Edgar M. Pera

Integrating Patterning Signals: Wnt/GSK3 Regulates the Duration of the BMP/Smad1 Signal

BMP receptors determine the intensity of BMP signals via Smad1 C-terminal phosphorylations. Here we show that a finely controlled cell biological pathway terminates this activity. The duration of the activated pSmad1(Cter) signal was regulated by sequential Smad1 linker region phosphorylations at conserved MAPK and GSK3 sites required for its polyubiquitinylation and transport to the centrosome. Proteasomal degradation of activated Smad1 and total polyubiquitinated proteins took place in the centrosome. Inhibitors of the Erk, p38, and JNK MAPKs, as well as GSK3 inhibitors, prolonged the duration of a pulse of BMP7. Wnt signaling decreased pSmad1(GSK3) antigen levels and redistributed it from the centrosome to cytoplasmic LRP6 signalosomes. In Xenopus embryos, it was found that Wnts induce epidermis and that this required an active BMP-Smad pathway. Epistatic experiments suggested that the dorsoventral (BMP) and anteroposterior (Wnt/GSK3) patterning gradients are integrated at the level of Smad1 phosphorylations during embryonic pattern formation.

Neural and Head Induction by Insulin-like Growth Factor Signals

Evidence is presented for a new pathway participating in anterior neural development. It was found that IGF binding protein 5 (IGFBP-5), as well as three IGFs expressed in early embryos, promoted anterior development by increasing the head region at the expense of the trunk in mRNA-injected Xenopus embryos. A secreted dominant-negative type I IGF receptor (DN-IGFR) had the opposite effect. IGF mRNAs led to the induction of ectopic eyes and ectopic head-like structures containing brain tissue. In ectodermal explants, IGF signals induced anterior neural markers in the absence of mesoderm formation and DN-IGFR inhibited neural induction by the BMP antagonist Chordin. Thus, active IGF signals appear to be both required and sufficient for anterior neural induction in Xenopus.

A direct screen for secreted proteins in Xenopus embryos identifies distinct activities for the Wnt antagonists Crescent and Frzb-1.

To determine the spectrum of secreted proteins that are present in the extracellular space of early Xenopus embryos, a direct secretion screen was performed. Surprisingly, 24% of previously identified bona fide secretory proteins corresponded to four secreted Wnt antagonists of the same family: frzb-1, sizzled, sfrp-2 and crescent. sfrp-2 and crescent are novel components of the growing cocktail of growth factor antagonists secreted by Spemann organizer cells in Xenopus. Crescent is first expressed at blastula, defining a deep endodermal region that may be homologous to the avian hypoblast. Unlike other members of this family of inhibitors, microinjection of crescent mRNA causes the development of cyclopic embryos, even though the amount of anterior neural tissue is normal. In crescent-injected embryos, studies with specific markers indicate that morphogenetic movements of the anterior midline are abnormal, resulting in a more posterior location of prechordal plate and ventral forebrain markers with respect to the developing eye field. The results are discussed in light of recent findings in zebrafish and Xenopus that suggest that Wnt signaling through non-canonical (non-beta-catenin dependent) pathways plays a pivotal role in the regulation of morphogenetic movements.

Biological Functions of Iduronic Acid in Chondroitin/Dermatan Sulfate.

The presence of iduronic acid in chondroitin/dermatan sulfate changes the properties of the polysaccharides because it generates a more flexible chain with increased binding potentials. Iduronic acid in chondroitin/dermatan sulfate influences multiple cellular properties, such as migration, proliferation, differentiation, angiogenesis and the regulation of cytokine/growth factor activities. Under pathological conditions such as wound healing, inflammation and cancer, iduronic acid has diverse regulatory functions. Iduronic acid is formed by two epimerases (i.e. dermatan sulfate epimerase 1 and 2) that have different tissue distribution and properties. The role of iduronic acid in chondroitin/dermatan sulfate is highlighted by the vast changes in connective tissue features in patients with a new type of Ehler–Danlos syndrome: adducted thumb‐clubfoot syndrome. Future research aims to understand the roles of the two epimerases and their interplay with the sulfotransferases involved in chondroitin sulfate/dermatan sulfate biosynthesis. Furthermore, a better definition of chondroitin/dermatan sulfate functions using different knockout models is needed. In this review, we focus on the two enzymes responsible for iduronic acid formation, as well as the role of iduronic acid in health and disease.

Isthmin is a novel secreted protein expressed as part of the Fgf-8 synexpression group in the Xenopus midbrain-hindbrain organizer

Patterning of the central nervous system is regulated by a signaling center located at the midbrain-hindbrain boundary (MHB), or isthmus organizer. Fibroblast growth factors secreted from the MHB are required and sufficient to direct the ordered growth and regionalization of the midbrain and anterior hindbrain. In an unbiased secretion cloning screen of Xenopus gastrula embryos we identified a novel gene, which we designated as Isthmin (xIsm) due to its prominent expression at the MHB. xIsm encodes a secreted protein of 449 amino acids containing one copy of the thrombospondin type 1 repeat (TSR). We also found orthologous Isthmin genes in human (hIsm) and mouse (mIsm), as well as a gene encoding an Isthmin-like human unknown protein (hIsm-l). The conservation of a unique carboxy-terminal region between hIsm and hIsm-l suggests that Isthmin is the founding member of a new family of secreted proteins. xIsm was strongly expressed maternally in the Xenopus egg and showed zygotic expression in the ventral blastopore lip, notochord, and MHB. Additional expression domains were detected in neural crest, ear vesicle, and developing blood islands. Interestingly, xIsm was co-expressed with Fibroblast growth factor-8 (xFgf-8) at multiple sites including the MHB, indicating that these two genes are part of a synexpression group which also includes sprouty and sef homologs.

The dual regulator Sufu integrates Hedgehog and Wnt signals in the early Xenopus embryo

Hedgehog (Hh) and Wnt proteins are important signals implicated in several aspects of embryonic development, including the early development of the central nervous system. We found that Xenopus Suppressor-of-fused (XSufu) affects neural induction and patterning by regulating the Hh/Gli and Wnt/β-catenin pathways. Microinjection of XSufu mRNA induced expansion of the epidermis at the expense of neural plate tissue and caused enlargement of the eyes. An antisense morpholino oligonucleotide against XSufu had the opposite effect. Interestingly, both gain- and loss-of-function experiments resulted in a posterior shift of brain markers, suggesting a biphasic effect of XSufu on anteroposterior patterning. XSufu blocked early Wnt/β-catenin signaling, as indicated by the suppression of XWnt8-induced secondary axis formation in mRNA-injected embryos, and activation of Wnt target genes in XSufu-MO-injected ectodermal explants. We show that XSufu binds to XGli1 and Xβ-catenin. In Xenopus embryos and mouse embryonic fibroblasts, Gli1 inhibits Wnt signaling under overexpression of β-catenin, whereas β-catenin stimulates Hh signaling under overexpression of Gli1. Notably, endogenous Sufu is critically involved in this crosstalk. The results suggest that XSufu may act as a common regulator of Hh and Wnt signaling and contribute to intertwining the two pathways.

Active signals, gradient formation and regional specificity in neural induction

The question of how the vertebrate embryo gives rise to a nervous system is of paramount interest in developmental biology. Neural induction constitutes the earliest step in this process and is tightly connected with development of the embryonic body axes. In the Xenopus embryo, perpendicular gradients of BMP and Wnt signals pattern the dorsoventral and anteroposterior body axes. Both pathways need to be inhibited to allow anterior neural induction to occur. FGF8 and IGF are active neural inducers that together with BMP and Wnt signals are integrated at the level of Smad 1/5/8 phosphorylation. Hedgehog (Hh) also contributes to anterior neural induction. Suppressor-of-fused plays an important role in intertwining the Hh and Wnt pathways. Distinct mechanisms are discussed that establish morphogen gradients and integrate retinoic acid and FGF signals during posterior development. These findings not only improve our understanding of regional specification in neural induction, but have profound implications for mammalian stem cell research and regenerative medicine.

Darmin is a novel secreted protein expressed during endoderm development in Xenopus.

Endoderm development is an area of intense interest in developmental biology, but progress has been hampered by the lack of specific markers for differentiated endodermal cells. In an unbiased secretion cloning screen of Xenopus gastrula embryos we isolated a novel gene, designated Darmin. Darmin encodes a secreted protein of 56 kDa containing a peptidase M20 domain characteristic of the glutamate carboxypeptidase group of zinc metalloproteases. We also identified homologous Darmin genes in other eukaryotes and in prokaryotes suggesting that Darmin is the founding member of a family of evolutionarily conserved proteins. Xenopus Darmin showed zygotic expression in the early endoderm and later became restricted to the midgut. By secretion cloning of Xenopus cleavage-stage embryos we isolated another novel protein, designated Darmin-related (Darmin-r) due to its sequence similarity with Darmin. Darmin-r was maternally expressed and showed at later stages expression in the lens and pronephric glomus. The endoderm-specific expression of Darmin makes this gene a useful marker for the study of endoderm development.

The serpin PN1 is a feedback regulator of FGF signaling in germ layer and primary axis formation

Germ layer formation and primary axis development rely on Fibroblast growth factors (FGFs). In Xenopus, the secreted serine protease HtrA1 induces mesoderm and posterior trunk/tail structures by facilitating the spread of FGF signals. Here, we show that the serpin Protease nexin-1 (PN1) is transcriptionally activated by FGF signals, suppresses mesoderm and promotes head development in mRNA-injected embryos. An antisense morpholino oligonucleotide against PN1 has the opposite effect and inhibits ectodermal fate. However, ectoderm and anterior head structures can be restored in PN1-depleted embryos when HtrA1 and FGF receptor activities are diminished, indicating that FGF signals negatively regulate their formation. We show that PN1 binds to and inhibits HtrA1, prevents degradation of the proteoglycan Syndecan 4 and restricts paracrine FGF/Erk signaling. Our data suggest that PN1 is a negative-feedback regulator of FGF signaling and has important roles in ectoderm and head development. Summary: The serpin PN1, which is transcriptionally activated by FGF signals, inhibits the protease HtrA1 and prevents syndecan-4 degradation to restrict FGF signaling during Xenopus head development.

Expression of the novel gene Ened during mouse and Xenopus embryonic development

We have recently identified 1110032E23Rik as a down-regulated target gene in Fgf receptor-signalling-deficient mouse embryoid bodies. Here, we present the expression pattern of this novel gene, designated Ened (Expressed in Nerve and Epithelium during Development), in mouse and Xenopus laevis embryos. Murine Ened transcripts were first seen at E9.5 in the heart and the gastrointestinal tract. At later stages of gestation, expression could be found in the floor plate, peripheral nervous system, lens epithelium, skin, midline dorsal aorta, lung, kidney and testis. In Xenopus, the expression of the Ened orthologue displayed common RNA distribution in several ectodermal and mesodermal tissues, but also distinct expression in locations including the brain, notochord and blood islands. We suggest that Ened might be a novel target gene of the Fgfr signalling pathway during embryonic development, and that its expression could be modulated by the basement membrane component laminin-111.