Analysis of Thyroid Hormone Receptor α-Knockout Tadpoles Reveals That the Activation of Cell Cycle Program Is Involved in Thyroid Hormone-Induced Larval Epithelial Cell Death and Adult Intestinal Stem Cell Development During Xenopus tropicalis Metamorphosis.

Background: There are two highly conserved thyroid hormone (triiodothyronine [T3]) receptor (TR) genes, TRα and TRß, in all vertebrates, and the expression of TRα but not TRß is activated earlier than T3 synthesis during development. In human, high levels of T3 are present during the several months around birth, and T3 deficiency during this period causes severe developmental abnormalities including skeletal and intestinal defects. It is, however, difficult to study this period in mammals as the embryos and neonates depend on maternal supply of nutrients for survival. However, Xenopus tropicalis undergoes a T3-dependent metamorphosis, which drastically changes essentially every organ in a tadpole. Of interest is intestinal remodeling, which involves near complete degeneration of the larval epithelium through apoptosis. Concurrently, adult intestinal stem cells are formed de novo and subsequently give rise to the self-renewing adult epithelial system, resembling intestinal maturation around birth in mammals. We have previously demonstrated that T3 signaling is essential for the formation of adult intestinal stem cells during metamorphosis. Methods: We studied the function of endogenous TRα in the tadpole intestine by using knockout animals and RNA-seq analysis. Results: We observed that removing endogenous TRα caused defects in intestinal remodeling, including drastically reduced larval epithelial cell death and adult intestinal stem cell proliferation. Using RNA-seq on intestinal RNA from premetamorphic wild-type and TRα-knockout tadpoles treated with or without T3 for one day, before any detectable T3-induced cell death and stem cell formation in the tadpole intestine, we identified more than 1500 genes, which were regulated by T3 treatment of the wild-type but not TRα-knockout tadpoles. Gene Ontology and biological pathway analyses revealed that surprisingly, these TRα-regulated genes were highly enriched with cell cycle-related genes, in addition to genes related to stem cells and apoptosis. Conclusions: Our findings suggest that TRα-mediated T3 activation of the cell cycle program is involved in larval epithelial cell death and adult epithelial stem cell development during intestinal remodeling.

Lack of GAS2L2 Causes PCD by Impairing Cilia Orientation and Mucociliary Clearance.

Primary ciliary dyskinesia (PCD) is a genetic disorder in which impaired ciliary function leads to chronic airway disease. Exome sequencing of a PCD subject identified an apparent homozygous frameshift variant, c.887_890delTAAG (p.Val296Glyfs∗13), in exon 5; this frameshift introduces a stop codon in amino acid 308 of the growth arrest-specific protein 2-like 2 (GAS2L2). Further genetic screening of unrelated PCD subjects identified a second proband with a compound heterozygous variant carrying the identical frameshift variant and a large deletion (c.867_∗343+1207del; p.?) starting in exon 5. Both individuals had clinical features of PCD but normal ciliary axoneme structure. In this research, using human nasal cells, mouse models, and X.laevis embryos, we show that GAS2L2 is abundant at the apical surface of ciliated cells, where it localizes with basal bodies, basal feet, rootlets, and actin filaments. Cultured GAS2L2-deficient nasal epithelial cells from one of the affected individuals showed defects in ciliary orientation and had an asynchronous and hyperkinetic (GAS2L2-deficient = 19.8 Hz versus control = 15.8 Hz) ciliary-beat pattern. These results were recapitulated in Gas2l2-/- mouse tracheal epithelial cell (mTEC) cultures and in X. laevis embryos treated with Gas2l2 morpholinos. In mice, the absence of Gas2l2 caused neonatal death, and the conditional deletion of Gas2l2 impaired mucociliary clearance (MCC) and led to mucus accumulation. These results show that a pathogenic variant in GAS2L2 causes a genetic defect in ciliary orientation and impairs MCC and results in PCD.

Tight junction-associated protein GEF-H1 in the neighbours of dividing epithelial cells is essential for adaptation of cell-cell membrane during cytokinesis.

Animal cells divide by a process called cytokinesis which relies on the constriction of a contractile actomyosin ring leading to the production of two daughter cells. Cytokinesis is an intrinsic property of cells which occurs even for artificially isolated cells. During division, isolated cells undergo dramatic changes in shape such as rounding and membrane deformation as the division furrow ingresses. However, cells are often embedded in tissues and thus are surrounded by neighbouring cells. How these neighbours might influence, or might themselves be influenced by, the shape changes of cytokinesis is poorly understood in vertebrates. Here, we show that during cytokinesis of epithelial cells in the Xenopus embryo, lateral cell-cell contacts remain almost perpendicular to the epithelial plane. Depletion of the tight junction-associated protein GEF-H1 leads to a transient and stereotyped deformation of cell-cell contacts. Although, this deformation occurs only during cytokinesis, we show that it originates from immediate neighbours of the dividing cell. Moreover, we show that exocyst and recycling endosome regulation by GEF-H1 are involved in adaptation of cell-cell contacts to deformation. Our results highlight the crucial role of tight junctions and GEF-H1 in cell-cell contact adaptation when cells are exposed to a mechanical stress such as cytokinesis.

Developmental neurogenesis in mouse and Xenopus is impaired in the absence of Nosip.

BACKGROUND: Genetic deletion of Nosip in mice causes holoprosencephaly, however, the function of Nosip in neurogenesis is currently unknown. RESULTS: We combined two vertebrate model organisms, the mouse and the South African clawed frog, Xenopus laevis, to study the function of Nosip in neurogenesis. We found, that size and cortical thickness of the developing brain of Nosip knockout mice were reduced. Accordingly, the formation of postmitotic neurons was greatly diminished, concomitant with a reduced number of apical and basal neural progenitor cells in vivo. Neurospheres derived from Nosip knockout embryos exhibited reduced growth and the differentiation capability into neurons in vitro was almost completely abolished. Mass spectrometry analysis of the neurospheres proteome revealed a reduced expression of Rbp1, a regulator of retinoic acid synthesis, when Nosip was absent. We identified the homologous nosip gene to be expressed in differentiated neurons in the developing brain of Xenopus embryos. Knockdown of Nosip in Xenopus resulted in a reduction of brain size that could be rescued by reintroducing human NOSIP mRNA. Furthermore, the expression of pro-neurogenic transcription factors was reduced and the differentiation of neuronal cells was impaired upon Nosip knockdown. In Xenopus as well as in mouse we identified reduced proliferation and increased apoptosis as underlying cause of microcephaly upon Nosip depletion. In Xenopus Nosip and Rbp1 are similarly expressed and knockdown of Nosip resulted in down regulation of Rbp1. Knockdown of Rbp1 caused a similar microcephaly phenotype as the depletion of Nosip and synergy experiments indicated that both proteins act in the same signalling pathway. CONCLUSIONS: Nosip is a novel factor critical for neural stem cell/progenitor self-renewal and neurogenesis during mouse and Xenopus development and functions upstream of Rbp1 during early neurogenesis.

Generation of a Xenopus laevis F1 albino J strain by genome editing and oocyte host-transfer.

Completion of the Xenopus laevis genome sequence from inbred J strain animals has facilitated the generation of germline mutant X. laevis using targeted genome editing. In the last few years, numerous reports have demonstrated that TALENs are able to induce mutations in F0 Xenopus embryos, but none has demonstrated germline transmission of such mutations in X. laevis. In this report we used the oocyte host-transfer method to generate mutations in both tyrosinase homeologs and found highly-penetrant germline mutations; in contrast, embryonic injections yielded few germline mutations. We also compared the distribution of mutations in several F0 somatic tissues and germ cells and found that the majority of mutations in each tissue were different. These results establish that X. laevis J strain animals are very useful for generating germline mutations and that the oocyte host-transfer method is an efficient technique for generating mutations in both homeologs.

Noggin is required for first pharyngeal arch differentiation in the frog Xenopus tropicalis.

The dorsal ventral axis of vertebrates requires high BMP activity for ventral development and inhibition of BMP activity for dorsal development. Presumptive dorsal regions of the embryo are protected from the ventralizing activity of BMPs by the secretion of BMP antagonists from the mesoderm. Noggin, one such antagonist, binds BMP ligands and prevents them from binding their receptors, however, a unique role for Noggin in amphibian development has remained unclear. Previously, we used zinc-finger nucleases to mutagenize the noggin locus in Xenopus tropicalis. Here, we report on the phenotype of noggin mutant frogs as a result of breeding null mutations to homozygosity. Early homozygous noggin mutant embryos are indistinguishable from wildtype siblings, with normal neural induction and neural tube closure. However, in late tadpole stages mutants present severe ventral craniofacial defects, notably a fusion of Meckel's cartilage to the palatoquadrate cartilage. Consistent with a noggin loss-of-function, mutants show expansions of BMP target gene expression and the mutant phenotype can be rescued with transient BMP inhibition. These results demonstrate that in amphibians, Noggin is dispensable for early embryonic patterning but is critical for cranial skeletogenesis.

no privacy, a Xenopus tropicalis mutant, is a model of human Hermansky-Pudlak Syndrome and allows visualization of internal organogenesis during tadpole development.

We describe a novel recessive and nonlethal pigmentation mutant in Xenopus tropicalis. The mutant phenotype can be initially observed in tadpoles after stage 39/40, when mutant embryos display markedly reduced pigmentation in the retina and the trunk. By tadpole stage 50 almost all pigmented melanophores have disappeared. Most interestingly, those embryos fail entirely to make pigmented iridophores. The combined reduction/absence of both pigmented iridophores and melanophores renders these embryos virtually transparent, permitting one to easily observe both the developing internal organs and nervous system; accordingly, we named this mutant no privacy (nop). We identified the causative genetic lesion as occurring in the Xenopus homolog of the human Hermansky-Pudlak Syndrome 6 (HPS6) gene, combining several approaches that utilized conventional gene mapping and classical and modern genetic tools available in Xenopus (gynogenesis, BAC transgenesis and TALEN-mediated mutagenesis). The nop allele contains a 10-base deletion that results in truncation of the Hps6 protein. In humans, HPS6 is one of the genes responsible for the congenital disease HPS, pathological symptoms of which include oculocutaneous albinism caused by defects in lysosome-related organelles required for pigment formation. Markers for melanin-producing neural crest cells show that the cells that would give rise to melanocytes are present in nop, though unpigmented. Abnormalities develop at tadpole stages in the pigmented retina when overall pigmentation becomes reduced and large multi-melanosomes are first formed. Ear development is also affected in nop embryos when both zygotic and maternal hsp6 is mutated: otoliths are often reduced or abnormal in morphology, as seen in some mouse HPS mutations, but to our knowledge not described in the BLOC-2 subset of HPS mutations nor described in non-mammalian systems previously. The transparency of the nop line suggests that these animals will aid studies of early organogenesis during tadpole stages. In addition, because of advantages of the Xenopus system for assessing gene expression, cell biological mechanisms, and the ontogeny of melanosome and otolith formation, this should be a highly useful model for studying the molecular mechanisms underlying the acquisition of the HPS phenotype and the underlying biology of lysosome-related organelle function.

TTC25 Deficiency Results in Defects of the Outer Dynein Arm Docking Machinery and Primary Ciliary Dyskinesia with Left-Right Body Asymmetry Randomization.

Multiprotein complexes referred to as outer dynein arms (ODAs) develop the main mechanical force to generate the ciliary and flagellar beat. ODA defects are the most common cause of primary ciliary dyskinesia (PCD), a congenital disorder of ciliary beating, characterized by recurrent infections of the upper and lower airways, as well as by progressive lung failure and randomization of left-right body asymmetry. Using a whole-exome sequencing approach, we identified recessive loss-of-function mutations within TTC25 in three individuals from two unrelated families affected by PCD. Mice generated by CRISPR/Cas9 technology and carrying a deletion of exons 2 and 3 in Ttc25 presented with laterality defects. Consistently, we observed immotile nodal cilia and missing leftward flow via particle image velocimetry. Furthermore, transmission electron microscopy (TEM) analysis in TTC25-deficient mice revealed an absence of ODAs. Consistent with our findings in mice, we were able to show loss of the ciliary ODAs in humans via TEM and immunofluorescence (IF) analyses. Additionally, IF analyses revealed an absence of the ODA docking complex (ODA-DC), along with its known components CCDC114, CCDC151, and ARMC4. Co-immunoprecipitation revealed interaction between the ODA-DC component CCDC114 and TTC25. Thus, here we report TTC25 as a new member of the ODA-DC machinery in humans and mice.

The aryl hydrocarbon receptor controls cyclin O to promote epithelial multiciliogenesis.

Epithelia function as barriers against environmental insults and express the transcription factor aryl hydrocarbon receptor (AhR). However, AhR function in these tissues is unknown. Here we show that AhR regulates multiciliogenesis in both murine airway epithelia and in Xenopus laevis epidermis. In air-exposed airway epithelia, induction of factors required for multiciliogenesis, including cyclin O (Ccno) and Multicilin (Mcidas), is AhR dependent, and air exposure induces AhR binding to the Ccno promoter. Submersion and hypoxic conditions impede AhR-dependent Ccno induction. This is mediated by the persistence of Notch signalling, as Notch blockade renders multiciliogenesis and Ccno induction by AhR independent from air exposure. In contrast to Ccno induction, air exposure does not induce the canonical AhR target cytochrome P450 1a1 (Cyp1a1). Inversely, exposure to AhR ligands induces Cyp1a1 but not Ccno and impeded ciliogenesis. These data indicate that AhR involvement in detoxification of environmental pollutants may impede its physiological role, resulting in respiratory pathology.

The positive transcriptional elongation factor (P-TEFb) is required for neural crest specification.

Regulation of gene expression at the level of transcriptional elongation has been shown to be important in stem cells and tumour cells, but its role in the whole animal is only now being fully explored. Neural crest cells (NCCs) are a multipotent population of cells that migrate during early development from the dorsal neural tube throughout the embryo where they differentiate into a variety of cell types including pigment cells, cranio-facial skeleton and sensory neurons. Specification of NCCs is both spatially and temporally regulated during embryonic development. Here we show that components of the transcriptional elongation regulatory machinery, CDK9 and CYCLINT1 of the P-TEFb complex, are required to regulate neural crest specification. In particular, we show that expression of the proto-oncogene c-Myc and c-Myc responsive genes are affected. Our data suggest that P-TEFb is crucial to drive expression of c-Myc, which acts as a 'gate-keeper' for the correct temporal and spatial development of the neural crest.

Sf3b4-depleted Xenopus embryos: A model to study the pathogenesis of craniofacial defects in Nager syndrome.

Mandibulofacial dysostosis (MFD) is a human developmental disorder characterized by defects of the facial bones. It is the second most frequent craniofacial malformation after cleft lip and palate. Nager syndrome combines many features of MFD with a variety of limb defects. Mutations in SF3B4 (splicing factor 3b, subunit 4) gene, which encodes a component of the pre-mRNA spliceosomal complex, were recently identified as a cause of Nager syndrome, accounting for 60% of affected individuals. Nothing is known about the cellular pathogenesis underlying Nager type MFD. Here we describe the first animal model for Nager syndrome, generated by knocking down Sf3b4 function in Xenopus laevis embryos, using morpholino antisense oligonucleotides. Our results indicate that Sf3b4-depleted embryos show reduced expression of the neural crest genes sox10, snail2 and twist at the neural plate border, associated with a broadening of the neural plate. This phenotype can be rescued by injection of wild-type human SF3B4 mRNA but not by mRNAs carrying mutations that cause Nager syndrome. At the tailbud stage, morphant embryos had decreased sox10 and tfap2a expression in the pharyngeal arches, indicative of a reduced number of neural crest cells. Later in development, Sf3b4-depleted tadpoles exhibited hypoplasia of neural crest-derived craniofacial cartilages, phenocopying aspects of the craniofacial skeletal defects seen in Nager syndrome patients. With this animal model we are now poised to gain important insights into the etiology and pathogenesis of Nager type MFD, and to identify the molecular targets of Sf3b4.

NF2/Merlin is required for the axial pattern formation in the Xenopus laevis embryo.

The NF2 gene product Merlin is a FERM-domain protein possessing a broad tumor-suppressing function. NF2/Merlin has been implicated in regulating multiple signaling pathways critical for cell growth and survival. However, it remains unknown whether NF2/Merlin regulates Wnt/ß-catenin signaling during vertebrate embryogenesis. Here we demonstrate that NF2/Merlin is required for body pattern formation in the Xenopus laevis embryo. Depletion of the maternal NF2/Merlin enhances organizer gene expression dependent on the presence of ß-catenin, and causes dorsanteriorized development; Morpholino antisense oligo-mediated knockdown of the zygotic NF2/Merlin shifts posterior genes anteriorwards and reduces the anterior development. We further demonstrate that targeted depletion of NF2 in the presumptive dorsal tissues increases the levels of nuclear ß-catenin in the neural epithelial cells. Biochemical analyses reveal that NF2 depletion promotes the production of active ß-catenin and concurrently decreases the level of N-terminally phosphorylated ß-catenin under the stimulation of the endogenous Wnt signaling. Our findings suggest that NF2/Merlin negatively regulates the Wnt/ß-catenin signaling activity during the pattern formation in early X. laevis embryos.

Differential requirement of F-actin and microtubule cytoskeleton in cue-induced local protein synthesis in axonal growth cones.

BACKGROUND: Local protein synthesis (LPS) via receptor-mediated signaling plays a role in the directional responses of axons to extrinsic cues. An intact cytoskeleton is critical to enact these responses, but it is not known whether the two major cytoskeletal elements, F-actin and microtubules, have any roles in regulating axonal protein synthesis. RESULTS: Here, we show that pharmacological disruption of either microtubules or actin filaments in growth cones blocks netrin-1-induced de novo synthesis of proteins, as measured by metabolic incorporation of labeled amino acids, implicating both elements in axonal synthesis. However, comparative analysis of the activated translation initiation regulator, eIF4E-BP1, revealed a striking difference in the point of action of the two elements: actin disruption completely inhibited netrin-1-induced eIF4E-BP1 phosphorylation while microtubule disruption had no effect. An intact F-actin, but not microtubule, cytoskeleton was also required for netrin-1-induced activation of the PI3K/Akt/mTOR pathway, upstream of translation initiation. Downstream of translation initiation, microtubules were required for netrin-1-induced activation of eukaryotic elongation factor 2 kinase (eEF2K) and eEF2. CONCLUSIONS: Taken together, our results show that while actin and microtubules are both crucial for cue-induced axonal protein synthesis, they serve distinct roles with F-actin being required for the initiation of translation and microtubules acting later at the elongation step.

Diverse functions of kindlin/fermitin proteins during embryonic development in Xenopus laevis.

The kindlin/fermitin family includes three proteins involved in regulating integrin ligand-binding activity and adhesion. Loss-of-function mutations in kindlins1 and 3 have been implicated in Kindler Syndrome and Leukocyte Adhesion Deficiency III (LAD-III) respectively, whereas kindlin2 null mice are embryonic lethal. Post translational regulation of cell-cell and cell-ECM adhesion has long been presumed to be important for morphogenesis, however, few specific examples of activation-dependent changes in adhesion molecule function in normal development have been reported. In this study, antisense morpholinos were used to reduce expression of individual kindlins in Xenopus laevis embryos in order to investigate their roles in early development. Kindlin1 knockdown resulted in developmental delays, gross malformations of the gut and eventual lethality by tadpole stages. Kindlin2 morphant embryos displayed late stage defects in vascular maintenance and angiogenic branching consistent with kindlin2 loss of function in the mouse. Antisense morpholinos were also used to deplete maternal kindlin2 protein in oocytes and eggs. Embryos lacking maternal kindlin2 arrested at early cleavage stages due to failures in cytokinesis. Kindlin3 morphant phenotypes included defects in epidermal ciliary beating and partial paralysis at tailbud stages but these embryos recovered eventually as morpholino levels decayed. These results indicate a remarkably diverse range of kindlin functions in vertebrate development.

The heterotaxy gene GALNT11 glycosylates Notch to orchestrate cilia type and laterality.

Heterotaxy is a disorder of left-right body patterning, or laterality, that is associated with major congenital heart disease. The aetiology and mechanisms underlying most cases of human heterotaxy are poorly understood. In vertebrates, laterality is initiated at the embryonic left-right organizer, where motile cilia generate leftward flow that is detected by immotile sensory cilia, which transduce flow into downstream asymmetric signals. The mechanism that specifies these two cilia types remains unknown. Here we show that the N-acetylgalactosamine-type O-glycosylation enzyme GALNT11 is crucial to such determination. We previously identified GALNT11 as a candidate disease gene in a patient with heterotaxy, and now demonstrate, in Xenopus tropicalis, that galnt11 activates Notch signalling. GALNT11 O-glycosylates human NOTCH1 peptides in vitro, thereby supporting a mechanism of Notch activation either by increasing ADAM17-mediated ectodomain shedding of the Notch receptor or by modification of specific EGF repeats. We further developed a quantitative live imaging technique for Xenopus left-right organizer cilia and show that Galnt11-mediated Notch1 signalling modulates the spatial distribution and ratio of motile and immotile cilia at the left-right organizer. galnt11 or notch1 depletion increases the ratio of motile cilia at the expense of immotile cilia and produces a laterality defect reminiscent of loss of the ciliary sensor Pkd2. By contrast, Notch overexpression decreases this ratio, mimicking the ciliopathy primary ciliary dyskinesia. Together our data demonstrate that Galnt11 modifies Notch, establishing an essential balance between motile and immotile cilia at the left-right organizer to determine laterality, and reveal a novel mechanism for human heterotaxy.

An essential and highly conserved role for Zic3 in left-right patterning, gastrulation and convergent extension morphogenesis.

Mutations in ZIC3 result in X-linked heterotaxy in humans, a syndrome consisting of left-right (L-R) patterning defects, midline abnormalities, and cardiac malformations. Similarly, loss of function of Zic3 in mouse results in abnormal L-R patterning and cardiac development. However, Zic3 null mice also exhibit defects in gastrulation, neural tube closure, and axial patterning, suggesting the hypothesis that Zic3 is necessary for proper convergent extension (C-E) morphogenesis. To further investigate the role of Zic3 in early embryonic development, we utilized two model systems, Xenopus laevis and zebrafish, and performed loss of function analysis using antisense morpholino-mediated gene knockdown. Both Xenopus and zebrafish demonstrated significant impairment of C-E in Zic3 morphants. L-R patterning was also disrupted, indicating that the role of Zic3 in L-R axis development is conserved across species. Correlation of L-R patterning and C-E defects in Xenopus suggests that early C-E defects may underlie L-R patterning defects at later stages, since Zic3 morphants with moderate to severe C-E defects exhibited an increase in laterality defects. Taken together, these results demonstrate a functional conservation of Zic3 in L-R patterning and uncover a previously unrecognized role for Zic3 in C-E morphogenesis during early vertebrate development.

Friend of GATA (FOG) interacts with the nucleosome remodeling and deacetylase complex (NuRD) to support primitive erythropoiesis in Xenopus laevis.

Friend of GATA (FOG) plays many diverse roles in adult and embryonic hematopoiesis, however the mechanisms by which it functions and the roles of potential interaction partners are not completely understood. Previous work has shown that overexpression of FOG in Xenopus laevis causes loss of blood suggesting that in contrast to its role in mammals, FOG might normally function to repress erythropoiesis in this species. Using loss-of-function analysis, we demonstrate that FOG is essential to support primitive red blood cell (RBC) development in Xenopus. Moreover, we show that it is specifically required to prevent excess apoptosis of circulating primitive RBCs and that in the absence of FOG, the pro-apoptotic gene Bim-1 is strongly upregulated. To identify domains of FOG that are essential for blood development and, conversely, to begin to understand the mechanism by which overexpressed FOG represses primitive erythropoiesis, we asked whether FOG mutants that are unable to interact with known co-factors retain their ability to rescue blood formation in FOG morphants and whether they repress erythropoiesis when overexpressed in wild type embryos. We find that interaction of FOG with the Nucleosome Remodeling and Deacetylase complex (NuRD), but not with C-terminal Binding Protein, is essential for normal primitive RBC development. In contrast, overexpression of all mutant and wild type constructs causes a comparable repression of primitive erythropoiesis. Together, our data suggest that a requirement for FOG and its interaction with NuRD during primitive erythropoiesis are conserved in Xenopus and that loss of blood upon FOG overexpression is due to a dominant-interfering effect.

The forkhead transcription factor FoxB1 regulates the dorsal-ventral and anterior-posterior patterning of the ectoderm during early Xenopus embryogenesis.

The formation of the dorsal-ventral (DV) and anterior-posterior (AP) axes, fundamental to the body plan of animals, is regulated by several groups of polypeptide growth factors including the TGF-ß, FGF, and Wnt families. In order to ensure the establishment of the body plan, the processes of DV and AP axis formation must be linked and coordinately regulated. However, the molecular mechanisms responsible for these interactions remain unclear. Here, we demonstrate that the forkhead box transcription factor FoxB1, which is upregulated by the neuralizing factor Oct-25, plays an important role in the formation of the DV and AP axes. Overexpression of FoxB1 promoted neural induction and inhibited BMP-dependent epidermal differentiation in ectodermal explants, thereby regulating the DV patterning of the ectoderm. In addition, FoxB1 was also found to promote the formation of posterior neural tissue in both ectodermal explants and whole embryos, suggesting its involvement in embryonic AP patterning. Using knockdown analysis, we found that FoxB1 is required for the formation of posterior neural tissues, acting in concert with the Wnt and FGF pathways. Consistent with this, FoxB1 suppressed the formation of anterior structures via a process requiring the function of XWnt-8 and eFGF. Interestingly, while downregulation of FoxB1 had little effect on neural induction, we found that it functionally interacted with its upstream factor Oct-25 and plays a supportive role in the induction and/or maintenance of neural tissue. Our results suggest that FoxB1 is part of a mechanism that fine-tunes, and leads to the coordinated formation of, the DV and AP axes during early development.

Skeletal muscle differentiation and fusion are regulated by the BAR-containing Rho-GTPase-activating protein (Rho-GAP), GRAF1.

Although RhoA activity is necessary for promoting myogenic mesenchymal stem cell fates, recent studies in cultured cells suggest that down-regulation of RhoA activity in specified myoblasts is required for subsequent differentiation and myotube formation. However, whether this phenomenon occurs in vivo and which Rho modifiers control these later events remain unclear. We found that expression of the Rho-GTPase-activating protein, GRAF1, was transiently up-regulated during myogenesis, and studies in C2C12 cells revealed that GRAF1 is necessary and sufficient for mediating RhoA down-regulation and inducing muscle differentiation. Moreover, forced expression of GRAF1 in pre-differentiated myoblasts drives robust muscle fusion by a process that requires GTPase-activating protein-dependent actin remodeling and BAR-dependent membrane binding or sculpting. Moreover, morpholino-based knockdown studies in Xenopus laevis determined that GRAF1 expression is critical for muscle development. GRAF1-depleted embryos exhibited elevated RhoA activity and defective myofibrillogenesis that resulted in progressive muscle degeneration, defective motility, and embryonic lethality. Our results are the first to identify a GTPase-activating protein that regulates muscle maturation and to highlight the functional importance of BAR domains in myotube formation.

Lhx1 is required for specification of the renal progenitor cell field.

In the vertebrate embryo, the kidney is derived from the intermediate mesoderm. The LIM-class homeobox transcription factor lhx1 is expressed early in the intermediate mesoderm and is one of the first genes to be expressed in the nephric mesenchyme. In this study, we investigated the role of Lhx1 in specification of the kidney field by either overexpressing or depleting lhx1 in Xenopus embryos or depleting lhx1 in an explant culture system. By overexpressing a constitutively-active form of Lhx1, we established its capacity to expand the kidney field during the specification stage of kidney organogenesis. In addition, the ability of Lhx1 to expand the kidney field diminishes as kidney organogenesis transitions to the morphogenesis stage. In a complimentary set of experiments, we determined that embryos depleted of lhx1, show an almost complete loss of the kidney field. Using an explant culture system to induce kidney tissue, we confirmed that expression of genes from both proximal and distal kidney structures is affected by the absence of lhx1. Taken together our results demonstrate an essential role for Lhx1 in driving specification of the entire kidney field from the intermediate mesoderm.