Graciela Pillemer

The dorsalizing and neural inducing gene follistatin is an antagonist of BMP-4

Specific signaling molecules play a pivotal role in the induction and specification of tissues during early vertebrate embryogenesis. BMP-4 specifies ventral mesoderm differentiation and inhibits neural induction in Xenopus, whereas three molecules secreted from the organizer, noggin, follistatin and chordin dorsalize mesoderm and promote neural induction. Here we report that follistatin antagonizes the activities of BMP-4 in frog embryos and mouse teratocarcinoma cells. In Xenopus embryos follistatin blocks the ventralizing effect of BMP-4. In mouse P19 cells follistatin promotes neural differentiation. BMP-4 antagonizes the action of follistatin and prevents neural differentiation. In addition we show that the follistatin and BMP-4 proteins can interact directly in vitro. These data provide evidence that follistatin might play a role in modulating BMP-4 activity in vivo.

Nested expression and sequential downregulation of the Xenopus caudal genes along the anterior-posterior axis.

Expression of the Xenopus Xcad-1 and Xcad-2 genes initiates during early gastrulation exhibiting a dorsoventral asymmetry in their domains of transcription. At mid-gastrulation the ventral preference becomes stronger and the caudal genes take up a posterior localization in their expression, which they will maintain until their downregulation along the dorsal midline. Comparison of the three Xenopus caudal genes revealed a temporal and spatial nested set of expression patterns. The transcription of the caudal genes is sequentially downregulated with the one expressed most caudally (Xcad-2) being shut down first, this sequence is most evident along the dorsal midline. This pattern of expression suggests a role for the caudal genes as posterior determinants along the anteroposterior axis. In chicken, mouse, man and Xenopus three members of the caudal family have been identified in the genome. Even though in Xenopus the Xcad-3 gene has been previously described, in order to obtain a better insight on the role of the caudal genes a comparative study of all three frog genes was performed.

Gbx2 interacts with Otx2 and patterns the anterior-posterior axis during gastrulation in Xenopus.

Anterior-posterior patterning of the embryo requires the activity of multiple homeobox genes among them Hox, caudal (Cdx, Xcad) and Otx2. During early gastrulation, Otx2 and Xcad2 establish a cross-regulatory network, which is an early event in the anterior-posterior patterning of the embryo. As gastrulation proceeds and the embryo elongates, a new domain forms, which expresses neither, Otx2 nor Xcad2 genes. Early transcription of the Xenopus Gbx2 homologue, Xgbx2a, is spatially restricted between Otx2 and Xcad2. When overexpressed, Otx2 and Xcad2 repress Xgbx2a transcription, suggesting their role in setting the early Xgbx2a expression domain. Homeobox genes have been shown to play crucial roles in the specification of the vertebrate brain. The border between the transcription domains of Otx2 and Gbx2 is the earliest known marker of the region where the midbrain/hindbrain boundary (MHB) organizer will develop. Xgbx2a is a negative regulator of Otx2 and a weak positive regulator of Xcad2. Using obligatory activator and repressor versions of Xgbx2a, we demonstrate that, during early embryogenesis, Xgbx2a acts as a transcriptional repressor. In addition, taking advantage of hormone-inducible versions of Xgbx2a and its antimorph, we show that the ability of Xgbx2a to induce head malformations is restricted to gastrula stages and correlates with its ability to repress Otx2 during the same developmental stages. We therefore suggest that the earliest known step of the MHB formation, the establishment of Otx2/Gbx2 boundary, takes place via mutual inhibitory interactions between these two genes and this process begins as early as at midgastrulation.

The Xcad-2 gene can provide a ventral signal independent of BMP-4

Patterning of the marginal zone in the Xenopus embryo has been attributed to interactions between dorsal genes expressed in the organizer and ventral-specific genes. In this antagonistic interplay of activities, BMP-4, a gene that is not expressed in the organizer, provides a strong ventralizing signal. The Xenopus caudal type homeobox gene, Xcad-2, which is expressed around the blastopore with a gap over the dorsal lip, was analyzed as part of the ventral signal. Xcad-2 was shown to efficiently repress during early gastrula stages the dorsal genes gsc, Xnot-2, Otx-2, XFKH1 and Xlim-1, while it positively regulates the ventral genes, Xvent-1 and Xvent-2, with Xpo exhibiting a strong positive response to Xcad-2 overexpression. Xcad-2 was also capable of inducing BMP-4 expression in the organizer region. Support for a ventralizing role for Xcad-2 was obtained from co-injection experiments with the dominant negative BMP receptor which was used to block BMP-4 signaling. Under lack-of-BMP-signaling conditions Xcad-2 could still regulate dorsal and ventral gene expression and restore normal development, suggesting that it can act downstream of BMP-4 signaling or independently of it. Xcad-2 could also inhibit secondary axis formation and dorsalization induced by the dominant negative BMP receptor. Xcad-2 was also shown to efficiently reverse the dorsalizing effects of LiCl. These results place Xcad-2 as part of the ventralizing gene program which acts during early gastrula stages and can execute its ventralizing function in the absence of BMP signaling.

Otx2 can activate the isthmic organizer genetic network in the Xenopus embryo.

Development and differentiation of the vertebrate caudal midbrain and anterior hindbrain are dependent on the isthmic organizer signals at the midbrain/hindbrain boundary (MHB). The future MHB forms at the boundary between the Otx2 and Gbx2 expression domains. Recent studies in mice and chick suggested that the apposition of Otx2- and Gbx2-expressing cells is instrumental for the positioning and early induction of the MHB genetic cascade. We show that Otx2 and Gbx2 perform different roles in this process. We find that ectopically expressed Otx2 on its own can induce a substantial part of the MHB genetic network, namely En2, Wnt1, Pax-2, Fgf8 and Gbx2, in a concentration-dependent manner. This induction does not require protein synthesis and ends during neurulation. In contrast, Gbx2 is a negative regulator of Otx2 and the MHB genes. Based on the temporal patterns of expression of the genes involved, we propose that Otx2 might be the early inducer of the isthmic organizer genetic network while Gbx2 restricts Otx2 expression along the anterior-posterior axis and establishes an Otx2 gradient.

The two Xenopus Gbx2 genes exhibit similar, but not identical expression patterns and can affect head formation

Gbx2 homeobox genes are important for formation and function of the midbrain/hindbrain boundary, namely the isthmic organizer. Two Gbx2 genes were identified in Xenopus laevis, differing in 13 amino acids, including a change in the homeodomain. Xgbx2a is activated earlier during gastrulation and reaches higher levels of expression while Xgbx2b is expressed later, at lower levels and has an additional domain in the ventral blood islands. Their overexpression results in microcephalic embryos with shortened axes and defects in brain and notochord formation. Both genes encode functionally homologous proteins, which differ primarily in their temporal and spatial expression patterns.

Negative autoregulation of Oct3/4 through Cdx1 promotes the onset of gastrulation

Gastrulation marks the onset of germ layer formation from undifferentiated precursor cells maintained by a network including the Pou5f1 gene, Oct3/4. Negative regulation of the undifferentiated state is a prerequisite for germ layer formation and subsequent development. A novel cross‐regulatory network was characterized including the Pou5f1 and Cdx1 genes as part of the signals controlling the onset of gastrulation. Of particular interest was the observation that, preceding gastrulation, the Xenopus Oct3/4 factors, Oct60, Oct25, and Oct91, positively regulate Cdx1 expression through FGF signaling, and during gastrulation the Oct3/4 factors become repressors of Cdx1. Cdx1 negatively regulates the Pou5f1 genes during gastrulation, thus contributing to the repression of the network maintaining the undifferentiated state and promoting the onset of gastrulation. These regulatory interactions suggest that Oct3/4 initiates its own negative autoregulation through Cdx1 up‐regulation to begin the repression of pluripotency in preparation for the onset of gastrulation and germ layer differentiation. Developmental Dynamics 240:796–807, 2011.