Oral-aboral axis specification in the sea urchin embryo - II. Mitochondrial distribution and redox state contribute to establishing polarity in Strongylocentrotus purpuratus

Stowers Institute for Medical Research, Kansas City, Kansas, United States
Developmental Biology (Impact Factor: 3.64). 10/2004; 273(1):160-71. DOI: 10.1016/j.ydbio.2004.06.005
Source: PubMed

ABSTRACT The initial asymmetry that specifies the oral-aboral (OA) axis of the sea urchin embryo has long been a mystery. It was shown previously that OA polarity can be entrained in embryos by imposing a respiratory asymmetry, with the most oxidizing side of the embryo tending to develop as the oral pole. This suggests that one of the earliest observable asymmetries along the incipient OA axis, a redox gradient established by a higher density and/or activity of mitochondria on the prospective oral side of the embryo, might play a causal role in establishing the axis. Here, we examine the origin and functional significance of this early redox gradient. Using MitoTracker Green, we show that mitochondria are asymmetrically distributed in the unfertilized egg of Strongylocentrotus purpuratus, and that the polarity of the maternal asymmetry is maintained in the zygote. Vital staining indicates that the side of the embryo that inherits the highest density of mitochondria tends to develop into the oral pole. This correlation holds when mitochondria are redistributed by centrifugation of eggs or by transfer of purified mitochondria into zygotes, indicating that an asymmetric mitochondrial distribution can entrain OA polarity, possibly through effects on intracellular redox state. In support of this possibility, we find that specification of oral ectoderm is suppressed when embryos are cultured under hypoxic conditions that enforce a relatively reducing redox state. This effect is reversed by overexpression of nodal, an early zygotic marker of oral specification whose localized expression suffices to organize the entire OA axis, indicating that redox state is upstream of nodal expression. We therefore propose that a threshold level of intracellular oxidation is required to effectively activate nodal, and that precocious attainment of this threshold within the blastomeres containing the highest density of mitochondria results in asymmetric nodal activity and consequent specification of the OA axis.

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Available from: James A. Coffman, Jul 30, 2015
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    • "Expression of the additional oral ectoderm marker goosecoid (gsc) as well as the aboral ectoderm marker tbx2/3 was also reduced (Figure 2I and Figure 2—figure supplement 1), as expected from the perturbed expression of nodal normally necessary for the establishment of the entire DV axis (Duboc et al., 2004). The variations in transcript abundance shown in Figure 2H,I could appear somewhat modest, although they are of the same order of magnitude as those reported by other authors (Agca et al., 2010; Bergeron et al., 2011; Coffman et al., 2004). A possible explanation is that the qPCR data refer to the whole embryo population , affected and not affected. "
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    ABSTRACT: eLife digest Embryos begin as a collection of identical cells. As the embryo develops further, the cells in different regions must take on different structures and roles in order to form the complex tissues and organs seen in the fully developed organism. Therefore, a key task in early development is to inform cells where they are in a developing embryo. Signaling proteins released by special groups of organizing cells are responsible for providing the information about where a cell is located. Networks of genes controlled by these proteins then inform embryonic cells of where they are and what they should, or should not, become. One such signaling protein is called Nodal, and is needed to perform a number of tasks in the developing embryo, including helping to form the basic tissues of the organism. Many animals depend on Nodal to develop correctly—from mice and humans, to zebrafish and sea urchins. During sea urchin development, Nodal establishes where the mouth of a larva forms, setting up what is called the dorsal/ventral axis of the embryo; this separates the front and back of the embryo. To do so, the Nodal protein is mostly produced at the front of the embryo. Although much is already known about the network of genes that the Nodal protein controls, the genes and proteins that ensure that the initial source of Nodal is present at the right time and place are largely unknown. Another protein called Hbox12 was also thought to be important for setting up the dorsal/ventral axis. Now, Cavalieri and Spinelli reveal that Hbox12 regulates Nodal during the development of a sea urchin embryo. In the early developing sea urchin, the gene that produces Hbox12 is activated in the region of the embryo that will become its back, directly opposite where Nodal is present. This activation normally occurs just before the gene that produces Nodal is turned on. If the hbox12 gene function is impaired, the Nodal protein is produced in both the front and the back sections of the embryo. Conversely, if Hbox12 is introduced into regions where Nodal is present, the amount of Nodal decreases. Furthermore, disrupting Hbox12 prevents any signs of the dorsal/ventral axis forming. Cavalieri and Spinelli propose that Hbox12 inhibits the production of Nodal by briefly inactivating another protein that is required to activate the nodal gene. By doing so, Hbox12 sets up the dorsal/ventral axis by restricting Nodal to the cells that will make up the front half of the embryo. Most complex organisms have asymmetric bodies, and failure to establish these body asymmetries can result in disease and other disorders in humans. Deciphering how the dorsal/ventral asymmetry in the sea urchin embryo is established should improve our understanding of how the mechanisms that form body shapes have evolved. DOI:
    eLife Sciences 12/2014; 3. DOI:10.7554/eLife.04664 · 8.52 Impact Factor
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    • "In sea urchin embryos, cell specifications take place along the two embryonic axes: the animal–vegetal (AV) axis and the oral–aboral (OA) axis. The AV axis can be traced back to the unfertilized egg (Boveri, 1901; Hörstadius, 1939), and the OA axis to the zygote (Coffman et al., 2004). By the end of the 4th division, three different sizes of blastomeres comprising eight mesomeres, four macromeres and four micromeres are formed and eventually become the ectoderm, the mesoendoderm and the mesoderm, respectively. "
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    ABSTRACT: Summary Sea urchin embryos initiate cell specifications at the 16-cell stage by forming the mesomeres, macromeres and micromeres according to the relative position of the cells in the animal-vegetal axis. The most vegetal cells, micromeres, autonomously differentiate into skeletons and induce the neighbouring macromere cells to become mesoendoderm in the β-catenin-dependent Wnt8 signalling pathway. Although the underlying molecular mechanism for this progression is largely unknown, we have previously reported that the initial events might be triggered by the Ca2+ influxes through the egg-originated L-type Ca2+ channels distributed asymmetrically along the animal-vegetal axis and through the stretch-dependent Ca2+channels expressed specifically in the micromere at the 4th cleavage. In this communication, we have examined whether one of the earliest Ca2+ targets, protein kinase C (PKC), plays a role in cell specification upstream of β-catenin. To this end, we surveyed the expression pattern of β-catenin in early embryos in the presence or absence of the specific peptide inhibitor of Hemicentrotus pulcherrimus PKC (HpPKC-I). Unlike previous knowledge, we have found that the initial nuclear entrance of β-catenin does not take place in the micromeres, but in the macromeres at the 16-cell stage. Using the HpPKC-I, we have demonstrated further that PKC not only determines cell-specific nucleation of β-catenin, but also regulates a variety of cell specification events in the early sea urchin embryos by modulating the cell adhesion structures, actin dynamics, intracellular Ca2+ signalling, and the expression of key transcription factors.
    Zygote 04/2014; 23(03):1-21. DOI:10.1017/S0967199414000033 · 1.32 Impact Factor
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    • "Many years ago, experiments showed that respiratory inhibitors could bias D/V axis orientation (Child, 1941; Coffman and Davidson, 2001; Coffman and Denegre, 2007; Czihak, 1963; Pease, 1941). More recently, it was shown that asymmetrically localized mitochondria in the egg might prefigure the ventral side of the embryo and localized microinjection of purified mitochondria on one side of the embryo caused that side to tend toward a ventral fate (Coffman et al., 2004). Furthermore, by simultaneously examining the spatial distribution of mitochondria in the early embryo and the pattern of pSmad2/3 by immunostaining, a modest but significant correlation between Nodal signaling activity and mitochondrial distribution was found (Coffman et al., 2009). "
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    ABSTRACT: The TGFβ family member Nodal is expressed early in the presumptive ventral ectoderm of the early sea urchin embryo and its activity is crucial for dorsal-ventral (D/V) axis specification. Analysis of the nodal promoter identified a number of critical binding sites for transcription factors of different families including Sox, Oct, TCF and bZIP, but in most cases the specific factors that regulate nodal expression are not known. In this study, we report that the maternal factor Oct1/2 functions as a positive regulator of nodal and that its activity is essential for the initiation of nodal expression. Inhibition of Oct1/2 mRNA translation produced embryos with severe axial defects similar to those observed following inhibition of Nodal function. We show that perturbing Oct1/2 function specifically disrupted specification of the ventral and dorsal ectodermal regions and that these effects were caused by the failure of nodal to be expressed early in development. Furthermore, we identified the key gene vg1/univin, which is also necessary for nodal expression, as an additional factor that was completely dependent on Oct1/2 for its zygotic expression. These data demonstrate that the maternal Oct1/2 protein plays an early and essential role in D/V axis specification by initiating the expression of nodal and vg1/univin, two genes that act at the top of the D/V ectoderm gene regulatory network.
    Developmental Biology 07/2011; 357(2):440-9. DOI:10.1016/j.ydbio.2011.07.005 · 3.64 Impact Factor
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