PFKFB4 control of AKT signaling is essential for premigratory and migratory neural crest formation.

Neural crest (NC) specification comprises an early phase, initiating immature NC progenitors formation at neural plate stage, and a later phase at neural fold stage, resulting in a functional premigratory NC that is able to delaminate and migrate. We found that the NC gene regulatory network triggers upregulation of pfkfb4 (6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 4) during this late specification phase. As shown in previous studies, PFKFB4 controls AKT signaling in gastrulas and glycolysis rate in adult cells. Here, we focus on PFKFB4 function in NC during and after neurulation, using time-controlled or hypomorph depletions in vivo We find that PFKFB4 is essential both for specification of functional premigratory NC and for its migration. PFKFB4-depleted embryos fail to activate n-cadherin and late NC specifiers, and exhibit severe migration defects resulting in craniofacial defects. AKT signaling mediates PFKFB4 function in NC late specification, whereas both AKT signaling and glycolysis regulate migration. These findings highlight novel and essential roles of PFKFB4 activity in later stages of NC development that are wired into the NC gene regulatory network.

PFKFB4 controls embryonic patterning via Akt signalling independently of glycolysis.

How metabolism regulators play roles during early development remains elusive. Here we show that PFKFB4 (6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 4), a glycolysis regulator, is critical for controlling dorsal ectoderm global patterning in gastrulating frog embryos via a non-glycolytic function. PFKFB4 is required for dorsal ectoderm progenitors to proceed towards more specified fates including neural and non-neural ectoderm, neural crest or placodes. This function is mediated by Akt signalling, a major pathway that integrates cell homeostasis and survival parameters. Restoring Akt signalling rescues the loss of PFKFB4 in vivo. In contrast, glycolysis is not essential for frog development at this stage. Our study reveals the existence of a PFKFB4-Akt checkpoint that links cell homeostasis to the ability of progenitor cells to undergo differentiation, and uncovers glycolysis-independent functions of PFKFB4.

Pax3 and Zic1 trigger the early neural crest gene regulatory network by the direct activation of multiple key neural crest specifiers.

Neural crest development is orchestrated by a complex and still poorly understood gene regulatory network. Premigratory neural crest is induced at the lateral border of the neural plate by the combined action of signaling molecules and transcription factors such as AP2, Gbx2, Pax3 and Zic1. Among them, Pax3 and Zic1 are both necessary and sufficient to trigger a complete neural crest developmental program. However, their gene targets in the neural crest regulatory network remain unknown. Here, through a transcriptome analysis of frog microdissected neural border, we identified an extended gene signature for the premigratory neural crest, and we defined novel potential members of the regulatory network. This signature includes 34 novel genes, as well as 44 known genes expressed at the neural border. Using another microarray analysis which combined Pax3 and Zic1 gain-of-function and protein translation blockade, we uncovered 25 Pax3 and Zic1 direct targets within this signature. We demonstrated that the neural border specifiers Pax3 and Zic1 are direct upstream regulators of neural crest specifiers Snail1/2, Foxd3, Twist1, and Tfap2b. In addition, they may modulate the transcriptional output of multiple signaling pathways involved in neural crest development (Wnt, Retinoic Acid) through the induction of key pathway regulators (Axin2 and Cyp26c1). We also found that Pax3 could maintain its own expression through a positive autoregulatory feedback loop. These hierarchical inductions, feedback loops, and pathway modulations provide novel tools to understand the neural crest induction network.

Protein tyrosine phosphatase 4A3 (PTP4A3) is required for Xenopus laevis cranial neural crest migration in vivo.

Uveal melanoma is the most common intraocular malignancy in adults, representing between about 4% and 5% of all melanomas. High expression levels of Protein Tyrosine Phosphatase 4A3, a dual phosphatase, is highly predictive of metastasis development and PTP4A3 overexpression in uveal melanoma cells increases their in vitro migration and in vivo invasiveness. Melanocytes, including uveal melanocytes, are derived from the neural crest during embryonic development. We therefore suggested that PTP4A3 function in uveal melanoma metastasis may be related to an embryonic role during neural crest cell migration. We show that PTP4A3 plays a role in cephalic neural crest development in Xenopus laevis. PTP4A3 loss of function resulted in a reduction of neural crest territory, whilst gain of function experiments increased neural crest territory. Isochronic graft experiments demonstrated that PTP4A3-depleted neural crest explants are unable to migrate in host embryos. Pharmacological inhibition of PTP4A3 on dissected neural crest cells significantly reduced their migration velocity in vitro. Our results demonstrate that PTP4A3 is required for cephalic neural crest migration in vivo during embryonic development.

Signaling and transcriptional regulation in neural crest specification and migration: lessons from xenopus embryos.

The neural crest is a population of highly migratory and multipotent cells, which arises from the border of the neural plate in vertebrate embryos. In the last few years, the molecular actors of neural crest early development have been intensively studied, notably by using the frog embryo, as a prime model for the analysis of the earliest embryonic inductions. In addition, tremendous progress has been made in understanding the molecular and cellular basis of Xenopus cranial neural crest migration, by combining in vitro and in vivo analysis. In this review, we examine how the action of previously known neural crest-inducing signals [bone morphogenetic protein (BMP), wingless-int (Wnt), fibroblast growth factor (FGF)] is controlled by newly discovered modulators during early neural plate border patterning and neural crest specification. This regulation controls the induction of key transcription factors that cooperate to pattern the premigratory neural crest progenitors. These data are discussed in the perspective of the gene regulatory network that controls neural and neural crest patterning. We then address recent findings on noncanonical Wnt signaling regulation, cell polarization, and collective cell migration which highlight how cranial neural crest cells populate their target tissue, the branchial arches, in vivo. More than ever, the neural crest stands as a powerful and attractive model to decipher complex vertebrate regulatory circuits in vivo.

Pfkfb (6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase) isoforms display a tissue-specific and dynamic expression during Xenopus laevis development.

Pfkfb (6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase) enzymes are bi-functional enzymes encoded by four different genes (pfkfb1, pfkfb2, pfkfb3, pfkfb4) in vertebrates. They are involved in the regulation of glycolysis: they catalyze the synthesis and the degradation of F-2,6-BP (fructose-2,6-bisphosphate), the most potent allosteric activator of phosphofructokinase 1 (Pfk1), a key glycolytic enzyme. By producing F-2,6-BP, Pfkfb enzymes allow glycolysis to proceed, while by degrading F-2,6-BP they block glycolysis. As major regulators of glycolysis, Pfkfb enzymes are involved in cancer: tumor cells have a higher glycolytic rate compared to normal cells, even in the presence of adequate oxygen levels (Warburg effect) and several cancer cell lines express elevated levels of Pfkfb enzymes. Glycolysis is also important for energy and metabolite production in proliferating cells. In embryos, however, the role of glycolysis and the expression of glycolysis regulators remain to be explored. Here, we provide a phylogenetic analysis of Pfkfb enzymes in vertebrates, and we detail the expression pattern of pfk1, pfkfb1, pfkfb2, pfkfb3, and pfkfb4 genes in Xenopus laevis embryos. We show that pfkfb transcripts expression is overlapping at blastula and gastrula stages and that from neurulation to tadpole stages, they display tissue-specific, complementary and dynamic expression patterns.

Tissue-specific expression of Sarcoplasmic/Endoplasmic Reticulum Calcium ATPases (ATP2A/SERCA) 1, 2, 3 during Xenopus laevis development.

Calcium-ATPase pumps are critical in most cells, to sequester calcium into intracytoplasmic stores and regulate general calcium signalling. In addition, cell-specific needs for calcium signals have been described and employ a diversity of calcium ATPases in adult tissues and oocytes. A major family of such calcium pumps is ATP2A/SERCA family, for Sarcoplasmic/Endoplasmic Reticulum Calcium ATPases. Although largely studied in adults, the developmental expression of the atp2a/serca genes remains unknown. Here, we provide genome organisation in Xenopuslaevis and tropicalis and phylogeny of atp2a/serca genes in craniates. We detail embryonic expression for the three X. laevis atp2a/serca genes. We found that the three atp2a/serca genes are strongly conserved among vertebrates and display complementary and tissue-specific expression in embryos. These expression patterns present variations when compared to the data reported in adults. Atp2a1/serca1 is expressed as soon as the end of gastrulation in a subset of the myod-positive cells, and later labels prospective slow muscle cells in the superficial part of the somite. In contrast atp2a2/serca2 is found in a larger subset of cells, but is not ubiquitous as reported in adults. Notably, atp2a2/serca2 is prominently expressed in the neural-related tissues, i.e. the neural plate, cement gland, but is excluded from premigratory neural crest. Finally, atp2a3/serca3 expression is restricted to the ectoderm throughout development.