Animal development entails the organization of specific cell types in space and time, and spatial patterns must form in a robust manner. In the zebrafish spinal cord, neural progenitors form stereotypic patterns despite noisy morphogen signaling and large-scale cellular rearrangements during morphogenesis and growth. By directly measuring adhesion forces and preferences for three types of endogenous neural progenitors, we provide evidence for the differential adhesion model in which differences in intercellular adhesion mediate cell sorting. Cell type–specific combinatorial expression of different classes of cadherins (N-cadherin, cadherin 11, and protocadherin 19) results in homotypic preference ex vivo and patterning robustness in vivo. Furthermore, the differential adhesion code is regulated by the sonic hedgehog morphogen gradient. We propose that robust patterning during tissue morphogenesis results from interplay between adhesion-based self-organization and morphogen-directed patterning.

The analysis of protein function is essential to modern biology. While protein function has mostly been studied through gene or RNA interference, more recent approaches to degrade proteins directly have been developed. Here, we adapted the anti-GFP nanobody-based system deGradFP from flies to zebrafish. We named this system zGrad and show that zGrad efficiently degrades transmembrane, cytosolic and nuclear GFP-tagged proteins in zebrafish in an inducible and reversible manner. Using tissue-specific and inducible promoters in combination with functional GFP-fusion proteins, we demonstrate that zGrad can inactivate transmembrane and cytosolic proteins globally, locally and temporally with different consequences. Global protein depletion results in phenotypes similar to loss of gene activity, while local and temporal protein inactivation yields more restricted and novel phenotypes. Thus, zGrad is a versatile tool to study the spatial and temporal requirement of proteins in zebrafish.

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An outstanding question in embryo development is how spatial patterns are formed robustly. In the zebrafish spinal cord, neural progenitors form stereotypic stripe-like patterns despite noisy morphogen signaling and large-scale cellular rearrangement required for tissue growth and morphogenesis. We set out to understand the mechanisms underlying this patterning robustness. Our adhesion assays revealed a preference for three neural progenitor types to stabilize contacts with cells of the same type. Genetic analysis uncovered a three-molecule adhesion code, composed of N-cadherin, Cadherin 11, and Protocadherin 19, with unique gene expression profiles for each cell type. Perturbation of the adhesion code results in loss of homotypic preference ex vivo and patterning errors in vivo. Both the cell fate and adhesion code are co-regulated by the common upstream morphogen signal Shh. We propose that robust patterning in tissues undergoing morphogenesis results from a previously unappreciated interplay between morphogen gradient-based patterning and adhesion-based self-organization.

25The analysis of protein function is essential to modern biology. While protein function has mostly 26 been studied through gene or RNA interference, more recent approaches to degrade proteins 27 directly have been developed. Here, we adapted the anti-GFP nanobody-based system 28 deGradFP from flies to zebrafish. We named this system zGrad and show that zGrad efficiently 29 degrades transmembrane, cytosolic and nuclear GFP-tagged proteins in zebrafish in an 30 inducible and reversible manner. Using tissue-specific and inducible promoters in combination 31 with functional GFP-fusion proteins, we demonstrate that zGrad can inactivate transmembrane, 32 cytosolic and nuclear proteins globally, locally and temporally with different consequences. 33Global protein depletion results in phenotypes similar to loss of gene activity while local and 34 temporal protein inactivation yields more restricted and novel phenotypes. Thus, zGrad is a 35 versatile tool to study the spatial and temporal requirement of proteins in zebrafish. 36 37 109 degree of GFP degradation as the ratio of sfGFP to mScarlet fluorescence intensity in the 110 cytoplasm and the nucleus. Consistent with the initial description (4), Ab-SPOP efficiently 111 degraded nuclear sfGFP-ZF1 (70% reduction, Figure 1 -figure supplements 3A-C) but not 112 cytoplasmic sfGFP-ZF1 (13% reduction, Figure 1 -figure supplements 3A-C). Abmut-SPOP did 113 not cause any detectable sfGFP-ZF1 degradation (Figure 1 -figure supplements 3A-C). As 114 reported previously (4), this confirms that the Ab-SPOP/FP-tag degradation system degrades 115 nuclear proteins efficiently but is not suitable for the degradation of non-nuclear proteins in 116 zebrafish.117 118 Fourth, we tested the deGradFP degradation system. This system uses the F-box domain from 119 the Drosophila Slimb adaptor protein fused to the anti-GFP nanobody vhhGFP4 to target FP-120 tagged proteins for degradation ( Figure 1A, B and (7)). We co-injected one-cell stage embryos 121 with sfGFP-ZF1 mRNA, deGradFP mRNA and mScarlet-V5 mRNA and assessed sfGFP-ZF1 122 degradation 9 hours later at the epiboly stage. As above, mScarlet-V5 served as an internal 123 standard and GFP degradation was assessed as the ratio of GFP to mScarlet fluorescence 124 intensity. We found that deGradFP reduced sfGFP-ZF1 only slightly (19% reduction, Figure 1C, 125 D). Since the fusion of the anti-GFP nanobody to the Cullin-binding domain from SPOP resulted 126 in efficient albeit only nuclear degradation of GFP, we reasoned that the anti-GFP nanobody 127 recognizes GFP-tagged proteins but that the Slimb F-box domain is not efficiently recruited to 128 the E3 ligase complex in zebrafish. We therefore replaced the Slimb F-box domain in deGradFP 129 with the homologuous F-box domain from zebrafish, reasoning that this should result in more 130 efficient GFP degradation in zebrafish. Based on sequence homology we identified the 131 zebrafish fbxw11b gene as the Drosophila slmb orthologue. We then replaced the Drosophila F-132 box domain from Slimb ...