Chk1 Inhibition of the Replication Factor Drf1 Guarantees Cell-Cycle Elongation at the Xenopus laevis Mid-blastula Transition.

The early cell divisions of many metazoan embryos are rapid and occur in the near absence of transcription. At the mid-blastula transition (MBT), the cell cycle elongates and several processes become established including the onset of bulk transcription and cell-cycle checkpoints. How these events are timed and coordinated is poorly understood. Here we show in Xenopus laevis that developmental activation of the checkpoint kinase Chk1 at the MBT results in the SCFß-TRCP-dependent degradation of a limiting replication initiation factor Drf1. Inhibition of Drf1 is the primary mechanism by which Chk1 blocks cell-cycle progression in the early embryo and is an essential function of Chk1 at the blastula-to-gastrula stage of development. This study defines the downregulation of Drf1 as an important mechanism to coordinate the lengthening of the cell cycle and subsequent developmental processes.

High-resolution analysis of gene activity during the Xenopus mid-blastula transition.

The Xenopus mid-blastula transition (MBT) marks the onset of large-scale zygotic transcription, as well as an increase in cell cycle length and a loss of synchronous cell divisions. Little is known about what triggers the activation of transcription or how newly expressed genes interact with each other. Here, we use high-resolution expression profiling to identify three waves of gene activity: a post-fertilisation wave involving polyadenylation of maternal transcripts; a broad wave of zygotic transcription detectable as early as the seventh cleavage and extending beyond the MBT at the twelfth cleavage; and a shorter post-MBT wave of transcription that becomes apparent as development proceeds. Our studies have also allowed us to define a set of maternal mRNAs that are deadenylated shortly after fertilisation, and are likely to be degraded thereafter. Experimental analysis indicates that the polyadenylation of maternal transcripts is necessary for the establishment of proper levels of zygotic transcription at the MBT, and that genes activated in the second wave of expression, including Brachyury and Mixer, contribute to the regulation of genes expressed in the third. Together, our high-resolution time series and experimental studies have yielded a deeper understanding of the temporal organisation of gene regulatory networks in the early Xenopus embryo.

Titration of four replication factors is essential for the Xenopus laevis midblastula transition.

The rapid, reductive early divisions of many metazoan embryos are followed by the midblastula transition (MBT), during which the cell cycle elongates and zygotic transcription begins. It has been proposed that the increasing nuclear to cytoplasmic (N/C) ratio is critical for controlling the events of the MBT. We show that four DNA replication factors--Cut5, RecQ4, Treslin, and Drf1--are limiting for replication initiation at increasing N/C ratios in vitro and in vivo in Xenopus laevis. The levels of these factors regulate multiple events of the MBT, including the slowing of the cell cycle, the onset of zygotic transcription, and the developmental activation of the kinase Chk1. This work provides a mechanism for how the N/C ratio controls the MBT and shows that the regulation of replication initiation is fundamental for normal embryogenesis.

Few Smad proteins and many Smad-interacting proteins yield multiple functions and action modes in TGFß/BMP signaling in vivo.

Signaling by the many ligands of the TGFß family strongly converges towards only five receptor-activated, intracellular Smad proteins, which fall into two classes i.e. Smad2/3 and Smad1/5/8, respectively. These Smads bind to a surprisingly high number of Smad-interacting proteins (SIPs), many of which are transcription factors (TFs) that co-operate in Smad-controlled target gene transcription in a cell type and context specific manner. A combination of functional analyses in vivo as well as in cell cultures and biochemical studies has revealed the enormous versatility of the Smad proteins. Smads and their SIPs regulate diverse molecular and cellular processes and are also directly relevant to development and disease. In this survey, we selected appropriate examples on the BMP-Smads, with emphasis on Smad1 and Smad5, and on a number of SIPs, i.e. the CPSF subunit Smicl, Ttrap (Tdp2) and Sip1 (Zeb2, Zfhx1b) from our own research carried out in three different vertebrate models.

The midblastula transition defines the onset of Y RNA-dependent DNA replication in Xenopus laevis.

Noncoding Y RNAs are essential for the initiation of chromosomal DNA replication in mammalian cell extracts, but their role in this process during early vertebrate development is unknown. Here, we use antisense morpholino nucleotides (MOs) to investigate Y RNA function in Xenopus laevis and zebrafish embryos. We show that embryos in which Y RNA function is inhibited by MOs develop normally until the midblastula transition (MBT) but then fail to replicate their DNA and die before gastrulation. Consistent with this observation, Y RNA function is not required for DNA replication in Xenopus egg extracts but is required for replication in a post-MBT cell line. Y RNAs do not bind chromatin in karyomeres before MBT, but they associate with interphase nuclei after MBT in an origin recognition complex (ORC)-dependent manner. Y RNA-specific MOs inhibit the association of Y RNAs with ORC, Cdt1, and HMGA1a proteins, suggesting that these molecular associations are essential for Y RNA function in DNA replication. The MBT is thus a transition point between Y RNA-independent and Y RNA-dependent control of vertebrate DNA replication. Our data suggest that in vertebrates Y RNAs function as a developmentally regulated layer of control over the evolutionarily conserved eukaryotic DNA replication machinery.

Smicl is required for phosphorylation of RNA polymerase II and affects 3'-end processing of RNA at the midblastula transition in Xenopus.

Smicl (Smad-interacting CPSF 30-like) is an unusual protein that interacts with transcription factors as well as with the cleavage and polyadenylation specificity factor (CPSF). Previous work has shown that Smicl is expressed maternally in the Xenopus embryo and is later required for transcription of Chordin. In this paper we search for additional targets of Smicl. We identify many genes whose onset of expression at the midblastula transition (MBT) requires Smicl and is correlated with the translocation of Smicl from cytoplasm to nucleus. At least one such gene, Xiro1, is regulated via 3'-end processing. In searching for a general mechanism by which Smicl might regulate gene expression at the MBT, we have discovered that it interacts with the tail of Rpb1, the largest subunit of RNA polymerase II. Our results show that Smicl is required for the phosphorylation of the Rpb1 tail at serine 2 of the repeated heptapeptide YSPTSPS. This site becomes hyperphosphorylated at the MBT, thus allowing the docking of proteins required for elongation of transcription and RNA processing. Our work links the onset of zygotic gene expression in the Xenopus embryo with the translocation of Smicl from cytoplasm to nucleus, the phosphorylation of Rpb1 and the 3'-end processing of newly transcribed mRNAs.

Xnrs and activin regulate distinct genes during Xenopus development: activin regulates cell division.

BACKGROUND: The mesoderm of the amphibian embryo is formed through an inductive interaction in which vegetal cells of the blastula-staged embryo act on overlying equatorial cells. Candidate mesoderm-inducing factors include members of the transforming growth factor type beta family such as Vg1, activin B, the nodal-related proteins and derrière. METHODOLOGY AND PRINCIPLE FINDINGS: Microarray analysis reveals different functions for activin B and the nodal-related proteins during early Xenopus development. Inhibition of nodal-related protein function causes the down-regulation of regionally expressed genes such as chordin, dickkopf and XSox17alpha/beta, while genes that are mis-regulated in the absence of activin B tend to be more widely expressed and, interestingly, include several that are involved in cell cycle regulation. Consistent with the latter observation, cells of the involuting dorsal axial mesoderm, which normally undergo cell cycle arrest, continue to proliferate when the function of activin B is inhibited. CONCLUSIONS/SIGNIFICANCE: These observations reveal distinct functions for these two classes of the TGF-beta family during early Xenopus development, and in doing so identify a new role for activin B during gastrulation.

Defining synphenotype groups in Xenopus tropicalis by use of antisense morpholino oligonucleotides.

To identify novel genes involved in early development, and as proof-of-principle of a large-scale reverse genetics approach in a vertebrate embryo, we have carried out an antisense morpholino oligonucleotide (MO) screen in Xenopus tropicalis, in the course of which we have targeted 202 genes expressed during gastrula stages. MOs were designed to complement sequence between -80 and +25 bases of the initiating AUG codons of the target mRNAs, and the specificities of many were tested by (i) designing different non-overlapping MOs directed against the same mRNA, (ii) injecting MOs differing in five bases, and (iii) performing "rescue" experiments. About 65% of the MOs caused X. tropicalis embryos to develop abnormally (59% of those targeted against novel genes), and we have divided the genes into "synphenotype groups," members of which cause similar loss-of-function phenotypes and that may function in the same developmental pathways. Analysis of the expression patterns of the 202 genes indicates that members of a synphenotype group are not necessarily members of the same synexpression group. This screen provides new insights into early vertebrate development and paves the way for a more comprehensive MO-based analysis of gene function in X. tropicalis.

The novel Smad-interacting protein Smicl regulates Chordin expression in the Xenopus embryo.

In this paper, we investigate the function of Smicl, a zinc-finger Smad-interacting protein that is expressed maternally in the Xenopus embryo. Inhibition of Smicl function by means of antisense morpholino oligonucleotides causes the specific downregulation of Chordin, a dorsally expressed gene encoding a secreted BMP inhibitor that is involved in mesodermal patterning and neural induction. Chordin is activated by Nodal-related signalling in an indirect manner, and we show here that Smicl is involved in a two-step process that is necessary for this activation. In the first, Smad3 (but not Smad2) activates expression of Xlim1 in a direct fashion. In the second, a complex containing Smicl and the newly induced Xlim1 induces expression of Chordin. As well as revealing the function of Smicl in the early embryo, our work yields important new insight in the regulation of Chordin and identifies functional differences between the activities of Smad2 and Smad3 in the Xenopus embryo.

Smicl is a novel Smad interacting protein and cleavage and polyadenylation specificity factor associated protein.

Ligand-bound receptors of the Transforming Growth Factor-beta (TGF-beta) family promote the formation of complexes between Smad proteins that subsequently accumulate in the nucleus and interact there with other transcriptional regulators, leading to modulation of target gene expression. We identified a novel nuclear protein, Smicl, which binds to Smad proteins. Smicl and Smads cooperate and enhance TGF-beta mediated activation of a Smad-responsive reporter gene. A domain with five CCCH-type zinc fingers in Smicl is structurally and functionally, at least in vitro, similar to a domain in CPSF-30, the 30 kDa subunit of Cleavage and Polyadenylation Specificity Factor (CPSF). Like CPSF-30, Smicl can associate with some other CPSF subunits characterized previously. Its effect on the induction of a reporter gene for TGF-beta requires the cleavage/polyadenylation signal downstream of the coding sequence of that gene. Thus, Smicl is a novel protein that displays CPSF-30-like activities, interacts in the nucleus with activated Smads, and potentiates in TGF-beta stimulated cells Smad-dependent transcriptional responses, possibly in conjunction with the activity of CPSF complexes.