Elevated Hoxb5b Expands Vagal Neural Crest Pool and Blocks Enteric Neuronal Development in Zebrafish

Neural crest cells (NCCs) are a migratory, transient, and multipotent stem cell population essential to vertebrate embryonic development, contributing to numerous cell lineages in the adult organism. While great strides have been made in elucidating molecular and cellular events that drive NCC specification, comprehensive knowledge of the genetic factors that orchestrate NCC developmental programs is still far from complete. We discovered that elevated Hoxb5b levels promoted an expansion of zebrafish NCCs, which persisted throughout multiple stages of development. Correspondingly, elevated Hoxb5b also specifically expanded expression domains of the vagal NCC markers foxd3 and phox2bb. Increases in NCCs were most apparent after pulsed ectopic Hoxb5b expression at early developmental stages, rather than later during differentiation stages, as determined using a novel transgenic zebrafish line. The increase in vagal NCCs early in development led to supernumerary Phox2b+ enteric neural progenitors, while leaving many other NCC-derived tissues without an overt phenotype. Surprisingly, these NCC-derived enteric progenitors failed to expand properly into sufficient quantities of enterically fated neurons and stalled in the gut tissue. These results suggest that while Hoxb5b participates in vagal NCC development as a driver of progenitor expansion, the supernumerary, ectopically localized NCC fail to initiate expansion programs in timely fashion in the gut. All together, these data point to a model in which Hoxb5b regulates NCCs both in a tissue specific and temporally restricted manner.


INTRODUCTION
As an embryonic stem cell population in vertebrates, neural crest cells (NCCs) are renowned for their remarkable migratory capacity, as well as their multipotency. Born from the dorsal neural tube, NCCs migrate along stereotypic routes throughout the early embryo and give rise to a wide range of diverse tissue lineages, such as craniofacial skeleton, portions of the peripheral nervous system, and pigment cells (Rocha et al., 2020). NCCs exhibit regional potential along the anteroposterior (AP) neuraxis such that they may be divided into four general populations: cranial, vagal, trunk, and sacral (Le Douarin, 1982;Le Douarin and Kalcheim, 1999). Each of these populations give rise to numerous discrete lineages, for example, cranial NCC largely give rise to cell lineages in the head. Particularly of interest are vagal NCCs, which contribute to several tissues, such as the cardiac outflow tract and nearly all of the enteric nervous system (ENS) (Tang et al., 2021) within the gut, and have been less well characterized than other populations (Hutchins et al., 2018). While the driving genetic factors which regulate the general pattern of NCC developmental trajectories have been well described (Simoes-Costa and Bronner, 2016;Martik and Bronner, 2017), we still have an incomplete understanding of what genes function in context of vagal NCC development and their subsequent differentiation.
Coincident with the anterior to posterior rise of NCC is the expression of Hox genes, a strongly conserved family of genes encoding for transcription factors most notable for their canonical role in body axis patterning (Amores et al., 1998;Pearson et al., 2005). Among their many roles, Hox transcription factors are known to play essential roles in establishing discrete partitions within the hindbrain, directing limb formation, regulating cardiac cell number, and guiding neural circuit formation within a variety of tissue contexts (Rancourt et al., 1995;Waxman et al., 2008;Minguillon et al., 2012;Breau et al., 2013;Di Bonito et al., 2013;Barsh et al., 2017). Organized in tight clusters in the genome, known as paralogy groups, Hox genes are labeled A-D to designate a particular chromosomal cluster, as well as by number, which represents the gene's chromosomal position within a particular cluster, ranging from 1-13 (Kudoh et al., 2001). Furthermore, not only are the Hox peptide sequences conserved between species, synteny of the Hox clusters is highly conserved (Santini et al., 2003). The role of Hox genes among vertebrates is also strongly conserved in their function, even among teleost fishes who have undergone a genome duplication during their evolutionary history (Amores et al., 1998;Parker et al., 2019). Each Hox gene is expressed along the anterior-posterior axis in nested domains collinear with their position in the chromosome, where they undergo complex regulatory interaction to establish discrete expression domains (Zhu et al., 2017). As such, earlier numbered Hox factors are commonly expressed within the cephalic tissues, while later numbered Hox factors are expressed more distally.
Within the context of cranial NCCs, Hox transcription factors have been shown to drive a number of NCC phenotypes. Overexpression of Hox factors in chicken, such as Hoxa2, Hoxa3, and Hoxb4, produces a variety of overlapping ablations of NCC-derived craniofacial skeleton (Creuzet et al., 2002). Similarly, the NCC-derived hyoid bones and presumptive thymic mesenchyme were greatly reduced or ablated in mice homozygous for single knockout for Hoxa-3, or double knockout of Hoxa-3 and Hoxd-3, while also causing homeotic transformations of other structures throughout the animal Capecchi, 1993, 1994). In addition to affecting formation of terminally differentiated craniofacial structures, earlier phases of NCC migration into pharyngeal arches and onset emigration of NCC from the neural tube are also acutely sensitive to changes in anterior Hox expression (Gouti et al., 2011;Parker et al., 2018). While the role of Hox transcription factors is less well characterized in posterior populations of NCC, vagal NCC in mice fail to colonize the gut following overexpression of Hoxa-4, which is endemic to the gut nervous network (Wolgemuth et al., 1989;Tennyson et al., 1993). The failure to form a complete enteric nervous system results in megacolon, a phenotype associated with human disease (Nagy and Goldstein, 2017). Overall, while we have learned much regarding Hox genes in the cranial NCC, the roles Hox transcription factors play within vagal and other posterior NCC populations requires further investigation.
One posterior Hox factor that has been implicated in vagal NCC development is Hoxb5. In mice, dominant negative abrogation of embryonic Hoxb5 activity alters vagal and trunk NCC development, with reduced NCCs observed en route to and along gut tissue, as well as decreased numbers of melanoblasts throughout the body (Lui et al., 2008;Kam and Lui, 2015). Additionally, Hoxb5 may regulate expression of key genes active in NCC development, notably Foxd3 (Kam et al., 2014a) and Phox2b (Kam and Lui, 2015). The orthologous gene in zebrafish, hoxb5b, which is the primary ortholog in teleost fishes (Jarinova et al., 2008), was detected in differentiating NCC lineages at both 48 and 68 h post fertilization (hpf) , suggesting it may also play a role during zebrafish NCC development. While characterization of zebrafish hoxb5b mRNA expression in situ has been pervasively characterized in several early embryonic contexts, such as the mesoderm and limb buds (Kudoh et al., 2001;Jarinova et al., 2008;Waxman et al., 2008;Hortopan and Baraban, 2011;van der Velden et al., 2012), the functional role of hoxb5b with respect to NCC development had not yet been examined.
Here, we postulated that hoxb5b functions as a potential driver of vagal NCC development. We provide evidence that overexpression of hoxb5b is sufficient to grossly expand NCC populations throughout the zebrafish embryo, in addition to ectopic expansion of vagal domains marked by foxd3 and phox2bb. The functional window of Hoxb5b activity was also restricted to a narrow developmental span, early during embryogenesis, rather than during NCC differentiation stages. The early expansion of NCC, however, did not lead to corresponding pan increases in NCC-derived tissues. Rather, elevated Hoxb5b activity expanded enteric neural progenitor cell pools along the gut, yet suppressed their subsequent expansion as they differentiated into enteric neurons, leading to overall fewer neurons along the gut. These data cumulatively support a model in which hoxb5b is a potent regulator of NCC expansion and cell number in zebrafish.

hoxb5b is Expressed in Post-Otic Vagal NCCs During Zebrafish Development
We examined hoxb5b expression in zebrafish embryos ( Figure 1A) using whole mount in situ hybridization (ISH). At 32 hpf, hoxb5b was expressed bilaterally immediately posterior to the otic vesicle (post-otic), along the foregut, in the hindbrain, and anterior spinal cord ( Figure 1B), as previously described (Jarinova et al., 2008). hoxb5b persisted in all three domains at 50 hpf ( Figure 1C), though the post-otic domains (POD) were slightly restricted and the hindbrain/spinal cord expression gained a more defined posterior boundary. By 77 hpf, hoxb5b expression remained largely in the hindbrain and foregut, with diminished yet persistent expression in the POD ( Figure 1D). Together, these ISH data reveal changing post-otic spatiotemporal expression patterns of hoxb5b during the first 4 days of development.
We next examined the relationship of hoxb5b expression to the vagal NCC population during NCC specification and migration phases. NCCs were assayed by using the zebrafish pan-NCC marker crestin (Luo et al., 2001), in combination with hoxb5b, via hybridization chain reaction (HCR) (Choi et al., 2010(Choi et al., , 2016(Choi et al., , 2018 , and crestin + /hoxb5b + regions were still observed along the dorsal neural tube. These data are consistent with our prior findings in which hoxb5b mRNA was present in posterior NCC at 48-50 hpf and 68-70 hpf . Collectively, the ISH and HCR data indicate that hoxb5b mRNA expression is coincident within the vagal NCCs area, persisting throughout the developmental window spanning NCC specification and well into their migration phase, highlighting hoxb5b's potential role as a driver of vagal NCC development.

Elevated Hoxb5b Activity Globally Promotes Expanded Localization and Increased Number of NCC in vivo
While other work has focused exclusively on the loss of function of Hoxb5 related genes (Lui et al., 2008;Dalgin and Prince, 2021), we have sought to understand the gain of function role of hoxb5b in the context of NCC development.
To examine the possible role of hoxb5b in NCC, we employed a hyperactive vp16-hoxb5b fusion construct (Waxman et al., 2008;Waxman and Yelon, 2009). Injection of vp16-hoxb5b mRNA resulted in expansion of NCC, when compared to control embryos at 32 hpf, as assayed with an ISH probe against crestin (Figures 2A,B). The expansion in NCC territory was prominent in the pre-otic region ( Figure 2B; white arrowheads), the POD ( Figure 2B; yellow arrowheads) Furthermore, quantification of the area occupied by POD NCCs corroborated the observed expansion of NCC localization ( Figure 2E).
Frontiers in Cell and Developmental Biology | www.frontiersin.org January 2022 | Volume 9 | Article 803370 Elevated Hoxb5b Activity is Sufficient to Expand Vagal NCC Marker Expression That vp16-hoxb5b expressing embryos contained an overabundance of NCC, and that the overproduced NCC were prominently enriched in the vagal axial levels suggests that excess Hoxb5b activity influences vagal NCC development in zebrafish.
To examine if vagal NCC specification was altered following excess Hoxb5b activity, we assayed the expression of canonical marker genes of the vagal NCC, foxd3 (Lister et al., 2006) and phox2bb (Elworthy et al., 2005). At 32 hpf, foxd3 expression serves as an indicator of multipotent NCC while phox2bb indicates NCCs which are now specified to an autonomic neural lineage, particularly the ENS (Pattyn et al., 1999;Uribe and Bronner, 2015). vp16-hoxb5b expression was sufficient to widen foxd3 expression domains at 32 hpf, principally along the anterior-posterior and mediolateral directions in the vagal region, when compared to control expression patterns ( Figures 3A,B; black bars). We found that vp16-hoxb5b expression expanded the POD foxd3 + area, leading to a 1.95 fold increase in the mean domain size compared to controls ( Figure 3E). Additionally, phox2bb was also greatly expanded in response to increased Hoxb5b activity along the hindbrain (Figures 3C,D; black bars), with expansion uniformly in both the anterior-posterior and mediolateral axis, similar to the expansion of the foxd3.
Measuring the hindbrain phox2bb + domain, we observed a 43% increase in the phox2bb expressed area throughout the hindbrain following elevated Hoxb5b ( Figure 3F). Lastly, POD phox2bb expression was dramatically altered by elevated Hoxb5b (Figures 3C,D; white arrow heads), with a 2.14 times mean increase in domain size compared to wild-type controls ( Figure 3G). In all, these data indicate that elevated Hoxb5b activity drastically expands vagal NCC marker expression along the embryo. In support of the specific expansion of POD localized phox2bb expression, we also observed a corresponding increase in the number of Phox2b + cells, via whole mount fluorescent Immunohistochemistry (IHC), using an antibody against Phox2b (Supplementary Figures S1A-C; Figure 3). At 32 hpf, Phox2b + cells were observed in the POD, with 16 cells on average ( Figure 3L; arrowhead, Figure 3K). Overexpression of Hoxb5b stimulated a dramatic expansion of POD Phox2b + cells (Figures 3I,J; arrowheads). The increase in POD Phox2b + cells was also concordant with an increase in vp16-hoxb5b dosage, with 77 and 107 mean POD Phox2b + cells per animal detected following injection with either 15 pg or 30 pg of mRNA, respectively ( Figure 3K). The quantifiable increase in POD Phox2b + cells is confirmatory of our prior qualitative observations regarding increased cell number ( Figure 2) and positions Hoxb5b as a potent driver of NCC number and localization.

Hoxb5b Overexpression Increases NCC Production During Early NCC Development
While we observed supernumerary NCCs ectopically localized, and expanded expression of vagal NCC specification factors following global expression of vp16-hoxb5b mRNA, exactly when during NCC development Hoxb5b may exert its influence was still unclear. To investigate the potential temporal role(s) of Hoxb5b during NCC development, we created and utilized a novel transgenic line, Tg (hsp70l:EGFP-hoxb5b;cryaa:dsRed) ci1014 (hereafter referred to as hsp70l:GFP-hoxb5b), which enables pan ectopic expression of a GFP-Hoxb5b protein fusion under the thermally inducible hsp70l promoter (Kwan et al., 2007) ( Figure 4A). The transgene can then be activated by rapidly transferring embryos to warm 37°C culture conditions, which drives strong global expression of the EGFP-Hoxb5b fusion protein throughout the embryonic tissues (Supplementary Figure S2A). EGFP-Hoxb5b demonstrated robust and distinctive nuclear localization, which was still detectable over 24 h after embryos were returned to 28°C (Supplementary Figures S2B,C).
Two early phases in NCC development were tested, with heat shocks conducted starting either at 14 hpf, during NCC specification, or 22 hpf, early during the migratory span of posterior NCCs ( Figure 4B). After heat shock at 14 hpf, whole mount ISH at 24 hpf showed enlargement of crestin + NCC domains in GFP-Hoxb5b + embryos, over the GFP-Hoxb5b − sibling controls (Figures 4C,D), particularly prevalent in the POD NCC ( Figure 4D; yellow arrowheads). This increase in area was also accompanied by a qualitative increase in the number of crestin + cells along the posterior dorsal length of the embryo, similar to the phenotype observed in the previous Hoxb5b mRNA overexpression assays (Figures 2A-E). Quantified area of crestin + PODs confirmed the expansion of vagal NCCs ( Figure 4I). Additionally, subtle expansion of cranial NCCs (Figures 4C,D, white arrowheads) and pre-otic NCCs (Figures 4C,D, red arrowheads) was also observed, though far less striking than that of the vagal population at this stage. These data indicate that NCC localization during early specification phase of NCC development is receptive to Hoxb5b activity.
GFP-Hoxb5b induction at 22 hpf also increased crestin staining by 26-28 hpf throughout the POD (Figures 4E-H, yellow arrowheads), which was especially prominent in the NCCs most proximal to the otic vesicles (Figures 4G,H, red arrowheads). As in the heat shock at 14 hpf, increased NCC localization was also observed in GFP-Hoxb5b + embryos across the cranial NCC populations (Figures 4E-H; white arrowheads). This induction of GFP-Hoxb5b also significantly expanded measurable vagal NCC area ( Figure 4J), with a 26% mean increase in area compared to GFP − siblings. The expansion in area coupled with the increase in crestin staining replicates the results obtained via the microinjection assays presented in Figure 2, albeit more subtly, as well as indicates that 14-22 hpf is a critical period during which Hoxb5b is sufficient to alter NCC localization in the developing vertebrate body.

Increased Hoxb5b Alters Specific Vagal NCC-Derived Tissues
Due to the multipotent nature of NCC, it is possible a wide diversity of tissues can be affected by even a small perturbation in NCC development. Because we observed an overproduction of NCC following increases in Hoxb5b activity, we wondered if downstream NCC derivatives were also affected. Therefore, we first investigated the effect of vp16-hoxb5b expression on vagal and trunk NCC-derived cell types; including, pigment cells (melanophores and iridophores) and neuronal derivatives. Embryos expressing vp16-hoxb5b were able to produce pigment cells without appreciable differences (Supplementary Figures S4A-D). Both dorsal root ganglia (DRG) and Superior Cervical Ganglion (SCG) are derived from the vagal/trunk NCC pool (Durbec et al., 1996). Neurons comprising the DRG and SCG were largely normal following vp16-hoxb5b injection (Supplementary Figures S4E-H). We next assayed the NCC-derived cell population along the gut length ( Figure 5A), enteric neural progenitors, which typically migrate to the midgut level by 2 dpf and will have colonized the hindgut by 3 dpf, giving rise to neurons of the enteric nervous system (Elworthy et al., 2005;Ganz, 2018). We found that by 50 hpf, Phox2b + enteric neural progenitors significantly increased after vp16-hoxb5b expression ( Figures  5C,D), compared to the controls ( Figure 5B Figure S4J). These findings indicate that elevated Hoxb5b elicits a global increase in enteric neural progenitor number through the first 2 days of development.
To determine if the supernumerary enteric cells were capable of differentiating into neurons later in development, we utilized -8.3phox2bb:kaede transgenic embryos which label enteric progenitors during their early neuronal differentiation (Harrison et al., 2014). Surprisingly, despite the increase in enteric progenitors at 50 hpf, enteric neurons by the 3 dpf were dramatically decreased in vp16-hoxb5b expressing embryos compared to controls. Kaede + cells successfully colonized the gut length by 3 dpf in control embryos ( Figure 5G; white arrowhead), many cells of which (42%) also co-expressed the pan neuronal marker Elavl3/4 (Figures 5G,K,L), signaling the onset of neuron differentiation. In contrast, vp16-hoxb5b expressing embryos at both doses displayed a drastic loss of Kaede + and Elavl3/4 + enteric cells ( Figures 5J,K), with the remaining Kaede + cells failing to localize past the level of the midgut (Figures 5H,I; white arrowheads). The fraction of Elavl3/4 + /Kaede + cells in both vp16-hoxb5b expressing conditions were reduced, at 0.31 and 0.28 respectively, when compared to control at 0.42 ( Figure 5L). While not reaching significance, when these data are taken together with the significant reduction in total enteric cell numbers along the gut ( Figures 5J,K), they likely indicate that general enteric progenitor pool depletion along the gut affects subsequent proper numbers of enteric neurons, following elevated Hoxb5b expression. Overall, these results suggest that while supernumerary enteric neural progenitors are present at and before 2 dpf after elevated Hoxb5b, they largely depleted by the 3 dpf.

Hoxb5b influences Enteric Colonization During Early Developmental Stages
In order to ascertain the timing during which excess Hoxb5b activity affects enteric nervous system development, we again leveraged the hsp70l:GFP-hoxb5b fish line. Embryos were heat shocked during NCC specification (14 hpf), migration (21 hpf), or differentiation (48 hpf), and all were fixed at 3 dpf, as schematized in Figure 6A. Embryos were assessed for enteric neuron abundance and localization via wholemount IHC, where Elavl3/4 + cells were counted along the gut tract (same region as in Figure 5A, box). When heat shocked at 14 or 21 hpf, GFP-Hoxb5b + embryos formed significantly fewer Elavl3/4 + cells, when compared to their GFP-Hoxb5b − sibling heat shock controls ( Figures 6B-E,H). After heat shock at 48 hpf, GFP-Hoxb5b + embryos did not significantly vary in number of Elavl3/ 4 + cells ( Figures 6F-H). The distribution of Elavl3/4 + cells was weighted more heavily toward the midgut, though cells could be detected along the entire length of the gut in GFP-Hoxb5b + embryos. This genetically encoded elevation of Hoxb5b activity during early NCC developmental phases corroborated the abrogation in Elavl3/4 + cells resulting from vp16-hoxb5b mRNA injection. Overall, these data indicate that the ability of GFP-Hoxb5b to affect enteric neuron number is limited to early stages of NCC development, but not thereafter.

Excess Hoxb5b Leads to Stalled Enteric Nervous System Development
We had thus far discovered that Hoxb5b was sufficient to strongly increase NCCs at 30 and 50 hpf, but suppressed the number of enteric neural progenitor cells by 3 dpf. The loss in cells could easily be explained by an acute wave of cell death during enteric neural progenitor migration. We tested this hypothesis with whole mount IHC probing for activated Caspase-3, a marker for apoptotic cells (Sorrells et al., 2013), as well as Phox2b to label enteric neural progenitors. In addition, we also conducted these experiments in the Tg (-4.9sox10:eGFP) embryos (hereafter referred to as sox10:GFP) (Carney et al., 2006), which marks migratory NCCs with cytoplasmic GFP, and relying on residual GFP signal post fixation to label the recently sox10 + enteric neural progenitor cells, as we have previously . Notably, Caspase-3 + cells were rare in all controls tissues examined from 33 to 66 hpf (Supplementary Figures  S3A,C,E,G). While small patches of apoptotic cells can be found proximal to the POD at 33 hpf and 55 hpf (Supplementary Figures S3B,D), there was not a detectable onset of cell death between 55 and 66 hpf along the entire vagal and gut region (Supplementary Figures S3F,H) to support the loss of enteric neural progenitors through apoptosis following Hoxb5b overexpression.
We next examined the progenitor state of enteric cells at 63 hpf, a window of development prior to the 3 dpf cut off, but after the 50 hpf NCC expansion noted previously. To this end, we asked if sox10: GFP + and/or Phox2b + cell numbers were reduced along the gut at 63 hpf. Intriguingly, vp16-hoxb5b did not lead to a significant change in the total number of GFP + cells along the gut tube at 63 hpf ( Figures 7A-C). Similarly, there was no change in the number of Phox2b + cells, compared with control embryos ( Figure 7C). This was in contrast to our earlier observation that enteric Phox2B + cells were increased at 50 hpf ( Figure 5). Furthermore, the fraction of sox10:GFP + cells expressing Phox2b was unchanged ( Figure 7D). Therefore, we found that by 63 hpf the enteric progenitors exhibited enteric differentiation capacity, despite their decreased abundance in the presence of excess Hoxb5b.
That we observed increased Phox2b + cells at 32 and 50 hpf, yet saw a dampening of their numbers by 63 hpf, this suggested that the kinetics of enteric progenitor expansion may have been adversely affected following elevated Hoxb5b. Plotting the total number of Phox2b + cells counted from each developmental stage assayed throughout this study ( Figure 7E) revealed a steady increase in control Phox2b + cells with developmental age, whereas Hoxb5b overexpressing animals presented a stalled curve at 63 hpf. As we previously have shown that the enteric cells are not cleared by apoptosis during the 63-80 hpf transition, these results indicated that Hoxb5b activity modulates enterically-fated NCC capacity to expand as a population along the gut. The Hoxb5b-dependent precocious expansion of NCC leads to a fixed number of available progenitors which are then unable to expand in sufficient numbers to lead to proper ENS formation. These findings position Hoxb5b as a fine-scale regulator of enteric NCC number. Frontiers in Cell and Developmental Biology | www.frontiersin.org January 2022 | Volume 9 | Article 803370

DISCUSSION
We discovered that throughout the course of NCC specification and migratory phases, hoxb5b is expressed within the vagal NCC domain along the post-otic/posterior zebrafish embryo. Enhancement of Hoxb5b activity was sufficient to dramatically expand NCC localization patterns, as well as their number, along the embryo. The expansion of NCCs was also accompanied by domain expansions in vagal NCC marker genes phox2bb and foxd3. Temporally-restricted pulses of ectopic Hoxb5b during early vagal NCC developmental phases was sufficient to swiftly expand vagal NCC populations, which also persisted well into the second day in development. While many vagal NCC derivatives were unaltered by 3 dpf, NCC derivatives along the enteric neural trajectory were dramatically impacted. While we observed an increase in enteric neural progenitors along the developing gut tube at 50 hpf, they failed to expand and colonize the gut efficiently, resulting in a marked decrease in the number of enteric neurons. The decrease in enteric neural progenitors following Hoxb5b induction appears to be due to a Hoxb5b- Frontiers in Cell and Developmental Biology | www.frontiersin.org January 2022 | Volume 9 | Article 803370 dependent modulation of the colonization capacity of enteric neural progenitors. Cumulatively these data position Hoxb5b as a potent regulator of NCC patterning and number during early embryonic development ( Figure 7F). The potential involvement of Hoxb5b in zebrafish NCC development has been suggested by previous expression analyses, with discrete expression along the dorsolateral neural tube and in the post-otic domain, posterior to rhombomere 8 (Kudoh et al., 2001;Jarinova et al., 2008;Waxman et al., 2008;Barsh et al., 2017). Additional expression domains are found within the lateral plate mesoderm and foregut by 24 hpf (Dalgin and Prince, 2021). As identified in a single cell atlas of posterior zebrafish NCC lineages at 48-50 and 68-70 hpf, hoxb5b was among the most pervasively expressed transcripts encoding for a Hox transcription factor in both the NCC and in neural fated lineages . Extending this prior work, our HCRs show for the first time at high resolution the persistent expression of hoxb5b in zebrafish posterior (post-otic) NCC through the early course of their development.
In our zebrafish model, despite the regional potential exhibited by NCCs (Rocha et al., 2020), elevated Hoxb5b uniformly expanded cranial, vagal, and trunk NCC progenitors, though more robustly among the posterior NCC populations. Whether the Hoxb5b-dependent increase in NCCs is driven by increased NCC specification from the neural tube or through upregulation of NCC proliferation remains unresolved and was beyond the current scope of this study. Regardless of the underlying mechanism, these findings clearly indicate that Hoxb5b participates in NCC development from an early position in the NCC gene regulatory network (Simoes-Costa and Bronner, 2016;Martik and Bronner, 2017). Further, the rapid sensitivity of vagal NCC to temporally restricted pulses of elevated Hoxb5b suggests competency of these cells to abruptly respond to Hox activity early in their development. Indeed, early NCCs appear sensitive not only to Hox mediated activity but also the amount of Hoxb5b present. In our injection experiments, POD NCCs increased coordinately with amount of vp16-hoxb5b mRNA delivered. As such, the number of cells fated in select NCC lineages, such as the enteric NCCs, appear to be influenced not only by the activation of Hoxb5b-dependent activity, but also in part through the levels of Hoxb5b expression. From data derived across multiple animal models, Hoxb5b and its orthologues are known to be under control of several classical morphogenic signals, including WNTs (Lengerke et al., 2008), NOTCH (Hortopan and Baraban, 2011), and Retinoic Acid (Waxman et al., 2008), which enables fine spatiotemporal tuning of the levels of Hox expression. Summarily, our data thus illuminate a model in which Hoxb5b serves a potent regulator of posterior NCC identity and cell number, dependent on additional unspecified cofactors as well as its expression level.
Our findings regarding Hoxb5b in zebrafish NCC is complementary to and extends the developmental understanding of mammalian Hoxb5. For example, dominant negative suppression of Hoxb5 activity in NCC lineages led to a depletion of several NCC-derived cell populations including DRGs, pigment cells, as well as enteric neural progenitors (Lui et al., 2008;Kam et al., 2014b). Complementing these prior loss of function studies, our gain of function data indicates that elevated Hoxb5b activity is sufficient to induce expansion of vagal NCC progenitors-that paradoxically also leads to severe ENS hypoganglionosis. While we did not observe corresponding changes in DRG and pigment populations in our experiments; early enteric progenitors were dramatically increased in response to Hoxb5b activity along the gut, prior to the onset of neurogenesis, yet failed to properly execute enteric neuronal differentiation. Somewhat counterintuitively, the increase in POD NCCs following increased Hoxb5b activity did not correspondingly manifest a pan increase in vagal-derived NCC lineages. While many vagal-derived lineages exhibited no discernable phenotypic change, we observed a dramatic decrease in the number of enteric neurons in animals with elevated Hoxb5b activity. The shift in abundance of enterically fated cells in embryos overexpressing Hoxb5b was not the result of NCC-specific apoptosis or abrogated differentiation potential. Indeed, there was no change in fractions of enteric NCCs which had initiated differentiation programs following elevated Hoxb5b. Rather this tissue specific cell decrease appears to be caused by a late-onset suppression of enteric neuroblasts expansion. Assays for apoptosis-mediated cell death revealed limited tissue death, which was not restricted to NCC populations. While we did not test cell loss by other modes of death, such as necrosis or pyroptosis, the numbers of enteric NCC-derived cells were more consistent with the arrest of cell division as opposed to changes in cell survival. The insufficiency of ectopic Hoxb5b to expand the DRG and pigment lineages suggests a separate regulatory mechanism for Hoxb5b gain of function activity in enteric NCC.
Several possible frameworks may explain the differential role of Hoxb5b to expand early NCCs but abrogate their later expansion in the ENS. First, the differential role may be correlated with the temporally dynamic expression of additional co-factors, such as other Hox or TALE-family transcription factors within the ENS linage. TALE-family factors in particular, such as Meis3 (Uribe and Bronner, 2015), or other factors such as Pbx3 (Di Giacomo et al., 2006) are both expressed within the early enteric neural lineage and may cooperate with Hoxb5b to facilitate functional specificity. Additional reflection on the emerging numerous descriptions of combinatorial "Hox Codes" which define NCC identity (Parker et al., 2019;Soldatov et al., 2019;Howard et al., 2021), reported functional similarity in certain tissues (Jarinova et al., 2008), as well as complex regulatory relationship between various members of the Hox gene family (Zhu et al., 2017), the prospect of a shared sensitivity in enteric NCCs to Hoxb5b and other posterior Hox transcription factors is possible, though beyond the context of this study. Another alternative hypothesis to explain Hoxb5b's differential role may be related to the precociousness of the expansion of neural crest itself. As more NCCs accumulate earlier in development, temporally restricted signals may be insufficiently timed to signal for NCCs to continue to expand in number. While the underlying mechanism remains to be fully elucidated, when the data are considered together with mammalian suppression of activity studies referenced above, our gain of function results suggest that the vertebrate embryo is exquisitely sensitive to perturbations in Hoxb5 activity, where either elevations or reductions in Hoxb5 lead to severe ENS defects. Collectively, we have discovered evidence in support of a model in which Hoxb5b plays an important role in NCC development, demonstrating the capacity to both expand vagal NCC localization and numbers. While additional questions still remain, these findings greatly inform our understanding of the role of posterior Hox genes in NCC development.

Generation of the Tg (hsp70l:EGFP-Hoxb5b) Transgenic Line
To generate EGFP-Hoxb5b, egfp was fused to the 5′-end of zebrafish hoxb5b with PCR. A sequence encoding a 7 amino acid linker was incorporated and the hoxb5b ATG was deleted to prevent alternative transcriptional initiation of hoxb5b downstream. To generate the hsp70l:EGFP-Hoxb5b transgene, standard Gateway methods were used (Kwan et al., 2007). The transgene includes a EGFP-Hoxb5b middle-entry vector and the reported p5E-hsp70l 5′-Entry and p3E-polyA 3′-entry vectors (Kwan et al., 2007), which were incorporated into the pDestTol2-acry:dsRed vector (Mandal et al., 2013). Sanger sequencing was used to confirm the proper orientation of the constructs within the destination vector and the sequence of EGFP-hoxb5b. Transgenic embryos were created by co-injecting wild-type embryos at the one-cell stage with 25 pg hsp70l:EGFP-hoxb5b vector and 25 pg of Tol2 mRNA (Kawakami, 2004;Kawakami et al., 2004). Embryos were raised to adulthood and screened for the present of dsRed in the lens at ∼3 days and the ability to induce robust EGFP expression following heat-shock (Supplementary Figure S2). Multiple founders for the Tg (hsp70l:EGFP-hoxb5b;acry:dsRed) ci1014 line were identified so the line that induced the most robust expression following heat-shock was retained. While some ectopic notochord expression was observed in non-heat shocked embryos they did not exhibit overt phenotypes and developed normally. For heat shock experiments, through routine outcrossing of transgenic animals to the wild type embryos of the AB/TL backgrounds, GFP-hoxb5b −/− siblings are produced with each subsequent breeding, which were heat shocked and processed in parallel.

Preparation and Injections of hoxb5b mRNA
Capped vp16-hoxb5b mRNA was prepared off a Not1 linearized pCS2+ plasmid containing the vp16-hoxb5b coding sequence using the Sp6 mMessage Kit (Ambion), as first reported in (Waxman et al., 2008). The vp16-hoxb5b construct encodes for a hyperactive form of Hoxb5b and allowed for lower doses of mRNA to be delivered (Waxman and Yelon, 2009). Embryos were injected prior to the four-cell stage with either 15 pg or 30 pg of mRNA and were empirically determined to produce similar phenotypes. Dosage of mRNA was determined per experimental condition. Uninjected wild type sibling embryos were cultured in parallel with injected animals and used for controls. Dead and grossly malformed embryos were removed from analysis.

In Situ Hybridization
In situ hybridizations were performed similarly to the protocol of Jowett and Lettice, 1994, which should be referenced for specific details. Briefly, antisense digoxigenin-labeled riboprobes were generated from previously characterized plasmids containing sequences for crestin (Luo et al., 2001), foxd3 (Odenthal and Nüsslein-Volhard, 1998;Hochgreb-Hagele and Bronner, 2013), phox2bb (Uribe and Bronner, 2015), and hoxb5b (Waxman et al., 2008). As per the protocol, PFA-fixed whole mount embryos stored in methanol were rehydrated in PBST, permeabilized with Proteinase K digestion (10 μg/ml), and post-fixed in 4% PFA. Embryos were incubated in probes overnight (∼16 h) at 65°C and washed sequentially in graded SSCT buffers. Riboprobes solutions were recovered and stored at −20°C for reuse, with multiple uses leading to minimal loss of signal. Probed embryos were blocked for 1-2 h at ambient temperature in 5% Goat sera in PBST before detection overnight (∼16 h) at 4°C using an anti-Digoxigenin-Fab fragments conjugated to Alkaline Phosphatase enzymes (1:1,000 dilution, Roche) in 5% Goat Sera in PBST. Finally, riboprobes were visualized with NBT/BCIP solution (3.5 μL each of NBT, BCIP stock solutions, Roche). Probes were validated prior to use on wildtype embryos to calibrate staining duration, with patterns compared to those curated on ZFIN (Howe et al., 2013;Ruzicka et al., 2019).
Whole Mount Immunohistochemistry, HCR, and WICHCR Immunohistochemistry (IHC), Hybridization Chain Reaction (HCR), and Whole mount Immuno-Coupled Hybridization Chain Reaction (WICHCR) protocols all were conducted according to the methods published in Ibarra-García-Padilla et al., 2021. All IHC assays conducted in blocking 5% Goat Sera in 1X PBST. Primary antibodies against the following Heat Shock Induction of the hsp70l: GFP-hoxb5b Transgene Adult zebrafish maintained as an outcross and positive for dsRed expression as larvae in the lens (see above method on description of the line) were bred to produce synchronously staged embryos. At the stage designated to begin the heat shock, embryos were rapidly transferred to 37°C E3 and maintained at that temperature. After a 1-h incubation, embryos were rapidly returned to 28°C. In the 1-3 h after heat shock GFP-Hoxb5b + embryos were sorted from GFP − siblings and cultured in parallel until the designated stage for tissue collection.

Imaging, Quantification, and Image Visualization
All embryos prior to imaging were cleared through graded washes of PBST/Glycerol to reach a final Glycerol content of 75%. Fluorescent Z-stacked images of IHC processed embryos were captured using an Olympus FV3000 point scanning confocal microscope supported by Fluoview Acquisition Software (version FV31S-SW). Images were stitched in FIJI (ImageJ version 1.53e) using the Grid/Collection applet as part of the Stitching plugin (Schindelin et al., 2012;Schneider et al., 2012;Rueden et al., 2017). Digital image files were converted with ImarisFileConverter Software (Bitplane) to three dimensional rendered images compatible with IMARIS (V9.4, V9.7, Bitplane). All images of fluorescent animals represented in this publication are derived from maximum intensity projections of the z-stacked image. Cells counts were conducted on volume images following an arithmetic background subtraction in IMARIS to ensure accurate counts, particularly when determining coincidence of labels. ISH processed embryos were imaged on a Nikon Eclipse Ni microscope equipped with a motorized stage. Z-stack images were acquired and extended depth of focus images were generated in the Nikon NIS-Elements BR software (v5.02.00). Areas of expression were measured in FIJI to include dark pixels in the post-otic or hindbrain domains. Quantifications were curated and analyzed in the Rstudio programming environment (v1.1.463). Images of pigmented embryos were captured similarly on the Nikon microscope with lateral illumination to distinguish iridophores, similar to (Petratou et al., 2021).

In vivo Confocal Microscopy
Embryos were sorted for RFP expression, anesthetized with 0.4% tricaine, and embedded in 1% low melting agarose in a 28.5°C chamber with a coverslip glass bottom. Care was taken to ensure embryo angled appropriately and proximal to the glass. Z-stack images were acquired approximately every half hour concurrently on the same Olympus FV3000 point scanning confocal microscope as above. Maximum intensity projections images were generated and exported from FIJI.

Statistical Analysis
An α of 0.05 was used as a cut off for all statistical tests. Normalcy of datasets was assessed by visual inspection of a density plot, a qqplot against a linear theoretical distribution, and Shapiro-Wilk test for Normalcy. Further, variance between each dataset was examined either with a Bartlett test for data which adhered to normalcy or with a Levene's test for non-normal data. Based on the normalcy and scedasticity conditions of the data, the appropriate statistical test was selected, as summarized in Table 1. All statistical analyses were carried out in the Rstudio (v1.1.463) programming environment, with key dependencies on the lawstat (v3.4) and stats (v3.6.3) packages. Plots were generated in Rstudio supported by the ggplot2 (v3.3.2) and ggsignif (v0.6.0) packages.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.

ETHICS STATEMENT
The animal study was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Rice University and Cincinnati Children's Hospital Medical Center.

ACKNOWLEDGMENTS
We offer our sincerest gratitude to Dr. Dan Wagner and to the entire Uribe Lab at Rice University for their insights and support throughout this project. We thank Dr. Budi Utama and the Rice University Shared Equipment Authority on IMARIS image analysis suite, which was indispensable toward this project. We thank Dr. Eric Bridenbaugh and Dr. Mariane Martinez at Olympus for their expert advice on confocal microscopy. We also thank Jonny Diaz and A. Augello Cook for technical assistance.

SUPPLEMENTARY MATERIAL
The the hindbrain exhibit coincidence of all three probes along the Hindbrain (Hb), Pharyngeal (Pa), and PODs (arrowheads). Kaede + cells which are negative for both mRNA and protein are detected at the dorsal most aspect of the motor neuron complex (star) as well as in anterior regions of the presumptive CNS. (C) Images highlighting the midgut axis reveals a perfect coincidence of all three enteric labels with very little background, demonstrating the efficacy of the Phox2b antibody as an enteric neural crest label. While nearly every cell is coincident, selected cells are annotated to aid in comparison (arrowheads). Scale bar A: 100 μm.
Supplementary Figure S2 | Characterization of novel hsp70l:GFP-hoxb5b transgenic zebrafish line. (A) GFP-Hoxb5b + overexpressing embryos at 24 hpf are easily distinguishable from GFP − siblings 1 hour after heat shock. GFP − and GFP + embryos are cultured and assayed under identical conditions to control for variations induced by growth at an elevated temperature. (B-C) GFP-Hoxb5b construct is localized to cell nuclei following heat shock and persists at least a day after initial induction (C). Scale bar B: 100 μM.
Supplementary Figure