Denaturing cross-linking immunoprecipitation to identify footprints for RNA-binding proteins.

The isolation of protein-RNA complexes in the "denaturing cross-linked RNA immunoprecipitation" (dCLIP) protocol is based on biotin-tagging proteins of interest, UV cross-linking RNA to protein in vivo, RNase protection assay, and isolating RNA-protein complexes under denaturing conditions over a streptavidin column. Insofar as conventional antibody-based CLIP assays have been challenging to apply to Polycomb complexes, dCLIP has been applied successfully and yields small RNA footprints from which de novo motif analysis can be performed to identify RNA binding motifs. For complete details on the use and execution of this protocol, please refer to Rosenberg et al. (2017).

Distinct stem cell populations regenerate the follicle and interfollicular epidermis.

The regeneration of the skin and its appendages is thought to occur by the regulated activation of a dedicated stem cell population. A population of cells in the bulge region of the hair follicle has been identified as the putative stem cell of both the follicle and the interfollicular epidermis. While this assertion is supported by a variety of surrogate assays, there has been no direct confirmation of the normal contribution of these cells to the regeneration of structures other than the cycling portion of the hair follicle. Here, we report lineage analysis revealing that the follicular epithelium is derived from cells in the epidermal placode that express Sonic hedgehog. This analysis also demonstrates that the stem cells resident in the follicular bulge that regenerate the follicle are neither the stem cells of the epidermis nor the source of the stem cells of the epidermis in the absence of trauma.

Epidermal stem cells arise from the hair follicle after wounding.

During normal development, the epidermis and hair follicle are distinct lineage compartments maintained by independent stem cell populations. Both epidermal and follicular keratinocytes are recruited to participate in epidermal repair in response to injury. However, it is generally thought that follicular cells contribute to the wound epidermis only transiently and are ultimately replaced by the progeny of stem cells derived from the original epidermal compartment prior to wounding. Here we use inducible and constitutive cre recombinase expressed from the Sonic hedgehog locus (Shh) for in vivo lineage tracing. This analysis confirms that follicular cells participate in the initial resurfacing of the wound but also reveals that their progeny persist in wound epidermis for months after the wound is healed. It further demonstrates that Shh is not induced in keratinocytes during the wound healing process. We conclude that follicular cells can undergo reprogramming to become long-term repopulating epidermal progenitors following wounding.

Temporal analysis of the early BMP functions identifies distinct anti-organizer and mesoderm patterning phases.

BMP signaling performs multiple important roles during early embryogenesis. Signaling through the BMP pathway is mediated by different BMP ligands expressed in partially overlapping temporal and spatial patterns. Assignment of different BMP-dependent activities to the individual ligands has relied on the patterns of expression of the various BMP genes. Temporal analysis of BMP signaling prior to and during gastrulation was performed using glucocorticoid-controlled Smad proteins. Overexpression of the BMP-specific Smad1 and Smad5 revealed that suppression of Spemann's organizer formation in Xenopus embryos can only take place by activating the BMP pathway prior to the onset of gastrulation. Blocking BMP signaling with the inhibitory Smad, Smad6, results in dorsalized embryos or secondary axis induction, only when activated up to early gastrula stages. BMP2 efficiently represses organizer-specific transcription from the midblastula transition onwards while BMP4 is unable to prevent the early activation of organizer-specific genes. Manipulation of the BMP pathway during mid/late gastrula affects mesodermal patterning with no external phenotypic effects. These observations suggest that the malformations resulting from inhibition or promotion of organizer formation, ventralized or dorsalized, respectively, are the result of a very early BMP function, through its antagonism of organizer formation. This function is apparently fulfilled by BMP2 and only at its latest phase by BMP4. Subsequently, BMP functions in the patterning of the mesoderm with no apparent phenotypic effects.

The competence of marginal zone cells to become Spemann's organizer is controlled by Xcad2.

The organizer in vertebrate embryos is responsible for the formation of the primary body axis. In amphibian embryos, the organizer forms in the dorsal marginal zone (prospective dorsal mesoderm) at a location determined by the point of sperm entry. Using inducible versions of axis-inducing proteins, it has been shown that, irrespective of the mode of secondary axis induction, organizer formation in the ventral marginal zone is temporally restricted from the midblastula transition to the onset of gastrulation. Here, we show that the competence of marginal zone cells to respond to organizer-inducing signals is under temporal control, one of the regulators being the homeobox transcription factor Xcad2. Overexpression of Xcad2 restricts the temporal competence for axis induction, whereas partial loss of function expands this competence, supporting our suggestion. We propose that Xcad2 competes with putative axis-inducing signals within the marginal zone to prevent expression of organizer-specific genes. Elimination of endogenous Xcad2 allows for the activation of organizer genes beyond the normal competence window during early/mid-gastrulation. We conclude that Xcad2, through its early expression in the ventrolateral marginal zone, terminates the competence of this embryonic region to respond to organizer-inducing signals by preventing the activation of organizer-specific genes.

Prenatal diagnosis of Down syndrome: ten year experience in the Israeli population.

Second trimester maternal serum biochemical markers, introduced between 1990 and 1995, were supplemented with new ultrasound methods at 14-16 weeks and first trimester biochemical markers between 1995 and 2000. This study evaluated the effectiveness of a Down syndrome (DS) prevention program among the Israeli Jewish population between 1990 and 2000. We collected data on the total number of prenatal tests performed on Israeli Jewish women, DS cases detected prenatally and DS livebirths in Israel during these years. We also studied the use of the newer screening tests in 1990, 1992, and 2000. Between 1990 and 1995, use of chromosomal studies for DS in this population increased from 11.3% to 21.6% and the percentage of cases detected prenatally from 53% to 70%. However, between 1996 and 2000, even with the new screening methods, the utilization rate remained similar (20.7% and 19.8%, respectively) and the percentage detected prenatally decreased to 61% in 2000. The total cost per case detected increased from $47,971 US dollars in 1990 to $75,229 US dollars in 1992, and to $190,171 US dollars in 2000. Between 1990 and 1995, improvement in the percentage of cases detected prenatally was associated with a significant increase in the amniocentesis rate-both are attributed to the introduction of second trimester maternal serum biochemical marker tests. Unexpectedly, the introduction between 1995 and 2000 of new genetic methods to assess the DS risk did not improve the percentage detected or reduce the amniocentesis rate, and was accompanied by an increased cost per case detected.