Jacqueline M. Veltmaat

Fgf10 expression identifies parabronchial smooth muscle cell progenitors and is required for their entry into the smooth muscle cell lineage.

Lineage formation in the lung mesenchyme is poorly understood. Using a transgenic mouse line expressing LacZ under the control of Fgf10 regulatory sequences, we show that the pool of Fgf10-positive cells in the distal lung mesenchyme contains progenitors of the parabronchial smooth muscle cells. Fgf10 gene expression is slightly repressed in this transgenic line. This allowed us to create a hypomorphic Fgf10 phenotype by expressing the LacZ transgene in a heterozygous Fgf10 background. Hypomorphic Fgf10 mutant lungs display a decrease inβ -galactosidase-positive cells around the bronchial epithelium associated with an accumulation of β-galactosidase-expressing cells in the distal mesenchyme. This correlates with a marked reduction of α smooth muscle actin expression, thereby demonstrating that FGF10 is mostly required for the entry of mesenchymal cells into the parabronchial smooth muscle cell lineage. The failure of exogenous FGF10 to phosphorylate its known downstream targets ERK and AKT in lung mesenchymal cultures strongly suggests that FGF10 acts indirectly on the progenitor population via an epithelial intermediate. We provide support for a role of epithelial BMP4 in mediating the formation of parabronchial smooth muscle cells.

Identification of the mammary line in mouse by Wnt10b expression

Mammogenesis in rabbit, rat, and human begins with the formation of an elevated ectodermal ridge in the embryo. Attempts to demonstrate a morphologically or histologically equivalent mammary line in the mouse have yielded controversial results. We show here that a mammary line exists in the mouse embryo at embryonic day (E) 11.25 as a concise line of Wnt10b expression and a broader band of Wnt6 expression in the surface ectoderm, between the subaxillary and suprainguinal region of each flank. Additional streaks of Wnt10b expression in the axillary and inguinal region join the mammary line on the flank slightly later. Expression of Wnt10b and Wnt6 becomes restricted to the placodes within 1.5 days. The ectoderm of the mammary line is organized as a pseudostratified epithelium connecting the developing mammary placodes at around E11.5, whereas all other surface ectoderm is single‐layered. Thus, the mammary line expressing Wnt10b defines a distinct ectodermal region that we consider the equivalent of the ectodermal ridge in, for example, rabbit. To date, the formation of the mammary line expressing Wnt10b is the earliest discernible ectodermal event in murine embryonic mammary gland development. Developmental Dynamics 229:349–356, 2004.

Pygo2 expands mammary progenitor cells by facilitating histone H3 K4 methylation

Recent studies have unequivocally identified multipotent stem/progenitor cells in mammary glands, offering a tractable model system to unravel genetic and epigenetic regulation of epithelial stem/progenitor cell development and homeostasis. In this study, we show that Pygo2, a member of an evolutionarily conserved family of plant homeo domain–containing proteins, is expressed in embryonic and postnatal mammary progenitor cells. Pygo2 deficiency, which is achieved by complete or epithelia-specific gene ablation in mice, results in defective mammary morphogenesis and regeneration accompanied by severely compromised expansive self-renewal of epithelial progenitor cells. Pygo2 converges with Wnt/β-catenin signaling on progenitor cell regulation and cell cycle gene expression, and loss of epithelial Pygo2 completely rescues β-catenin–induced mammary outgrowth. We further describe a novel molecular function of Pygo2 that is required for mammary progenitor cell expansion, which is to facilitate K4 trimethylation of histone H3, both globally and at Wnt/β-catenin target loci, via direct binding to K4-methyl histone H3 and recruiting histone H3 K4 methyltransferase complexes.

Gli3-mediated somitic Fgf10 expression gradients are required for the induction and patterning of mammary epithelium along the embryonic axes.

Little is known about the regulation of cell fate decisions that lead to the formation of five pairs of mammary placodes in the surface ectoderm of the mouse embryo. We have previously shown that fibroblast growth factor 10 (FGF10) is required for the formation of mammary placodes 1, 2, 3 and 5. Here, we have found that Fgf10 is expressed only in the somites underlying placodes 2 and 3, in gradients across and within these somites. To test whether somitic FGF10 is required for the formation of these two placodes, we analyzed a number of mutants with different perturbations of somitic Fgf10 gradients for the presence of WNT signals and ectodermal multilayering, markers for mammary line and placode formation. The mammary line is displaced dorsally, and formation of placode 3 is impaired in Pax3ILZ/ILZ mutants, which do not form ventral somitic buds. Mammary line formation is impaired and placode 3 is absent in Gli3Xt-J/Xt-J and hypomorphic Fgf10 mutants, in which the somitic Fgf10 gradient is shortened dorsally and less overall Fgf10 is expressed, respectively. Recombinant FGF10 rescued mammogenesis in Fgf10-/- and Gli3Xt-J/Xt-J flanks. We correlate increasing levels of somitic FGF10 with progressive maturation of the surface ectoderm, and show that full expression of somitic Fgf10, co-regulated by GLI3, is required for the anteroposterior pattern in which the flank ectoderm acquires a mammary epithelial identity. We propose that the intra-somitic Fgf10 gradient, together with ventral elongation of the somites, determines the correct dorsoventral position of mammary epithelium along the flank.

Fibroblast growth factor 10 is critical for liver growth during embryogenesis and controls hepatoblast survival via β‐catenin activation

Fibroblast growth factor (FGF) signaling and β‐catenin activation have been shown to be crucial for early embryonic liver development. This study determined the significance of FGF10‐mediated signaling in a murine embryonic liver progenitor cell population as well as its relation to β‐catenin activation. We observed that Fgf10−/− and Fgfr2b−/− mouse embryonic livers are smaller than wild‐type livers; Fgf10−/− livers exhibit diminished proliferation of hepatoblasts. A comparison of β‐galactosidase activity as a readout of Fgf10 expression in Fgf10+/LacZ mice and of β‐catenin activation in TOPGAL mice, demonstrated peak Fgf10 expression from E9 to E13.5 coinciding with peak β‐catenin activation. Flow cytometric isolation and marker gene expression analysis of LacZ+ cells from E13.5 Fgf10+/LacZ and TOPGAL livers, respectively, revealed that Fgf10 expression and β‐catenin signaling occur distinctly in stellate/myofibroblastic cells and hepatoblasts, respectively. Moreover, hepatoblasts express Fgfr2b, which strongly suggests they can respond to recombinant FGF10 produced by stellate cells. Fgfr2b−/−/TOPGAL+/+ embryonic livers displayed less β‐galactosidase activity than livers of Fgfr2b+/+/TOPGAL+/+ littermates. In addition, cultures of whole liver explants in Matrigel or cell in suspension from E12.5 TOPGAL+/+mice displayed a marked increase in β‐galactosidase activity and cell survival upon treatment with recombinant FGF10, indicating that FGFR (most likely FGFR2B) activation is upstream of β‐catenin signaling and promote hepatoblast survival. Conclusion: Embryonic stellate/myofibroblastic cells promote β‐catenin activation in and survival of hepatoblasts via FGF10‐mediated signaling. We suggest a role for stellate/myofibroblastic FGF10 within the liver stem cell niche in supporting the proliferating hepatoblast. (HEPATOLOGY 2007.)

Next Stop, the Twilight Zone: Hedgehog Network Regulation of Mammary Gland Development

The hedgehog signal transduction network is a critical mediator of cell-cell communication during embryonic development. Evidence also suggests that properly regulated hedgehog network function is required in some adult organs for stem cell maintenance or renewal. Mutation, or misexpression, of network genes is implicated in the development of several different types of cancer, particularly that of skin, brain, lung, and pancreas. Recent studies in the mouse mammary gland have demonstrated roles for hedgehog network genes at virtually every phase of mammary gland development where it regulates such diverse processes as embryonic mammary gland induction, establishment of ductal histoarchitecture, and functional differentiation in lactation. Further, studies suggest a role for misregulated network function in the progression of breast cancer.

FGF10 controls the patterning of the tracheal cartilage rings via Shh

During embryonic development, appropriate dorsoventral patterning of the trachea leads to the formation of periodic cartilage rings from the ventral mesenchyme and continuous smooth muscle from the dorsal mesenchyme. In this work, we have investigated the role of two crucial morphogens, fibroblast growth factor 10 and sonic hedgehog, in the formation of periodically alternating cartilaginous and non-cartilaginous domains in the ventral mesenchyme. Using a combination of gain- and loss-of-function approaches for FGF10 and SHH, we demonstrate that precise spatio-temporal patterns and appropriate levels of expression of these two signaling molecules in the ventral area are crucial between embryonic day 11.5 and 13.5 for the proper patterning of the cartilage rings. We conclude that the expression level of FGF10 in the mesenchyme has to be within a critical range to allow for periodic expression of Shh in the ventral epithelium, and consequently for the correct patterning of the cartilage rings. We propose that disturbed balances of Fgf10 and Shh may explain a subset of human tracheomalacia without tracheo-esophageal fistula or tracheal atresia.

Gli3Xt−J/Xt−J mice exhibit lambdoid suture craniosynostosis which results from altered osteoprogenitor proliferation and differentiation

Gli3 is a zinc-finger transcription factor whose activity is dependent on the level of hedgehog (Hh) ligand. Hh signaling has key roles during endochondral ossification; however, its role in intramembranous ossification is still unclear. In this study, we show that Gli3 performs a dual role in regulating both osteoprogenitor proliferation and osteoblast differentiation during intramembranous ossification. We discovered that Gli3Xt-J/Xt-J mice, which represent a Gli3-null allele, exhibit craniosynostosis of the lambdoid sutures and that this is accompanied by increased osteoprogenitor proliferation and differentiation. These cellular changes are preceded by ectopic expression of the Hh receptor Patched1 and reduced expression of the transcription factor Twist1 in the sutural mesenchyme. Twist1 is known to delay osteogenesis by binding to and inhibiting the transcription factor Runx2. We found that Runx2 expression in the lambdoid suture was altered in a pattern complimentary to that of Twist1. We therefore propose that loss of Gli3 results in a Twist1-, Runx2-dependent expansion of the sutural osteoprogenitor population as well as enhanced osteoblastic differentiation which results in a bony bridge forming between the parietal and interparietal bones. We show that FGF2 will induce Twist1, normalize osteoprogenitor proliferation and differentiation and rescue the lambdoid suture synostosis in Gli3Xt-J/Xt-J mice. Taken together, we define a novel role for Gli3 in osteoblast development; we describe the first mouse model of lambdoid suture craniosynostosis and show how craniosynostosis can be rescued in this model.

Prenatal Morphogenesis of Mammary Glands in Mouse and Rabbit

Our understanding of prenatal morphogenesis of mammary glands has recently greatly advanced. This review focuses on morphogenesis proper, as well as cellular processes and tissue interactions involved in the progression of the embryonic mammary gland through sequential morphogenic stages in both the mouse and rabbit embryo. We provide a synthesis of both historical and more recent studies of embryonic mammary gland development, as well as arguments to revise old concepts about mechanisms of mammary line and rudiment formation. Finally, we highlight outstanding issues that remain to be addressed.

High-resolution association mapping of number of teats in pigs reveals regions controlling vertebral development

BackgroundSelection pressure on the number of teats has been applied to be able to provide enough teats for the increase in litter size in pigs. Although many QTL were reported, they cover large chromosomal regions and the functional mutations and their underlying biological mechanisms have not yet been identified. To gain a better insight in the genetic architecture of the trait number of teats, we performed a genome-wide association study by genotyping 936 Large White pigs using the Illumina PorcineSNP60 Beadchip. The analysis is based on deregressed breeding values to account for the dense family structure and a Bayesian approach for estimation of the SNP effects.ResultsThe genome-wide association study resulted in 212 significant SNPs. In total, 39 QTL regions were defined including 170 SNPs on 13 Sus scrofa chromosomes (SSC) of which 5 regions on SSC7, 9, 10, 12 and 14 were highly significant. All significantly associated regions together explain 9.5% of the genetic variance where a QTL on SSC7 explains the most genetic variance (2.5%). For the five highly significant QTL regions, a search for candidate genes was performed. The most convincing candidate genes were VRTN and Prox2 on SSC7, MPP7, ARMC4, and MKX on SSC10, and vertebrae δ-EF1 on SSC12. All three QTL contain candidate genes which are known to be associated with vertebral development. In the new QTL regions on SSC9 and SSC14, no obvious candidate genes were identified.ConclusionsFive major QTL were found at high resolution on SSC7, 9, 10, 12, and 14 of which the QTL on SSC9 and SSC14 are the first ones to be reported on these chromosomes. The significant SNPs found in this study could be used in selection to increase number of teats in pigs, so that the increasing number of live-born piglets can be nursed by the sow. This study points to common genetic mechanisms regulating number of vertebrae and number of teats.