Biologia dello sviluppo

Development of Xenopus and Zebrafish Embryos

In this way, it has been possible to follow the process of early stages development of Xenopus. Fish embryos too can be colored with dyes to be observed. In the development of zebrafish, the vegetal pole (formed by only one cell) corresponds to the yolk, containing the nutrients needed by cells dividing (blastoderm) in the animal pole. Gastrula is formed when the blastula completes a 50% epiboly, so called because half of the egg is covered, as in an epidermis formation, with blastoderm. These cells move from the animal to the vegetal pole, forming a platform called shield on the dorsal side at this stage (5hps), from where cells will start to differentiate. The shield is a fundamental structure for the head formation (its missing leads to the missing of the head). Labelling of midbrain structures using fluorescent dye (GFP) can allow observation of midbrain formation.

in20hps (hours post fertilization). Labelling anterior forebrain cells, the formation of telencephalon can be followed, just back them we can label cells that will become eyes and so on. In situ hybridization technique is also used to see which cells express which genes that might also be involved with their development. In this way scientists discovered that an homeobox gene involved in the eye formation is Rx, which loosing leads to the lacking of eyes. The gene Pax2, then, is fundamental for the midbrain formation. Only genes that are expressed in the early stages can be labelled to perform these investigations. Computational reconstruction of gastrulation can be done too, and single cell fate mapping is termed lineage tracing. The most sophisticated technique is to follow developmental movement in real time with timelapse. This observation are carried out by microscopes, taking a picture every half our and in a few days picture can be put together. In fish and other animals a just

1. fertilized egg with only one cell, yoke can be labelled with mRNAs, dyes, morpholinos and many other things to observe cells.
2. H2B-GFP can be used to label hystones (DNA)
3. Morpholinos are nucleotide analogs that recognize and bind short sequences (about 25 nucleotides) at the transcription start site or at splice sites of pre-mRNAs, and thus block the translation or proper splicing of the mRNA
4. Gastrulation movement are conserved in all the animal kingdom:
   • Epiboly, moving the animal cells to the vegetal pole, covering the entire yoke cell in the end and moving through the shield
   • Convergence to the midline of the cell
   • Involution in the margin of the blastopore
   • Extension, to form longitudinal structures
5. Problems with medaka: embryo is hard and calcium wave contracts the embryo. First

Commentato [e10]: Morpholinos are nucleotide analogs that recognize and bind short sequences (about 25 nucleotides) at the transcription start site or at splice sites of pre-mRNAs, and thus block the translation or proper splicing of the mRNA

Commentato [e11]: https://www.youtube.com/watch?v=vC72_ZSZkd8

pictures were taken labelling cells one by one and taking photos on hand. Take-home message: many processes in embryonic development in animal kingdom are highly comparative, leading to the study of comparative biology field.

Cells divide in different ways and blastoderm is formed in distinguished methods, so as the initiation of the gastrulation. Nieuwkoop center formation activates the cell to form the dorsal/ventral axe, kicking in a gastrulation event and inducing the formation of the SMO (Speeman-Mangold Organizer). Cells movement are very conserved in animals. Embryos too look similar even if they will form completely different animals, that will be defined only in the last stages of the development. Movements happen around all the structure, even if not everywhere with the same importance. 09/10/2019

In the chick embryo, 12 hrs are necessary to the primitive streak to be considered the elongated blastopore lip of amphibians, through which gastrulation takes place. Cells of the streak are derived from

The amniotic cavity, filled with fluid to prevent drying out, will form the organism, while hypoblast will be consumed, and Trophoblast develops into the chorion (a part of placenta). Basically, all of our cells derive from one of the layers, plus the germ lines, formed during gastrulation:

• Ectoderm, the outer layer composing epidermis, nervous system and pigment cells;
• Mesoderm, positioned in the middle and forming such as muscles, kidneys, connective tissue and blood;
• Endoderm, corresponding to the inner layer that will originate gut, lungs and liver;
• Germ cells, that will form eggs and sperm.

Fate maps obtained with gastrulation are very much comparable through vertebrates. Early cells are still multipotent and flexible as cell fate is not yet determined, so cells that are transplanted from one part of the gastrula will become cells whose destiny is the one of cells in the zone where they are implanted, and cells that are removed can grow back again. If a transplant from late gastrulation

To plate happens, structures from the germ layers will form, because cells started to differentiate. Ectodermal cells are called animal cap cells: they can be combined with other cells in a gastrula to test which factors can induce differentiation in cells.

The 4 main cell movements during gastrulation are:

1. Invagination
2. Epiboly
3. Convergent extension
4. Cell intercalation

Techniques that can be applied to generate an animal fate map include:

• Lineage tracing
• Fluorescent labeling

Two cell types derived from each of the three germ layers are:

• Ectoderm: neurons and skin cells
• Mesoderm: muscle cells and blood cells
• Endoderm: liver cells and lung cells

3. Animals

3.1. Drosophila

Neurexin is a presynaptic protein that helps to connect neurons and synapse that regulate sleep in Drosophila. In humans, genes encoding neurexins are implicated in disease such as autism, Tourette's syndrome, and many other cognitive diseases. Morgan, Sturtevant, Bridges, and Muller discovered in Drosophila that chromosomes must contain genes, arranged in a linear way in chromosomes, and that x-rays cause genetic damage and chromosomal rearrangements. For these reasons, many Nobel prizes have been won, also in 1995, when Nusslein.

development: they were clustered into groups of genes according to their time of action and phenotype caused when mutated.

Homeobox genes determine the position of developing structure. They are evolutionary conserved in their basic chromosomal arrangement and function, even if their number varies from one specie to another. Trees of evolution that show similarities of all metazoan can be composed observing 16S rRNA: more similar sequences mean more familiar organisms.

Drosophila has a life cycle of 10 days, during which cleavage, gastrulation, hatching and metamorphosis happen. Its life span is about 30 days, during which 400 to 2000 eggs per female can be produced. Its genome is formed by four pairs of chromosomes: three autosomes and one pair of sexual chromosomes. Its genome has been sequenced in 2000, discovering that 60% genes are conserved. Body axes are mainly determined maternally before fertilization. Some of the advantages on using this model are that cost is minimum (they use apple agar).

To survive and produce lots of eggs), the times to obtain a generation is short, it has a small and completely known genome and it allows genetic screenings. On the other hand, it is far from human reality, it is not transparent, it doesn't allow transplantation and its behaviour is not easily tested. Antibodies for many proteins are present for Drosophila, that can be used also to target and observe ovarioles, that contain oocytes of different stages and are often used for research to have descendants of the same adult.

Oogenesis in Drosophila starts with each oocyte being descendant from one germ cell called oogonium. Oogonium divides 4 times into 16 cells with an incomplete cytokinesis that allows for the generation of ring canals. The 15 nurse cells are metabolically active and make mRNA, which is transported on MTs through the ring canals (that allow communication between cells) to the one oocyte at the posterior of the egg. This cell develops a MT organising centre (MTOC) and the