Histological Analyses Demonstrate the Temporary Contribution of Yolk Sac, Liver, and Bone Marrow to Hematopoiesis during Chicken Development

<div><p>The use of avian animal models has contributed to the understanding of many aspects of the ontogeny of the hematopoietic system in vertebrates. However, specific events that occur in the model itself are still unclear. There is a lack of consensus, among previous studies, about which is the intermediate site responsible for expansion and differentiation of hematopoietic cells, and the liver's contribution to the development of this system. Here we aimed to evaluate the presence of hematopoiesis in the yolk sac and liver in chickens, from the stages of intra-aortic clusters in the aorta-genital ridges-mesonephros (AGM) region until hatching, and how it relates to the establishment of the bone marrow. <i>Gallus gallus domesticus</i> L. embryos and their respective yolk sacs at embryonic day 3 (E3) and up to E21 were collected and processed according to standard histological techniques for paraffin embedding. The slides were stained with hematoxylin-eosin, Lennert's Giemsa, and Sirius Red at pH 10.2, and investigated by light microscopy. This study demonstrated that the yolk sac was a unique hematopoietic site between E4 and E12. Hematopoiesis occurred in the yolk sac and bone marrow between E13 and E20. The liver showed granulocytic differentiation in the connective tissue of portal spaces at E15 and onwards. The yolk sac showed expansion of erythrocytic and granulocytic lineages from E6 to E19, and E7 to E20, respectively. The results suggest that the yolk sac is the major intermediate erythropoietic and granulopoietic site where expansion and differentiation occur during chicken development. The hepatic hematopoiesis is restricted to the portal spaces and represented by the granulocytic lineage.</p></div

Scheme of hematopoiesis in the yolk sac, liver, and bone marrow during chicken development.

<p>Bars indicate the temporal distribution of this activity in the AGM region, yolk sac (YS), liver (L), and bone marrow (BM). Black dotted lines indicate the presence of both erythropoiesis and granulopoiesis in the YS (<b>C</b>–<b>F</b>) and BM (<b>E</b>–<b>G</b>). The blue dotted line is to draw attention to the granulopoiesis in the L. (<b>A</b>) At E4, immature erythropoietic cells, incomplete erythropoietic foci, and rare granulocytes are distributed in the YS. (<b>B</b>) From E6, the YS shows complete erythropoietic foci (all maturation stages are seen) and some granulocytes. (<b>C</b>) From E7, complete erythropoietic and granulopoietic foci are distributed in the YS. (<b>D</b>) At E10, erythropoietic and granulopoietic foci are seen in the YS. Granulocytes at different stages of maturation are noted in the BM. At E11, basophilic cells are also seen in the BM. (<b>E</b>) At E15, both erythropoietic and granulopoietic foci are frequently observed in the YS and BM. In this phase, granulopoiesis begins in the L around the portal vessels. (<b>F</b>) At E17, hematopoiesis is reduced in the YS. Granulopoiesis persists in the L portal spaces, and both erythropoietic and granulopoietic activities are noted in the BM. (<b>G</b>) At E21, granulopoietic foci are seen in L connective tissues, both erythrocytic and granulocytic activities are observed in the BM, and the YS is no longer a hematopoietic site.</p

(A–H) Panoramic view of chicken yolk sacs between E3 and E19.

<p>The embryonic day (E) is indicated in the upper right corner in each picture. Histological sections of yolk sacs show endoderm (End), vessels (Art), and areas of hematopoietic foci (arrows). (<b>A</b>–<b>E</b>) The areas occupied by these foci are shown gradually increasing in the photomicrographs between E3 and E12, and (<b>F</b>–<b>H</b>) decreasing in onwards stages. (<b>G, H</b>) Atrophic vessels (arrowheads). (<b>F</b>–<b>H</b>) Yolk (Y). Hematoxylin-eosin. Bars 100 µm.</p

Chicken liver development without hematopoietic activity from E9 to E 14.

<p>The embryonic day (E) is indicated in the upper right corner in each picture. Hp, hepatoblasts; CV, central vein; sinusoidal capillaries (asterisks); PV, portal vein; CT, connective tissue. (<b>A</b>) Lennert's Giemsa and (<b>B</b>–<b>F</b>) Hematoxylin-eosin stains. Bars 20 µm.</p

Establishment of hematopoiesis in the diaphysis of long bones during chickens' developmental period.

<p>The embryonic day (E) is indicated in the upper right corner in each picture. (<b>A</b>) Leukocytes with eosinophil granules (arrowheads) are shown in the marrow stroma (St) in the beginning of the bone marrow formation. Metamyelocyte (black arrow) into vessel (V). Osteoclast (white arrow). (<b>B</b>) Granulocytes (white arrows) and basophilic cells with eccentric nucleus and prominent nucleolus (black arrows) are shown in the marrow stroma. Mitosis in erythrocyte (arrowhead). (<b>C</b>) Basophilic cell (arrowhead). Granulocytes at different stages of maturation in the stroma including promyelocytes (arrows). (<b>D</b>) These cells are shown in high magnification (arrows). (<b>E</b>) Granulocytes in the stroma (St) and erythropoiesis (limited by arrows) in sinusoidal capillaries, shown (<b>F</b>) in high magnification. (<b>G</b>) Panoramic view of a transversal section of diaphysis. The bone marrow (limited by arrows) shows areas of erythropoiesis (blue color) and granulopoiesis (red color). (<b>H</b>) Erythropoiesis (Ery) in sinusoidal capillaries showing immature hematopoietic cells (arrows) and mitosis in erythrocyte (arrowhead). Granulopoiesis (Gr) in the marrow stroma. HC, hypertrophic cartilage; SC, sinusoidal capillary; BT, bone tissue. (<b>A</b>) Sirius Red at pH 10.2, and (<b>B</b>–<b>H</b>) Lennert's Giemsa stain. Bars 20 µm.</p

Chicken liver development without hematopoietic activity from E3 to E8.

<p>The embryonic day (E) is indicated in the upper right corner in each picture. (<b>A</b>, <b>C</b>, <b>D</b>) Numerous mitosis (arrows) are seen in hepatoblasts (Hp). (<b>B</b>) Mitosis in circulating erythrocyte (arrow). (<b>E</b>) Immature hematopoietic circulating cells (arrows) in sinusoidal capillaries (vessels located between hepatoblast cords, Hp). Note the large and irregular lumen of the sinusoidal capillaries. (<b>F</b>) Foci of immature erythropoietic cells in circulation (limited by arrows). Hp, hepatoblasts; VD, venous duct; sinusoidal capillaries (asterisks). (<b>A</b>, <b>C</b>, <b>F</b>) Hematoxylin-eosin and (<b>B</b>, <b>D</b>, <b>E</b>) Lennert's Giemsa stains. Bars 20 µm.</p

(A–K) Erythropoiesis (Ery) and (E–L) granulopoiesis (Gr) in chicken yolk sac between E3 and E20.

<p>The embryonic day (E) is indicated in the upper right corner in each picture. Thin arrows show mitosis in erythrocytes (<b>A</b>, <b>D</b>, <b>E</b>, <b>I</b>, <b>K</b>) and in a granulocyte (<b>H</b>). (<b>A</b>) Predominance of basophilic cells with a slight acidophily. (<b>B</b>, <b>C</b>) Numerous pro-erythroblasts and basophilic erythroblasts (Ery) between artery (Art) and endoderm (End). (<b>C</b>) Note a cell band leukocyte (arrowhead). (<b>D</b>) Erythrocytic (Ery) focus showing mature erythrocyte (arrowhead). (<b>F</b>, <b>G</b>) Cell band leukocyte (arrowhead). (<b>H</b>) Eosinophil granules into granulocytic cells at different stages of maturation show the cytoplasm of these cells in red-orange color. (<b>I</b>) Promyelocyte (arrowhead). (<b>J</b>, <b>K</b>) Foci of erythrocytic (white asterisks) and granulocytic (black asterisks) differentiation are present in equivalent numbers at this stage. (<b>K</b>) Mature leukocyte (arrowhead). (<b>L</b>) Myelocyte (arrowhead). (<b>E</b>–<b>K</b>) Note that granulocytic and erythrocytic lineages do not mix. End, endoderm; Art, artery. (<b>A</b>–<b>E</b>, <b>G</b>, <b>I</b>, <b>L</b>) Lennert's Giemsa, (<b>F</b>, <b>J</b>, <b>K</b>) Hematoxylin-eosin, and (<b>H</b>) Sirius Red stains at pH 10.2. Bars 20 µm.</p

Structural investigation of C6/36 and Vero cell cultures infected with a Brazilian Zika virus - Fig 6

<p><b>C6/36 cells infected with ZIKV analyzed by transmission electron microscopy (TEM) at different time points post-infection (A-B: 48 hr p.i., C-D: 72 hr p.i.).</b> Several large viroplasm-like perinuclear compartments (V) (A-B) and ZIKV particles (*) measuring approximately 40–50 nm in diameter in the endoplasmic reticulum cisternae (RER) (A, B) and in lysosomes (L) (C-D) were observed. Nucleocapsids were observed inside the rER (D). Thickening of the nuclear membrane and rough endoplasmic reticulum cisternae (rER) (black head arrow) (C), numerous lysosomes (L) (C, D) and vesicular compartments associated with rER (arrow) (C) measuring approximately 100 nm in diameter were observed. Nucleus (N).</p

Uninfected C6/36 cells (negative controls) at different time points in culture.

<p><b>No cellular ultrastructural alterations were observed.</b> (A–B) 24 hr of culture; (C) 48 hr of culture; (D) 72 hr of culture. Rough endoplasmic reticulum cisternae (rER), nucleus (N).</p

Detection of ZIKV RNA and viral load quantification from C6/36 and Vero cell supernatants at 24, 48 and 72 hr p.i. by quantitative real-time RT-PCR.

<p>Detection of ZIKV RNA and viral load quantification from C6/36 and Vero cell supernatants at 24, 48 and 72 hr p.i. by quantitative real-time RT-PCR.</p