This study describes one of the first systematic demonstrations of the developmental GRNs activated in blastomeres that emit and respond to embryonic endomesoderm induction signals. We ectopically activated the Pmar1-dependent PMC-GRN in nonvegetal cells and demonstrate that adjacent blastomeres are specified as endomesoderm through a responding GRN that includes genes encoding the core factors Z13, Eve, and FoxA. In contrast, a significant part of the sea urchin EM-GRN, consisting of the regulatory outputs of Wnt8, Blimp1, and Brachyury, is not activated through Pmar1-mediated signals. Two members of this Pmar1-insensitive set, Wnt8 and Blimp1, have recently been proposed to constitute a self-sustaining cis
-regulatory subcircuit that maintains nuclearization of β-catenin in endomesoderm progenitors [23
]. Since ectopic Pmar1-misexpressing cells do not detectably induce wnt8
expression in adjacent animal blastomeres, our work implies that these responding cells are specified as endomesoderm without accumulating β-catenin in their nuclei. This conclusion is consistent with previous studies demonstrating the absence of detectable nuclear β-catenin in animal blastomeres specified as endomesoderm through ectopic micromere induction signals [13
expression, the molecular hallmark of micromere-mediated endomesoderm specification [16
], we show through localized loss-of-function analyses that the TGF-β cytokine, ActivinB, is required in both Pmar1-expressing cells that send endomesoderm-inducing signals as well as in the responding EM-GRN in blastomeres that transduce this signal. The requirement for ActivinB in Pmar1-dependent endomesoderm induction strongly suggests that it plays a similar essential role in endomesoderm formation in response to signals from ectopic micromeres, a phenomenon first described almost 70 y ago in the sea urchin embryo [16
]. ActivinB signaling through ALK4/5/7 during early cleavage and blastula stages also is necessary for endogenous vegetal activation of almost the entire E-EM/En-GRN induced by ectopic Pmar1 expression. This critical finding, along with the fact that, like micromere progeny, ActivinB is required for normal endo16
expression in veg2 endomesoderm progenitors, and for timely gastrulation, strongly suggests that this TGFβ ligand plays an essential role in early micromere-dependent endomesoderm specification.
In contrast to the Pmar1-dependent EM-GRN factors, ActivinB is not required for normal vegetal expression of any of the Pmar1-insensitive EM-GRN core factors. Furthermore, both the Delta-dependent secondary mesoderm–GRN and the late endoderm-GRN (17 to 30 h p.f.) that is subsequently activated in veg2 and veg1 macromere progeny are independent of ActivinB function. Thus, the primary cellular targets of ActivinB signaling during pregastrular endomesoderm development are the more vegetal (veg2) macromere derivatives. The exact state of specification of these cells in ActivinB morphants is not clear, but some of them transfate to pigment cells, presumably as a result of reduced FoxA activity [29
]. Whether any of these cells adopts other mesodermal fates is not yet known. If indeed they do not contribute to definitive endoderm, then the gut that eventually forms would arise from veg1 progeny that are specified much later than veg2 derivatives [44
], through the regulatory outputs of the ActivinB-independent late endoderm-GRN. Alternatively, if veg2 endoderm progenitors in ActivinB morphants do not acquire other secondary mesoderm fates, then they probably participate in gut formation. In this case, either they eventually express all of the core EM-GRN factors through micromere-independent mechanisms or, in a later vegetal developmental context, the Wnt8/Blimp1/nuclear β-catenin subcircuit [23
] is sufficient to support this process. Either scenario would require considerable regulatory cross talk between GRN components in order to achieve threshold concentrations of critical core factors that drive definitive endoderm development.
Recent models of pregastrular development in echinoderm embryos have proposed that a single micromere signal specifies veg2 macromere progeny as endo16
-expressing endomesoderm progenitors and also causes the gradual clearance of the β-catenin antagonist, SoxB1, from nuclei of these blastomeres [21
]. This is an attractive model because it invokes a causal linkage between activation and maintenance of the EM-GRN that depends on nuclear β-catenin accumulation and removal of an antagonist. However, the results of several experiments presented here argue strongly that micromere-mediated early endomesoderm induction and micromere-regulated SoxB1 removal are independent processes. First, SoxB1 down-regulation occurs normally in embryos lacking the early endomesoderm-inducing (endo16
-inducing) signal ActivinB. Second, SoxB1 clearance is not required for activation of the early EM-GRN because macromere progeny express early Pmar1-responsive core factors in this subnetwork before SoxB1 protein levels are detectably reduced in nuclei of the same cells. Third, ectopic micromere-mediated EM-GRN activation and complete archenteron formation occur in cells that lack detectable nuclear β-catenin [13
] but still clear SoxB1 [21
]. Our findings are consistent with other studies showing that endoderm specification can occur without SoxB1 clearance [40
]. The first phase of SoxB1 clearance in presumptive secondary mesoderm also does not depend on other known micromere signals, because it occurs normally in either Delta (this work) or Wnt8 morphants [27
]. Therefore, although SoxB1 clearance depends on an early micromere signal(s), it is a gradual process that is not completed until the mesenchyme blastula stage and relies on an unknown pathway(s) distinct from the one that regulates endo16
induction through ActivinB (). Therefore, we favor the view that fine-scale patterning of endoderm and mesoderm in different blastomere tiers probably requires micromere-dependent regulation of the level and duration of SoxB1 expression, but early micromere-mediated endomesoderm specification is independent of this process ().
Pregastrular EM-GRN Subnetworks Mediating Micromere-Mediated Endomesoderm Induction
This work is the first report, to our knowledge, of the requirement of ActivinB in the earliest steps of endomesoderm specification in any developmental model system and of the involvement of a TGFβ cytokine of the Activin/Nodal/TGFβ class in endomesoderm formation in an invertebrate embryo. Activin was initially proposed to be a mesoderm-inducing agent because exogenous Activin can induce mesoderm in amphibian animal cap explants [45
]. Activin can also direct endoderm differentiation in human and mouse embryonic stem cells, which, interestingly, transit through an endomesoderm-like primordium prior to expressing markers of definitive endoderm [46
]. Recent studies in which ActivinB was knocked down with MOs in amphibian embryos suggest that it does play some role in axial mesoderm formation, possibly by regulating convergent extension movements of gastrulation through the activities of other mesoderm-inducing factors [48
] and/or the timing of cell cycle transitions in involuting dorsal axial mesoderm [49
]. However, even though ActivinB is expressed during early development of amphibian embryos [50
], there is, at present, no evidence suggesting that it functions in early endomesoderm specification per se.
By analyzing the EM-GRN response to ActivinB, we show that it is exclusively required for the earliest steps of endomesoderm specification, eventually leading to the formation of definitive endoderm and timely gastrulation in the sea urchin embryo. This work, therefore, provides a critical in vivo model of the requirement of ActivinB function in early embryogenesis. Furthermore, the remarkable developmental plasticity of the sea urchin embryo allows us to demonstrate that ActivinB is the cardinal primary mesenchyme-derived signal that can activate an ectopic endomesoderm GRN. We thus describe an important experimental paradigm that elucidates the GRNs that drive endogenous and ectopic endomesoderm induction.
By defining major regulatory connections between individual networks of the overall sea urchin EM-GRN, we have built a new framework for future studies of endomesoderm specification. In the sea urchin embryo, endomesoderm development is highly regulative and eventually occurs even in the absence of micromeres [18
]. Similarly isolated macromeres also express endoderm markers [51
]. Our identification of a Pmar1-insensitive set of core regulatory factors may significantly inform future work addressing how the network responds in these situations, potentially leading to an understanding of the molecular basis of such regulative endomesoderm development. Our work also postulates the existence of an uncharacterized micromere signal that is emitted during early cleavage stages to clear the β-catenin antagonist SoxB1 from endomesoderm precursors. Identifying this signal and its GRN properties will be necessary to achieve a comprehensive understanding of pregastrular development in echinoderm embryos. We show that endomesoderm is ectopically induced through the regulatory outputs of a set of Pmar1-responsive EM-GRN core factors, making it important to understand how this core set activates and stabilizes downstream GRN circuits. Since this Pmar1-responsive GRN potentially drives formation of a complete archenteron without detectable nuclear β-catenin, it is also conceivable that additional uncharacterized β-catenin–independent GRNs exist. Interestingly, recent studies have demonstrated the existence of such a β-catenin-independent JNK/Axin dorsalization pathway in zebrafish embryos (e.g., [52
]). The sea urchin embryo, with its well-known developmental plasticity, would be an ideal system for characterizing such nascent GRNs and examining their interactions with the currently understood endomesoderm regulatory networks.