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Armadillo repeat and Armadillo-like helical domain containing proteins form a large family with diverse and fundamental functions in many eukaryotes. Herein we investigated the spatiotemporal expression pattern of Armadillo-like helical domain containing 4 (or Armh4) as an uncharacterized protein coding mouse gene, within the mouse embryo during the initial stages of heart morphogenesis. We found Armh4 is initially expressed in both first heart field as well as the second heart field progenitors and subsequently within predominantly their cardiomyocyte derivatives. Armh4 expression is initially cardiac-restricted in the developing embryo and is expressed in second heart field subpharyngeal mesoderm prior to cardiomyocyte differentiation, but Armh4 diminishes as the embryonic heart matures into the fetal heart. Armh4 is subsequently expressed in craniofacial structures and neural crest-derived dorsal root and trigeminal ganglia. Whereas lithium chloride-induced stimulation of Wnt/β-catenin signaling elevated Armh4 expression in both second heart field subpharyngeal mesodermal progenitors and outflow tract, right ventricle and atrial cardiomyocytes, neither a systemic loss of Islet-1 nor an absence of cardiac neural crest cells had any effect upon Armh4 expression. These results confirm that Wnt/β-catenin-responsive Armh4 is a useful specific biomarker of the FHF and SHF cardiomyocyte derivatives only., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Midkine (MDK) and Pleiotrophin (PTN) belong to a group of heparin-binding growth factors that has been shown to have pleiotropic functions in various biological processes during development and disease. Development of the vertebrate eye is a multistep process that involves coordinated interactions between neuronal and non-neuronal cells, but very little is known about the potential function of MDK and PTN in these processes. In this study, we demonstrate by section in situ hybridization, the spatiotemporal expression of MDK and PTN during ocular development in chick and mouse. We show that MDK and PTN are expressed in dynamic patterns that overlap in a few non-neuronal tissues in the anterior eye and in neuronal cell layers of the posterior eye. We show that the expression patterns of MDK and PTN are only conserved in a few tissues in chick and mouse but they overlap with the expression of some of their receptors LRP1, RPTPZ, ALK, NOTCH2, ITGβ1, SDC1, and SDC3. The dynamic expression patterns of MDK, PTN and their receptors suggest that they function together during the multistep process of ocular development and they may play important roles in cell proliferation, adhesion, and migration of neuronal and non-neuronal cells., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Brain functions are the products of dynamic interactions between multiple genes and environments. Accordingly, there are large differences among mouse strains at the behavioral and neurobiological levels. Therefore, it is crucial to manipulate genes on the same and homogenous genetic background and then to analyze and compare the phenotypes of various genetically modified mice. Furthermore, a conditional gene targeting to restrict the gene knockout to specific cells and time is a powerful tool to investigate the molecular basis of higher brain functions such as learning and memory. We have developed a system employing Cre-progesterone receptor fusion recombinase for temporal regulation of gene targeting and Flp/frt recombination system for elimination of marker genes. Importantly, both the recombinase lines and target mice have been produced with embryonic stem cells derived from the C57BL/6 strain suitable for brain function analysis. Thus, we have established an inducible and neuron-specific gene targeting system on the pure C57BL/6 genetic background.

Androgen-induced growth factor (AIGF/FGF-8) has purified from a mouse mammary carcinoma cell line and its cDNA cloning revealed that AIGF is the eighth member of the fibroblast growth factor family. To clarify a biological role of FGF-8, Northern blot analysis of various adult tissues was carried out, but no expression was found. However, whole mount in situ hybridization of 7.5- to 14.5-day gestation (E7.5-14.5) embryos revealed six predominant expression domains: [1]primitive streak region (E7.5-9.75); [2]midbrain-hindbrain border (E8.0-10.5); [3]rostral forebrain (E8.0-10.5); [4]limb ectoderm and apical ectodermal ridge (E9.25-13.5); [5]nasal placode and epithelium (E9.5-12.5); and [6]branchial arch ectoderm (E8.5-11.5). The embryonic expression pattern suggests a unique role of FGF-8 in mouse development, especially in gastrulation, brain development, and limb and facial morphogenesis.

Objective: Our objective was to identify novel genes that are expressed in temporally and spatially restricted patterns during mouse embryonic development and organogenesis., Study Design: Two genetic trapping reporter constructs that lack transcriptional regulatory sequences were introduced independently into transcriptionally active gene loci by electroporation into mouse embryonic stem cells. Patterns of host gene-reporter construct expression were investigated in differentiated embryonic stem cells, embryoid bodies, and chimeric embryos at various stages of development., Results: Three patterns of host gene-reporter construct expression were observed from the developmental analysis of four vector-integrated cell lines. Reporter expression patterns reflecting developmental regulation, constitutive activity, and developmental inactivation of the host genes were observed., Conclusions: Two vector-integrated gene loci from cell lines CCE-1C1 and D3-B44 have expression patterns not previously described by genetic trapping. Molecular characterization of these interrupted genes will shed light on their developmental function.

Differences are described in the effects of treatment of preimplantation mouse embryos with low levels (0.01-1 nM) of phorbol myristate acetate (PMA), during three different periods of a 48-h culture from the 2-cell stage, on pre- and postimplantation development. Treatment of embryos with PMA for 48 h (first group) or 24 h (second group) from the 2-cell stage caused premature cavitation (prior to the 16-cell stage) and it also reduced the size and alkaline phosphatase (ALPase) activity of inner cell masses (ICMs), as well as the numbers of cells in blastocysts, in a dose-dependent manner. Treatment of early morulae with PMA for 24 h (third group) did not have the above mentioned effects on embryos but inhibited the formation and subsequent enlargement of the blastocoel. The blastocysts that were allowed to develop in the three treatment groups were examined for postimplantation development. Implantation was unaffected in all groups. The survival rate after implantation was low in the first and second groups but relatively high in the third group. The results indicate that an embryo exposed to PMA for 24 h from the 2-cell stage forms a premature blastocoel, and, in such an embryo, quantitative and qualitative differentiation into the ICM is blocked but qualitative differentiation into trophectoderm is uninhibited. Consequently, the embryo can implant but does not survive for a long time. When embryos were exposed to PMA for 24 h from the early morula stage, the formation and enlargement of the blastocoel were inhibited even though the treatment had a minimal effect on other developmental events.(ABSTRACT TRUNCATED AT 250 WORDS)

The patterns of development of T cells from the very early stem cells that settle in the embryonic thymus have been studied. For this purpose, mouse embryonic thymuses (14 days) depleted of thymocytes were reconstituted with hemopoietic stem cells from fetal liver (FL) and yolk sac (YS) and T-cell development was followed in vitro in organ culture. It was found that cells derived from FL and YS of 10- to 14-day-old embryos were capable of reconstituting depleted thymic explants and exhibiting membrane markers in a pattern similar to that of thymocytes developing in intact thymic explants. Furthermore, these cells responded to concanavalin A in proliferative and cytotoxic assays as measured by limiting-dilution analysis. Thus, lymphohemopoietic stem cells emerging in the embryo prior to thymus lymphoid development are capable of differentiation in the thymus microenvironment into T cells, identified by phenotypic markers and functions that are characteristic of cells developing in the intact embryonic thymus.

The possibility has been repeatedly raised that erythropoiesis results from clonal succession--the differentiation of one or a very small number of the most primitive stem cells that are sequentially activated to proliferate forming clones of differentiated cells and then eventually decline, to be replaced by new stem cell clones. We studied this possibility in chimeric mice made by combining embryos from two different strains so that they would have two distinct stem cell populations, each of which produces a different hemoglobin type (d and s). These were compared with F1 hybrids in which every stem cell produces both types. We measured the percentage of type d in seven to ten serial samples of circulating reticulocytes taken at three- to seven-day intervals and found that the variability in percent of this hemoglobin was only slightly higher in the chimeric mice than in F1 controls; SD ranged from 2.7% to 5.5% in the chimeric mice and from 3.4% to 3.9% in the controls. Using the binomial formula, the numbers of new clones formed during the reticulocyte life span, approximately three days, ranged from 33 to 118 in the individual chimeric mice. However, these numbers are underestimates because estimated numbers of clones depend inversely on variabilities, and the calculations did not exclude the contribution of experimental error to the overall variability. Total percentages of type d hemoglobin were also measured in seven to nine successive serial samples at 60- to 136-day intervals. These gave mean values similar to measures of newly synthesized hemoglobin in the same mice, but SD were larger, ranging from 5.3% to 8.4%. This reflects experimental error, both because of excess day-to-day variability found in this type of measurement and because there could not be fewer primitive stem cells activated to form clones of erythrocytes during the 45-day erythrocyte life span than during the three-day life span of reticulocytes. Since most and maybe all of the variation between successive samples in the same chimeric mouse appear to result from experimental error, many or even all of the primitive stem cells may simultaneously contribute to erythropoiesis.

A simian adenovirus SA7 (C-8) DNA was microinjected into fertilized mouse eggs. Thirty-five mice derived from eggs injected with SA7 DNA were screened for the presence of the adenovirus genome in their liver DNA. Eighteen of these mice contained the virus-specific sequences. SA7 DNA was detected in some tissues, but in all cases, viral sequences were absent from muscle and heart DNA. Viral DNA was inherited by 50-70% of the next generation. One mouse that contained about 1 copy of SA7 DNA per haploid genome has been shown to pass it on to five generations, although the integrated viral DNA sustained a considerable structural change between F1 and F3. RNA analysis in various organs of 12 mice has shown the transcription of SA7 DNA to be very infrequent: only in the kidney of one mouse and in the spleen of another did RNA contain SA7-specific sequences.