Ophir D. Klein

A reserve stem cell population in small intestine renders Lgr5 -positive cells dispensable

The small intestine epithelium renews every 2 to 5 days, making it one of the most regenerative mammalian tissues. Genetic inducible fate mapping studies have identified two principal epithelial stem cell pools in this tissue. One pool consists of columnar Lgr5-expressing cells that cycle rapidly and are present predominantly at the crypt base. The other pool consists of Bmi1-expressing cells that largely reside above the crypt base. However, the relative functions of these two pools and their interrelationship are not understood. Here we specifically ablated Lgr5-expressing cells in mice using a human diphtheria toxin receptor (DTR) gene knocked into the Lgr5 locus. We found that complete loss of the Lgr5-expressing cells did not perturb homeostasis of the epithelium, indicating that other cell types can compensate for the elimination of this population. After ablation of Lgr5-expressing cells, progeny production by Bmi1-expressing cells increased, indicating that Bmi1-expressing stem cells compensate for the loss of Lgr5-expressing cells. Indeed, lineage tracing showed that Bmi1-expressing cells gave rise to Lgr5-expressing cells, pointing to a hierarchy of stem cells in the intestinal epithelium. Our results demonstrate that Lgr5-expressing cells are dispensable for normal intestinal homeostasis, and that in the absence of these cells, Bmi1-expressing cells can serve as an alternative stem cell pool. These data provide the first experimental evidence for the interrelationship between these populations. The Bmi1-expressing stem cells may represent both a reserve stem cell pool in case of injury to the small intestine epithelium and a source for replenishment of the Lgr5-expressing cells under non-pathological conditions.

The branching programme of mouse lung development

Mammalian lungs are branched networks containing thousands to millions of airways arrayed in intricate patterns that are crucial for respiration. How such trees are generated during development, and how the developmental patterning information is encoded, have long fascinated biologists and mathematicians. However, models have been limited by a lack of information on the normal sequence and pattern of branching events. Here we present the complete three-dimensional branching pattern and lineage of the mouse bronchial tree, reconstructed from an analysis of hundreds of developmental intermediates. The branching process is remarkably stereotyped and elegant: the tree is generated by three geometrically simple local modes of branching used in three different orders throughout the lung. We propose that each mode of branching is controlled by a genetically encoded subroutine, a series of local patterning and morphogenesis operations, which are themselves controlled by a more global master routine. We show that this hierarchical and modular programme is genetically tractable, and it is ideally suited to encoding and evolving the complex networks of the lung and other branched organs.

In vitro generation of human pluripotent stem cell derived lung organoids

Recent breakthroughs in 3-dimensional (3D) organoid cultures for many organ systems have led to new physiologically complex in vitro models to study human development and disease. Here, we report the step-wise differentiation of human pluripotent stem cells (hPSCs) (embryonic and induced) into lung organoids. By manipulating developmental signaling pathways hPSCs generate ventral-anterior foregut spheroids, which are then expanded into human lung organoids (HLOs). HLOs consist of epithelial and mesenchymal compartments of the lung, organized with structural features similar to the native lung. HLOs possess upper airway-like epithelium with basal cells and immature ciliated cells surrounded by smooth muscle and myofibroblasts as well as an alveolar-like domain with appropriate cell types. Using RNA-sequencing, we show that HLOs are remarkably similar to human fetal lung based on global transcriptional profiles, suggesting that HLOs are an excellent model to study human lung development, maturation and disease. DOI: http://dx.doi.org/10.7554/eLife.05098.001

Secretion of Shh by a Neurovascular Bundle Niche Supports Mesenchymal Stem Cell Homeostasis in the Adult Mouse Incisor

Mesenchymal stem cells (MSCs) are typically defined by their in vitro characteristics, and as a consequence the in vivo identity of MSCs and their niches are poorly understood. To address this issue, we used lineage tracing in a mouse incisor model and identified the neurovascular bundle (NVB) as an MSC niche. We found that NVB sensory nerves secrete Shh protein, which activates Gli1 expression in periarterial cells that contribute to all mesenchymal derivatives. These periarterial cells do not express classical MSC markers used to define MSCs in vitro. In contrast, NG2(+) pericytes represent an MSC subpopulation derived from Gli1+ cells; they express classical MSC markers and contribute little to homeostasis but are actively involved in injury repair. Likewise, incisor Gli1(+) cells, but not NG2(+) cells, exhibit typical MSC characteristics in vitro. Collectively, we demonstrate that MSCs originate from periarterial cells and are regulated by Shh secretion from an NVB.

An FGF signaling loop sustains the generation of differentiated progeny from stem cells in mouse incisors.

Rodent incisors grow throughout adult life, but are prevented from becoming excessively long by constant abrasion, which is facilitated by the absence of enamel on one side of the incisor. Here we report that loss-of-function of sprouty genes, which encode antagonists of receptor tyrosine kinase signaling, leads to bilateral enamel deposition, thus impeding incisor abrasion and resulting in unchecked tooth elongation. We demonstrate that sprouty genes function to ensure that enamel-producing ameloblasts are generated on only one side of the tooth by inhibiting the formation of ectopic ameloblasts from self-renewing stem cells, and that they do so by preventing the establishment of an epithelial-mesenchymal FGF signaling loop. Interestingly, although inactivation of Spry4 alone initiates ectopic ameloblast formation in the embryo, the dosage of another sprouty gene must also be reduced to sustain it after birth. These data reveal that the generation of differentiated progeny from a particular stem cell population can be differently regulated in the embryo and adult.

Inhibition of Wnt signaling by Wise (Sostdc1) and negative feedback from Shh controls tooth number and patterning

Mice carrying mutations in Wise (Sostdc1) display defects in many aspects of tooth development, including tooth number, size and cusp pattern. To understand the basis of these defects, we have investigated the pathways modulated by Wise in tooth development. We present evidence that, in tooth development, Wise suppresses survival of the diastema or incisor vestigial buds by serving as an inhibitor of Lrp5- and Lrp6-dependent Wnt signaling. Reducing the dosage of the Wnt co-receptor genes Lrp5 and Lrp6 rescues the Wise-null tooth phenotypes. Inactivation of Wise leads to elevated Wnt signaling and, as a consequence, vestigial tooth buds in the normally toothless diastema region display increased proliferation and continuous development to form supernumerary teeth. Conversely, gain-of-function studies show that ectopic Wise reduces Wnt signaling and tooth number. Our analyses demonstrate that the Fgf and Shh pathways are major downstream targets of Wise-regulated Wnt signaling. Furthermore, our experiments revealed that Shh acts as a negative-feedback regulator of Wnt signaling and thus determines the fate of the vestigial buds and later tooth patterning. These data provide insight into the mechanisms that control Wnt signaling in tooth development and into how crosstalk among signaling pathways controls tooth number and morphogenesis.

Lgr5-Expressing Cells Are Sufficient and Necessary for Postnatal Mammary Gland Organogenesis

Mammary epithelial stem cells are vital to tissue expansion and remodeling during various phases of postnatal mammary development. Basal mammary epithelial cells are enriched in Wnt-responsive cells and can reconstitute cleared mammary fat pads upon transplantation into mice. Lgr5 is a Wnt-regulated target gene and was identified as a major stem cell marker in the small intestine, colon, stomach, and hair follicle, as well as in kidney nephrons. Here, we demonstrate the outstanding regenerative potential of a rare population of Lgr5-expressing (Lgr5(+)) mammary epithelial cells (MECs). We found that Lgr5(+) cells reside within the basal population, are superior to other basal cells in regenerating functional mammary glands (MGs), are exceptionally efficient in reconstituting MGs from single cells, and exhibit regenerative capacity in serial transplantations. Loss-of-function and depletion experiments of Lgr5(+) cells from transplanted MECs or from pubertal MGs revealed that these cells are not only sufficient but also necessary for postnatal mammary organogenesis.

Hedgehog signaling regulates the generation of ameloblast progenitors in the continuously growing mouse incisor

In many organ systems such as the skin, gastrointestinal tract and hematopoietic system, homeostasis is dependent on the continuous generation of differentiated progeny from stem cells. The rodent incisor, unlike human teeth, grows throughout the life of the animal and provides a prime example of an organ that rapidly deteriorates if newly differentiated cells cease to form from adult stem cells. Hedgehog (Hh) signaling has been proposed to regulate self-renewal, survival, proliferation and/or differentiation of stem cells in several systems, but to date there is little evidence supporting a role for Hh signaling in adult stem cells. We used in vivo genetic lineage tracing to identify Hh-responsive stem cells in the mouse incisor and we show that sonic hedgehog (SHH), which is produced by the differentiating progeny of the stem cells, signals to several regions of the incisor. Using a hedgehog pathway inhibitor (HPI), we demonstrate that Hh signaling is not required for stem cell survival but is essential for the generation of ameloblasts, one of the major differentiated cell types in the tooth, from the stem cells. These results therefore reveal the existence of a positive-feedback loop in which differentiating progeny produce the signal that in turn allows them to be generated from stem cells.

Sox2 marks epithelial competence to generate teeth in mammals and reptiles

Tooth renewal is initiated from epithelium associated with existing teeth. The development of new teeth requires dental epithelial cells that have competence for tooth formation, but specific marker genes for these cells have not been identified. Here, we analyzed expression patterns of the transcription factor Sox2 in two different modes of successional tooth formation: tooth replacement and serial addition of primary teeth. We observed specific Sox2 expression in the dental lamina that gives rise to successional teeth in mammals with one round of tooth replacement as well as in reptiles with continuous tooth replacement. Sox2 was also expressed in the dental lamina during serial addition of mammalian molars, and genetic lineage tracing indicated that Sox2+ cells of the first molar give rise to the epithelial cell lineages of the second and third molars. Moreover, conditional deletion of Sox2 resulted in hyperplastic epithelium in the forming posterior molars. Our results indicate that the Sox2+ dental epithelium has competence for successional tooth formation and that Sox2 regulates the progenitor state of dental epithelial cells. The findings imply that the function of Sox2 has been conserved during evolution and that tooth replacement and serial addition of primary teeth represent variations of the same developmental process. The expression patterns of Sox2 support the hypothesis that dormant capacity for continuous tooth renewal exists in mammals.

A genome-wide association study identifies susceptibility loci for nonsyndromic sagittal craniosynostosis near BMP2 and within BBS9

Sagittal craniosynostosis is the most common form of craniosynostosis, affecting approximately one in 5,000 newborns. We conducted, to our knowledge, the first genome-wide association study for nonsyndromic sagittal craniosynostosis (sNSC) using 130 non-Hispanic case-parent trios of European ancestry (NHW). We found robust associations in a 120-kb region downstream of BMP2 flanked by rs1884302 (P = 1.13 × 10−14, odds ratio (OR) = 4.58) and rs6140226 (P = 3.40 × 10−11, OR = 0.24) and within a 167-kb region of BBS9 between rs10262453 (P = 1.61 × 10−10, OR = 0.19) and rs17724206 (P = 1.50 × 10−8, OR = 0.22). We replicated the associations to both loci (rs1884302, P = 4.39 × 10−31 and rs10262453, P = 3.50 × 10−14) in an independent NHW population of 172 unrelated probands with sNSC and 548 controls. Both BMP2 and BBS9 are genes with roles in skeletal development that warrant functional studies to further understand the etiology of sNSC.