Synergistic integration of Netrin and ephrin axon guidance signals by spinal motor neurons.

During neural circuit assembly, axonal growth cones are exposed to multiple guidance signals at trajectory choice points. While axonal responses to individual guidance cues have been extensively studied, less is known about responses to combination of signals and underlying molecular mechanisms. Here, we studied the convergence of signals directing trajectory selection of spinal motor axons entering the limb. We first demonstrate that Netrin-1 attracts and repels distinct motor axon populations, according to their expression of Netrin receptors. Quantitative in vitro assays demonstrate that motor axons synergistically integrate both attractive or repulsive Netrin-1 signals together with repulsive ephrin signals. Our investigations of the mechanism of ephrin-B2 and Netrin-1 integration demonstrate that the Netrin receptor Unc5c and the ephrin receptor EphB2 can form a complex in a ligand-dependent manner and that Netrin-ephrin synergistic growth cones responses involve the potentiation of Src family kinase signaling, a common effector of both pathways.

Loss of LKB1 leads to impaired epithelial integrity and cell extrusion in the early mouse embryo.

LKB1/PAR-4 is essential for the earliest polarization steps in Caenorhabditis elegans embryos and Drosophila oocytes. Although LKB1 (also known as STK11) is sufficient to initiate polarity in a single mammalian intestinal epithelial cell, its necessity in the formation and maintenance of mammalian epithelia remains unclear. To address this, we completely remove LKB1 from mouse embryos by generating maternal-zygotic Lkb1 mutants and find that it is dispensable for polarity and epithelia formation in the early embryo. Instead, loss of Lkb1 leads to the extrusion of cells from blastocyst epithelia that remain alive and can continue to divide. Chimeric analysis shows that Lkb1 is cell-autonomously required to prevent these extrusions. Furthermore, heterozygous loss of Cdh1 exacerbates the number of extrusions per blastocyst, suggesting that LKB1 has a role in regulating adherens junctions in order to prevent extrusion in epithelia.

Initiation of Hippo signaling is linked to polarity rather than to cell position in the pre-implantation mouse embryo.

In the mouse embryo, asymmetric divisions during the 8-16 cell division generate two cell types, polar and apolar cells, that are allocated to outer and inner positions, respectively. This outer/inner configuration is the first sign of the formation of the first two cell lineages: trophectoderm (TE) and inner cell mass (ICM). Outer polar cells become TE and give rise to the placenta, whereas inner apolar cells become ICM and give rise to the embryo proper and yolk sac. Here, we analyze the frequency of asymmetric divisions during the 8-16 cell division and assess the relationships between cell polarity, cell and nuclear position, and Hippo signaling activation, the pathway that initiates lineage-specific gene expression in 16-cell embryos. Although the frequency of asymmetric divisions varied in each embryo, we found that more than six blastomeres divided asymmetrically in most embryos. Interestingly, many apolar cells in 16-cell embryos were located at outer positions, whereas only one or two apolar cells were located at inner positions. Live imaging analysis showed that outer apolar cells were eventually internalized by surrounding polar cells. Using isolated 8-cell blastomeres, we carefully analyzed the internalization process of apolar cells and found indications of higher cortical tension in apolar cells than in polar cells. Last, we found that apolar cells activate Hippo signaling prior to taking inner positions. Our results suggest that polar and apolar cells have intrinsic differences that establish outer/inner configuration and differentially regulate Hippo signaling to activate lineage-specific gene expression programs.

FGF4 is a limiting factor controlling the proportions of primitive endoderm and epiblast in the ICM of the mouse blastocyst.

The primitive endoderm (PE) and epiblast (EPI) are two lineages derived from the inner cell mass (ICM) of the E3.5 blastocyst. Although it has been shown that FGF signaling is necessary and sufficient for PE specification in the ICM, it is unknown what mechanisms control the PE/EPI proportion in the embryo. Because modulation of FGF signaling alone is sufficient to convert all ICM cells to either PE or EPI, a model has been proposed in which the amount of FGF in the embryo controls the PE/EPI proportion. To test this model, we reduced the amount of FGF4, the major FGF in the preimplantation embryo, using various genotypes of Fgf4 mutants. We observed a maternal contribution of Fgf4 in PE specification, but it was dispensable for development. In addition, upon treatment of Fgf4 mutant embryos with exogenous FGF4, we observed a progressive increase of PE proportions in an FGF4 dose dependent manner, regardless of embryo genotype. We conclude that the amount of FGF4 is limited and regulates PE/EPI proportions in the mouse embryo.

Twist1 activity thresholds define multiple functions in limb development.

The basic helix-loop-helix transcription factor Twist1 is essential for normal limb development. Twist1(-/-) embryos die at midgestation. However, studies on early limb buds found that Twist1(-/-) mutant limb mesenchyme has an impaired response to FGF signaling from the apical ectodermal ridge, which disrupts the feedback loop between the mesenchyme and AER, and reduces and shifts anteriorly Shh expression in the zone of polarizing activity. We have combined Twist1 null, hypomorph and conditional alleles to generate a Twist1 allelic series that survives to birth. As Twist1 activity is reduced, limb skeletal defects progress from preaxial polydactyly to girdle reduction combined with hypoplasia, aplasia or mirror symmetry of all limb segments. With reduced Twist1 activity there is striking and progressive upregulation of ectopic Shh expression in the anterior of the limb, combined with an anterior shift in the posterior Shh domain, which is expressed at normal intensity, and loss of the posterior AER. Consequently limb outgrowth is initially impaired, before an ectopic anterior Shh domain expands the AER, promoting additional growth and repatterning. Reducing the dosage of FGF targets of the Etv gene family, which are known repressors of Shh expression in anterior limb mesenchyme, strongly enhances the anterior skeletal phenotype. Conversely this and other phenotypes are suppressed by reducing the dosage of the Twist1 antagonist Hand2. Our data support a model whereby multiple Twist1 activity thresholds contribute to early limb bud patterning, and suggest how particular combinations of skeletal defects result from differing amounts of Twist1 activity.

Specification of motor axon trajectory by ephrin-B:EphB signaling: symmetrical control of axonal patterning in the developing limb.

Studies of the innervation of limb muscles by spinal motor neurons have helped to define mechanisms by which axons establish trajectories to their targets. Related motor axons select dorsal or ventral pathways at the base of the limb, raising the question of how these alternate trajectories are specified. EphA signaling has been proposed to control the dorsal trajectory of motor axons in conjunction with other signaling systems, although the respective contributions of each system to motor axon guidance are unclear. We show that the expression of EphB receptors by motor axons, and ephrin-B ligands by limb mesenchymal cells, directs the ventral trajectory of motor axons. Our findings reveal symmetry in the molecular strategies that establish this aspect of nerve-muscle connectivity. The involvement of ephrin:Eph signaling in guiding both sets of motor axons raises the possibility that other signaling systems function primarily to refine or modulate a core Eph signaling program.

Identification of genes controlled by LMX1B in the developing mouse limb bud.

In the developing limb, dorsal-ventral patterning is controlled by the transcription factor LMX1B, expressed in the dorsal mesenchyme. Loss of Lmx1b function in mice or humans results in the loss of dorsal limb structures and Nail-Patella syndrome, but the effectors through which LMX1B controls limb patterning are virtually unknown. Using microarrays to analyze the differential expression of mRNAs in wild-type vs. Lmx1b(-/-) limb buds, we have identified hundreds of genes as putative LMX1B targets. Analysis of a subset of these candidates by in situ mRNA localization has identified eight genes previously unknown to require Lmx1b for their dorsal-ventral restriction of expression in the limb. Furthermore, our results suggest that LMX1B controls different targets along the proximal-distal axis of the limb, and suggest the existence of a dorsal proximal limb region that is rich in mRNAs requiring Lmx1b for their expression.

Altered Twist1 and Hand2 dimerization is associated with Saethre-Chotzen syndrome and limb abnormalities.

Autosomal dominant mutations in the gene encoding the basic helix-loop-helix transcription factor Twist1 are associated with limb and craniofacial defects in humans with Saethre-Chotzen syndrome. The molecular mechanism underlying these phenotypes is poorly understood. We show that ectopic expression of the related basic helix-loop-helix factor Hand2 phenocopies Twist1 loss of function in the limb and that the two factors have a gene dosage-dependent antagonistic interaction. Dimerization partner choice by Twist1 and Hand2 can be modulated by protein kinase A- and protein phosphatase 2A-regulated phosphorylation of conserved helix I residues. Notably, multiple Twist1 mutations associated with Saethre-Chotzen syndrome alter protein kinase A-mediated phosphorylation of Twist1, suggesting that misregulation of Twist1 dimerization through either stoichiometric or post-translational mechanisms underlies phenotypes of individuals with Saethre-Chotzen syndrome.