395This effect seems to be mediated through E-cadherin. E-cadherin is normally down-
regulated as PGCs begin to polarize and disperse, but this does not occur in PGCs
depleted of Dnd. PGCs remain clustered in groups and maintain close cell–cell
contacts (Richardson and Lehmann 2010 ). During migration to the gonadal ridges,
PGCs remain in close contact with neighboring somatic cells. Such cell-cell interac-
tion is governed by extracellular matrix components. Particularly important are the
heparin sulfate glycosaminoglycans (HS-GAGs), which are present in the surround-
ing area of migrating PGCs. They play a critical role in guidance, survival, and
localization of the migrating PGCs (Wei and Liu 2014 ). During germ cell specifica-
tion, zebrafish PGCs have a smooth, round morphology and lack migratory activity
(1k-cell stage or 3 hpf). PGCs begin to extend small cellular projections in multiple
directions at 3.5 hpf. These projections disappear during mitosis. At the end of blas-
tula (4.5 hpf), PGCs become polarized and extend broader projections at the guiding
edge. This step is dependent on transcription and the Dead-end protein, and is nec-
essary for the cells to respond to SDF1A (Richardson and Lehmann 2010 ).
8.2.5 Genetic Approaches
Perhaps the main advantage of using zebrafish to study germ cell specification is the
availability of transgenic and mutant lines that allow functional approaches at any
time point along the zebrafish life-cycle. In order to make any genetically manipu-
lated organism, changes must be made in germ cells. Therefore it is of critical
importance to establish an efficient technology for high specific PGC-targeted gene
manipulation in vertebrates. New, sophisticated and affordable genetic approaches
have recently emerged, allowing the manipulation of virtually any gene in a diverse
range of organisms, including zebrafish (Auer and Del Bene 2014 ; Gaj et al. 2013 ;
Dong et al. 2014 ). This core technology exploits different approaches, permitting
the generation of transgenic and mutant lines. One well known strategy to obtain
transgenic zebrafish is using the Cre/loxP recombinase and the Gal4/UAS transcrip-
tion system for induction and regulation of genes of interest. PGCs are ideal target
cells for genetics since they can be specifically labeled and manipulated during
early development (Ciruna et al. 2002 ; Weidinger et al. 2003 ; Hsiao and Tsai 2003 ).
Most importantly, they will generate adult gametes that provide genetic materials
necessary to form a whole organism. The use of the Gal4/UAS system was opti-
mized in zebrafish, allowing a tissue-specific expression of an improved Gal4 tran-
scriptional activator (KalTA4), thus promoting gene expression driven by upstream
activation sequence (UAS) (Distel et al. 2009 ).
The use of a specific germ cell promoter, allowing the localized expression of
Gal4 in the germ cell lineage, or the fusion of the gene of interest to the 3′ UTR of
the germ cell marker nanos, promoting the targeting of the newly transcribed mRNA
mainly in germ cells, are new approaches used to improve transgenesis efficiency
(Blaser et al. 2005 ; Xiong et al. 2013 ). Novel technologies will allow investigators
not only to generate transgenic line with labeled PGCs, but will also have the
8 Mechanisms of Vertebrate Germ Cell Determination