The pluripotency factor NANOG controls primitive hematopoiesis and directly regulates Tal1

Progenitors of the first hematopoietic cells in the mouse arise in the early embryo from Brachyury-positive multipotent cells in the posterior-proximal region of the epiblast, but the mechanisms that specify primitive blood cells are still largely unknown. Pluripotency factors maintain uncommitted cells of the blastocyst and embryonic stem cells in the pluripotent state. However, little is known about the role played by these factors during later development, despite being expressed in the postimplantation epiblast. Using a dual transgene system for controlled expression at postimplantation stages, we found that Nanog blocks primitive hematopoiesis in the gastrulating embryo, resulting in a loss of red blood cells and downregulation of erythropoietic genes. Accordingly, Nanog-deficient embryonic stem cells are prone to erythropoietic differentiation. Moreover, Nanog expression in adults prevents the maturation of erythroid cells. By analysis of previous data for NANOG binding during stem cell differentiation and CRISPR/Cas9 genome editing, we found that Tal1 is a direct NANOG target. Our results show that Nanog regulates primitive hematopoiesis by directly repressing critical erythroid lineage specifiers.This work was supported by the Spanish government (grant BFU2014-54608-P and BFU2017-84914-P to MM; grants RYC-2011-09209 and BFU-2012-35892 to JI). The Gottgens and Nichols laboratories are supported by core funding from the Wellcome Trust and MRC to the Wellcome and MRC Cambridge Stem Cell Institute. The CNIC is supported by the Spanish Ministry of Science, Innovation and Universities (MINECO) and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015-0505)S

Nanog regulates Pou3f1 expression at the exit from pluripotency during gastrulation.

Pluripotency is regulated by a network of transcription factors that maintain early embryonic cells in an undifferentiated state while allowing them to proliferate. NANOG is a critical factor for maintaining pluripotency and its role in primordial germ cell differentiation has been well described. However, Nanog is expressed during gastrulation across all the posterior epiblast, and only later in development is its expression restricted to primordial germ cells. In this work, we unveiled a previously unknown mechanism by which Nanog specifically represses genes involved in anterior epiblast lineage. Analysis of transcriptional data from both embryonic stem cells and gastrulating mouse embryos revealed Pou3f1 expression to be negatively correlated with that of Nanog during the early stages of differentiation. We have functionally demonstrated Pou3f1 to be a direct target of NANOG by using a dual transgene system for the controlled expression of Nanog Use of Nanog null ES cells further demonstrated a role for Nanog in repressing a subset of anterior neural genes. Deletion of a NANOG binding site (BS) located nine kilobases downstream of the transcription start site of Pou3f1 revealed this BS to have a specific role in the regionalization of the expression of this gene in the embryo. Our results indicate an active role of Nanog inhibiting neural regulatory networks by repressing Pou3f1 at the onset of gastrulation.This article has an associated First Person interview with the joint first authors of the paper.This work was funded by the Spanish government [grant BFU2017-84914-P to M.M.]. The Gottgens laboratory is supported by core funding from the Wellcome Trust and Medical Research Council to the Wellcome and Medical Research Council Cambridge Stem Cell Institute. The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia, Innovación y Universidades (MCNU) and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence [SEV-2015-0505]

Pluripotency factors regulate the onset of Hox cluster activation in the early embryo

Pluripotent cells are a transient population of the mammalian embryo dependent on transcription factors, such as OCT4 and NANOG, which maintain pluripotency while suppressing lineage specification. However, these factors are also expressed during early phases of differentiation, and their role in the transition from pluripotency to lineage specification is largely unknown. We found that pluripotency factors play a dual role in regulating key lineage specifiers, initially repressing their expression and later being required for their proper activation. We show that Oct4 is necessary for activation of HoxB genes during differentiation of embryonic stem cells and in the embryo. In addition, we show that the HoxB cluster is coordinately regulated by OCT4 binding sites located at the 3′ end of the cluster. Our results show that core pluripotency factors are not limited to maintaining the precommitted epiblast but are also necessary for the proper deployment of subsequent developmental programs

Basal oxidation of conserved cysteines modulates cardiac titin stiffness and dynamics

Titin, as the main protein responsible for the passive stiffness of the sarcomere, plays a key role in diastolic function and is a determinant factor in the etiology of heart disease. Titin stiffness depends on unfolding and folding transitions of immunoglobulin-like (Ig) domains of the I-band, and recent studies have shown that oxidative modifications of cryptic cysteines belonging to these Ig domains modulate their mechanical properties in vitro. However, the relevance of this mode of titin mechanical modulation in vivo remains largely unknown. Here, we describe the high evolutionary conservation of titin mechanical cysteines and show that they are remarkably oxidized in murine cardiac tissue. Mass spectrometry analyses indicate a similar landscape of basal oxidation in murine and human myocardium. Monte Carlo simulations illustrate how disulfides and S-thiolations on these cysteines increase the dynamics of the protein at physiological forces, while enabling load- and isoform-dependent regulation of titin stiffness. Our results demonstrate the role of conserved cysteines in the modulation of titin mechanical properties in vivo and point to potential redox-based pathomechanisms in heart disease.This work was supported by the Ministerio de Ciencia e Innovación grants BIO2014-54768-P, BIO2017-83640-P, RYC-2014-16604 to JAC and PGC2018-097019-B-I00 to JV, the Regional Government of Madrid grants S2018/NMT-4443 and PEJ16/MED/TL-1593 to JAC and the Instituto de Salud Carlos III (Fondo de Investigación Sanitaria grant PRB3 (PT17/0019/0003- ISCIII-SGEFI /ERDF, ProteoRed), and “la Caixa” Banking Foundation (project code HR17-00247) to JV. We acknowledge funding from the European Research Area Network on Cardiovascular Disease through grant MINOTAUR to SS (The Austrian Science Fund – FWF, I3301) and JAC (ISCIII-AC16/00045). The CNIC is supported by ISCIII, the Ministerio de Ciencia e Innovación and the Pro CNIC Foundation, and was a Severo Ochoa Center of Excellence (SEV-2015-0505). IMM was the recipient of a CNIC-ACCIONA Masters Fellowship and holds a fellowship from “La Caixa” Foundation (ID 100010434, fellowship code LCF/BQ/DR20/11790009). CSC is the recipient of an FPI-SO predoctoral fellowship BES-2016-076638. We thank Wolfgang A. Linke and Pablo García-Pavía for critical feedback. We are also thankful for the insights of three anonymous reviewers.S

<em>Bambi</em> and <em>Sp8</em> Expression Mark Digit Tips and Their Absence Shows That Chick Wing Digits 2 and 3 Are Truncated

<div><p>An often overlooked aspect of digit development is the special nature of the terminal phalanx, a specialized structure with characteristics distinct from other phalanges, for example the presence of ectodermal derivatives such as nails and claws. Here, we describe the unique ossification pattern of distal phalanges and characteristic gene expression in the digit tips of chick and duck embryos. Our results show that the distal phalanx of chick wing digit 1 is a genuine tip with a characteristic ossification pattern and expression of <em>Bambi</em> and <em>Sp8</em>; however, the terminal phalanx of digits 2* and 3 is not a genuine tip, and these are therefore truncated digits. <em>Bambi</em> and <em>Sp8</em> expression in the chick wing provides a direct molecular assessment of digit identity changes after experimental manipulations of digit primordia. In contrast, digits 1 and 2 of the duck wing both possess true tips. Although chick wing-tip development was not rescued by application of Fgf8, this treatment induced the development of extra phalanges. Grafting experiments show that competence for tip formation, including nails, is latent in the interdigital tissue. Our results deepen understanding of the mechanisms of digit tip formation, highlighting its developmental autonomy and modular nature, with implications for digit reduction or loss during evolution. * Numbering of wing digits is 1, 2, 3 from anterior to posterior.</p> </div

Fgf8 induces elongation and extra phalanges in digits 1 and 3, but not digit 2, of the chick wing.

<p>Beads soaked in Fgf8 (1 mg/ml) were applied to the first (B,C) or second (D,E) interdigital spaces of HH27 wings. Five days after the operation embryos were collected and stained with alcian green to reveal skeletal elements. A: Scheme showing the position of the beads at the time of operation. Text refers to the panels showing each experiment. B, C: Application to the first interdigital space induced elongation of digit 1 (B, arrow), sometimes with the production of a complete extra phalange (C, arrow) with a new joint (asterisk). In both cases, the tip is normal (arrowheads). D: Application to the second interdigital space did not induce elongation of digit 2, but caused widening (D, arrow). E: In contrast, this treatment induced elongation of digit 3 with the formation of an extra phalanx (arrow) with a new joint (asterisk). Operated wings are shown on the right in panels B,C and on the left in panels D,E (all dorsal views).</p

Expression of <i>Sp8</i> and <i>Bambi</i> in digit tips.

<p>A: <i>Sp8</i> in situ hybridization in mouse digits at the indicated developmental stages. (a-g) Whole-mount hind limbs. Note that <i>Sp8</i> is expressed in the AER up to E13 (a,b, arrows) and later reappears in the tip ectoderm (d-f). Expression is stronger dorsally (g) and examination of stained paraffin sections shows that it is restricted to the ectoderm (h). B: In situ hybridization for <i>Sp8</i> in chick digits at the indicated Hamburger and Hamilton stages. <i>Sp8</i> is expressed in the AER of all leg digits up to HH32 (a,b, arrows) and later reappears in the tips of digits (d-f). Expression is limited to the tip, showing a sharp boundary (g), and is restricted to the ectoderm (h, paraffin section). (i) In the wing, <i>Sp8</i> is expressed at the tip of digit 1 (arrow) but is absent from the ends of digits 2 and 3. C: <i>Bambi</i> in situ hybridisation in chick digits at the indicated stages (a-e, legs; f-j, wings). Note the strong initial expression in the interdigital space mesenchyme (a, f, arrows) that subsequently fades away, and the expression in the digit tips as the last phalanx forms. This terminal expression is observed in all toes (e) but is only present in the tip of digit 1 in the wing (j). Within digits, <i>Bambi</i> is sharply restricted to the tip (k) and is expressed only in the ectoderm (l, paraffin section). <i>Bambi</i> is also expressed (arrows) in leg scales (m) and feather buds (n,o).</p

Ossification pattern of the terminal phalanx in chick digits.

<p>A: Ossification in the tip of leg digit 3 on the indicated days of development, as detected by alizarin red staining. Note the ventral initiation of ossification (arrowhead) and the progression towards the distal end. A proximal phalanx (phalanx 1 of digit 3, P1D3, at E10), with an evident central ossification ring, is shown for comparison (arrows). The bottom right panel shows a stained longitudinal section in which alizarin red is evident at the distal extreme of the tip phalanx; other panels show whole-mount specimens. B: Ossification of the terminal phalanges of chick wing digits. Ossification in digit 1 is similar to that in leg tips, starting ventrally and progressing distally (arrowheads). In contrast, the ossification of digit 2 resembles a proximal/intermediate phalange, with the presence of a central ossification ring (arrows).</p

Interdigital tissue can form ectopic digits with tips and claws.

<p>Explants of distal interdigital space 2 (2ID: A,B) or distal digital ray 3 (D3: C,D) were taken from HH27 legs (distal 150 µm plus AER) and grafted to the somites of HH20 hosts. 2ID and D3 explants both formed ectopic digits with phalanges (alcian blue staining, A,C) and claws (white keratinized area in fresh specimens, B,D), detected 8–10 days after transplantation.</p

<i>Bambi</i> tip expression marks changes of digit identity triggered by surgical manipulations.

<p>Two surgical manipulations were performed in HH27 wings to transform the identity of digit 2 towards digit 1. Seven days after the operation, <i>Bambi</i> expression was detected by in situ hybridisation. A: In type I experiments, the posterior part of the digit 2 primordium and the posterior interdigital space 2 were removed (see scheme in the inset in a). (a) The remnant digit 2 primordium has developed into a shorter digit (transformed digit 2*) with a tip positive for <i>Bambi</i> expression (arrow). (b) Magnified view of <i>Bambi</i> expression in the tip of digit 1 in the operated limb. (c) <i>Bambi</i>-negative tip of digit 2 in the control (non-operated) wing. (d) <i>Bambi</i> expression in the tip of the operated and transformed digit 2*. B: In type II experiments the digit 2 primordium was bisected (see scheme in the inset in a). (a) The extra digit formed from the anterior half of the digit 2 primordium (transformed digit 2*) shows expression of <i>Bambi</i> in the tip (arrow). (b) Magnified view of <i>Bambi</i> expression in the tip of digit 1 in the operated limb. (c) <i>Bambi</i>-negative tip of digit 2 in the control wing. (d) <i>Bambi</i> expression in the tip of the operated and transformed digit 2*. C: Digits from the specimen in A (and additionally the digit 1 from the control wing) were sectioned after the <i>Bambi</i> in situ hybridisation and stained with haematoxylin & eosin to detect transformation of cartilage elements. The length of phalanx 2 (P2) relative to phalanx 1 (P1) in transformed digit 2* is reduced, resembling the proportions in digit 1. (e) Magnified view of the boxed area in d (rotated 90°). Note the expression of <i>Bambi</i> in the tip ectoderm of the transformed digit 2* (arrows).</p