption of the normal asymmetric patterns of expression of nodal, lefty-2 and Pitx2 .

Nodal Signaling Uses Activin and Transforming Growth Factor-β Receptor-regulated Smads

Nodal, a member of the transforming growth factor β (TGF-β) superfamily, is implicated in many events critical to the early vertebrate embryo, including mesoderm formation, anterior patterning, and left-right axis specification. Here we define the intracellular signaling pathway induced by recombinant nodal protein treatment of P19 embryonal carcinoma cells. Nodal signaling activates pAR3-Lux, a luciferase reporter previously shown to respond specifically to activin and TGF-β. However, nodal is unable to induce pTlx2-Lux, a reporter specifically responsive to bone morphogenetic proteins. We also demonstrate that nodal induces p(CAGA)12, a reporter previously shown to be specifically activated by Smad3. Expression of a dominant negative Smad2 significantly reduces the level of luciferase reporter activity induced by nodal treatment. Finally, we show that nodal signaling rapidly leads to the phosphorylation of Smad2. These results provide the first direct biochemical evidence that nodal signaling is mediated by both activin-TGF-β pathway Smads, Smad2 and Smad3. We also show here that the extracellular cripto protein is required for nodal signaling, making it distinct from activin or TGF-β signaling.

Nodal Signaling Recruits the Histone Demethylase Jmjd3 to Counteract Polycomb-Mediated Repression at Target Genes

Developmental signaling integrates with epigenetic processes to control the expression of Nodal target genes. Regulatory Two-Step During the development of metazoans, lineage-specific patterns of gene expression that become stably maintained across many cell generations are established in response to transient intercellular signals. One such signal is the transforming growth factor–β (TGF-β)–related ligand, Nodal, which is required for the formation of mesoderm and endoderm and for body patterning. The established gene expression programs are still responsive to further developmental cues, and this transcriptional plasticity arises due to epigenetic regulation at the level of chromatin. Trimethylation of Lys27 (K27) of histone H3 (H3K27me3) is mediated by Polycomb, which results in repression of gene expression. Histone demethylases, such as Jmjd3, target H3K27me3, which renders Polycomb-mediated repression highly dynamic; however, how the activity of Polycomb is linked to developmental, intercellular signaling pathways is unclear. Dahle et al. showed that the expression of Nodal, which depends on Nodal activity, was repressed by Polycomb-mediated deposition of H3K27me3 and that Nodal signaling, through the proteins Smad2 and Smad3 (Smads2/3), recruited Jmjd3 to the Nodal locus to counteract the effects of Polycomb. In the absence of Polycomb, the expression of Nodal and another Nodal target, Brachyury, was independent of Nodal signaling. Together, these findings suggest how transcriptional plasticity can arise through cooperation between the epigenetic machinery and developmental signaling pathways. Both intercellular signaling and epigenetic mechanisms regulate embryonic development, but it is unclear how they are integrated to establish and maintain lineage-specific gene expression programs. Here, we show that a key function of the developmentally essential Nodal-Smads2/3 (Smad2 and Smad3) signaling pathway is to recruit the histone demethylase Jmjd3 to target genes, thereby counteracting repression by Polycomb. Smads2/3 bound to Jmjd3 and recruited it to chromatin in a manner that was dependent on active Nodal signaling. Knockdown of Jmjd3 alone substantially reduced Nodal target gene expression, whereas in the absence of Polycomb, target loci were expressed independently of Nodal signaling. These data establish a role for Polycomb in imposing a dependency on Nodal signaling for the expression of target genes and reveal how developmental signaling integrates with epigenetic processes to control gene expression.

Essential Role of Chromatin Remodeling Protein Bptf in Early Mouse Embryos and Embryonic Stem Cells

We have characterized the biological functions of the chromatin remodeling protein Bptf (Bromodomain PHD-finger Transcription Factor), the largest subunit of NURF (Nucleosome Remodeling Factor) in a mammal. Bptf mutants manifest growth defects at the post-implantation stage and are reabsorbed by E8.5. Histological analyses of lineage markers show that Bptf−/− embryos implant but fail to establish a functional distal visceral endoderm. Microarray analysis at early stages of differentiation has identified Bptf-dependent gene targets including homeobox transcriptions factors and genes essential for the development of ectoderm, mesoderm, and both definitive and visceral endoderm. Differentiation of Bptf−/− embryonic stem cell lines into embryoid bodies revealed its requirement for development of mesoderm, endoderm, and ectoderm tissue lineages, and uncovered many genes whose activation or repression are Bptf-dependent. We also provide functional and physical links between the Bptf-containing NURF complex and the Smad transcription factors. These results suggest that Bptf may co-regulate some gene targets of this pathway, which is essential for establishment of the visceral endoderm. We conclude that Bptf likely regulates genes and signaling pathways essential for the development of key tissues of the early mouse embryo.

Loss of SUMO1 in mice affects RanGAP1 localization and formation of PML nuclear bodies, but is not lethal as it can be compensated by SUMO2 or SUMO3

Conjugation of the small ubiquitin-like modifier (SUMO) to target proteins regulates numerous biological processes and has been implicated in tumorigenesis and metastasis. The three SUMO isoforms in vertebrates, SUMO1 and the highly similar SUMO2 and SUMO3, can be conjugated to unique as well as overlapping subsets of target proteins. Yet, it is still not clear whether roles for each family member are distinct or whether redundancy exists. Here we describe a mutant mouse line that completely lacks SUMO1, but surprisingly is viable and lacks any overt phenotype. Our study points to compensatory utilization of SUMO2 and/or SUMO3 for sumoylation of SUMO1 targets. The ability of SUMO isoforms to substitute for one another has important implications for rational targeting of the SUMO pathway.

Mutation of SENP1/SuPr-2 reveals an essential role for desumoylation in mouse development.

ABSTRACT The covalent modification of proteins by the small ubiquitin-like protein SUMO has been implicated in the regulation of numerous biological processes, including nucleocytoplasmic transport, genomic stability, and gene transcription. Sumoylation occurs by a multienzyme process similar to ubiquitination and, in Saccharomyces cerevisiae, is reversed by desumoylating enzymes encoded by the Ulp1 and Smt4/Ulp2 genes. The physiological importance of desumoylation has been revealed by mutations in either gene, which lead to nonoverlapping defects in cell cycle transition and meiosis. Several mammalian Ulp homologues have been identified, but, to date, nothing is known of the phenotypic effects of their loss of function. Here, we describe a random retroviral insertional mutation of one homolog, mouse SENP1/SuPr-2. The mutation causes increased steady-state levels of the sumoylated forms of a number of proteins and results in placental abnormalities incompatible with embryonic development. Our findings provide the first insight into the critical importance of regulating sumoylation in mammals.

The Nedd4-binding partner 1 (N4BP1) protein is an inhibitor of the E3 ligase Itch

Nedd4-binding partner-1 (N4BP1) has been identified as a protein interactor and a substrate of the homologous to E6AP C terminus (HECT) domain-containing E3 ubiquitin–protein ligase (E3), Nedd4. Here, we describe a previously unrecognized functional interaction between N4BP1 and Itch, a Nedd4 structurally related E3, which contains four WW domains, conferring substrate-binding activity. We show that N4BP1 association with the second WW domain (WW2) of Itch interferes with E3 binding to its substrates. In particular, we found that N4BP1 and p73α, a target of Itch-mediated ubiquitin/proteasome proteolysis, share the same binding site. By competing with p73α for binding to the WW2 domain, N4BP1 reduces the ability of Itch to recruit and ubiquitylate p73α and inhibits Itch autoubiquitylation activity both in in vitro and in vivo ubiquitylation assays. Similarly, both c-Jun and p63 polyubiquitylation by Itch are inhibited by N4BP1. As a consequence, genetic and RNAi knockdown of N4BP1 diminish the steady-state protein levels and significantly impair the transcriptional activity of Itch substrates. Notably, stress-induced induction of c-Jun was impaired in N4BP1−/− cells. These results demonstrate that N4BP1 functions as a negative regulator of Itch. In addition, because inhibition of Itch by N4BP1 results in the stabilization of crucial cell death regulators such as p73α and c-Jun, it is conceivable that N4BP1 may have a role in regulating tumor progression and the response of cancer cells to chemotherapy.

The expression pattern of nodal and lefty in the mouse mutant Ft suggests a function in the establishment of handedness

We have investigated the expression of left/right (L/R) asymmetry markers, nodal and lefty, in the situs inversus mouse mutant Fused toes (Ft). Both genes exhibited bilateral expression in the lateral plate mesoderm (LPM) at developmental stages whereas in wildtype embryos these genes were found to be expressed exclusively in the left LPM. Inspection of tail location and primitive heart tube looping, structures known to be handed in their orientation, documented a random orientation of these structures. Crossing of the Ft mutation into a different genetic background resulted in a strong reduction of this random orientation. Although the major fraction of these individuals still displayed nodal and lefty on both sides of the LPM, expression was almost always found to be weaker in the right LPM. These results suggest that the establishment of asymmetry is independent of nodal or lefty signals. However, handed asymmetry, which means consistent L/R differences, such as the dextral looping of the primitive heart tube or the right-oriented tail, is directed by differences in the L/R expression pattern of these two genes.

FGFR1 function at the earliest stages of mouse limb development plays an indispensable role in subsequent autopod morphogenesis

Fibroblast growth factors (FGFs) and their receptors have been implicated in limb development. However, because of early post-implantation lethality associated with fibroblast growth factor receptor 1 (FGFR1) deficiency, the role of this receptor in limb development remains elusive. To overcome embryonic lethality, we have performed a conditional knockout of Fgfr1 using the Cre-LoxP approach. We show that Cre-mediated deletion of Fgfr1 in limb mesenchyme, beginning at a time point slightly after the first sign of initial budding, primarily affects formation of the first one or two digits. In contrast, deletion of Fgfr1 at an earlier stage, prior to thickening of limb mesenchyme, results in more severe defects, characterized by malformation of the AER, diminished Shh expression and the absence of the majority of the autopod skeletal elements. We show that FGFR1 deficiency does not affect cell proliferation. Instead, it triggers cell death and leads to alterations in expression of a number of genes involved in apoptosis and digit patterning, including increased expression of Bmp4, Dkk1 and Alx4, and downregulation of MKP3. These data demonstrate that FGF/FGFR1 signals play indispensable roles in the early stages of limb initiation, eliciting a profound effect on the later stages of limb development, including cell survival, autopod formation and digit patterning.