A spatio-temporally constrained gene regulatory network directed by PBX1/2 acquires limb patterning specificity via HAND2.

A lingering question in developmental biology has centered on how transcription factors with widespread distribution in vertebrate embryos can perform tissue-specific functions. Here, using the murine hindlimb as a model, we investigate the elusive mechanisms whereby PBX TALE homeoproteins, viewed primarily as HOX cofactors, attain context-specific developmental roles despite ubiquitous presence in the embryo. We first demonstrate that mesenchymal-specific loss of PBX1/2 or the transcriptional regulator HAND2 generates similar limb phenotypes. By combining tissue-specific and temporally controlled mutagenesis with multi-omics approaches, we reconstruct a gene regulatory network (GRN) at organismal-level resolution that is collaboratively directed by PBX1/2 and HAND2 interactions in subsets of posterior hindlimb mesenchymal cells. Genome-wide profiling of PBX1 binding across multiple embryonic tissues further reveals that HAND2 interacts with subsets of PBX-bound regions to regulate limb-specific GRNs. Our research elucidates fundamental principles by which promiscuous transcription factors cooperate with cofactors that display domain-restricted localization to instruct tissue-specific developmental programs

Abnormal Placental Development and Early Embryonic Lethality in EpCAM-Null Mice

BACKGROUND: EpCAM (CD326) is encoded by the tacstd1 gene and expressed by a variety of normal and malignant epithelial cells and some leukocytes. Results of previous in vitro experiments suggested that EpCAM is an intercellular adhesion molecule. EpCAM has been extensively studied as a potential tumor marker and immunotherapy target, and more recent studies suggest that EpCAM expression may be characteristic of cancer stem cells. METHODOLOGY/PRINCIPAL FINDINGS: To gain insights into EpCAM function in vivo, we generated EpCAM -/- mice utilizing an embryonic stem cell line with a tacstd1 allele that had been disrupted. Gene trapping resulted in a protein comprised of the N-terminus of EpCAM encoded by 2 exons of the tacstd1 gene fused in frame to betageo. EpCAM +/- mice were viable and fertile and exhibited no obvious abnormalities. Examination of EpCAM +/- embryos revealed that betageo was expressed in several epithelial structures including developing ears (otocysts), eyes, branchial arches, gut, apical ectodermal ridges, lungs, pancreas, hair follicles and others. All EpCAM -/- mice died in utero by E12.5, and were small, developmentally delayed, and displayed prominent placental abnormalities. In developing placentas, EpCAM was expressed throughout the labyrinthine layer and by spongiotrophoblasts as well. Placentas of EpCAM -/- embryos were compact, with thin labyrinthine layers lacking prominent vascularity. Parietal trophoblast giant cells were also dramatically reduced in EpCAM -/- placentas. CONCLUSION: EpCAM was required for differentiation or survival of parietal trophoblast giant cells, normal development of the placental labyrinth and establishment of a competent maternal-fetal circulation. The findings in EpCAM-reporter mice suggest involvement of this molecule in development of vital organs including the gut, kidneys, pancreas, lungs, eyes, and limbs

Limb Development: The Rise and Fall of Retinoic Acid

SummaryRetinoic acid was thought to play a key instructive role during limb bud initiation and subsequent patterning. New results argue instead that its role is permissive: retinoic acid is essential only to antagonize early axial Fgf signals that otherwise inhibit the limb field

The Organizer-Associated Chick Homeobox Gene, Gnot1, Is Expressed before Gastrulation and Regulated Synergistically by Activin and Retinoic Acid

AbstractGnot1, a Not (for notochord) family homeobox gene, is expressed in the chick pregastrulation blastoderm. Gnot1 expression in the epiblast is upregulated as the posteriorly derived hypoblast moves forward anteriorly to form a layer beneath it, which is of particular interest considering the known inductive role of the hypoblast in axis formation in the chick. Both activin and retinoic acid are able to activate Gnot1 expression in cultured blastodermal cells and show a strong synergistic effect when applied in combination. Strong superinduction of Gnot1 transcripts in the presence of cycloheximide also indicates the presence of a potent and labile intracellular inhibitor capable of modulating Gnot1 expression. During gastrulation, Gnot1 transcripts become localized specifically to tissues associated with "organizer" function (Hensen's node, head process, notochord). The expression data and the response to mesoderm inducing factors and axial "caudalizing" signals suggest that Gnot1 may be involved in specification of the embryonic body axis and could play a part in regulating features of the trunk/tail organizer in the chick embryo

Bmp2CreER, Osr1Cre and Gdf5Cre have distinct and partly complementary distributions of recombinase activity.

<p>Bmp2CreER activity after single dose tamoxifen (Tam) administration at 2 different times (E11.5, A-B or E13.5, C-D) compared to Osr1Cre (E-F) and Gdf5Cre (G-H) in E15.5 limbs. In each case, whole mount and adjacent longitudinal sections through dorsoventral axis of forelimb (FL) or hindlimb (HL) are oriented with distal at top, and anterior-to-posterior (digit 1–5) from left-to-right. Hatch marks in panels of longitudinal sections show the levels at which cross-sections were taken (rightmost panel sets; distal to proximal). Cross-sections are all oriented with dorsal at top and anterior-to-posterior (digit 1–5) from left-to-right. Note that Bmp2CreER labels LacZ-positive descendants mainly in interdigital mesenchyme, including collateral and joint ligaments, whereas Osr1Cre descendent cells reside in subcutaneous tissue and joint ligaments, and Gdf5Cre labels cells lining the joint regions and immediately adjacent subarticular and perichondrial cells at E15.5.</p

Tissue localization of Bmp2CreER activity during the course of limb development.

<p>Upper panels: 24-hour collection (A-J). Tissue localization of Bmp2CreER activity at 24 hours after tamoxifen (Tam) treatment on consecutive days from E10.5–14.5. Panels A,C,E,G,I (forelimb, FL) and B,D,F,H,J (hindlimb, HL) show longitudinal sections through limb dorsoventral axis oriented with distal at top, and anterior-to-posterior (digit 1–5) from left-to-right. Hatch marks in panels C-J show the levels at which cross-sections below panels C-J were taken (distal to proximal). Cross-sections are all oriented with dorsal at top and anterior-to-posterior (digit 1–5) from left-to-right. Lower panels: E13.5 collection (K-N). Tissue distribution of LacZ activity at E13.5 after tamoxifen (Tam) treatment at E10.5 (K,L) or E11.5 (M,N). For each treatment time, whole mount and adjacent longitudinal and cross-sections of forelimb (FL) or hindlimb (HL) are shown. Hatch marks in panels of longitudinal sections show the levels at which cross-sections through digit region were taken (distal to proximal). Longitudinal and cross-sections are oriented as for Upper Panels. Whole mount images are oriented the same as longitudinal sections.</p

Tissue distribution of LacZ activity at E15.5 following tamoxifen (Tam) treatment at different stages from E10.5—E14.5.

<p>Panels A,C,E,G (forelimb, FL) and B,D,F,H (hindlimb, HL) show longitudinal sections through limb dorsoventral axis oriented with distal at top, and anterior-to-posterior (digit 1–5) from left-to-right. Hatch marks in panels A-H show the levels at which cross-sections below panels A-H were taken (distal, proximal). Cross-sections are all oriented with dorsal at top and anterior-to-posterior (digit 1–5) from left-to-right. Note that tamoxifen treatment at E13.5 results in LacZ staining restricted to collateral ligament. Lower panel cartoon shows a diagram of an E15.5 digit cross-section with cartilage (red), tendon (blue) and ligament (green) structures annotated for reference.</p

Bmp2CreER activity 24 hours after tamoxifen (Tam) treatment surveyed at daily intervals during the course of limb development.

<p>A. LacZ activity at 48 hours after tamoxifen treatment at E9.5 with arrow pointing to weak activation in distal forelimb (FL inset). Only trace expression is seen in hindlimb (HL inset). (No appreciable LacZ staining was observed at 24 hours after tamoxifen treatment; data not shown). B. LacZ activity assayed at 24 hrs after tamoxifen treatment at times indicated above each panel. All insets to the right show forelimb (FL) and hindlimb (HL) buds oriented with distal at top, and anterior-to-posterior (digit 1–5) from left-to-right. Note that no LacZ staining is evident in tissues outside of the limb.</p

Comparison of Bmp2CreER activity levels and distribution assayed at E15.5 in LacZ-positive descendants, following tamoxifen (Tam) treatment between stages E11.25 to E11.75.

<p>For each tamoxifen (Tam) treatment time, whole mount and adjacent longitudinal sections through dorsoventral axis of forelimb (FL) or hindlimb (HL) are oriented with distal at top, and anterior-to-posterior (digit 1–5) from left-to-right. Hatch marks in panels of longitudinal sections show the levels at which cross-sections through digit region were taken (rightmost panel sets; distal to proximal). Cross-sections are all oriented with dorsal at top and anterior-to-posterior (digit 1–5) from left-to-right. Note that Cre activity transitions from digit condensations and interdigital mesenchyme to primarily interdigits and collateral ligaments during this time window of tamoxifen administration.</p