In the vertebrate neural tube, regional Sonic hedgehog (Shh) signaling invokes a time-and concentration-dependent induction of six different cell populations mediated through Gli transcriptional regulators. Elsewhere in the embryo, Shh/Gli responses invoke different tissue-appropriate regulatory programs. A genome-scale analysis of DNA binding by Gli1 and Sox2, a pan-neural determinant, identified a set of shared regulatory regions associated with key factors central to cell fate determination and neural tube patterning. Functional analysis in transgenic mice validates core enhancers for each of these factors and demonstrates the dual requirement for Gli1 and Sox2 inputs for neural enhancer activity. Furthermore, through an unbiased determination of Gli-binding site preferences and analysis of binding site variants in the developing mammalian CNS, we demonstrate that differential Gli-binding affinity underlies threshold-level activator responses to Shh input. In summary, our results highlight Sox2 input as a contextspecific determinant of the neural-specific Shh response and differential Gli-binding site affinity as an important cis-regulatory property critical for interpreting Shh morphogen action in the mammalian neural tube.

During Caenorhabditis elegans embryogenesis, anteroposterior (AP) polarity is established by a hierarchy of PAR proteins (for a review, see Kemphues, 2000). Many of these PAR proteins are asymmetrically localized at the cortex along the AP axis. The asymmetric distribution of PAR proteins determines the position of the first mitotic spindle and the asymmetric localization of key cytoplasmic determinants (for reviews, see Cowan and Hyman, 2004;Lyczak et al., 2002). This results in two daughters that are different in both size and developmental fate: the larger anterior somatic cell, AB, and the smaller posterior germline blastomere, P1. P1 then undergoes a series of three more asymmetric divisions, each giving rise to a germline blastomere (sequentially P2 to P4, termed the P lineage) and a corresponding somatic cell (Fig. 1).PAR proteins regulate spindle position via a G-protein signaling pathway (Colombo et al., 2003;Hess et al., 2004), and cytoplasmic polarity via two maternally supplied proteins, MEX-5 and MEX-6 (Schubert et al., 2000). MEX-5 and MEX-6 are closely related proteins and are both preferentially localized toward the anterior cytoplasm of the one-cell embryo and are enriched in the somatic daughter after the division of each germline blastomere (Cuenca et al., 2003;Schubert et al., 2000). Whereas mex-5 mutants exhibit 100% embryonic lethality, mex-6 mutant embryos are 100% viable with no observable defects (Schubert et al., 2000). However, many molecular defects in mex-5 mutant embryos are dramatically enhanced when mex-6 is also mutated or depleted, suggesting partially redundant functions for these two genes (Schubert et al., 2000). For simplicity, unless specifically noted, we will use MEX-5/6 to refer to MEX-5 and MEX-6.One major function of MEX-5/6 is to restrict the localization of maternally supplied germline proteins, such as PIE-1, POS-1 and MEX-1, to germline blastomeres (Guedes and Priess, 1997;Mello et al., 1996;Schubert et al., 2000;Tabara et al., 1999). In the onecell embryo, as MEX-5/6 become asymmetrically localized anteriorly, PIE-1 becomes localized posteriorly (Cuenca et al., 2003;Mello et al., 1996;Schubert et al., 2000). After cell division, PIE-1 is enriched in P1, and this pattern reiterates in each subsequent Plineage division. The small amount of PIE-1 segregated to the somatic sister after each division is degraded by a ZIF-1-containing CUL-2 E3 ligase complex (DeRenzo et al., 2003;Reese et al., 2000). Both asymmetric distribution of PIE-1 before division, as well as asymmetric degradation after division, require the function of MEX-5/6 (DeRenzo et al., 2003;Schubert et al., 2000). MEX-5/6 are themselves also substrates for this ZIF-1-containing E3 ligase complex (DeRenzo et al., 2003).Before meiosis II, high levels of both PIE-1 and MEX-5/6 proteins are detected uniformly throughout the cytoplasm of oocytes and one-cell embryos ( Fig. 1) (Cuenca et al., 2003;Schubert et al., 2000). This suggests that localization of PIE-1 by MEX-5/6 is a developmentally regulated event ...

In C. elegans, four asymmetric divisions, beginning with the zygote (P0), generate transcriptionally repressed germline blastomeres (P1–P4) and somatic sisters that become transcriptionally active. The protein PIE-1 represses transcription in the later germline blastomeres, but not in the earlier germline blastomeres P0 and P1. We show here that OMA-1 and OMA-2, previously shown to regulate oocyte maturation, repress transcription in P0 and P1 by binding to and sequestering in the cytoplasm TAF-4, a component critical for assembly of TFIID and the pol II preinitiation complex. OMA-1/2 binding to TAF-4 is developmentally regulated, requiring phosphorylation by the DYRK kinase MBK-2, which is activated at meiosis II following fertilization. OMA-1/2 are normally degraded after the first mitosis, but ectopic expression of wildtype OMA-1 is sufficient to repress transcription in both somatic and later germline blastomeres. We propose that phosphorylation by MBK-2 serves as a developmental switch, converting OMA-1/2 from oocyte to embryo regulators.

Oocyte maturation and fertilization initiates a dynamic and tightly regulated process in which a non-dividing oocyte is transformed into a rapidly dividing embryo. We have shown previously that two C. elegans CCCH zinc finger proteins, OMA-1 and OMA-2, have an essential and redundant function in oocyte maturation. Both OMA-1 and OMA-2 are expressed only in oocytes and 1-cell embryos, and need to be degraded rapidly after the first mitotic division for embryogenesis to proceed normally. We report here a distinct redundant function for OMA-1 and OMA-2 in the 1-cell embryo. Depletion of both oma-1 and oma-2 in embryos leads to embryonic lethality. We also show that OMA-1 protein is directly phosphorylated at T239 by the DYRK kinase MBK-2, and that phosphorylation at T239 is required both for OMA-1 function in the 1-cell embryo and its degradation after the first mitosis. OMA-1 phosphorylated at T239 is only detected within a short developmental window of 1-cell embryos, beginning soon after the proposed activation of MBK-2. Phosphorylation at T239 facilitates subsequent phosphorylation of OMA-1 by another kinase, GSK-3, at T339 in vitro. Phosphorylation at both T239 and T339 are essential for correctly-timed OMA-1 degradation in vivo. We propose that a series of precisely-timed phosphorylation events regulates both the activity and the timing of degradation for OMA proteins, thereby allowing restricted and distinct functions of OMA-1 and OMA-2 in the maturing oocyte and 1-cell embryo, ensuring a normal oocyte-to-embryo transition in C. elegans.

The oocytes of most sexually reproducing animals arrest in meiotic prophase I. Oocyte growth, which occurs during this period of arrest, enables oocytes to acquire the cytoplasmic components needed to produce healthy progeny and to gain competence to complete meiosis. In the nematode Caenorhabditis elegans, the major sperm protein hormone promotes meiotic resumption (also called meiotic maturation) and the cytoplasmic flows that drive oocyte growth. Prior work established that two related TIS11 zinc-finger RNA-binding proteins, OMA-1 and OMA-2, are redundantly required for normal oocyte growth and meiotic maturation. We affinity purified OMA-1 and identified associated mRNAs and proteins using genome-wide expression data and mass spectrometry, respectively. As a class, mRNAs enriched in OMA-1 ribonucleoprotein particles (OMA RNPs) have reproductive functions. Several of these mRNAs were tested and found to be targets of OMA-1/2-mediated translational repression, dependent on sequences in their 3′-untranslated regions (3′-UTRs). Consistent with a major role for OMA-1 and OMA-2 in regulating translation, OMA-1-associated proteins include translational repressors and activators, and some of these proteins bind directly to OMA-1 in yeast two-hybrid assays, including OMA-2. We show that the highly conserved TRIM-NHL protein LIN-41 is an OMA-1-associated protein, which also represses the translation of several OMA-1/2 target mRNAs. In the accompanying article in this issue, we show that LIN-41 prevents meiotic maturation and promotes oocyte growth in opposition to OMA-1/2. Taken together, these data support a model in which the conserved regulators of mRNA translation LIN-41 and OMA-1/2 coordinately control oocyte growth and the proper spatial and temporal execution of the meiotic maturation decision.